Carbohydrate Chemistry v.15 - Part II

Carbohydrate Chemistry Volume 15 Part II A Specialist Periodical Report Carbohydrate Chemistry Volume 15 Part II Mac...

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Carbohydrate Chemistry Volume 15 Part II

A Specialist Periodical Report

Carbohydrate Chemistry Volume 15 Part II Macromolecules A Review of the Literature Published during 1981 Senior Reporter J. F. Kennedy, University of Birmingham Reporters

D. P. Atkins, University of Birmingham

1. M. Morrison, Hannah Research Institute, Ayr C. M. Sturgeon, University of Edinburgh R. J. Sturgeon, Heriot- Watt University, Edinburgh C. A. White, University of Birmingham

The Royal Society of Chemistry Burlington House, London W I V OBN

ISBN 0-85186-152-0 ISSN 0576-7172

Copyright @ 1986 The Royal Society of Chemistry All Rights Reserved No part of this book may be reproduced or transmitted in any form or by any means graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems - without written permission from The Royal Society of Chemistry

-

Printed in Great Britain by Whitstable Litho Ltd., Whitstable, Kent

Preface This Report, t h e f i f t e e n t h o f t h i s T i t l e , is, l i k e its predecessors, a c o m p r e h e n s i v e summary of p u b l i c a t i o n s , b o t h p r i m a r y and s e c o n d a r y , p r e s e n t e d i n a c o n c i s e and r e a d a b l e form. In s p i t e o f t h e s u p p o r t i v e comments we h a v e r e c e i v e d for t h i s T i t l e from r e a d e r s i n v a r i o u s p a r t s o f t h e w o r l d , r e f e r e n c e must be made t o t h e 1985 d e c i s i o n o f t h e Royal S o c i e t y o f C h e m i s t r y t o cease p u b l i c a t i o n o f ' C a r b o h y d r a t e C h e m i s t r y ' P a r t I1 i n i t s p r e s e n t form.

T h i s d e c i s i o n h a s been b a s e d on

i n s u f f i c i e n t income t o s u p p o r t t h e h i g h c o s t o f p r o d u c t i o n .

Such a c e s s a t i o n

was h i n t e d a t i n t h e p r e f a c e t o Volume 14 a s b e i n g a p o s s i b i l i t y .

It is

r e g r e t t a b l e n o t o n l y s i n c e t h e c a r b o h y d r a t e macromolecular c h e m i s t r y may no l o n g e r b e a v a i l a b l e i n s u c h a n a c c e s s i b l e form b u t a l s o s i n c e so much e f f o r t h a s b e e n p u t i n t o t h e change t o t h e p r o d u c t i o n o f camera-ready c o p y d e s p i t e t h e problems a s s o c i a t e d w i t h t h e c o m p l e x i t i e s o f c a r b o h y d r a t e names and s t r u c t u r e s and a b b r e v i a t e d forms t h e r e o f . I n t h e p r o d u c t i o n o f t h i s volume I h a v e been s u p p o r t e d o n c e a g a i n by c o l l e a g u e s who have u n s t i n t i n g l y c o n t r i b u t e d t o many volumes o f t h i s T i t l e and by Drs C . A. White and D. P. A t k i n s who j o i n e d i n t h e p r o d u c t o n o f t h i s volume t o e a s e t h e work-load.

I a g a i n v e r y g r a t e f u l l y acknowledge t h e c o n s i d e r a b l e

e f f o r t p u t i n by e v e r y r e p o r t e r , and t h e h e l p a n d a d v i c e t h e y have g i v e n . F i n a l l y , t h e e d i t o r i a l and o t h e r a s s i s t a n c e p r o v i d e d by Mrs R.H.

Pape,

A s s i s t a n t E d i t o r , Books, a t t h e Royal S o c i e t y o f C h e m i s t r y i n t h e p r o d u c t i o n o f t h i s Report i s much a p p r e c i a t e d . A p r i l 1986

JOHN F. KENNEDY

Contents Chapter 1

Introduction By J . F. Kennedy

1

Chapter 2

G e n e r a l Methods By R. J. S t u r g e o n

3

1

Gas-Li q u i d Chromatography

3

2 Column and Ion-exchange Chromatography

5

3 T h i n - l a y e r Chromatography

5

4 H i g h - p r e s s u r e L i q u i d Chromatography

6

5 Electrophoresis

9

6 A n a l y t i c a l Methods

10

S t r u c t u r a l Methods N. M. R. S p e c t r o s c o p y Mass S p e c t r o m e t r y M i s c e l l a n e o u s Methods

13 13 14 15

P l a n t a n d Algal P o l y s a c c h a r i d e s

21

1

Introduction

21

2

Starch

21

3 Fruct a n s

31

4 Ce 11u l o s e

32

5 H e m i c e l l u l oses

39

6 Pectins

48

7 Gums a n d M u c i l a g e s

53

8 Algal Polysaacharides

56

7

Chapter 3

By I. M. M o r r i s o n

Microbial P o l y s a c c h a r i d e s By C. A. Whibe

70

1

Teichoic Acids

70

2

Peptidoglycans

77

Chapter 4

viii

Carbohydrate Chemistry 3

Lipopolysaccharides

89

4

Capsular Polysaccharides

107

5

E x t r a c e l l u l a r and I n t r a c e l l u l a r P o l y s a c c h a r i d e s

119

6

Miscellaneous B a c t e r i a l Polysaccharides

136

7

Fungal Polysaccharides D - G l ucan s D-Ma nn a n s Chitin Miscellaneous Cell-wall Polysaccharides

141 145 150 157 158

C l y c o p r o t e i n s , G l y c o p e p t i d e s , P r o t e o g l y c a n s , and Animal P o l y s a c c h a r i d e s By R . J. S t u r g e o n

168

1

Microbial Glycoproteins Fungal G l y c o p r o t e i n s Viral Glycoproteins

168 168 169

2

Plant Glycoproteins

178

Chapter 5

3 Lectins

179

4

Fibronectin

193

5

Collagen

197

6

Glycogen

202

7

Glycosaminoglycans and Proteoglycans Analysis O c c u r r e n c e , I s o l a t i o n , and S t r u c t u r e Biosynthesis Pa t h o l o g y

203 20 3 205 21 6 219

8 Cell and T i s s u e G l y c o p r o t e i n s

222

9

Cell-surface Glycoproteins

234

10

G l y c o p r Q t e i n Hormones

249

11

Milk G l y c o p r o t e i n s

253

12

Serum G l y c o p r o t e i n s

25 8

13

Immunoglobulins

269

14

Erythrocyte Glycoproteins

284

15

S a l i v a r y a n d Mucous G l y c o p r o t e i n s

292

16

U r i n a r y G l y c o p r o t e i n s , G l y c o p e p t i d e s , and 01 i g o s a c c h a r i d e s

298

17

Avian C l y c o p r o t e i n s

30 1

18 M i s c e l l a n e o u s C l y c o p r o t e i n s a n d C h i t i n

307

Contents

Chapter

19 Analysis of Glycoproteins

310

20 Biosynthesis of Glycoprotelns

314 347

6 Enzymes By J. F. Kennedy 1

Introduction General Aspects and Nomenclature Methods of Assay Kinetics Mechanisms o f Action Applications Enzyme Immobilization

2

fi-D-2-Acetamido-2-deoxygalactosidases, B-D-2-Acetamido-2-deoxyglucosidases, 6-D-2-Acetamido-2-deoxyhexosidases

3 a-L-Arabinofuranosidases

347 347 347 348 348 348 349 and

and a-L-Arabinopyranosidases

350 376

4 B-D-Fructofuranosidases

378

5 D-Fructose-1.6-bisphosphatase

383

6 B-D- and a-L-Fucosidases

384

7

aa-

and 8-D-Galactosidases and D-Galactolipid-oriented and B-D-Galactosidases

390

8

a-

and B-D-Glucosidases

414

9 B-D-Glucuronidases

441

10 a-L-Iduronidases

445

11

446

a-

and B-D-Uannosidases

12 Ne ur ami n id a ses ( Sia 1 ida ses 1

450

13 6-D-Xylosidases

455

14

e8-D-2-Ace tamido-2-deoxygluc anases

456

15 Agarases

457

16 Alginases and Alginate Lyases

458

17 a4mylases

458

18 6-Amylases

477

19 Amylo-l,6-D-glucosidases

481

20 Cellulases

482

21

497

Chitinases

22 Dextranases 23

( 1-3 1-aCD-Glucanases

498 499

Carbohydrate Chemistry

X

500

24 endo-( 1-3)-~-D-Glucanases 25

exo- ( 1 -

-

502

4) B-D-Gl ucanases

-

503

endo-(1-6)-B-D-Glucanases

506

28 D-Glucanases (Miscellaneous)

507

26 endo-( 1-4)-~-D-Glucanases 27

29

G 1ucoamy 1a se s

509

30

Glycanases (Miscellaneous)

513

31

Heparin Hydrolases

514

32

Hyaluronidases

515

33

Inulinases

518

34

Isoamylases

35

Laminaranases

519

36

Ly so2 yme s

519

37

Oligo-1 ,6-D-glucosidases

528

3a

Pectate, Pectin, and Poly-D-galacturonate

39

Poly-D-galacturonases

40

exo-Poly-D-galacturonate

41

Pullulanases

534

42

Sucrose-a-D-glucohydrolase

535

Lyases

529 531

Lyases

534

43 aa- and B B-Trehalases

536

44

endo- ( 1*4) -8-D-Xyl -

537

45

Xylanases (Miscellaneous)

539

46

Carbohydrate Isomerases D-Xylose Isomerases (D-Glucose Isomerase) L-Ribose Isomerases

54 1 54 1 544

47

Carbohydrate Oxidases D-Glucose Oxidases L-Galactonolactone Oxidases L-Gulonolac tone Oxidases Cellobiose Oxidases L-Fructose Oxidases D-Galactose Oxidases

545 545 54 a 549 549 549 54 9

anases

48 Carbohydrate T r an sferases

550

49

L-Iduronic Acid 2-Sulphate Sulphatases

555

50

Arylsulphatases

556

xi

Contents 558

51

2-Acetamido-2-deoxy-D-glucose

52

M i s c e l l a n e o u s Enzymes Dextransucrase Syn t h a s e s Lya se s De h y d r o g e n a s e s Levansucrase Pectinesterases

558 558 560 562 562 563 564

G l y c o l i p i d s and G a n g l i o s i d e s By I. M. M o r r i s o n

578

1

Introduction

578

2

A n a l y t i c a l and G e n e r a l Methods

578

3

Gangliosides

579

4

Animal G l y c o l i p i d s

592

5

Plant Glycolipids

605

6

Microb i a 1 G l y c o l i p i d s

609

Chemical S y n t h e s i s and M o d i f i c a t i o n o f O l i g o s a c c h a r i d e s , P o l y s a c c h a r i d e s , G l y c o p r o t e i n s , Enzymes, and G l y c o l i p i d s By C. M. S t u r g e o n

61 8

Chapter 7

Chapter 8

6-Sulphate Sulphates

1 S y n t h e s i s of P o l y s a c c h a r i d e s , O l i g o s a c c h a r i d e s , G l y c o p r o t e i n s , G l y c o p e p t i d e s , and G l y c o l i p i d s Polysacchar i d e s Oligosaccharides Glycoproteins G 1y c o p e p t i d e s Glycolipids 2

3

M o d i f i c a t i o n o f P o l y s a c c h a r i d e s and O l i g o s a c c h a r i d e s a n d Uses of M o d i f i e d P o l y s a c c h a r i d e s and O l i g o s a c c h a r i d e s Introduction Agarose Amylose Cellulose Chitin Chitosan Cycloamyloses De x t r a n s D-Glucan s G 1ycosami n o g l y c a n s D-Mannans M i s c e l l a n e o u s P o l y s a c c h a r i d e s , O l i g o s a c c h a r i d e s , and G 1y c o l i p i d s Starch M o d i f i c a t i o n of G l y c o p r o t e i n s and Uses of Modified G 1y c o p r o t e i n s Albumins Antibodies Phytohaemagglutinins Transferrin

61 8 61 8 61 9 630 63 1 632 633 633 6 36 675 676 67 9 680 68 1 683 685 685 685 686 687 687 688 688 68 9 689


xii Miscellaneous Glycoproteins Immobilized D e r i v a t i v e s of Glycoproteins Immobilized C e l l s

4 Modification of Enzymes and Uses of Modified Enzymes Acid Phosphatase B-D-Fucosidase B-D-Galactosidase Glucoamylase D-Glucose Isomerase Hya lur onid a s e Lysozyme Miscellaneous Glycoenzymes Immobilized Derivatives of Enzymes

Author Index

689 690 71 4 718 71 8 718 71 8 718 718 718 71 8 719 719 772

Abbreviations

The f o l l o w i n g a b b r e v i a t i o n s have been u s e d : C h e m i c a l s a n d Chemical Groups AD P adenosine 5 I-diphosphate AMP adenosine 5'-phosphate ATP adenosine 5'-triphosphate CDP c y t i d i n e 5'-diphosphate CM P c y t i d i n e 5'-phosphate DEAE N,N-diethylaminoethyl DMF E,N-dimethylformamide DM SO dimethyl sulphoxide DNA deoxyribonucleic acid GDP guanosine 5'-phosphate RNA ribonucleic acid THF tetrahydrofuran RIS trimethylsilyl UDP u r i d i n e 5'-diphosphate P h y s i c o c h e m i c a l Methods c.d. c i r c u l a r d i c h r o is m d.s.c. d i f f e r e n t i a l scanning calorimetry e.s.r. e l e c t r o n s p i n resonance gas-liquid chromatography-mass spectrometry g c -m. s g.1.c. gas-liquid chromatography g.p.c. g e l permeation chromatography h.p.1.c. high-performance l i q u i d chromatography i.r. infrared m.s. mass s p e c t r o m e t r y n u c l e a r magnetic resonance n .m.r. 0.r .d. optical rotatory dispersion t.1.c. thin-layer chromatography U.V. ultraviolet

..

.

1

Introduction BY J. F. KENNEDY

The o b j e c t i v e s o f t h e P a r t I 1 R e p o r t h a v e r e m a i n e d i n t h e a r e a o f p r o v i d i n g a summary o f t h e l i t e r a t u r e o f c a r b o h y d r a t e - c o n t a i n i n g and c a r b o h y d r a t e - d i r e c t e d macromolecules,

but s i n c e t h e i n c e p t i o n of

C a r b o h y d r a t e Chemistry’ f o u r t e e n volumes and t h e r e f o r e f o u r t e e n y e a r s ago,

t h e coverage has n e c e s s a r i l y changed.

T h i s change has o f t e n

b e e n n o t h i n g more t h a n k e e p i n g up t o d a t e w i t h i m p r o v e m e n t s i n t h e s c i e n t i f i c u n d e r s t a n d i n g o f t h e m a c r o m o l e c u l e s concerned.

However,

s i n c e w h a t h a v e come t o be c a l l e d b i o t e c h n o l o g i c a l p r o c e s s e s the l i f e aspects o f

molecules

-

-

i.e.

h a v e become a d r i v i n g f o r c e i n

t o d a y ’ s s c i e n t i f i c t h i n k i n g , s o i n t h i s T i t l e we h a v e c o n t i n u e d t o c i t e a p p l i c a t i o n s of

t h e m a c r o m o l e c u l e s as w e l l as t o r e p o r t on t h e

b a s i c science. The i n t r o d u c t i o n t o

Volume 14 P a r t

comprehensive and s e t t h e scene f o r Report.

111 w a s n e c e s s a r i l y

subsequent

volumes o f t h i s

I n i t , t h e f o l l o w i n g a s p e c t s were covered:

1.

Scope and C o v e r a g e o f t h e R e p o r t ,

2.

O r g a n i z a t i o n , N o m e n c l a t u r e , a n d Use o f t h e R e p o r t ,

3.

S i g n i f i c a n t Advances i n M a c r o m o l e c u l a r C a r b o h y d r a t e

4.

C o n c l u s i o n s and R e a d e r s h i p ,

Chemistry,

and r e a d e r s o f t h i s volume a r e advised t o c o n s u l t t h e p r e v i o u s one t o d e r i v e maximum b e n e f i t . March

1982 saw

Carbohydrate Research

-

the

e a r l y on i n i t s l i f e t i m e , important carbohydrate

publication

of

a n o t a b l e landmark.

the

100th volume

This publication,

of

very

became a p r i m a r y p u b l i c a t i o n c h a n n e l f o r work,

particularly

academic,

and i t i s

p l e a s i n g t o see t h a t t h e j o u r n a l l o o k s e a s i l y s e t f o r a n o t h e r one h u n d r e d volumes.

The s i s t e r j o u r n a l ,

p u b l i s h e s a r t i c l e s more i n t h e f i e l d s research,

Carbohydrate of

Polymers,

which

a p p l i c a t i o n and i n d u s t r i a l

h a s a l o n g way t o go t o c a t c h t h i s u p ,

because i t i s a

2


much y o u n g e r j o u r n a l .

N e v e r t h e l e s s t h e r e i s scope f o r b o t h j o u r n a l s

t o c o n t i n u e s u c c e s s f u l l y w i t h t h e c o n t i n u a l s i g n i f i c a n t advances i n b o t h a c a d e m i c and i n d u s t r i a l c a r b o h y d r a t e c h e m i s t r y . A s p e c i a l i s s u e o f C a r b o h y d r a t e R e s e a r c h 2 was p u b l i s h e d t o

honour

Professor

Sumio

Umezawa

and

his

work

on

antibiotics,

p a r t i c u l a r l y i n the f i e l d o f sugar-containing antibiotics. Re f e r e n c e s 1.

Kennedy,

J.F.

(Macromolecules), Reports), 2.

i n ed.

‘Carbohydrate

Chemistry’,

Kennedy

(Specialist

J.F.

The R o y a l S o c i e t y o f C h e m i s t r y ,

p.

1.

T.

Tsuchiya,

Carbohydr.

e., 1982,

109,

London,

1.

Part

I1

Periodical

1983,

Vol.

14,

2

General Methods BY R. J. STURGEON 1 G a s - L i q u i d Chromatoqraphy

An i m p r o v e d g.1.c.

method f o r t h e simultaneous d e t e r m i n a t i o n o f

a l d i t o l a c e t a t e s o f n e u t r a l and amino-sugars

has been r e p o r t e d . ’

S h o r t r e t e n t i o n t i m e s and b a s e l i n e s e p a r a t i o n b e t w e e n Q - g l u c o s e a n d a-galactose

were

recorded.

The

method

has

been

used

for

the

a n a l y s i s o f s i a l o g l y c o p r o t e i n s f r o m bone. A c h i r a l p o l y s i l o x a n e s t a t i o n a r y phase has been used i n t h e

9.1. c.

analysis o f mixtures o f neutral

s e p a r a t i o n of 3-g-methylg.1.c.

and a m i n o - s u g a r s . 2

Complete

t h e a l d i t o l a c e t a t e s o f t h e n e u t r a l sugars,

including

and 4 - ~ - m e t h y l - ~ - g l u c i t o l s , was a c c o m p l i s h e d .

Capillary

s e p a r a t i o n o f monosaccharides as t h e i r a l d i t o l a c e t a t e s has

been r e p ~ r t e d . ~

2-Amino-2-deoxy-Q-galactose

and 2-amino-2-deoxy-~-glucose,

a f t e r r e d u c t i o n w i t h sodium b o r o h y d r i d e t o t h e c o r r e s p o n d i n g amino a l c o h o l s , and c o n v e r s i o n t o t h e t r i f l u o r o a c e t y l d e r i v a t i v e s , have b e e n s e p a r a t e d b y g.1.c. flame thermionic

and d e t e c t e d u s i n g a n i t r o g e n - s p e c i f i c

detector.4

The m e t h o d may

be a p p l i e d t o

the

d e t e r m i n a t i o n o f amino-sugars i n glycoconjugates. The q u a n t i t a t i v e g.1.c.

a n a l y s i s o f sucrose i n t h e presence o f

t h e o x i m e s o f Q - g l u c o s e and Q - f r u c t o s e has been r e p o r t e d u s i n g a b u f f e r e d o x i m a t i o n reagent.5

No h y d r o l y s i s o f t h e s u c r o s e t a k e s

p l a c e d u r i n g t h e o x i m a t i o n procedure,

n o r do t h e r e a g e n t s a f f e c t t h e

subsequent s i l y l a t i o n o f t h e d i s a c c h a r i d e . l - M e t h y l - i m i d a z o l e has been used as a s o l v e n t and a c a t a l y s t

f o r t h e p r e p a r a t i o n o f a l d o n o n i t r i l e a c e t a t e s o f aldoses.6 methods have been m o d i f i e d f o r t h e a n a l y s i s o f g l y c o s e s , sugars,

and S m i t h d e g r a d a t i o n p r o d u c t s by u s i n g g.1.c.

of t h e i r aldononitrile acetate

derivative^.^

Existing amino-

determination

M o n o s a c c h a r i d e s h a v e been c o n v e r t e d t o p r o d u c e one d e r i v a t i v e f o r each aldose,

as d e t e c t e d by g.l.c.,

the formation o f aldoximes,

i n a sequence w h i c h i n v o l v e s

t h e i r r e d u c t i o n w i t h borane t o t h e

4


corresponding aminopolyols,

l-

and s u b s e q u e n t c o n v e r s i o n t o t h e

e t hox y c a r bo n y 1-0t r i m e t hy 1s i1y 1 d e r iv a t iv e s

.

diastereomers

formation

upon d e r i v a t i z a t i o n .

The

K e t o s es pr o d uc e t w o

o f side

and

decomposition products i s n e g l i g i b l e . The m o n o s a c c h a r i d e c o m p o s i t i o n o f d e t e r m i n e d b y g.1.c.-m.s.

of

g l y c o c o n j u g a t e s has been

the g - t r i m e t h y l s i l y l

ethers

after

l i b e r a t i o n o f t h e component s u g a r s , E - d s a c e t y l a t i o n , and d e a m i n a t i o n o f amino-sugars

and n e u r a m i n i c acid.9

6-Deoxyhexoses,

hexose, a n d

p e n t o s e s , d e r i v e d f r o m c a r d i a c g l y c o s i d e s , have been e s t i m a t e d by g.1.c.-m.s. may

also

of be

their g-trimethylsilyl

c a p i l l a r y columns.12 convenient

r e s o l u t i o n g.1.c.

Monosaccharides

These d e r i v a t i v e s have been f o u n d

alternative

quantitation o f

ethers.lO,ll

as 2 - t r i m e t h y l s i l y l a l d i t o l s on f u s e d - s i l i c a

separated

to

the

g-acetate

acid hydrolysates

of

to offer

derivatives

for

polysaccharides.

a

the High

s e p a r a t i o n s o f a l d i t o l a c e t a t e s on f u s e d - s i l i c a

w a l l - c o a t e d open t u b u l a r col u mns ha ve been achieved.13 A d i f f e r e n t i a l g.1.c. i n carbohydrate procedure,

me t h od f o r d e t e r m i n a t i o n o f u r o n i c a c i d s

mixtures

has

be en

deve10ped.l~

I n a two-step

n e u t r a l s u g a r s a r e f i r s t d e t e r m i n e d by g.1.c.

a l d o n o n i t r i l e acetates,

of their

b e f o r e t h e u r o n i c a c i d s i n t u r n a r e reduced

and c o n v e r t e d t o t h e same d e r i v a t i v e s .

The r e l a t i v e p r o p o r t i o n s o f

Q - m a n n u r o n i c a c i d and C - g u l u r o n i c a c i d s i n a l g i n i c a c i d have been d e t e r m i n e d i n a g.1.c.

me t h od . 1 5

After lowering the viscosity o f

t h e a l g i n i c a c i d by l i m i t e d a c i d h y d r o l y s i s ,

c a r b o x y l groups a r e

f i r s t e s t e r i f i e d b y r e a c t i o n w i t h l-ethyl-3-{3-(dimethylamino)propyll-carbodi-imide,

t h e n r e d u c e d w i t h so dium b o r o h y d r i d e , end t h e

r e s u l t i n g m i x t u r e o f hexosans i s c o n v e r t e d by a c i d h y d r o l y s i s t o monosaccharides.

The m o n o s a c c h a r i d e s i n t u r n a r e r e d u c e d w i t h s o d i u m

b o r o h y d r i d e t o h e x i t o l s w h i c h can be a n a l y s e d as t h e b u t a n e b o r o n i c esters. New c h i r a l s t a t i o n a r y p h a s e s f o r t h e g.1.c. enantiomers

of

monosaccharides

amines, have

amino-alcohols,

been

described.l6

separation o f the hydroxy-acids,

The

separation

and

af

m ono s a c c h a r id e e n a n t i o m e r s as t h e 0 - t r i f l u o r a a c e t a t e a is a c h i e v e d on a s t a t i o n a r y phase by a t t a c h m e n t o f ~ - v a l y i - S - a - p h e n y l s t a t i o n a r y phase.

ethylamide t o the p o l y s i l o x a n e U r i n a r y p o l y o l s have been e s t i m a t e d by g.1.c. of

t h e i r acetates.17

I n t e r f e r e n c e i n t h e chromatographic

the

functionalized cyanoethyl

side

chains

of

p a t t e r n s due

t o m o n o s a c c h a r i d e s was o v e r c o m e b y f o r m i n g m e t h y l o x i m e - a c e t a t e d e r i v a t i v e s o f r e d u c i n g sugars.

2: General Methods

5

2 Column a n d I o n - e x c h a n g e C h r o m a t o g r a p h y

Macroporous m i c r o s p h e r i c a l c e l l u l o s e h a s been used as a s t a t i o n a r y p h a s e i n t h e c h r o m a t o g r a p h y o f w a t e r - s o l u b l e h i g h and low m o l e c u l a r weight carbohydrates.18 D i s t r i b u t i o n c o e f f i c i e n t s of t h e polymer molecules depend on t h e hydrodynamic d i a m e t e r s o f t h e polymer molecules. The h y d r o d y n a m i c b e h a v i o u r o f r e d u c e d g l y c o p o l y p e p t i d e s h a s b e e n s t u d i e , d by g e l f i l t r a t i o n i n g u a n i d i n e h y d r o c h l o r i d e i n c o n j u n c t i o n w i t h h.p.1.c.” Even t h o u g h c a r b o h y d r a t e - r i c h g l y c o p e p t i d e s may o c c a s i o n a l l y y i e l d a n u n d e r e s t i m a t e d m o l e c u l a r w e i g h t value, t h e method a p p e a r s t o be u s e f u l f o r t h e r a p i d e s t i m a t i o n of molecular weights of simple polypeptides. The s e p a r a t i o n o f 2-amino-2-deoxy-Q-glucose from 2-amino-2deoxy-n-galactose i n t h e r o u t i n e amino-acid a n a l y s i s of p l a n t glycop r o t e i n s h a s b e e n i m p r o v e d by t h e u s e o f l o n g e r c o l u m n s o n t h e a m i n o - a c i d a n a l y s e r . 2 0 An i m p r o v e d c h r o m a t o g r a p h i c s e p a r a t i o n o f s u g a r s and s u g a r a l c o h o l s on c a t i o n - e x c h a n g e r e s i n s (Ca2+) i s reported.21 The a d d i t i o n o f s m a l l a m o u n t s o f t r i e t h y l a m i n e t o the eluant catalyses the mutarotation of reducing sugars, resulting i n r e d u c e d p e a k w i d t h s w i t h o u t a f f e c t i n g t h e e l u t i o n times. T h e s e p a r a t i o n of s t r o n g l y basic ion-exchange r e s i n s f o r t h e s e p a r a t i o n o f a l d i t o l s f r o m m o n o s a c c h a r i d e s has p r e v i o u s l y b e e n r e p o r t e d a s a method f o r t h e e s t i m a t i o n o f t h e d e g r e e o f p o l y m e r i z a t i o n o f n e u t r a l o l i g o - and p o l y - s a c c h a r i d e s . I n t h i s p r o c e d u r e , t h e a l d i t o l s were r e p o r t e d n o t t o b i n d t o t h e r e s i n , a n d were e s t i m a t e d a f t e r I t h a s now b e e n s h o w n t h a t i n a n u m b e r o f d e r i v a t i z a t i o n by g.1.c. cases a l d i t o l s do b i n d t o t h e r e s i n s , b u t t h e i r e l u t i o n from t h e r e s i n i s i n f l u e n c e d by m e t h a n o l , ammonium a c e t a t e , a n d ammonium forrnate.22 Cation-exchange r e s i n s i n t h e s i l v e r form r e t a i n o l i g o saccharides t o a g r e a t e r e x t e n t t h a n t h e calcium forms o f t h e r e s i n s , r e s u l t i n g i n a g r e a t e r number o f o l i g o s a c c h a r i d e s b e i n g s e p a r a t e d . 23

3 Thin-layer Chromatography

A c o n t i n u o u s - f l o w t.1.c. s y s t e m h a s b e e n d e v e l o p e d f o r t h e i d e n t i f i c a t i o n a n d m e a s u r e m e n t o f u r i n a r y c a r b o h y d r a t e s i m p r e g n a t e d on p a p e r . 24 M u r a m i c a c i d , 2-am i n o - 2 - d e o x y -Q-gl u c o s e , 2-am i n o - 2 - d e o x y Q-galactose,and t h e i r corresponding aminodeoxyalditols have been s e p a r a t e d b y t w o - d i m e n s i o n a l t.1.c. as t h e i r 2 , 4 - d i n i t r o p h e n y l


6

derivative^.'^

Some o f

the

factors

influencing the derivatization

and s e p a r a t i o n o f t h e s e s u g a r s a r e r e p o r t e d .

4 H i q h - p r e s s u r e L i q u i d Chromatography A r e v i e w o f t h e d i f f e r e n t t y p e s o f s i l i c a and m o b i l e phases t h a t a r e

u s e d i n t h e h.p.1.c.

of

s u g a r s has been published.26

column p r e p a r a t i o n , pre-column d e r i v a t i z a t i o n ,

Methods o f

and d e t e c t i o n methods

are discussed. C h r o m a t o g r a p h y on b o r o n i c a c i d s i l i c a r e d u c e s t h e s e p a r a t i o n t i m e of

diol-containing

compared

to

less

p o l y m e t h a c r y l i c acid.27 the

separation

glycoproteins.

of

s u b s t a n c e s w i t h o u t loss o f

r i g i d

polymers

such

as

r e s o l u t i o n when cellulose

substances

Proteoglycans

such

as

catecholamines

from bovine nasal,

and

bovine a r t i c u l a r

and r a t c h o n d r o s a r c o m a c a r t i l a g e h a v e been a n a l y s e d by h.p.1.c. silica-based

and

T h i s s t a t i o n a r y p h a s e h a s been p r o p o s e d f o r

m a t e r i a l bonded w i t h an a m i d e phase.28

on a

The b i o c h e m i c a l

i n t e g r i t y o f t h e proteoglycans i s r e t a i n e d d u r i n g t h i s procedure. High-performance obtain

data

for

size-exclusion

hydrodynamic

c h r o m a t o g r a p h y h a s been u s e d t o

molecular

radii

of

dextrans.29

G l y c o p r o t e i n hormones and g l y c o p r o t e i n a l l e r g e n s f r o m p l a n t and a n i m a l o r i g i n h a v e been a n a l y s e d by s i z e - e x c l u s i o n c h r o m a t o g r a p h y o n t h e TSK s e r i e s o f m o d i f i e l d s i l i c a s u n d e r h.p.1.c.

condition^.^^

S i l i c a g e l c h e m i c a l l y m o d i f i e d w i t h aminopropyl groups, s i l i c a g e l w i t h 1,4-diaminobutane effective

i n the

h.p.l.~.~l

The

separation o f method

has

n e u t r a l o l i g o s a c c h a r i d e s by

been

used

p u r i f i c a t i o n o f neutral oligosaccharides b r a n c h i n g and d e r i v e d f r o m

or

p r e s e n t i n t h e m o b i l e phase, i s successfully

with d i f f e r e n t

dolichol-linked

in

the

degrees o f

oligosaccharide

intermediates. O l i g o s a c c h a r i d e s (up t o d.p. hydrolysis of

s t a r c h have

12) o b t a i n e d f r o m a c i d and e n z y m i c

been s e p a r a t e d by

reversed-phase

h. p. 1. c. 32 A method f o r t h e r a p i d s e p a r a t i o n o f a n i o n i c o l i g o s a c c a r i d e s has been d e v e l o p e d u s i n g an anion-exchange s u p e r i o r speed,

column which o f f e r s

r e s o l u t i o n , and y i e l d when c o m p a r e d w i t h o t h e r i o n -

exchange m e t h o d s o r h i g h - v o l t a g e

p a p e r e l e c t r o p h ~ r e s i s . ~B~a s e l i n e

r e s o l u t i o n o f anionic oligosaccharides which d i f f e r i n t h e i r net negative charge i s achieved,

r e g a r d l e s s o f whether t h e charge i s

i m p a r t e d by n e u r a m i n o s y l o r p h o s p h a t e m o i e t i e s .

7

2: General Methods A

h.p.1.c.

procedure

for

the

separation

and i s o l a t i o n o f

h a s been d e v e l o p e d u s i n g an i s o c r a t i c

neuraminosyl-oligosaccharides

s y s t e m t o g i v e b a s e l i n e s e p a r a t i o n o f n e u r a m i n i c a c i d , 3’neuraminosyl-lactose.34

A complex m i x t u r e o f

a n d 6‘-

oligosaccharides

o b t a i n e d b y a l k a l i n e b o r o h y d r i d e d e g r a d a t i o n o f human,

bronchial

mucous

on

g l y c o p r o t e i n s has

been s e p a r a t e d

by

h.p.1.c.

bonded

p r i m a r y a m i n e p a c k i n g u s i n g a l i n e a r g r a d i e n t s o l v e n t system.35

The

s e p a r a t i o n o f i s o m e r i c o l i g o s a c c h a r i d e s i s achieved, suggesting t h a t t h e t o t a l number o f h y d r o x y l g r o u p s , as w e l l as t h e i r c o n f i g u r a t i o n , d e t e r m i n e s t h e r e t e n t i o n t i m e o f each o l i g o s a c c h a r i d e . A m e t h o d h a s been d e v e l o p e d f o r s e p a r a t i o n o f r e d u c e d ,

o l i g o s a c c h a r i d e s b y h . p . l . ~ . ~ ~p - G l u c i t o l oligosaccharides

containing

b e t w e e n one

neutral

and r e d u c e d p - g l u c o and t w e n t y

Q-glucosyl

r e s i d u e s have been s e p a r a t e d . The m e t h o d h a s a l s o b e e n a p p l i e d t o t h e analysis

and p r e p a r a t i v e

isolation o f

glycoprotein-derived

o l i g o s a c c h a r i d e s o b t a i n e d by e n z y m i c r e l e a s e by e n d o g l y c o s i d a s e s o r by c h e m i c a l r e l e a s e by h y d r a z i n o l y s i s .

Subnanomolar q u a n t i t i e s o f

o l i g o s a c c h a r i d e s may be d e t e c t e d when s o d i u m b o r o t r i t i d e i s u s e d i n t h e r e d u c t i o n step. The u r i n a r y e x c r e t i o n o f i s o m e r i c c h o n d r o i t i n s u l p h a t e s h a s been m o n i t o r e d by h.p.1.c.

o f t h e unsaturated disaccharides produced

by d i g e s t i o n w i t h c h o n d r o i t i n s u l p h a t e lyases.37 v a r i a t i o n i n t h e c o m p o s i t i o n o f t h e h.p.1.c.

A systematic

m o b i l e p h a s e was u s e d

for the selection o f the optimal conditions for separation. Endo-B-p-2-acetamido-2-deoxy-P-glucanase

a c t i v i t y h a s been a s s a y e d

using the dansyl derivative o f the L-asparaginyl oligosaccharide

(11-

Mane)5-(Q-GlceNAc)2-L-Asn

the

as

the

substrate.38

Analysis

of

product, dansyl 2-acetamido-2-deoxy-~-glucosyl-~-asparagine, has method.

The l o w e r l i m i t

nmol o f dansyl glycopeptides.

Reducing sugars

b e e n a c h i e v e d b y a r e v e r s e - p h a s e h.p.1.c. of

d e t e c t i o n i s 0.1

h a v e been r e a c t e d w i t h d a n s y l h y d r a z i n e p r i o r t o t h e i r s e p a r a t i o n by F l u o r e s c e n t d e t e c t i o n p r o v i d e s a s e n s i t i v i t y o f 10 p m o l

h.p.l.c.39

p e r i n j e c t i o n f o r t h e monosaccharide components o f g l y c o p r o t e i n s . Numerous a l k y l mannosides,

and a r y l P - g l u c o s i d e s ,

Q-glucosiduronic acids,and

d e r i v a t i v e s have been s e p a r a t e d by h . p . l . ~ . ~ ’ h.p.1.c.

been d e s c r i b e d . 4 1

sides.42

Q-

A rapid isocratic

method f o r t h e e s t i m a t i o n o f n e u r a m i n i c a c i d i n serum has

u s i n g 0.003M h.p.1.c.

Q-galactosides,

t h e i r a c e t y l and b e n z o y l

Separation i s achieved on a cation-exchange r e s i n

s u l p h u r i c a c i d as t h e m o b i l e phase.

Reversed-phase

h a s b e e n u s e d i n t h e s e p a r a t i o n o f some m e t h y l g l y c o -

I n t h e aldohexose series,

the pyranosides e l u t e before

8


furanosides,

but i n the aldopentose series the furanosides e l u t e

first. Mono- a n d d i - s a c c h a r i d e s separated

by

h.p.1.c.

on

and p o l y h y d r i c a l c o h o l s have been

silica

columns

e t h ~ l e n e p e n t a m i n e . ~ The ~ method,

modified with

tetra-

which o f f e r s a h i g h degree o f

r e s o l u t i o n , r e q u i r e s low o p e r a t i n g temperatures. T r y p t i c glycopeptides from t o g r a p h e d u s i n g reverse-phase a hydrophilic for the

p a i r i n g agent,

the analysis of enzymic

reduced,

digestion

chondroitin sulphate,

v i r a l s o u r c e s have been chroma-

ion-pair

p a r t i t i o n chromatography w i t h

phosphoric acid.44

An h.p.1.c.

method

unsaturated disaccharides derived from

and

sodium

borohydride

dermatan sulphate,

h e p a r i n h a s been d e s c r i b e d . 4 5

reduction of

heparan s u l p h a t e , and

The m e t h o d a v o i d s t h e p o s s i b i l i t y o f

o b t a i n i n g anomeric forms o f t h e unsaturated disaccharides. An i m p r o v e d s e p a r a t i o n o f m o n o s a c c h a r i d e s by h.p.1.c. achieved using porous m i c r o p a r t i c l e

Fourteen oligosaccharylpyrophosphoryl dolichols, L-asparagine-linked separated

by

precursors o f

the

o l i g o s a c c h a r i d e s o f g l y c o p r o t e i n s , have been

h.p.1.c.

on

p r o d u c t s were shown t o enzyme-catalysed

h a s been

c a r b o h y d r a t e columns.46

silica

Some

retain their

reactions.

t h e i r b o r a t e c o m p l e x e s by h.p.

activity

of as

the

resolved

substrates

in

M o n o s a c c h a r i d e s h a v e been s e p a r a t e d as a n i o n - e x c h a n g e c h r ~ m a t o g r a p h y . ~The ~

s u g a r s a r e d e t e c t e d f l u o r i m e t r i c a l l y a f t e r s e p a r a t i o n w i t h 2-cyanoacetamide.

Monosaccharide

residues of

glycoproteins,

after

c o n v e r s i o n t o 2 - a m i n o p y r i d y l d e r i v a t i v e s , h a v e been p u r i f i e d by i o n exchange c h r o m a t o g r a p h y and t h e n s e p a r a t e d by h.p.1.c. phase columns.49

on reversed

A l t h o u g h i t was d i f f i c u l t t o d e t e c t mono- a n d d i -

s a c c h a r i d e d e r i v a t i v e s because o f i n t e r f e r e n c e f r o m 2 - a m i n o p y r i d i n e and o t h e r f l u o r e s c e n t m a t e r i a l s ,

higher oligosaccharides

can be

separated. I m p r o v e m e n t s i n a d e t e c t o r f o r l i q u i d c h r o m a t o g r a p h y b a s e d on o p t i c a l a c t i v i t y o f t h e components a l l o w

100 n g o f mono-

or di-

s a c c h a r i d e t o be d e t e ~ t e d . ~ ' S i m u l t a n e o u s d e t e r m i n a t i o n o f s i x c a r b o h y d r a t e c o m p o n e n t s o f human u r i n e ,

which i s i n j e c t e d a f t e r

d e i o n i z a t i o n , h a s been a c c o m p l i s h e d .

I m p r o v e m e n t s h a v e been made t o

the sensitivity o f refractive-index

d e t e c t o r s by e l i m i n a t i n g n o i s e

c a u s e d by p o o r t e m p e r a t u r e c o n t r o l o f t h e d e t e c t o r a n d e l u a n t . 5 1 The u s e o f 2 - c y a n o a c e t a m i d e i n p o s t - c o l u m n d e t e c t i o n o f a l d o s e s has

been

evaluated.52

The

condensation reactions obtained

h e a t i n g carbohydrates w i t h 3-methyl-2-benzothiazolinone alkali

containing

2-methoxyethanol

have

been

by

hydrazone i n described.53

2: General Methods

9

Monosaccharide

aldoses

and

ketoses

and

disaccharides

chromogens w h i c h can be measured a t 390 nm.

t h e method f o r p o s t - c o l u m n d e t e c t i o n o f c a r b o h y d r a t e s h.p.1.c.

produce

The p o t e n t i a l i n u s i n g s e p a r a t e d by

i s discussed.

5 Electrophoresis The a c c u r a c y o f m o l e c u l a r w e i g h t e s t i m a t e s f o r g l y c o p r o t e i n s h a s been e v a l u a t e d

by

SDS-pore

gradient

electrophoresis

of

glyco-

p r o t e i n s o f known m o l e c u l a r w e i g h t and known a m i n o - a c i d

and

c a r b o h y d r a t e c o m p ~ s i t i o n . ~A ~ c o m p a r i s o n was made o f T r i s - g l y c i n e and Tris-borate-e.d.t.a. by 2 - m e r c a p t o e t h a n o l . formation of

b u f f e r s y s t e m s w i t h and w i t h o u t

reduction

A l t h o u g h b o t h t h e g r a d i e n t g e l system and t h e

b o r a t e complexes w i t h t h e i n d i v i d u a l c a r b o h y d r a t e

m o i e t i e s of the g l y c o p r o t e i n c o n t r i b u t e t o t h e p r e c i s i o n o f t h e molecular weight estimate,

i t i s t h e thermodynamic and h y d r o d y n a m i c

p r o p e r t i e s o f t h e i n d i v i d u a l g l y c o p r o t e i n which determine

its

m o b i l i t y on g e l e l e c t r o p h o r e s i s . L e c t i n s have been us ed f o r transferred separation

to

on

the

d e t e c t i o n of

n i t r o c e l l u l o s e sheets polyacrylamide

asialoglycopeptides

have

gels.55

been

after

glycoproteins

electrophoretic

Fluorescein-derivatized

resolved

by

polyacrylamide

gel

e l e c t r o p h ~ r e s i s . ~When ~ t h e method i s used i n c o n j u n c t i o n w i t h s p e c i f i c exoglycosidase d i g e s t i o n o f the glycopeptides, correlation

i s

observed

between

the

relative

a linear

electrophoretic

m o b i l i t y and t h e number o f m o n o s a c c h a r i d e r e s i d u e s removed by t h e enzymes. a-Glucuronic

a c i d and I - i d u r o n i c

a c i d may be s e p a r a t e d by

e l e c t r o p h o r e s i s on T i t a n I11 c e l l u l o s e a c e t a t e p l a t e s . 5 7

The method

has been a p p l i e d t o t h e a n a l y s i s o f u r o n i c a c i d s i n c h o n d r o i t i n 4and 6 - s u l p h a t e s

and dermatan s u l p h a t e .

A n a l y t i c a l i s o t a c h o p h o r e s i s has been used i n t h e e s t i m a t i o n o f n e u r a m i n i c acid.58

Under t h e c o n d i t i o n s o f assay n e u r a m i n i c a c i d

a p p e a r s as a t r o u g h b e t w e e n t w o buffer.

U.V.

i m p u r i t y components o f t h e

The t r o u g h l e n g t h i s p r o p o r t i o n a l

t o t h e amount

of

n e u r a m i n i c a c i d i n j e c t e d i n t o t h e system. E l e c t r o p h o r e t i c and c h r o m a t o g r a p h i c methods f o r s e p a r a t i n g c a r b o h y d r a t e s have been r e v i e w e d i n t h e l a t e s t e d i t i o n o f a t r e a t i s e on t h e c h e m i s t r y and b i o c h e m i s t r y o f

carbohydrate^.^'


10 6 A n a l y t i c a l Methods Modifications o f

the

Park-Johnson

ferricyanide

submicromethod

for

t h e assay o f r e d u c i n g g r o u p s i n c a r b o h y d r a t e s have been r e p o r t e d . 6 0 O x a l i c a c i d i s used as a s o l v e n t f o r f e r r i c f e r r o c y a n i d e i n p l a c e o f sodium d o d e c y l s u l p h a t e , w h i c h g i v e s t u r b i d s o l u t i o n s . When a l d o s e s

are determined i o d o m e t r i c a l l y

magnesium o x i d e ,

f o r m a t i o n o f i o d a t e ions.61

i n t h e presence o f

t a k e s p l a c e w i t h t h e minimum

no o v e r - o x i d a t i o n

C o l o r i m e t r i c analyses o f carbohydrates

have been c a r r i e d o u t on a l i q u i d s c i n t i l l a t i o n c o u n t e r o v e r a w i d e r r a n g e o f c o n c e n t r a t i o n s t h a n can be measured w i t h a c o n v e n t i o n a l l i g h t spectrophotometer.62 hydroxybenzoic

The u s e o f a l k a l i n e s o l u t i o n s o f 4 -

acid hydrazide for the determination o f reducing

s u g a r s i n e x t r a c t s o f p l a n t t i s s u e s has been e v a l u a t e d . 6 3 w i t h a s e n s i t i v i t y range o f

An e n z y m i c m i c r o a s s a y f o r l a c t o s e , 12.5-500

n m o l p e r sample, h a s been developed.64

upon t h e h y d r o l y s i s o f l a c t o s e t o 4 - g l u c o s e

The method i s based

and 4 - g a l a c t o s e

by

0-4-

g a l a c t o s i d a s e f o l l o w e d b y t h e c o l o r i m e t r i c a s s a y o f t h e f r e e Qglucose u s i n g t h e Q-glucose oxidase procedure. A s i m p l e d e t e c t i o n method f o r 9 - g a l a c t o s e phosphate i n blood-impregnated screening

test

for

and Q - g a l a c t o s e

1-

f i l t e r p a p e r has been d e v e l o p e d as a

galactosaemia.65

When

Q-galactose

( o r Q-

g a l a c t o s e 1-phosphate a f t e r t r e a t m e n t w i t h phosphatase) i s o x i d i z e d by a - g a l a c t o s e d e h y d r o g e n a s e ,

t h e r e s u l t i n g NADH may be m e a s u r e d

I n a s i m i l a r approach t h e m i c r o d e t e r m i n a t i o n o f

fluorimetrically.

Q - g a l a c t o s e and 1 - g a l a c t o s e 1 - p h o s p h a t e i n d r i e d b l o o d s p o t s has been achieved.66 galactose determined phosphate

Q-Galactose i s determined f l u o r i m e t r i c a l l y

dehydrogenase using ester

the with

whilst

same

the

Q-galactose

procedure

alkaline

after

phosphatase.

with

1-phosphate

4i s

hydrolysis

of

I n Nelson’s

Somogyi

method f o r t h e d e t e r m i n a t i o n o f r e d u c i n g s u g a r s , r e a g e n t has been r e p l a c e d by t h e F o l i n - C i o c a l t e u

the

t h e arsenomolybdate p h e n o l reagent.67

The method compares f a v o u r a b l y w i t h t h e o r i g i n a l method i n s t a b i l i t y o f c o l o u r and r e p r o d u c i b i l i t y . a n a l y t i c a l element,

A new t y p e o f m u l t i l a y e r f i l m

consisting o f a spreading layer,

a blocking

l a y e r , an e n z y m e l a y e r ( c o m p o s e d o f a - g l u c o s e o x i d a s e ,

peroxidase

and

dye),

and

a

transparent

layer,

d e t e r m i n a t i o n o f 1-glucose i n blood.68 i n d r o p p e d on t h e f i l m ,

has

been d e v e l o p e d

for

the

A f t e r a spot o f whole b l o o d

the a-glucose concentration i s determined

without further manipulations. S u l p h y d r y l r e a g e n t s such as c y s t e i n e and g l u t a t h i o n e i n t e r -

11

2: General Methods fere

with

the

P-glucose

oxidase

assay

for

Q-glucose.69

This

i n t e r f e r e n c e c a n be e l i m i n a t e d by u s e o f N - e t h y l m a l e i m i d e .

Q-

G l u c o s e has been e s t i m a t e d e n z y m i c a l l y

using a commercial analyser

a f t e r e l u t i o n f r o m b l o o d s p o t t e d on t o

filter

paper.70

An oxygen-

s t a b i l i z e d enzyme e l e c t r o d e has been d e s i g n e d f o r t h e a n a l y s i s o f Qg l u c o s e i n f e r m e n t a t i o n broths.71 I n a new e n z y m a t i c method f o r t h e d e t e r m i n a t i o n o f [)-glucose human s e r a ,

t h e h y d r o g e n p e r o x i d e p r o d u c e d by Q - g l u c o s e o x i d a s e

measured f r o m

in

is

t h e change i n absorbance due t o o x i d a t i o n o f NAD(P)H

i n t h e presence o f catalase,

a l d e h y d e dehydrogenase, and a h i g h

c o n c e n t r a t i o n o f ethanol.72 S e v e r a l a d v a n t a g e s o f t h i s m e t h o d , a s c o m p a r e d w i t h o t h e r Qg l u c o s e o x i d a s e methods c o u p l e d w i t h l e u c o dyes, estimation

o f Q-glucose

hexokinase-Q-glucose

are recorded.

i n haemolysed b l o o d samples

The

using the

6-phosphate dehydrogenase method has been

described.73 Two

new

systems

have

been

developed

measurement o f pH i n s m a l l volumes.74

for

the d i f f e r e n t i a l

The a p p a r a t u s i s c a p a b l e o f

f o l l o w i n g t h e pH change caused by h e x o k i n a s e - c a t a l y s e d Q - g l u c o s e a n d ATP, 18 m M .

r e a c t i o n of

when t h e s u g a r c o n c e n t r a t i o n i s i n t h e r a n g e 1-

The s y s t e m can be used t o m o n i t o r [ ) - g l u c o s e c o n c e n t r a t i o n s i n

biological fluids.

Optimal

p r o p e r t i e s o f D-glucose been s t u d i e d

i n order

d e t e r m i n a t i ~ n . ~The ~

reaction conditions

and k i n e t i c

dehydrogenase f r o m B a c i l l u s m e q a t e r i u m have to

develop a method

Em

value

of

the

for

serum q - g l u c o s e

enzyme f o r

Q-glucose

i n f l u e n c e d by t h e pH o f t h e medium and by i o n i c s t r e n g t h . conditions end-point

f o r t h e use o f R-glucose assay

i s

Suitable

dehydrogenase i n r a t e assay and

methods were i d e n t i f i e d .

a-Glucose

concentrations

d e t e r m i n e d i n serum u s i n g t h e s e methods show good c o r r e l a t i o n w i t h t h o s e o b t a i n e d w i t h t h e h e x o k i n a s e method.

n-Glucose

has been c o v a l e n t l y a t t a c h e d t o n y l o n t u b i n g . 7 6

dehydrogenase

The a c t i v i t y and pH

o p t i m u m o f t h e bound enzyme depends on t h e t r a n s i t i o n m e t a l s u s e d as s p a c e r s b e t w e e n t h e enzyme and t h e s u p p o r t . A

fluorimetric

r e l e a s e d from

method f o r t h e measurement

glycoproteins

by a - L - f u c o s i d a s e

o f a-l-fucopyranose has been r e p o r t e d . 7 7

The I - f u c o s e i s o x i d i z e d w i t h I - f u c o s e d e h y d r o g e n a s e a n d NAD, a n d t h e NADH p r o d u c e d i s used i n c o n j u n c t i o n w i t h d i a p h o r a s e t o c o n v e r t the nonfluorescent

dye r e s a z u r i n t o t h e h i g h l y f l u o r e s c e n t

product

resorufin. I-Fucose

has been measured r a d i o m e t r i c a l l y

u s i n g a method

c a p a b l e o f m e a s u r i n g q u a n t i t i e s o f t h e s u g a r a s l o w a s 25 p m 0 1 . ~ ~


12

The a s s a y c o u p l e s I - f u c o s e d e h y d r o g e n a s e t o I = - g l u t a m a t e d e h y d r o genase and L - g l u t a m a t e d e c a r b o x y l a s e t o y i e l d 14C02 w h i c h i s d e r i v e d f r o m {l-14C)-a-ketoglutarate.

L-Fucose

dehydrogenase has been used

p r e v i o u s l y i n f l u o r e s c e n c e assays o f t h e sugar, limited

when

the

sample

to

be

assayed c o n t a i n s

fluorescent

substances,

commonly

glycopeptides

and membrane

preparations

on c o lu m n s h a v i n g a r e l a t i v e l y

b u t t h e method i s

observed

endogenous

i n

hydrolysed

a t low operating pressures

l e n g t h y l i f e and h i g h l i n e a r sample

capacity. The p h l o r o g l u c i n o l m e t h o d f o r t h e e s t i m a t i o n o f Q - x y l o s e i n u r i n e has been a s s e s s e d w i t h r e s p e c t t o t h e i n t e r f e r e n c e f r o m l o w l e v e l s o f ~-glucose.'g A micromethod f o r t h e d e t e r m i n a t i o n o f f r e e o r g l y c o p r o t e i n bound n e u r a m i n i c a c i d on t h e b a s i s o f o x i d a t i o n w i t h p e r i o d i c a c i d and

reaction

with

thiobarbiturate

has

been

A

described.80

o f the t h i o b a r b i t u r i c acid technique for the d e t e r m i n a t i o n o f n e u r a m i n i c a c i d ha s been developed.81 Elimination modification

o f i n t e r f e r e n c e caused by 2 - d e o x y - P - r i b o s e

without

loss o f n e u r a m i n i c a c i d ,

extraction

of

the

chromogen.

i s

The

and s e v e r a l o t h e r sugars, achieved

by

neuraminic

pH-dependent

acid

in

level

s i a l o g l y c o c o n j u g a t e s ha s been measured i n a new e n z y m i c method a f t e r r e l e a s e f r o m g l y c o p r o t e i n s w i t h n eu rami n i da se.82

Neuraminic a c i d i s

cleaved t o release pyruvate using N-acetylneuraminic followed peroxide

by o x i d a t i o n o f which

pyruvate oxidase t o

i s measured c o l o r i m e t r i c a l l y

chlorophenol-4-am

i n o - a n t i p y r i n e m e t ho d.

a c i d aldolase,

produce

by t h e

hydrogen

peroxidase-4-

C o r r e l a t i o n o f r e s u 1t s

w i t h t h o s e o b t a i n e d u s i n g a c h e m i c a l m e t h o d o f a n a l y s i s was reported. Free neuraminic a c i d s p l i t from red-blood-cell receptors t o N e w c a s t l e d i s e a s e v i r u s has been d e t e r m i n e d d u r i n g s i m u l t a n e o u s e l u t i o n and h a e m o l y s i s o f t h e c e l l s . 8 3 The p r o c e d u r e depends on t h e presence o f neuramin id ase a c t i v i t y on t h e preadsorbed v i r i o n s . F l u o r e s c a m i n e 14 -p h e ny 1s p ir o ( f u r a n - 2 - ( 3H ),1' -( 3 ' H ) furan)-3,3'-dione}

- i so b e n z o -

may be a p p l i e d t o t h e q u a n t i t a t i v e a n d q u a l i -

t a t i v e a n a l y s i s o f amino-sugars.84

The m e t h o d h a s b e e n a p p l i e d t o

of acid hydrolysis of chitin. Methods have been d e v e l o p e d f o r t h e i d e n t i f i c a t i o n o f t h e g l y c o s i d i c

the

analysis

of

the

product

l i n k a g e s b e t w e e n amino-sugars

a nd I - a s p a r a g i n e ,

C - s e r i n e , and

C,-

t h r e o n i n e i n g l y c o p r ~ t e i n s . ~A~f t e r a l k a l i n e e l i m i n a t i o n i n t h e p r e s e n c e o f s o d iu m b o r o t r i t i d e , sugar

components

separation

o f

of the

a c i d hydrolysis,and

0-glycosyl dansyl

linkages

are

hexosaminitols

dansylation, identified by

the

after

thin-layer

13

2: General Methods electrophoresis

.

The 1i n k age co m po un d 4! -( 2 -ace t a m ido -2 -deo x y -S-E

glycosidic

linkage

region of

glycoproteins

by

-

L-

g l u c o p y r a n o s y l ) hydrogen I - a s p a r a g i n a t e i s i s o l a t e d from t h e

partial acid

h y d r o l y s i s and i d e n t i f i e d as i t s d a n s y l d e r i v a t i v e . The m a n n i t o l l e v e l s i n serum samples f r o m dogs a f t e r m a n n i t o l i n f u s i o n have been measured u s i n g t h e s p e c t r o p h o t o m e t r i c measurement o f t h e i n i t i a l r a t e o f NADH f o r m a t i o n when t h e sugar i s o x i d i z e d by a b a c t e r i a l m a n n i t o l dehydrogenase p r e p a r a t i o n . 8 6

7 S t r u c t u r a l Methods A comprehensive

review

o f p h y s i c a l methods used f o r s t r u c t u r a l

a n a l y s i s o f c a r b o h y d r a t e s has been p u b l i s h e d . 5 9 high-resonance

n.m.r.

spectroscopy,

spectroscopy, and X - r a y Spectroscopy.-A

N.m.r.

m.s.,

The methods i n c l u d e

polarimetry,

U.V.

and i.r.

diffraction. r e v i e w d e a l i n g w i t h 13C n.m.r.

spectroscopy

o f p o l y s a c c h a r i d e s has been p ~ b l i s h e d . ~ ' The p r i m a r y s t r u c t u r e s o f p o l y s a c c h a r i d e s and t h e i r d e r i v a t i v e s have

been

investigated

c r o s s p o l a r i z a t i o n n.m.r.

using

magic-angle

13C

spectroscopy.88

spinning-

Although the r e s o l u t i o n o f

t h e i n d i v i d u a l c a r b o n r e s o n a n c e s was n o t p e r f e c t when x a n t h a n gum and i t s o c t y l a m i d e d e r i v a t i v e w e r e u s e d , considerably

the technique provided

more d e t a i l t h a n t h e c o r r e s p o n d i n g 13C n.m.r.

i n an e q u i v a l e n t p e r i o d o f t i m e .

A 1 3 C n.m.r.

studies

a n a l y s i s method has

been d e s c r i b e d f o r t h e i d e n t i f i c a t i o n o f c a r b o h y d r a t e r e s i d u e s i n

n-

g a l a c t o s i d e s a n d Q - g l u c o ~ i d e s . ~A ~ s s i g n m e n t s c a n be made t o t h e a n o m e r i c c o n f i g u r a t i o n and t o t h e r i n g s i z e o f t h e s u g a r s . of

13C

s p e c t r o s c o p y i s a p r a c t i c a l method f o r f o l l o w i n g t h e k i n e t i c s

N.m.r.

enzymic

digestion

of

individual

carbohydrate

residues

of

g l y c o p e p t i d e s and f o r d e t e r m i n i n g t h e s t r u c t u r e o f t h e p r o d u c t s o f p a r t i a l d i g e ~ t i o n . ~ ' I n a s t u d y o f t h e h y d r o l y s i s o f hen o v a l b u m i n g l y c o p e p t i d e s by a-n-mannosidase,

t h e method showed t h a t some a-n-

mannopyranosyl-(1 + 3)-P-mannopyranosyl faster

r a t e than the corresponding ( 1

The r e s o l u t i o n - e n h a n c e d

*

500 MHz 'H

oligo-(a-mannopyranosyl)-l-asparagines are

chemical-shift

mainly

n.m.r.

spectra o f three

N4-

can be i n t e r p r e t e d i n t e r m s o f

t h e complete p r i m a r y s t r u c t u r e s o f these structures

linkages are cleaved a t a

6 ) linkages.

compound^.^^

b a s e d upon, i n t e r p r e t a t i o n o f

The p r o p o s e d the

sets o f

v a l u e s o f H - 1 s and H-2s o f c o n s t i t u t i n g P-mannopyran-

14


o s y l residues.

The d a t a a r e s e n s i t i v e t o t h e t y p e and c o n f i g u r a t i o n

of t h e g l y c o s i d i c l i n k a g e o f t h e Q - m a n n o p y r a n o s y l r e s i d u e and t o t h e p o s i t i o n o f t h a t su ga r i n t h e c h a i n . M as s S p e c t r o m e t r y , -

Carbohydrates,

have

using

been

measured

a

mass

a f t e r s e p a r a t i o n by h.p.l.c., detector.92

The

solvent

i s

evaporated a f t e r n e b u l i z a t i o n i n a heated column producing f i n e l y d i v i d e d s o l u t e p a r t i c l e s w h i c h p a s s t h r o u g h a l i g h t beam.

Light

s c a t t e r e d f r o m t h e p a r t i c l e s i s d e t e c t e d by a p h o t o m u l t i p l i e r and a m p l i f i e d on a c h a r t r e c o r d e r . with refractive-index

The mass d e t e c t o r ,

detection,

when c o m p a r e d

showed a t e n - f o l d

increase i n

s e n s i t i v i t y , i m p r o v e d s t a b i l i t y , a n d an a b i l i t y t o d e t e c t p r o d u c t s s e p a r a t e d by g r a d i e n t e l u t i o n . By c a r e f u l s e l e c t i o n o f d e r i v a t i v e s a n d a n a l y s i s c o n d i t i o n s , m.s.

has

been

applied

dodecasaccharide

to

derived

a n a l y s i s b y m.s.

of

oligosaccharides

has

methylated aldoses

the

from

a

microscale

some s y n t h e t i c f u l l y been

have

reported.94

been

sequencing

g l y ~ o l i p i d . ’ ~ The

separated

of

a

structural

methylated I-rhamno-

A

number

of

partially

by

g.1.c.

on

capillary

columns as t h e i r t r i m e t h y l s i l y l a t e d d i e t h y l d i t h i o a c e t a l d e r i v a tives.”

S i n g l e peaks f o r each d e r i v a t i v e a r e o b t a i n e d and m.s.

u s e d i n t h e i d e n t i f i c a t i o n o f peaks.

is

Thirteen methyl ethers o f

m e t h y l N-acetyl-N-methyl-a-Q-neuraminate

m e t h y l g l y c o s i d e have been

N-

i d e n t i f i e d b y g.1.c.-m.s.

a f t e r p a r t i a l methylation o f methyl

acetyl-u-g-neuraminate

methyl g l y ~ o s i d e . ’ ~ Chemical-ionization

(methane) m.s. of

partially

has been used t o enhance t h e s e n s i t i v i t y o f d e t e c t i o n methylated

alditol

acetates

of

neutral

and

a m i n d - s ~ g a r s . ~The ~ mass s p e c t r a o f 5 2 p a r t i a l l y m e t h y l a t e d a n d acetylated methyl

glycosides

of

a-galactose,

a-mannose,

Q-glucose

and 2-ace t a m id o - 2 - d eo xy - Q - g l u cose have been d e t e r m i n e d . 98

A scheme

f o r t h e r a p i d d e t e r m i n a t i o n o f t h e p o s i t i o n o f t h e m e t h y l and a c e t y l r e s i d u e s is proposed. Field-desorption

m.s.

has

been

used

to

analyse

s a c c h a r i d e s c o n t a i n i n g f i v e t o f o u r t e e n he xose u n i t s , derivatization.”

The

molecular

weight

o f the oligosaccharide

be d e t e r m i n e d by means o f t h e a bu nd an t q u a s i - m o l e c u l a r t y p e MNa+,

MH’,

MNa22+,and

MNa33+.

structural elucidation of

x

i o n s of

can the

The me t h od has t h e p o t e n t i a l f o r

oligosaccharides

p r o d u c t s u p t o a r a n g e o f 3.5

oligo-

without p r i o r

lo3

and t h e i r

mass u n i t s .

reaction

Neuraminic a c i d

i s o l a t e d f r o m e r y t h r o c y t e g h o s t s h a s b e e n e s t i m a t e d by m e t h a n e chemical-ionization

m.s.

of

the 2 - t r i m e t h y l s i l y l

methyl glycoside

15

2: General Methods

-

u s in g p h e n y 1 2 - a c e t am ido - 2 d e o xy -( 3,4,6- tr i-0- t r i m e t h y 1s i 1y 1)-a-Q g l u c o p y r a n o s i d e as i n t e r n a l s t a n d a r d . l o O sugar

can

be

detected

i n

the

-

Nanogram l e v e l s o f t h e

presence o f

other

contaminating

substances. The a n o m e r i c c o n f i g u r a t i o n o f a 2 - a c e t a m i d o - 2 - d e o x y - Q glucopyranosyl residue linked t o

a

secondary

h y d r o x y l group

a n o t h e r s u g a r r e s i d u e h a s b e e n e s t a b l i s h e d f r o m g.1.c.-m.s.

of

data

o b t a i n e d when s u c h a d i s a c c h a r i d e is s u c c e s s f u l l y t r e a t e d w i t h periodate,

s o d i u m b o r o h y d r i d e , and a c e t i c anhydride.”’

Miscellaneous

Methods.-A

modification

o f

the

Hakomori

m e t h y l a t i o n r e a g e n t has been r e p o r t e d , u s i n g p o t a s s i u m h y d r i d e i n place o f

sodium

hydride.lo2

The

preparation o f

the

potassium

m e t h y l s u l p h e n y l m e t h i d e i s f a s t e r a n d may b e p e r f o r m e d a t a m b i e n t temperature.

Significantly

fewer

impurities

i n

the

reaction

p r o d u c t s a r e a l s o observed. I n t e r n a l 8-2-substituted

N-glycolylneuraminic

m e t h y l a t e d s a c c h a r i d e s undergo d e - N - a c y l a t i o n f u l l y methylated N-glycolylneuraminic

acid residues o f

whereas t h e t e r m i n a l

a c i d r e s i d u e s do n o t u n d e r t h e

conditions o f m e t h a n o l y s i ~ . ~ ~ T h~i s, ~d i~f f~e r e n c e i n t h e s t a b i l i t y o f N - a c y l g r o u p s h a s been o b s e r v e d i n p o l y s i a l o s y l c h a i n s c o m p r i s i n g either N-glycolylneuraminic

a c i d or N-acetylneuraminic acid.

On m e t h y l a t i o n and s u b s e q u e n t s a p o n i f i c a t i o n ,

a-a-glucopyranosyluronic galactopyranosyluronic

.

acid)-Q-xylose

acid)-a-xylose

2-2-(4-g-methyland

4-g-(a-Q-

g i v e f o u r and t w o

methyl

g l y c o s ides, r e s p e c t ive l y lo5 I n v e s t i g a t i o n o f t h e i r s t r u c t u r e s u s i n g t h e H a k o m o r i m e t h y l a t i o n p r o c e d u r e showed t h a t t h e 4 - 2 - s u b s t i t u t e d uronic acid residues afford 6-elimination products i n addition t o the permethylated derivatives. u n s u b s t i t u t e d 4’-OH

The a l d o b i o u r o n i c a c i d w i t h a n

g r o u p gave t h e p r o d u c t s o f d i r e c t m e t h y l a t i o n .

The h y d r a z i n o l y s i s - n i t r o u s

acid deamination o f glycopeptides

leads t o the s p e c i f i c cleavage o f 2-acetamido-2-deoxy-~-glucosyl l i n k a g e s . lo6 The 2,5-anhy d r o -P-mannose-con t a i n i n g

o l i g o s a c c h a r i des

thus o b t a i n e d a r e r e d u c e d w i t h sodium b o r o h y d r i d e , m e t h y l a t e d , and a n a l y s e d by

g.1.c.-m.s.

P a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s have been s e p a r a t e d by g.1.c.

on g l a s s c a p i l l a r y columns.107

column and t h e

application to

s a c c h a r i d e s are r e p o r t e d .

Some c h a r a c t e r i s t i c s o f t h e

structural analysis of

poly-

Methylation techniques i n the s t r u c t u r a l

a n a l y s i s o f g l y c o p r o t e i n s and g l y c o l i p i d s h a v e been r e v i e w e d . l o 8 Kinetic

data

for

eleven

common

methyl glucopyranosidea

i n u n b u f f e r e d s o d i u m p e r i o d a t e h a v e b e e n reported:”

The r e s u l t s

16


i n d i c a t e t h e e x t e n t o f v a r i a t i o n s i n r e a c t i v i t y t h a t can a r i s e f r o m differences i n configuration, i l l u s t r a t e the r o l e

o v e r a l l reaction rates. branches

on

the

e s p e c i a l l y a t t h e anomeric centre,

intermediate

0.f

The s i t e o f s u b s t i t u t i o n o f p e r i p h e r a l

core

Q-mannosyl

residues

carbohydrate u n i t s o f glycoproteins

of

complex-type

have been s t u d i e d by S m i t h

By u s i n g g l y c o p e p t i d e s o f known s t r u c t u r e

degradation.’”

i s s h o w n t h a t t h e s i t e o f a t t a c h m e n t o f t h e (1

( 1 ) it 4)-linked, t h i r d

+

b r a n c h t o t h e Q-mannose c o r e i n t r i - and t e t r a - a n t e n n a r y may be i d e n t i f i e d . used

for

and

hemiacetals i n determining

the

cleavage

of

the

structures

i s the concentration o f a c i d

The c r i t i c a l f a c t o r

periodate-oxidized

product.

The

r e s u l t s support t h e concept t h a t i n g l y c o p r o t e i n s from most sources the

(1 + 4 ) - l i n k e d

branch i s attached t o

the

(1

+

3)-linked

mannosyl r e s i d u e o f t h e core p o r t i o n i n t h e complex-type The

possible

conformations

analysed using semi-empirical neuraminic

acid

may

hydroxymethyl group of i s

consistent

with

preponderantly i n the

m e t h y l acarboxyl

’H- a n d 13C-n.m.r. C.d.

and B - a - g l y c o s i d e s

of

a r o u n d 220 nm a r i s e group

and a r e

p o s i t i v e f o r B-linked

acid

i n

been

different

the

terminal

The p r e s e n t m o d e l

spectroscopic

data,

but

s p e c t r a have been r e c o r d e d f o r

neuraminic acid.’l2

from

negative

have

I n solution,

two

orientation of

t h e g l y c e r o l s i d e chain.

d i f f e r s from e a r l i e r models. effects

neuraminic

p o t e n t i a l f u n c t i o n s . 11’

exist

conformations which d i f f e r

of

Q-

structures.

t h e ,” for

+

H*

The C o t t o n

transition of

a-q-linked

glycosides

the and

glycosides.

The r e s u l t s o f X - r a y d i f f r a c t i o n a n a l y s i s o f t h e c r y s t a l l i n e c o n f o r m a t i o n o f homo- and r e g u l a r h e t e r o - Q - g l u c a n

c h a i n s have been

used t o e s t a b l i s h t h e h y p o t h e s i s t h a t t h e g l y c o s i d i c l i n k a g e t y p e i s t h e d e t e r m i n a n t o f p o l y s a c c h a r i d e c o n f o r m a t i o n . 11’

I n t h i s respect,

p o l y s a c c h a r i d e s a r e more l i k e s y n t h e t i c p o l y m e r s t h a n p r o t e i n s o r n u c l e o t i d e s . I n t h e l a t t e r i t is v a r i a t i o n s i n t h e s u b s t i t u e n t s w h i c h are responsible f o r the conformational diversity.

The c o n t i n u i n g

c o m p i l a t i o n o f c r y s t a l s t r u c t u r e s o f carbohydrates,

n u c l e o s i d e s , and

n u c l e o t i d e s has been p u b l i s h e d . An i m p r o v e d m e t h o d f o r

the determination o f

the molecular

w e i g h t s o f o l i g o s a c c a r i d e s by a c o m b i n a t i o n o f p a p e r e l e c t r o p h o r e s i s and

h.p.1.c.

reported.l15

of

their

2-aminopyridyl

derivatives

has

been

The e l e c t r o p h o r e t i c m o b i l i t i e s o f t h e d e r i v a t i v e s a r e

i n d e p e n d e n t o f l i n k a g e p o s i t i o n s , a n o m e r i c c o n f i g u r a t i o n s , and t h e p r e s e n c e o f a c e t a m i d o groups, 0-Q-Glucans,

but

not

up t o m o l e c u l a r w e i g h t s o f 1.7 x their

oligo-

or

lo3.

mono-saccharide

17

2: General Methods

R - B - D - G ~ ~ ~ N A-t~ 2)-a-D-Manp-(l - ( ~ -m

R-D-GlcpNAc-(1 -

-

+

6)-B-D-Manp=

3 4 1

-f

4)-a-D-Manp =

2

+

1 6-D-G1 c ~ N A c =

( 1 ) R = o t h e r glycosyl r e s i d u e s

+

R

18


degradation products, interact with Congo Red.116 Radial diffusion of 6-Q-glucanases into a substrate-bearing gel slab has been used a s a sensitive method for assay o f the enzymes, by measuring the clearing zone formed by enzyme action on the substrate. The use of glycosyltransferases in assessing oligosaccharide structure has been reviewed."' Re fe rences 1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 23 24

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25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

2: General Methods

42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79

80 81 82 83 84 85 86 87 88 89 90 91 92

19

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m,

20

93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 ll5 116 117


M.E.Brek, G.C . H a n s s a n , K.-A.Karlsm, H .&if ler, W .Pimlott,and B.E.Samuelsson, FEES Lett., 1981, 124,299. V.KovbEik, P.KodE, and A.LiplAk, Carb0hydr.Res. ,1981, 98, 242. S.Honda, M.Nagata,and K.Kakehi, J.ChraMtogr., 1981, 209, 299. C.Bnnrier, Y.Leroy, J.MontreUi1, B.Fournet, and J.P.Kamerliq, J.Qlrcmatogr. , 1981, 210, 487. R.A.Laine, Anal.Biochan., 1981, 116, 383. B.Fournet, G.Strecker, Y.Leroy, and J.Montreui1, AnaLBiochem., 1981,,&I 489. M.Linscheid, J . D a n g o ~ , A.L.Burlingam, A.Dell,and C.E.Ballou, Proc.Natl .Acad.Sci .USA, 1981, 78, 1471. J.Ashraf , DAButterfield, J.Jarnef elf and RALaine, J.Lipid Res., 1980,2& 1137. S.R.Sarfati, Chrbohydr. Res., 1981, 94, 236. L.R.Phillips and B.A.Fraser, Carbohydr.Res., 1981, 90, 149. S.Inoue and G.Matsumura, FEE!S Lett., 1980, 12, 33. S.I~~W? and M.Iwasaki, Biochen.Biophys.Res.Carm., 1980, 2, 162. K.Shimizu, Carbohydr.Res., 1981, 92, 65. G.Strecka, A.PieraeCretel, B.Fournet, G.Spik, and J.Montreui1, 17. Anal.Bi&an., 1981, I&, N.Shihya, J.chramatogr., 1981, 208, 96. H.Rauvala, J . F h , T.Krusius, J.Karkkainen,and J.Jarnefelt, Adv.Carbohyr.ch&.Bicchan., 1981, 38, 389: K.M.Aalmo and T.J. Painter, Carbohydr.Res., 1981, 89, 73. T.Krusius and J.Finne, Carbohydr.Res., 1981, 90, 203. K.Veluraja and V.S.R.Rao, Biochim.Biophys.Acta, 1980, 630, 442. H.Ogum and K.Furuhata,'Tetrahedron Lett., 1981, 22, 4265. R.H.Marchessault and Y.Deslandes, Chrbohydr.Polymers, 1981, I, 31. G.A.Jeffrey and M.Sundaralingan, Adv.Carbohydr.Chem.Bi&en., 1981I 38, 417. S.Hase, T.Ikenaka,and Y.Matsushima, J.Bio&an.(Tokyo), 1981, 90, 127'5. P.J.Wocd, Carbohydr.Res., 1981, 94, C19. T.A.Beyer, J.E.Sadler, J.I.Rearick, J.C.Paulson, and R.L.Hil1, Adv.Enzp1 .Related Areas Mol .Biol , 1981, 52, 23.

.

3

Plant and Algal Polysaccharides BY I. M. MORRISON 1 Introduction

volume of the comprehensive treatise on the biochemistry of plants has been devoted to the structure and function of carbohydrates.' The proceedings of a symposium on the mechanism of saccharide polymerization and depolymerization have been published with several articles relevant to this chapter.' The state of macromolecular chemistry at the present time, with particular reference to the pulp and paper industry, has been r e ~ i e w e d . ~ A

2 Starch

The structure of the hydrated amylose-iodine complex has been determined by combined methods of X-ray diffraction and stereochemical packing analysis and shown to be an orthorhombic 13.60, = 23.42,and 2 (the fibre unit cell with dimensions of 2 Two amylose chains pass through the unit cell repeat) = 8. and iodide ions were present as an almost linear chain in the centre of the six-fold, left-handed amylose helix. Eight molecules of hydration per unit cell were located in good hydrogen-bonding positions between the amylose helices. The crystal structure of lJh amylose has been refined by similar methods to show another orthorhombic unit cell of dimensions ,a = 13.65, b = 23.70,and 2 = 8.05i.5 The chain conformation is a left-handed, six-fold helix with 0-6 in the position Bauche to both 0-5 and C-4. The water molecules are located inside the helical channel of the amylose and in the interstitial spaces, forming an intensive hydrogen-bonded network. Starch has been degraded by plasma and thermolysis in the ionization chamber of a mass spectrometer, and the s u g h and oligosaccharides formed were characterized by direct chemical ionization mass spectrometry.6 The technique, especially with negative ions, extends mass spectrometry to polymers. The molecular-size distribution of modified corn starch has

22


been determined by size-exclusion chromatography. The high performance liquid chromatography of malto-oligosaccharides has been reported. The degree of mechanical damage imparted to starch granules during flour milling has been correlated with various absorbancies in the near -infrared reflectance spectrum. The wavelengths of importance corresponded to overtones and combinations of vibration frequencies due to free and hydrogen-bonded hydroxy groups in the starch. A resonance Raman spectroscopic study has been made of the blue Similar spectra were colouring of iodine/iodide in amylose.l o obtained regardless of the KI and I 2 concentrations, degree of polymerization, and excitation wavelengths, but the relative intensities of the lines changed. It was concluded that the basic unit changed from 162- to I 2- through I s2- with decreasing KI concentration. A simple straight-line relationship has been obtained between average chain length of linear amyloglucans and extent of iodine staining." The wavelength of maximal optical absorbance, the maximal absorbance,and the iodine-binding capacity per chain are all linearly related to the chain length. The sorption of water and water-soluble alcohols by starch granules in aqueous suspension has been studied,and potato and corn starches absorb 33 and 28% water, respectively, at pH 7.'* The method was not suitable for cationic starches. Alcohols were also absorbed in large amounts. The kinetics of adsorption of 2,3-dialdehydostarch can be expressed by an exponential equation uniform heterogeneous which is applicable for energetically cellulose surfaces.13 The activation energy did not depend on the initial concentration of starch in solution but increased linearly with the increased amount of starch adsorbed. Some physicochemical properties of heat-moisture treatment of starches have been determined. The water-binding capacity and enzyme susceptibility increased, while the swelling power decreased. The starches with the highest moisture content before heating had the lowest swelling powers. The same treatments had adverse effects on the functional properties and baking potential o f the starches.15 An enzymatic and an acidic hydrolysis procedure have been compared f o r the analysis of starch in feeds and digesta.16 Comparable results were obtained for purified starches and feeds with high starch contents. Only in treated materials like digesta

23

3: Plant and Algal Polysaccharides

with more than 17% cellulose did problems arise due to hydrolysis of cellulose by the acidic treatment. The enzymes do not need to be highly purified to be used in starch determination even in the presence of cellulose and hemicelluloses. A series of n.m.r. spectra of whole seeds of various types have been obtained by using cross-polarization magic-angle spinning techniques. Selected signals provided a means of comparing the protein content relative to the starch content within a group of seed varieties. The multi-branched nature of amylose samples from several plant sources has been revealed by methods for estimating reducing and non-reducing residues. The isoamylase of a Pseudomonas partially split the branch linkage in potato amylose. The concurrent action of pullulanase from Aerobacter and @-amylase from sweet potato hydrolysed the amylose completely. An enzymatic method of determining the A and B chains in amylopectin has led to a ratio of 1 : 1 , not 2:l as previously suggested. 2o Partial debranching with pullulanase gave results A chains are consistent with earlier suggestions that predominantly and selectively removed by this enzyme. The changes in physicochemical properties of starches isolated from maize, barley,and triticale grains which had been allowed to sprout for various lengths of time have been determined.2’ The water-binding capacity initially decreased but then increased with increasing time whereas swelling power decreased and solubilities and enzyme susceptibilities both increased with time, A procedure adapted for the rapid analysis of total starch and amylose has been used on a series of 37 barley genotypes.22 The amylose content was not related to either starch content or grain yield. Investigations of the starch granules of wheat throughout their development have suggested that there are two distinct populations of granules which are initiated at separate stages of kernel d e v e l ~ p m e n t . ~This ~ theory was supported by results using a Coulter counter as well as light and electron microscopy and chemical analysis. In studies on the characterization of legume starches, the fine structures of amylose and amylopectin have been determined by the use of pullulanase, 6-amylase, and glucoamylase .24 The results indicated tbat there were some 1+6 substituted a-D-glucopyranosyl residues in amylose. Similar studies were carried out on

’

24

Carbohydrate Chemktry

acid-treated starches.25 A fast hydrolysis of amorphous and gel phases occurred with a slow hydrolysis of crystalline regions. The results were consistent with the structure being a cluster type In attempts to separate the starch and protein fractions of legumes, the starch fraction was found to contain between 7.7 and 20.1% protein while the protein fraction contained 0.0-4.6% starch.2 6 During germination of dry beans at 22OC, the total starch, amylose, and amylopectin contents all declined over the first 6-day period when the amy1ose:amylopectin ratio also declined.27 The solubility and swelling characteristics of the starch from the Great Northern bean have been found to be both temperature and pH dependent. 2 8 The solubility was highest at pH 6.0. Oxidation increased the solubility as a function of temperature whereas acetylation decreased the solubility. Acetylation and oxidation both reduced the swelling while addition of free fatty acids reduced the viscosity and raised the gelatinization temperature. The gelatinization temperature of the starch from the winged bean was 60-70°C and it exhibited single-stage swelling and low s ~ l u b i l i t y . ~It ~ was very soluble in DMSO and had an amylose content of 38.5%. The gelatinized starch was more susceptible to a-amylase and glucoamylase than the native starch. The morphological and physicochemical properties of starch from mature green and ripe bananas have been deter~nined.~' The granules were irregularly shaped with spheroid and elongated forms predominating. Other parameters were similar to those of the starch from mung beans. The starch from mature, unripe breadfruit has been isolated and characterized by the use of pullulanase and The @-amylase followed by gel-permeat ion chromatography. granules were 10-20 pm in size with an amylose content of 18.2% and 8-amylolysis limit of 58%. The debranched amylopectin had chains of 15 and 38-45 residues,respectively, in the ratio 4.4:l. The relationships between contents of dry matter, starch, total fermentables, and specific gravity in cassava storage roots have = a been determined.32 A curvilinear relationship of the form + 5 was found to exist between dry matter and starch content and a similar one for specific gravity and starch. The native starch from fermented cocoa beans has been isolated by solvent extraction and flocculation of non-starch material at pH 2.7.33 The egg-shaped granules had a mean size of 4 . 4 pm, an

-

'

x-'


25

amylose content of 30.4%, a gelatinization temperature of 52.5in 1M KOH. 68°C and a limiting viscosity number of 166.2 ml l'g Kuzu starch contains 21% amylose and 124 mg kg-' p h o ~ p h o r u s . ~ ~ The B-amylolysis limits of the amylose and amylopectin were 76 and 57, respectively, with 20.5 being the average chain length of the amylopectin. The granules were of the Ca crystalline type. A non-aqueous procedure, using glycerol and 3-chloro-1,2-propanediol, has been developed for the isolation from maize of starch granules with associated metabolite^.^^ Electron microscopy failed to demonstrate the presence of the amyloplast's membrane but it may be dried onto the granule surface. Soluble metabolites were found which may derive from the stroma of amyloplasts. The distribution of starch components of maize (Zea mays) and their properties have been investigated by gel filtration after debranching and by exhaustive hydrolysis of the granules with glucoamylase .36 Based on the results, the relationship between genotypes and properties of starch components was discussed. For gene produced example, introduction of the amylose-extender ( E ) amylopectins of longer average chain lengths. The starch and phytoglycogen fractions from normal and sugary (E) maize have been compared, and negligible differences were observed between 2 inbred lines and 2 9~ conversions of the lines studied.37 The phytoglycogen was shown to be distributed in a soluble fraction and a particulate fraction which also contained some amylose. These observations are consistent with a pattern of conversion of starch into phytoglycogen in endosperm. The starch of wild rice (Zizania aquatica) has been isolated and is a very small granule (2-7 urn) of angular and polygonal shape.38 It has an A-type pattern on X-ray diffraction but an amylose content of only 2%. Other properties were compared with those of wheat starch. Normal- and waxy-type starches have been isolated from two types of Amaranthus hypochondriacus and found to be round granules ( 1 urn diameter) with similar X-ray diffraction diagrams to other A-types. 39 The amylose contents were 1 4 and O%, respectively,while the amylopectins were similar to those from rice and maize starches. The elephant yam (Amorphophallus campanulatus) has yielded a starch which exhibited single-stage swelling and moderate solubility in water but low resistance to solubilization in

26


DMS0.40 The amylose content was 24.5%. The amylolytic susceptibility of the native starch was low but that of the gelatinized starch was high. Oxidation of malto-oligosaccharides has yielded the series of oligomers with aldonolactones at the reducing end position and these give diamides with alkylenediamines in 30-755 yield.41 The effect of these derivatives on the enzymatic activity of potato phosphorylase was determined. The digestion of hydroxypropyl wheat starches by porcine pancreatic a-amylase has been examined.42 The digestibility of the of gelatinized starches decreased with increasing degree substitution, and the action pattern favoured a non-repetitive attack whereas the digestibility of the native starches increased with extent of modification. The hydrolysates from the above digestion of starches with degrees of substitution from 0-0.17 were separated into total digest, oligo- and polysaccharide fractions and further analysed .43 Increasing the degree of substitution caused a reduction in the reducing value, an increase in the average degree of polymerization, and a greater proportion of oligosaccharides in the hydrolysate. The oligosaccharide fraction had double the hydroxypropyl content of the undigested starch. The oligosaccharide fraction was further analysed by h.p. 1.c. on a u-BondapakR carbohydrate column .44 The proportion of oligosaccharides with three and four ;-glucose residues increased and those with five !-glucose residues decreased as the degree of substitution increased. The changes in molecular weight of amylose and amylopectin during thermal dispersion in water have been followed by paper Grain disaggregation and mo lecular chromatography.4 5 disintegration of amylopectin proceeded more rapidly than in amylose. The effect of sodium hydroxide was more marked on amylose than amylopectin. Aqueous suspensions of starch at neutral pH values were heated by microwave energy in sealed tubes.46 The colour of the hydrolysates darkened with increasing time and pressure. The total acidity increased and oligosaccharides in the range monomer to octamer were determined. The amounts of glyceraldehyde, dihydroxyacetone, and 2-hydroxyl,+propanedial induced in maize starch by Y-radiation have been determined by a g.c. procedure,and the influence of several .parameters such as dose and temperature has been in~estigated.~’

27


This is the first report of 2-hydroxy-1,3-propanedial from irradiated starch. The influence of the polysaccharide structure on the oxidation process with Ce4+, Mn3+, and V 5 + ions has been studied,and it was confirmed that the rates were highest for compounds containing a-glycol groups.48 The nature of the functional groups and the conformation of the macromolecular unit exert an influence on the oxidation rate. An enzymatic procedure has been developed to measure starch in cereal product^.^' The precision of the test is 2 1.5% and is applicable to a wide range of products. The amylases from pearl millet have an action pattern similar to those of other cereal a-amylases but are able to hydrolyse wheat starch more readily than millet starch.50 The immature wheat endosperm aleurone with seed coat and embryo detached has produced considerably less a-amylase activity than immature whole or de-embryonated wheat kernels. The isoenzyme composition of the incubated endosperm aleurone was unique suggesting that the seed coat may contain factors required for normal a-amylase isoenzyme synthesis. An enzymatic assay for the determination of a-amylase in serum has been developed which employs maltoheptaose as substrate and a coupled enzymatic indicator system.52 H.p.1.c. was used to establish the action pattern of hydrolysis of the maltoheptaose. The stoichiometric equations for the action of a-amylase alone and a-amylase and a-glucosidase together were derived. The multiple-attack model for the kinetics of hog pancreatic a-amylase action has been a n a l y ~ e d . ~The ~ model explains the dependence of ,V and 2 on the degree of polymerization of substrates. The distribution in the degree of polymerization of the hydrolysis products has been used to calculate the number of unitary movements of the enzyme during the single enzymesubstrate meeting. A mixture of 6-amylase and pullulanase from Bacillus cereus has been used to produce maltose from a variety of starches which had previously been liquefied by the a-amylase of Bacillus ~ u b t i l i s . ~ ~ The maximum yield was 87-88% obtained at pH 6.0-8.0, 55"C,and 30h hydrolysis of a 5% solution. Similar results were obtained if acidic conditions were used for the liquefaction. Two inbred lines of rye (Secale cereale), whose kernels had only 1-3% of the 6-amylase activity of normal lines, have been

'

28


investigated using an anti-wheat B-amylase immune serum which cross-reacted with the rye enzyme.55 The results indicated that the reduced activity was due neither to the presence of an inhibitor nor to the production of inactive enzymes. An inducible cell-bound glucoamylase, which degrades cyclodextrins, has been isolated from a Flavobacterium species. 56 The final degradation product in all cases was ;-glucose with small amounts of maltose. Amylopectin and glycogen were very poor substrates. The enzymes of starch metabolism in the developing grains of a high-lysine barley mutant have been determir~ed.~’The activity of ADP-E-g1ucose:starch synthetase was lower while those of UDP-Eglucose-pyrophosphorylase and starch phosphorylase were higher than in normal barley. The structural changes in starch molecules during malting of barley have been investigated by comparing a normal malt where 14% of the starch had been lost with an over-modified malt where 68% of the starch had been lost.58 In the normal malt, 14% of the large granules were eroded but the small granules remained intact whereas 38% of the large granules were eroded in the over-modified malt with a marked reduction in the small granules. Malting resulted in an apparent increase in the amylose content. There was no apparent difference in the amylopectin structure between the two malts. A reduction in light intensity during starch synthesis in developing wheat grains has reduced the starch synthetase I activity but not the starch phosphorylase activity. 59 The low starch production was not due to an insufficient supply of assimilates. [ 35S]-Methionine has been incorporated into B-amylase from germinating rice seeds.60 The specific radioactivity of the enzyme derived from scutellum was significantly greater than that from the endosperm. These results indicate that, at the onset of germination, 6-amylase is synthesized in the scutellum and only at a later stage is an inactive, latent form of the enzyme activated in the endosperm. This latter form becomes dominant during the later stages of germination. The role of the recessive amylose-extender ( E ) allele in the biosynthesis of maize starch has been investigated.61 Multiple forms of starch synthetase were observed, some of them appearing at the same time as the rapid increase in amylose content. The

29


presence of modified amylopectin synthesized by the enzymes which were affected by the ae allele was suggested. Debranching of these amylopectins followed by analysis of the products was attempted to confirm this theory, but the results were inconclusive. Starch granule-bound D-glucan synthetase has been isolated from cotton leaves.63 On further separation, amylopectin was the only glycosidically linked with protein and this had E-glucan Q-glucan synthetase activity. The amylose component was free from covalently bound protein. On germination, both in the dark and light, a DMSO-soluble P-glucan is synthesized in the cotyledons of lupins .64 Chemical analysis indicated that it was starch-like but its chromatographic behaviour differed from that of amylopectin. The concentrations of starch synthetase and debranching enzyme from developing seeds of Pisum sativum have been mea~ured.'~ Primed starch synthetase activity increased from day 8 to day 14 after anthesis but then decreased by 50% at 26 days,whereas citrate-stimulated starch synthetase activity was highest at 10 days after anthesis thereafter falling to a low value. Debranching activity increased from day 8 to day 18 after anthesis. The changes in starch synthetase and debranching activity resulted from changes in the concentration of a few enzyme forms and not from the appearance of different enzyme forms. The @-amylase activities of some soybean varieties are times lower than others yet they have similar starch metabolism. It was therefore concluded that starch metabolism was independent of the 8-amylase activity. The starch contents of plantains and bananas, ripened under similar conditions, have been determined with a content of 1% in ripe bananas and 9% in ripe plantain^.'^ These values fell to zero and 3%,respectively,in over-ripe material. Phosphorylase I1 from potato tubers has been purified and characterized.68 No separation of primed from unprimed activity was achieved. The molecular weight was 9.6 x l o 4 and the native enzyme was a dimer. The reaction product was a protein-bound P-glucan which possessed long ( 1+4)-a-E-glucan chains. 6 9 Since protein was also present in amylose from potato starch grains, it is suggested that these findings may be further evidence of a from a precursor common biosynthetic pathway for (1+4)-a-D-glucans protein. The rate of starch breakdown in isolated chloroplasts from

'*

''


30

spinach is similar to that found in whole leaves. These rates have been measured both in the dark and in light and compared with the rates of starch synthesis in the light.’l’ Illumination has little inhibitory effect on breakdown at high phosphate levels but 67% inhibition was observed at low phosphate levels. Since C02 evolution is prevented by illumination, the oxidative pentose phosphate pathway must have been inhibited. The balance between accumulation and breakdown is very sensitive to the rate at which carbon is withdrawn from the chloroplast in exchange for inorganic phosphate. The unusually high solution torque developed by the starch from the Hinoat variety of oats when a hot paste is cooled to 75-80°C has been abolished in the presence of acetic acid at pH 3.0.’12 Salts at 0.1M concentration reduced the torque value as did increasing sucrose concentrations. The gelatinization of wheat starch as modified by xanthan, guar and cellulose gums has been investigated and each gum hastens the onset of initial paste viscosity.’13 The reaction seemed to be a strong association with the amylose component since the B-amylolysis limit of the amylose is reduced. The starch-xanthan gum exhibited a pseudoplastic behaviour whereas the starch-guar gum resisted shearing at low shear rates. The syneresis of curdlan gel has been repressed by the addition of starch before heating but not by other sugars.74 The role of starch in this process was discussed. A number of starch esters of herbicides have been prepared from activated pregelatinized starch and evaluated as slow release agents.’15 With sterile soil a picloram ester was phytotoxic for 48 days. Unmodified starch esters were hydrolysed at a slower rate than gelatinized starch esters. A Flavobacterium from tap water was able to grow on tap water but the addition of starch greatly enhanced its growth.76 X-Ray and neutron-diffraction studies on hexa-amylose hexahydrate crystal forms A and B have given an indication of the chain-like and circular arrangement of hydrogen bonds within the molecules. ’17 In the circles, homodromic and antidromic orientations of the hydrogen bonds are observed, with the homodromic circles being more stable by 8% per hydrogen bond. The changes in electronic charges on H and 0 atoms are greater in homodromic circles and the dipole moments are only ~2.30 in the chain-like and antidromic homodromic circles but 6-8D in

-


31

arrangements. -X-Ray analysis of the hexa-amylose-3-nitroaniline ( 1 : l ) hexahydrate complex has shown that the hexa-amylose molecules are stacked along the z-axis in a head-to-tail fashion to form a channel-type structure .78 A stereo-drawing of the packing feature of the complex with its associated water molecules was shown. Similar studies have been conducted on the hexa-amylose-benzaldehyde ( 1 : l ) hexahydrate complex and a similar packing was observed with the hexa-amylose molecule being tilted at 11.5" against the channel axis." The benzene ring is inserted into the hexa-amylose ring from the secondary hydroxy side while the carbonyl group is in Van der Waals contact with the primary hydroxy side of the next hexa-amylose molecule. A correlation between the penetration depth of the C atoms of a variety of phenols in the cavity of hexa-amyloseand the changes in chemical shifts has been investigated.80 The their 1 3 C n.m.r. formation and molecular dynamics of cycloamylose inclusion complexes with phenylalanine have been investigated by 1 3 C n.m.r. spectroscopy.8 The influence of the cavity size was determined and the cyclohepta-amylose-phenylalanine system was most strongly coupled. The cyclohexa-amylose coupling was shallow and loose while that of the octa-amylose was deep and loose. The induced c.d. and U.V. spectra ofhepta-amylose-disubstituted benzene systems has been studied with respect to their molecular structures.82 The dipole moment was correlated with rotational intensity and the energy-level transition calculated by molecular orbital methods. The use of a hexa-amylose mobile phase in the separation of mono-substi tuted phenols by t .I. c. has been reported .83 The llf values depended on both the structural features of the phenolic compounds and the concentration of hexa-amylose in the mobile phase. A selective attack of dichlorocarbene at the 4-position of phenolate ion was achieved with hexa-amylose as catalyst.84 The mechanism was studied by lH and 13C n.m.r. spectroscopy.

3 Fructans The has and

crystalline conformation of inulin [(2+1)-B-E-fructofurananl been determined by conformational analysis coupled with g-ray electron- diffraction data.85 The only two models possible

32


correspond to 5-fold helices, one left-handed and the other right-handed. Steric interactions of the substituents and the exanomeric effect were believed to be responsible for the observed conformational differences. An invertase and an inulase from germinating garlic (Allium sativum) bulbs have been separated and purified.86 The invertase had no effect on inulin whereas the inulase, as well as hydrolysing inulin, could hydrolyse sucrose and raffinose. The enzymes had similar molecular weights (7.6 x l o 4 ) . In the early stages of development the activities increased in paralle1,but at later stages the invertase activity was higher. A pathway for fructan trisaccharide synthesis from sucrose has been reported.87 Two non-reducing hexasaccharides ( 1 ) and (2) have been isolated from the roots of Asparagus officinalis and their structures confirmed by the usual methods.88 A novel tetrasaccharide ( 3 ) has been isolated from Chinese milk vetch honey.89 It is probably synthesised 2 erlose which is produced from sucrose in nectar.

4 Cellulose A history of the study of cellulose structure has been published which also considered how this knowledge has been extended to synthetic polymers. A hydrate of cellulose I1 has been prepared and its crystal structure determined.91 The unit cell contains disaccharide sections of two chains,and an antiparallel arrangement of adjacent chains was assumed. The chains are stacked in the same way as in cellulose I1 with the water molecules between the stacks,but it was not possible to position the water molecules in a regular arrangement. The treatment of cotton and rayon with ethylenediamine resulted in the transformation of cellulose IV to cellulose I11 as determined by X-ray analy~is.’~ Thus ethylenediamine-methanol can affect lattice modifications. The transformation of cellulose I into cellulose I11 in Valonia macrophysa, preformed in ethylenediamine, has maintained the external appearance of the microfibrils as shown by electron microscopy. 93 This conversion must have involved a fracturing and fibrillation of the initial crystals. ‘ H n.m.r. spectroscopy has shown that both ramie and cotton of native have a unique phase structure characteristic

33


B-~-Fruf-(2+l)-~-~-Fruf-(2-tl)-~-~-Fruf-(

-

1 .f

2 B-D-Fruf -

(1)

B-E-Fruf- (2+1)-B-E-Fruf-(2+6)-a-D-Glcp- (1+2)-B-!-Fruf -

1

.f

B-D-Fruf-(Z+l (2)

2 )-$-E-Fruf

34

Carbohydrate Chemiitry

cellul0se.9~ The non-crystalline component did not exhibit micro-Brownian segmental motion even in the swollen state. The phase structure was distinctly different from that of regenerated cellulose. An n.m.r. spectroscopic study of the interactions between cadoxen and cellulose has been carried out using signals from 'H, 13C, and 113Cd.95 A well recorded 13C spectrum was obtained which could be assigned by comparison with the spectra of simpler sugars. The 13Cd and 13C results were not consistent with the formation of chelate-alcoholate complexes with the C-2 and C-3 hydroxy groups. Hydrogen bonding is probably the dominant interaction. It is further suggested that the metal ion serves the dual purpose of holding two amino groups in a favourable orientation to hydrogen bond with a pair of equatorial hydroxy groups on the cellulose. The solubilization of cellulose and other plant structural carbohydrates has been achieved in a mixture of 4-methylmorpholine g-oxide and DMSO.' The separation and purification of homogeneous polgsaccharide fractions could then be made. Hydroxymethylcellulose has been prepared and characterized by i.r. and n.m.r. s p e c t r o s ~ o p y . ~To ~ achieve dissolution of cellulose, a high degree of molecular substitution was required, but once solution was achieved the molecular substitution could be lowered, the actual value of precipitation depending on the solvent. A rapid separation of cello-oligosaccharides (degree of polymerization 2-9) has been obtained on a cation-exchange resin in 60 min with water as eluant.98 The cellulose content of raw and cooked vegetables has been determined by extraction procedures using detergent solutions.9 9 The U.V. spectra of extracts of hydrocellulose with the hydroxides of Li', Na', K ' , Ca2+, Sr2+,and Ba2+ were identical."' The curves of yellowing absorption coefficients against loss in weight of hydrocellulose in boiling solutions of these alkalis were linear, the slopes giving apparent molar absorption coefficients in the order Na+= K+>Li+>Ba2+>Sr2+>Ca2+.The divalent cations reduced the yield of chromogen from the 4-deoxy-~-glycero-2,3-hexodiulose released from reducing end groups of the hydrocellulose. In the same systems, greater weight losses and production of acidity were obtained with the alkalineearth cations than alkali-metal cations when the OH- ion

35


concentration was 0.02M while the reverse was found at 1.OM. l o ' The results were explained in terms of a hypothesis that divalent cations exercise their catalytic effects through enhanced ionization of the ;-glucose moiety at the reducing terminus of cellulose chains. The selectivity (viscosity at a given Kappa number) of softwood Kraft pulp has been shown to increase dramatically when soaked with S02-H20 in the presence of diethylenetriaminepentamethylene phosphonic acid before oxygen bleaching in the presence of magnesium sulphate. O 2 Addition of manganese(VI1) sulphate to soaked pulps led to a severe loss in selectivity while to dry pulps a slight increase was obtained. Addition of the phosphonic acid to manganese(VI1)-treated pulps caused a large increase in selectivity. Aqueous sulphur dioxide treatment of cellulose pulp of and cotton linters dramatically reduced the degree polymerization of the cellulose but did not affect its enzymatic digestibility or decrease its crystallinity as previously reported. 1 0 3 The acetyl content of cellulose acetate and a variety of woods has been determined by aminolysis with pyrrolidine followed by g.c. of the resultant I-acetylpyrrolidine. O 4 The distribution of the sulphate ester groups in the natural cellulose sulphate of Buccinum undatum has been determined by 1 3 C n.m.r.; the results were compared with those from a randomly The relative substituted synthetic cellulose sulphate.' O5 simplicity of the natural cellulose sulphate spectrum indicated that this polymer consisted of blocks of disulphated (1+4)-B-;-glucan chains separated by blocks of unsulphated chains. (1+4)-~-Q-glucan A variety of sulphoalkylcelluloses have been synthesized in NaOH solutions at 65°C. l o 6 Increasing temperature and NaOH concentrations increased the sulphoalkylation as well as the yield of water- and alkali-soluble fractions. The relative oxyanion formation at hydroxy groups of q-glucosides and cellulose has been reviewed O7 Native cellulose and pulps from a variety of hardwoods have been fractionated by the nitric acid rnethod.'O8 A regression analysis showed a direct correlation between the age of the tree and the degree of polymerization of the cellulose. The values were a useful indicator of the papermaking potential of the wood. Cellulose powders obtained by mechanical disintegration have

'

'

.'

36


larger average particle sizes and broader size distributions than those obtained by hydrolytic degradation.l o g Microcrystallization of bleached sulphate pulp from beechwood and reed increased the crystallinity index from 66 to 71% and 56 to 61%, respectively, as determined by X-ray analysis, but decreased the yield from 86 to 75% and 74 to 67%, respectively.'" The average length of the microcrystalline pulp fibres was 55 um compared with 220 urn for the control fibres. By the use of polarizing microscopy, the microfibrils in the spherulites formed in the pellicle of bacterial cellulose have been shown to be radially or tangentially orientated. Some of the results indicated that the initial stage of nucleation may be a function of intermediate lipid-carbohydrate compounds as in the lyotropic liquid crystal of the soap micelle. When virulent strains of Agrobacterium tumefaciens bind to carrot cell walls, fibrils develop which surround the bacteria and anchor them to the cell surface.'12 The fibrils are composed of cellulose and are synthesized by the bacteria since they are produced on dead as well as living carrot cells. Within this entrapped mass, the bacteria are still able to multiply and form large clusters. On pyrolysis of cellulose under various conditions, 82 compounds have been identified as volatile product^."^ The maximum yield in a nitrogen atmosphere was achieved at 600-650°C while in air a 50-200°C lower temperature was required. The volatile products were qualitatively similar to those found in cigarette smoke. Combined q.c. gas-phase thermal fragmentation has been developed for the separation and identification of pyrolysis The compounds were products from bark polysaccharides.' identified by their fragmentation pattern which was useful in demonstrating the thermal stability of the parent compounds. Various polymer initiating systems, especially redox systems involving cerium(II1) and iron(I1) salts,had little effect on the thermal properties of cotton cellulose.' l 5 The cellulose from Acetobacter xylinum has been used for visualizing the action of the cellulase enzyme system from Trichoderma reesei by high-resolution electron microscopy.' l 6 The enzymes initially bind to the cellulose ribbon, but within 10 min the ribbon is split along its longitudinal axis into bundles of microfibrils which are thinned until they are dissolved,which takes about 30 min. Separation of the various enzyme components

'''

'


37

produced less dramatic changes confirming the synergistic mode of action of these enzymes. The cellulase from T. viride has been immobilized on SepharoseR 4B.l17 The immobilized enzyme had a similar Km to the free enzyme, the activity increased up to 50°C and did not decrease after 4 cycles of batch use and recovery. Treatment of finely divided Pinus densiflora with the cellulase from T. viride at 40°C and pH 4.0 resulted in hydrolysis of 80-90% of the carbohydrate present .I18 A series of mono- and oligo-saccharides were obtained from bagasse and pith by cellulase hydrolysis."' The presence of pentoses and their oligosaccharides in hydrolysates of alkali-treated bagasse showed that hemicelluloses had not been completely removed by this treatment. The action of the cellulase from Penicillium funiculosum on a variety of cellulosic substrates has been investigated by scanning electron microscopy and X-ray diffraction analysis. 120 The differences were interpreted in terms of the a-cellulose content, surface area, degree of crystallinity, and crystallite dimensions of the substrate. The kinetics of hydrolysis of ball-milled cotton linters by eight cellulase complexes from Trichoderma, Geotrichum, and Asperuillus species have been studied under steady-state and nonThe steady-state rate of D-glucose steady-state conditions. 12' formation was proportional to the endo-(1-+4)-B-D-glucanase content. A mechanism was proposed in which the enzyme which first attacks the insoluble substrate is this m - P - g l u c a n a s e . This contradicts the accepted view for a prehydrolytic C1 enzyme. The C 1 role is filled by the endo-g-glucanase. Partial acid hydrolysis has been investigated as a pretreatment to enhance the enzymatic hydrolysis of cellulosic materials. 2 2 Up to 100% of the potential E-glucose could be obtained,and this was due to removal of hemicellulose, reduction in the degree of polymerization, and changes in the crystal structure of the cellulose. The purification of a cellulase isoenzyme from kidney bean abscission zones has been carried out by affinity chromatography. 1 2 3 Antibodies raised against this cellulase (PI = 9.5) did not react with the cellulase (PI = 4.5) obtained from 2,bE-treated abscission zones. examined, only 3 1 were Of 60 species of soil fungi


38

cellulolytic.1 2 4 The- cellulolytic activity of 25 species of pyrenomycetous fungi has been determined with 18 being able to utilize cellulose. 125 A Bacteroides species, of human faecal origin, was able to degrade isolated cell walls of peanuts.' 2 6 When autospores of Oocystis solitaria were treated with Congo Red or Calcofluor White a more diffuse fibrillar material was obtained [which could be chains of (1+4)-B-D-glucanl rather than the discrete microfibrils of untreated cells. 2 7 Using intact cotton fibres, it has been shown by pulse-chase experiments that (1+3)-8-~-glucans (callose) have a high rate of turnover and are likely to be intermediates in cellulose biosynthesis at the stage of secondary cell-wall formation. 28 Protoplasts from cultured soybean (Glycine max) cells have been used to study polysaccharide synthesis during the early stages of cell-wall regeneration. 29 Label from !-glucose was rapidly incorporated into cellulose but neither UDP- nor GDP-P-glucose was rapidly incorporated into (1+3)-B-~-glucans, Calcofluor White ST can increase the rate of ;-glucose polymerization into the cellulose of Acetobacter xylinum.' 30 At a concentration of >O.lmM, the rate was four times the control but the assembly of crystalline cellulose I microfibrils was disrupted. It is concluded that polymerization and crystallization are cell-directed, coupled processes and that the rate of crystallization determines the rate of polymerization. Coupling must be maintained for biogenesis of cellulose I. Addition of coumarin (1 g kg-') to Phaseolus vulgaris leaves inhibited the synthesis of cellulose I but stimulated the synthesis of a-cel1ulose.l3' A persisting cellulosic wall has been demonstrated by light microscopy in microspore mother cells during microsporogenesis in Allium tuberosum and Cyclamen persicum. 1 3 2 Cellulose metabolism by the flagellate Trichonympha, isolated from a termite, has been studied.'33 The protozoon, in culture, harboured no endosymbiotic bacteria and produced acetate, CO,, and H, from cellulose. The cellulolytic activity is therefore an inherent property. A new method of determining the extent of binding of an ionic dye to a polyelectrolyte in solution has used the fact that dyes can adsorb to a cellulose dialysis membrane.'34 The permeability of cellulosic walls to macromolecules may

'

'

'

39


limit the ability of enzymes to alter the properties of these walls. 135 Since proteins of molecular weight 6 x lo4 can permeate a substantial portion of the wall, previous results may have underestimated this property. The fermentation of cellulose by Acetivibrio cellulolyticus, Methanosarcina barkeri, and a Desulphovibrio species has been culture allowed a rapid studied. 3 6 Only a three-component conversion to C02 and CH4 and utilized 85% of the cellulose present. This value was increased to 90% by increasing the concentrations of M. barkeri.

5 Hemicelluloses

A number of sugar residues were found in hydrolysates of the water-soluble hemicelluloses of rice brans. 3 7 Although L-arabinose was the major sugar residue, there was no direct evidence for the presence of an L-arabinan. The intracellular site of the synthesis of 4-arabinose-containing cell-wall precursors in suspension-cultured tobacco cells has been determined as the Golgi bodies with transport being vesicles in the low-density fraction.1 3 8 A D-galactan has been isolated from the leaves of Aloe barbadensis by hot-water extraction and separation from contaminating pectin, ;-gluco-q-mannan, and 4-arabinan. 1 3 ’ The (l+4)-linked ;-galactan had one branch point at 0-6 for every 26 residueq and the molecular weight of the acetylated polysaccharide was 3.7 x 104. The formation of monodispersed polysaccharides on Smith degradation of the acidic k-arabino-;-galactan from rapeseed (Brassica campestris) strongly suggests a molecular structure composed of regularly repeated subunits. 1 4 * The molecular weight of the subunit was 1.2 x lo4 and the resistance of the polysaccharide to subsequent Smith degradations suggested that the main chain was a (1*3)-?-galactan. An 4-arabino-2-galactan-protein has been released by sonication of a crude cell organelle fraction from hypocotyl tissue of bean seedlings (Phaseolus vulj?aris) and suspension cultured cells. 1 4 ’ The macromolecule (mol. wt. 1.4 x l o 5 ) contained 90% carbohydrate and 10% protein with the major sugar residues being ;-galactose, L-arabinose,and uronic acids and the major amino-acids present being k-hydroxyproline, &-serine,and L-alanine. The carbohydrate

’

-


40

was linked the first two amino-acids and also to 4-threonine with the carbohydrate portion of the linkage regions containing residues of L_-arabinose,q-galactose , 8-glucose,and k-rhamnose .14' The L-hydroxyproline glycosides were different from those in nonextractable cell-wall protein but have similarities with those in the wall proteins of the algae. The changes in the 5-arabino-q-galactan-proteins of the carrot have been studied during germination and growth.143 An extracellular k-arabino-8-galactan-protein has been obtained from suspension-cultured tobacco cells. 44 The complex (mol. wt. 2.24 x l o 5 ) contained 5.5% protein and the sugar moiety was a A B-q-glucopyranosyluronic typical L-arabino-(1+3)-8-galactan. acid residue was present at the non-reducing terminus attached to 0-6 of a P-galactopyranosyl residue. Auxin-induced changes in the sugar composition, intrinsic weight distribution of matrix viscosity, and molecular polysaccharides in the epicotyl cell wall of Vigna angularis have been determined. 1 4 5 The greatest change was the reduction in 4-arabino-q-galactan content, but other changes in pectin and E-xylo-g-glucan content were also observed. The linkage regions in cell walls of rice coleoptiles have been characterized as involving 0 _ 3 - ~ - g a l a c t o p y r a n o s y l - ~ - s e r i n e and N4 - ( 2-ac etam i do 2- d eoxy- B -E- g1ucopyran0s y1 ) hydrogen LThe linkage region in the water-soluble asparaginate. 1 4 6 4-arabino-E-galactan-protein complex from wheat endosperm has been characterized as g4 B ga1acto p yrano s y1 4 -trans hyd ro xy proline. 14' Methylation analysis of the glycopeptide linkage region from cell walls of Chlamydomonas reinhardii has revealed that g-glycosidically linked g-galactofuranosyl residues are present. 1 48 Bacteroides thetaiotaomicron, a polgsaccharide degrading bacterium from the human colon,can use larch L-arabino-;-galactan as its carbon source with yields similar to those obtained on p-glucose. 149 The crystal structure of a regenerated form of (1+3)-a-D-glucan has been determined by X-ray diffraction analysis and stereochemical model refinement as an orthorhombic unit cell. 50 The chain conformation is almost completely extended and is very close to a 2/1 helix even though the dimer residue is the crystallographic repeat unit. The vacuum c.d. spectrum of (1+6)-~-~-glucan has a single

-

c

-

---

-

-

-

41


positive band near 177 nm.151 As gels formed, a negative band at 190 nm appeared followed by a blue shift in both bands with ageing. The specificity of the interaction of direct dyes with polysaccharides has been studied by changes in solubility and in the fluorescence and absorption spectra of the dyes.’52 The strongest interactions were shown by polysaccharides with (l+4)-linked-B-Q-glucopyranosyl residues, but contiguous (1+3)-8-P-glucans exhibited some complex formation. Dyes differed in their affinity. The direct dye, Calcofluor, has been used in a fluorimetric assay for (1+3)(1+4)-B-D-glucans in beer, wort, malt, and barley. The B-E-glucan content of developing and germinating kernels of several varieties of barley has been determined 5 4 The E-glucan content varied considerably but did not seem to be related to the B-g-glucanase activity of the variety. A g-glucan is present in the cotyledons of Mirabilis jalapa seeds.155 Methylation, periodate oxidation, and graded and enzymatic hydrolysis studies have indicated 34 (l+4)- and 3 ( l + 3 ) linkages for every 38 ;-glucose residues. The branch point appears at 0-2. In some places, at least three (1+3)-linked P-glucopyranosyl residues are in sequence. Both a and 8-glycosidic linkages are present. The hemicellulose fraction accounted for 47% of the pollen tube wall of Camellia japonica and comprised essentially c-glucose The polysaccharide had a ( 1 +3)-B-;-glucan chain with residues. 15‘ a degree of polymerization of 21. The pollen tube wall of C. sasanqua, C. sinensis, Tulipa gesneriana,and Lilium longiflorum also consisted mostly of ;-glucose residues,and the hemicellulose fractions were essentially pure D - g l u c a n ~ . ’ ~The ~ deposition of (1+3)-8-;-glucan during megagametogenesis in two species of Oenothera is partly in continuum with the wall and partly in the degenerating cytoplasm. The addition of B-g-glucanase before the use of u-amylase has been used to aid filtration in the measurement of fibre contents by detergent procedures. 59 The fact that fungal g-glucans will stimulate soybeans to accumulate phytoalexins has prompted an investigation into the (1+3)-B-~-glucanases and B-P-glucosidases of soybean and their hydrolytic patterns.16’ The B-9-glucosylase I enzyme has been purified. Several lines of evidence suggest that the

.’

’

’

42

Carbohydrate Chemisrry

(1+3)-8-;-glucanase and 8-P-glucosidase activities exhibited by I preparations are catalysed by the same enzyme. B-g-glucosylase Treatment of the 2-glucan elicitor from the walls of Phytophthora megasperma by E-glucosylase I results in extensive hydrolysis and loss of 94% of its activity.lbl It is unlikely that some other enzyme(s) may be involved in the overall reaction. The endo- and exo-8-P-glucanases from Zea mays have been partially purified. l b 2 The endo-8-q-glucanase (mol. wt. 2.6 x lo4> showed a marked preference for substrates with mixed linkages,and it probably initiated the solubilization of wall g-glucan. The exo-8-E-glucanase (mol. wt. 6.0 x lo4) was required for extensive hydrolysis in vitro. During coleoptile development, the autolytic activity of the cell wall increased dramatically but did not parallel the growth potential of the tissue. Conditions which induce a transmembrane electrical potential, positive with respect to the inside of the membrane vesicles, have resulted in a 4-12-fold stimulation of the activity of membraneassociated 8-q-glucan synthetases in a preparation from developing cotton (Gossypium hirsutum) fibres.1 6 3 The products were mainly (1+3)-8-;-glucans but some increase in (1-+4)-B-D-glucan content was also found. No (1+4)-a-q-glucan was formed. The products of 8-q-glucan synthetases have been characterized using specific -9-glucan hydrolases. 6 4 The B-Q-glucan synthesis by particulate enzymes from suspension-cultured endosperm cell walls from ryegrass has been studied. 6 5 The 8-E-glucan synthetase activity of the fungus Saprolemia monoica has been assayed and a product is observed which contains The both 1 +3- and 1 -+blinked B-g-glucopyranosyl residues. 6 6 presence of Mg2+ ions affects the structure of the product. In the absence of Mg2+, only a (1-+3)-8-P-glucan is formed, but as the substrate concentration is reduced and Mg2+ ion concentration is increased the proportion of lj3-linked residues in the product is reduced and a pure ( 1-+4)-B-Q-glucan is formed. High molecular weight E-glucans have been isolated from mycelial walls of Fusarium oxysporum.' 6 7 The P-glucans contained both 1+3- and 1-+6-linked residues in contrast to the E-glucans from Colletotrichum lindemuthianum, which contained 1+3- and 1-+4-linkedresidues. However, the two types of 9-glucans had very similar effects in eliciting browning and phytoalexin production in green-bean cotyledons. An exo-(l+3)-B-;-glucanase derived from Sclerotinia libertiana

'


has

induced

growth

43

of Avena sativa coleoptiles and degraded the No endo-( 1+4)or endo-( 1+3)-B-pglucanase activity could be detected. The exo-(1+3)-8-;-glucanase from a Basidiomycete did not induce growth. The metabolism of a e-glucan from the cactus Opuntia bigelovii has been studied in a desert en~ir0nment.l~’The D-glucan content increased slightly during a 9-week drought period but then decreased during the irrigation period. The ;-glucan is probably storage material. The g-xylo-;-glucan from the walls of etiolated mung beans has been studied by acidic and enzymatic hydrolyses, the latter by a preparation from Aspergillus oryzae. 170 Cellobiose, cellotriose, and cellotetraose were obtained from the acid hydrolysate while isoprimeverose ( 4 ) and the pentasaccharide ( 5 ) were characterized from the enzymic hydrolysate. Similar studies using the cellulase from Trichoderma viride produced three types of oligosaccharide, hepta-, nona-, and deca-saccharides, and their structures were partially characterized. 1 7 ’ The structure of a cell-wall polysaccharide isolated from cotyledons of tora bean has been established by methylation analysis, Smith degradation, and enzymatic fragmentation. 1 7 2 The main features were those of a E-galacto-E-xylo-D-glucan where a (1+4)-8-P-glucan main chain is substituted at 0-6 by and 8-8-galactopyranosyl-E-xylopyranosyl residues. D-xylopyranosyl A further feature was the presence of (1+5)-a-L-arabinan side chains (degree of polymerization = 8 ) at some 0-3 positions of the main chain. A Q-xylo-E-glucan has been isolated from the cell walls of potato (Solanum tuberosum), and the methylation analysis and fragmentation results confirmed the usual structure. 73 Some of the g-xylose residues carried further substitution at 0-2 with L-arabinofuranosyl and 9-galactopyranosyl residues. The biosynthesis of the P-xylo-P-glucan in cultured soybean cells has been studied. 7 4 Two types were obtained from cultured cell walls (mol. wts. 1.8 x l o 5 and 6 x l o 4 ) and one from the culture medium, the molecular weight of which shifted from 6 x l o 4 to 3 x l o 4 during the progress of cultivation. The compositions were very similar and two kinds of oligosaccharide repeating units were present, namely a heptasaccharide (Q-xy1ose:D-glucose 3:4) and a nonasaccharide (?-galactose:&-fucose:;-xylose:Q-glucose (1:1:3:4)). Incubation of a particulate fraction from soybean ( 1 + 3 ) (1+4)-8-p-glucans. -

44



45

cells with UDP-14C-Q-glucose or UDP-I C-E-xylose produced a A. oryzae enzymes gave a labelled polymer.175 Digestion with labelled disaccharide characterized as isoprimeverose (4) by the usual methods. Since isoprimeverose is the smallest structural unit of D-xylo-q-glucan, the polymer can be synthesized from these nucleotide sugars. The results have been used as the basis of an 76 They have also assay for Q-xylo-P-glucan:q-xylosyltransferase.l been used to suggest that this transferase can be distinguished from other polysaccharide synthetase activities. The metabolism of polysaccharides by pea stem segments treated with and without auxin has been studied by a centrifugation technique.’ 7 7 Auxin enhanced the levels of e-xylose and ;-glucose in ethanol-insoluble polysaccharide, and these sugars were in a Q-xylo-;-glucan. Similar studies using ethene showed that this treatment lowered the amount of Q-xylo-9-glucan in ethanol-insoluble polysaccharides. 78 The vacuum u.v.-c.d. spectra of guar, tara, and carob p-galacto-E-mannans, which had D-galactose contents of 3 9 , 25,and 198, respectively,have been re~0rded.l~’A positive band at 169 nm and a negative band at 149 nm were observed whose relative intensities increased systematically with decreasing ;-galactose content for solid films. Some of the differences were attributed to conformational restriction of ;-galactose residues by chain packing with a consequent increase in the c.d. intensities from these residues and a cancellation of q-mannan backbone contributions. High-performance size-exclusion chromatography of guar gum has indicated that the molecular weight was >2.0 x 1O6.l8’ The seeds of AstraRalus sinicus contain a polysaccharide which ratio of 1:2.3 and a molecular weight has a p-galactose:p-mannose of 1.6 x 104.181 The results of methylation and Smith degradation analyses indicated that the P-galactopyranosyl residues were attached to 0-6 of the main chain but that both (1+3)-and (1+4)-linkages were present in the main c-mannan chain. Treatment of guar gum with an a-E-galactosidase has produced a polymer in which 75% of the E-galactose residues have been removed, but there was no reduction in viscosity.182 Further treatment did reduce the viscosity and led to D-mannan-type precipitates. The interaction of guar gum with xanthan gum is increased by removal of the Q-galactose residues. The chemical structure of the 2-galacto-q-mannan component of

46


Trypanosoma cruzi has been determined by 13C n.m.r. spectroscopy and confirmed that B-IJ-galactofuranosyl residues were linked at the non-reducing terminus to 0-3 of P-mannose residues.’83 E-Galacto-g-mannans are the major storage reserve carbohydrate in the embryo of fenugreek but they are not mobilized during the first 24 h of germination. 84 A pure D-gluco-g-mannan has been recovered from the hot-water extract from Aloe barbadensis .185 The methylated polysaccharide (mol. wt. 1.5 x l o 4 ) contained residues of 2,3,4,6-tetra-, 2,3,6-tri-, and 2,3-di-g-methyl-P-mannose and 2,3,6-tri-g-methylD-glucose in the ratios 1.3:18.3:1.2:1.0. ;-Mannose was observed in hydrolysates of the holocellulose of Panicum coloratum leaves.186 The material containing this sugar residue was particularly resistant to extraction from the a-cellulose fraction. The conditions for the preparation of a rigid and homogeneous gel from Konjac mannan have been p ~ b 1 i s h e d . l ~ ~ Fractionation of cell-wall preparations from grass leaves has shown that the less dense fractions had a higher ;-xylan content as well as a higher content of lignin and acetyl residues. 88 Fully methylated P-xylan-type oligosaccharides have been analysed by mass spectrometry and the fragmentation patterns compared.189 Within the series of di- and tri-saccharides studied, the criteria proposed permitted reliable determinations of any of the theoretically possible branching points. The structure of the water-soluble 4-arabino-Q-xylan from delignified bark of Cinnamomum zeylanicum has been established.”’ The main (1+4)-B-;-xylan chain is substituted both at 0-2 and 0-3 with b-arabinofuranosyl and 3-C+a-~-xylopyranosyl-~-arabinofuranosyl groups. The isolation, purification,and partial characterization of a g-glucurono-L-arabino-;-xylan, a previously unobserved component of the primary cell wall of dicotyledonous plants, have been described.lgl It represents 5% of the wall and has the usual structural features of this type of hemicellulose. A procedure has been developed for the fractionation of the non-starchy polysaccharides of wheat bran.’ 9 2 Determination of the activity of Ca2+ ions bound to carboxy groups in the E-xylan from white willow has shown that 4-~-methyl-~-glucopyranosyluronic acid groups are concentrated in blocks where at least every second a-Q-xylopyranosyl residue

’


47

contains a single uronic acid residue linked by a ( 1 + 2 ) bond.lg3 These acidic blocks alternate with blocks of unsubstituted D-xylose units which are three times as long. The hemicellulose contents of leaves, nodes,and internodes of wheat straw were similar.lg4 Ozone and sulphur dioxide at 70°C for 72 h solubilized virtually all the wheat hemicelluloses. 9 5 Results were also presented on the digestion of the hemicelluloses by rumen micro-organisms. Similar studies with rumen organisms have been carried out on NaOH-treated straw. l g 6 The hemicellulose content of cotton straw is reduced by 50% on treatment with ozone. l g 7 Auxin was found to have no effect on the structure of the L-arabino-q-xylans isolated from Avena coleoptiles. 9 8 The pentosans of pearl millet (Pennisetum americanum) have been separated into four fractions and each was found to contain varying amounts of seven sugar residues.lg9 Each fraction also contained protein. A mechanism for the oxidative gelation of the water-soluble pentosan of wheat flour has been proposed that suggests an attack by addition of a protein radical to the activated double bond of a ferulic acid residue.200 The substrate-binding site of the endo-(1-+4)-8-~-xylanase from the yeast Cryptococcus albidus has been investigated using a series of (1+4)-8-;-xylan oligosaccharides which were labelled with tritium at the reducing end.201 The substrate binding site is composed of four subsites with the catalytic groups located in the centre. As a consequence of an assymetric distribution of negative values of affinity around the binding site, the enzyme displays a strong preference to attack near the reducing end of the substrate. The mechanism of action was found to involve pathways other than a simple hydrolytic cleavage.202 Some IJ-xylosyl transfer occurred to increase the molecular size of the substrate and the transfer came from the non-reducing end of the molecule. All features of the degradation of oligosaccharides by this E-xylanase were consistent with the lysozyme-type reaction mechanism Particulate enzyme preparations obtained from homogenates of differentiated xylem cells from sycamore trees have catalysed the synthesis of a D_-xylan from UDP-q-xylose. 203 The polysaccharide had the expected structure. Various properties of the enzyme and its requirements were determined. The specific activity of the

.

48


enzyme system increased markedly as cells differentiated from the vascular cambium to the xylem.204 The increase was closely correlated with the enhanced deposition of D-xylan occurring during the formation of secondary thickening. The control of hemicellulose synthesis during differentiation of vascular tissue in bean callus and hypocotyl has been investigated and the E-xylan synthetase induction correlated with the induction of phenylalanine ammonia-lyase and with lignin synthesis.*05 A cell-wall degrading agent has been isolated from Chlorella 206 and characterized as a (1+4)-8-Q-xylanase. Residual carbohydrates have been removed almost completely from milled wood lignin preparations of birchwood by treating with NaOH-dioxane. 2 0 7 Treatment with HC1-dioxane did not decrease the carbohydrate content. Studies using i.r. and n.m.r. spectroscopy indicated that a benzyl ester type of linkage existed between lignin and 'the carbohydrates. The lignin-carbohydrate complex from the milled wood lignin fraction of Pinus densiflora contained three sub-fractions on gel filtration.2 0 8 Two of the fractions were homogeneous and their compositions were determined. Methylation analysis showed that the carbohydrate moiety exhibited multiple branching with the major backbone being composed of (1+4)-p-mannan chains. A water-soluble lignin-carbohydrate complex has been released from jute (Corcharus capsularis) fibre by reduction with borohydride. * 0 9 Analysis of the pxylans from the original and borohydride-treated fibres indicated that 34% of the acidic side chains of the g-xylan were linked to lignin by ester bonds. Lignin-carbohydrate complexes have been released from wheat straw by prolonged hot-water extraction.2 The bound sugars were identified as g-glucose, 4-arabinose, q-xylose,and ;-galactose residues with uronic acids being absent. The higher molecular weight fractions were richer in !-glucose residues with the lower molecular weight ones being richer in E-xylose and 4-arabinose residues.

6 Pectins

A

high-performance gel-permeation chromatographic method has been developed to determine the molecular weight distribution of pectins.211 High-methoxy, low-methoxy,and amidated pectins can be analysed within 15 min.


49

An investigation of oligo- and poly-galacturonic acids has been carried out by potentiometry and c.d.212 It was shown that no Ca2+ ion is ever fixed in excess of stoichiometry. The c.d. spectra, in spectroscopic data, suggested that, in agreement with 1 3 C n.m.r. dilute solutions, the polymer adopts two conformations. The gelation of pectin under conditions of low water activity has been investigated by c.d., competitive inhibition, and mechanical properties. 2 1 3 The optimum degree of esterification for pectin gelation under conditions of low water activity is 70%. The reduction in gel strength at lower degrees of esterification is not merely due to increased charge density but also results from the loss of a significant contribution which the ester group makes to the stability of the interchain junctions. Gel permeation chromatography, c.d., and viscosity measurements showed that appreciable aggregations of aqueous solutions of pectin occur at low concentrations in pectins of both low and high ester content . 2 1 The aggregation is independent of divalent cations. The Arrhenius slopes were greater at lower temperatures and they increase with conditions such as pH, ionic strength, concentration, and degree of esterification that are known to favour interchain association of pectin. The c.d. spectra at various pH values and in the presence of additives indicated that two modes of interchain association contribute to the network formation in calcium pectate gels.215 The first is by interchain chelation of Ca2+ ions and the second is by non-ionic interchain associations analogous to those in pectin gels with low water activity. The contribution of the latter type increases on lowering the pH, as the Ca2+ ion binding capacity is diminished and interchain electrostatic repulsions are minimized. The mechanical properties and thermal stability of a gel are a function of the proportion of each type. A relationship has been sought between the ability of various ions to inhibit growth and their ability to cause gelation of isolated pectins, their binding affinity for isolated cell walls, and their binding affinity for purified pectin.216 A good correspondence was found with the second factor. Pectic gel formation was not involved in cation-induced growth inhibition. The previously determined activity coefficients of Ca2+ ion in oligo-g-galacturonates were plotted and extrapolated to infinity.217 In molecular disperse solutions the binding of Ca2+ ions was electrostatic, whereas in aggregated molecules a chelate


50

intermolecular binding also took place. The non-reducing terminal uronic acid residues bind Ca2+ ions less firmly than to the inner units of the chain. Lemon pectin and pectic acids have been hydrolysed into acid-soluble and acid-insoluble products and separated by gel filtration.*18 The latter product had a molecular weight of 3.03.2 x l o 4 whereas the former could be separated into two components (mol. wts. 2.2 -2.5 x l o 4 and < 6 x l o 3 ) . The larger of these two components contained only (1+4)-a-Q-galacturonan chains while the smaller contained residues of E-galacturonic acid and L_-rhamnose as well as other neutral sugars. Cell-wall material has been isolated from cabbage (Brassica oleracea) and methylated.21 Hydrolysis of the product and analysis of the fragments revealed some of the structural features many of which were derived from the pectin present. Two pectic fractions, comprising 52% of the cell wall, have been isolated from potatoes. 2 2 0 Both fractions contained a range of molecular types differing in composition. The more easily extracted fraction had a high 4-arabinose:E-galactose ratio as well as a high D-galacturonic acid content. The more easily extractable fraction was probably held by Ca2+ ion bonds. An endo-poly-2-galacturonanase from Aspergillus japonica has been used for the isolation and characterization of cell-wall pectic Methylation analysis of the four substances from potato tubers. fractions obtained by gel filtration was used to elucidate their structure. The two highest molecular size fragments had very similar compositions while the intermediate fraction had a very complex structure including P-galacturonic acid residues which were branched at C-3. Sequential extractions of Rosa glauca cell walls showed that two different types of acidic polysaccharide were present . 2 2 2 One was extracted with chelating agents while the other remained in close association with the a-cellulose fraction. A structurally complex pectic polysaccharide, k-rhamno-Pgalacturonan I, has been isolated from the primary cell wall of suspension-cultured sycamore cells. 223 The polysaccharide (mol. wt. 2 x l o 5 ) has the usual pectin backbone to which is attached a wide variety of side chains containing &-arabinosyl and/or P-galactosyl residues. Tomato pectin esterase has been used to prepare a high molecular weight pectin from citrus.224 The product from the free

’”

51


ca.

enzyme had a degree of esterification of 2% whereas an immobilized enzyme gave a value of z . 1 9 8 . The molecular weight was lowered by less than 5% by the enzymatic treatments. The steric course of the methyl esterification reaction of a has been studied using 5-adenosylmethionine and an p-galacturonan enzyme preparation from Phaseolus a ~ r e u s .The ~ ~methyl ~ group of the methionine derivative was chiral, possessing 'H, 2H, and 3H substituents,and it was shown that transfer of the methyl group to the oxygen of the carboxy group proceeds with inversion of the configuration of the methyl group. mg g-l) was found in a wide variety of pectin Silica ( 1 . 4 - 2 . 3 samples. 226 Pectic substances were the major components of cell-wall material from pea flour and concentrates. 227 Detergent methods were shown to underestimate the pectin contents in a range of dietary fibres.228 For example, 50% of orange pectin was recovered in the acid-detergent residue and 38% in the neutral-detergent residue. Many of the physical properties of the dietary fibre from apples were shown to be due to the high pectin content.229 The interactions of bile acids and their conjugates with pectins of different degrees of esterification in the pH range 3.5-7.3 have spectroscopy, but no molecular been investigated by 'H n.m.r. interactions were apparent .23 O The pH-dependent interaction between pea cell-wall polymers possibly involved in wall deposition and growth has been investigated and there occurs, at neutral pH values, a pH- and time-dependent binding of soluble pectin, in the walls, to a heat-labile protein component of the wall.231 The reaction is also observed in water extracts of walls. The reaction is inhibited at low pH values and by Ca2+ ions and is lectin-like in nature. An elicitor of phytoalexin accumulation has been solubilized from cell walls of soybeans.232 The active material contains residues of E-galacturonic acid, L-rhamnose,and E-xylose and is probably pectic in origin. Similar elicitors could be prepared from suspension-cultured tobacco, sycamore, and wheat cells. A pectic polysaccharide, solubilized by the action of a fungal endo-(1+4)-a-~-galacturonanase from sycamore cell walls, possesses activity similar to the proteinase-inhibitor inducing factor isolated from tomato leaves.2 3 3 The synthesis and accumulation of proteinase inhibitor I in excised tomato leaves can be induced with oligosaccharides obtained by endo-(1+4)-a-D-galacturonanase -

52


action of a pectic polysaccharide from tomato leaves .234 These oligosaccharides released by enzymic action at a wound or site of infection may play a hormone-like role in regulating plant defence responses in unwounded tissue some distance from the site of release. Pectic multienzyme preparations have been separated by h.p.1.c. using both isocratic and linear-gradient conditions.235 Three D_-galacturonanases have been isolated from carrots, two from root tissue and one from cell-suspension cultures.236 All three enzymes were of similar size (mol. wt. 4.8 x l o 4 > and were exo-enzymes. The only major differences were in the binding properties of the enzymes to cell-wall material. Pectic enzymes have been extracted from haustoria of Cassytha filiformis growing on Nerium indicum and Hibiscus rosa-sinensis and both strains contained exo-(1+4)-a-~-galacturonanase and exo-pectin methylgalacturonanase activity. 2 3 7 The two strains differed markedly in the production of lyase enzymes: the Nerium strain produced endo-pectic acid lyase while the Hibiscus strain produced endo-pectin lyase. 4 A g-galacturonanase (mol. wt. 3.6 x 10 ) has been isolated from Sodium citrus fruit infected with Penicillium italicum. 238 P-galacturonan was a better substrate than citrus pectin. The P-galacturonanase activity in seed cavity tissue from harvested cucumber fruit increases 20-fold after the fruit produces a transient burst of ethene during maturation.239 A single pectic enzyme, g-galacturonanase, is produced by Rhizoctonia solani in culture and during infection of cotton seedlings.240 The enzyme (mol. wt. 4.2 x l o 4 > has a pH optimum of 5.2.

The P-galacturonanase from Rhizopus stolonifer has been shown to be an elicitor of casbene synthetase activity in castor bean seedlings.241 The enzyme is a glycoprotein (mol. wt. 3.2 x lo4) and contains 20% carbohydrate. The principal sugar residues present are E-mannose (92%) and 2-amino-2-deoxy-~-glucose (8%) and the enzyme is an e n d o - h y d r ~ l a s e . ~ ~Other ~ features of the enzyme were reported. The active site of the endo-q-galacturonanase from tomato has been studied using oligo-2-galacturonic acids and their aldehydo derivatives.243 The relationship of the substrate chain length to the initial and maximum reaction rates was determined. The topology of the active site was suggested from the results. The


53

;-galacturonanase of ripe tomatoes has been extracted in two isoenzyme forms, I and II.244 The enzymes have different properties although their polypeptides are similar. Green fruit contains a factor which can convert form I1 into form 1,and the amount of this factor increases during ripening. The characteristics of the pectin methylesterase from potato have been determined and the role of the enzyme in commercial dehydrated mashed-potato production has been examined. 2 4 5 Ca2+ ions. but not Mg2+ ions, retard solubilization. Texture measurements suggested a firming effect which could be attributed to Ca2+ ion release during pre-cooking and stabilization of Ca2+ ion bridges during cooling. The changes in pectinesterase activity and other pectin changes in mango varieties during storage and ripening have been investigated.246 In a study of the microbiology of wetwood, the importance of Clostridia species in the degradation of pectin has been assessed.247 High levels of pectinolytic enzymes were produced in culture filtrates of Phoma exigua and Graphium penicillioides isolated from decaying seeds of Phaseolus aureus and Cyamopsis tetragonalobus,respectively.248 The susceptibility of strawberry varieties to breakdown in sulphate liquor by the m-g-galacturonanase from Zygomycete spoilage fungi has been in~estigated.~~' Fruit waste containing de-esterified pectin as a thickener has been assessed for its potential use in canned products.250

7 Gums and Mucilages The carbohydrate gum from bael (AeRle marmelos) seeds has been resolved into four glycoprotein fractions. 2 5 1 One of these contained residues of :-galactose, !-glucose, L-arabinose, and &-rhamnose in the molar ratios 6:2:8:3. The linkages amongst these residues were determined as well as the anomeric configuration of the glycosyl residues. The structure of the linkage region was established as Q3-4-arabinopyranosyl-4-threonine. A histological and a histochemical comparison of the mucilages on the root tips of several grasses has been made,and it was concluded that there were two types.252 One is a gelatinous material originating from the root cap and the other is a firm uniformly thick mucilage overlying the columnar epidermal cells.

54


Reactions of the outer layer and cell wall indicated that carboxy groups were present and these were absent from the inner mucilage. A mucous polysaccharide has been isolated from the bulbs of Narcissus t a ~ e t t a . ~It~ was ~ homogeneous and contained g-mannose and g-glucose residues in the ratio 5:l with a molecular weight of 1.19 x lo’. The g-acetyl content was 22.79,and these groups were present on C - 6 and on C-2 and C-6 of most p-mannose residues. The main chain is a (1+4)-B-g-hexopyran. The glycoprotein from neem (Azadirachta indica) gum (mol. wt. 1.3 x lo4) contained 50% carbohydrate.254 The sugar residues present were Q-mannose, 2-acetamido-2-deoxy-D-glucose, p-galactose, 4-arabinose, 4-fucose, q-xylose,and g-glucose in the ratios 14:13:5:3:1:1:1. The linkage between the carbohydrate and the protein was an y 4 - (2-acetamido-2-deoxy-8-D-glucopyranosyl) hydrogen L-asparaginate bond. Another glycoprotein from the same This material contained source contained only 19% ~arbohydrate.~’~ the same sugar residues in the ratio 4:3:2:3:2:1:1. The linkage region was the same. The mucilage of Opuntia ficus-indica and the partially degraded mucilage have been investigated by methylation analysis and periodate oxidation.256 The results show that the mucilage is composed of l,4-substituted a-D-galactopyranosyluronic acid and 1,2-substituted B-L-rhamnopyranosyl residues to which short chains of 1,6-substituted B-P-galactopyranosyl residues were attached at 0-4 of all of the &-rhamnopyranosyl residues. Most of the 9-galactosyl residues carry branches at 0-3 while some are branched at 0-4. Partial acid hydrolysis of this mucilage has e its polymeryielded ~ - ~ --~ - g a l a c t o p y r a n o s y l - ( 1 ’ 6 ) - ~ - g a l a c t o sand homologous trimer as well as fourteen oligosaccharides that contain L-arabinose residues and most of which have D-xylosyl residues at the non-reducing terminus.257 The oligosaccharides ( 6 ) and (7) were the most abundant, Similar studies have been reported on the mucilage of 0. aurantiaca with similar results.258 The gum exudates from some Prosopis species have been analysed by the Smith degradation procedure. 259 The degraded gums have chain was molecular weights of 6 x lo3. A simple (1+3)-!-galactan obtained after only two degradations showing that the skeleton was similar to those of Acacia gums. Autohydrolysis of the gum exudate from the Spondias dulcis tree gave a gum containing !-galactose, ;-arabinose,and D-galacturonic acid in the ratio 3:3:1.260 Three neutral and three acidic


55

@-P-Xylp-(1+5)-a-&-AraL-(l-+5)-L-Araf (7)


56

oligosaccharides were obtained and their structures characterized to suggest a sequence for the repeating unit. Chromium(V1) trioxide oxidation was used to determine the anomeric configuration of the sugar residues. 8 Algal Polysaccharides

Based on chemical shifts and C-H coupling constants from model molecules, the structural conformation of aqueous agarose solutions and gels has been determined from the 1 3 C n.m.r. spectra. 2 6 1 Similar studies on the agaroses of Pterocladia species and Gracilaria secundata have been reported and all signals in the spectra were assigned to various ;-galactose derivatives and agaro-oligosaccharides.26 The chemical composition and rheological properties of the agar isolated from Gelidium purpurascens before and after alkaline treatment have been reported .263 The D_-xylose, ;-glucose, and p-glucuronic acid residues in the agar were removed on alkaline treatment together with 86% of the protein. Sequential extraction of the alga accounted for low yields of agar as losses incurred on precipitation with ethanol. A method has been presented for the determination of the backbone conformations of polymer chains conforming to a given helical type.264 The method is illustrated by its application to agarose. The high resolution 13C n.m.r. spectra of slightly depolymerized alginates have been interpreted.265 The sequence of the monomer units k-guluronate (G) and p-mannuronate (M) markedly influences the chemical shifts. Some of the individual carbon resonances were resolved into four lines,which was evidence for a dependence on the identities of the units immediately before and after them in the polymer chain. The relative intensities of the signals permitted a rapid computation of the monomeric composition (M/G ratio), the composition of end units (M/G ratio), and the monomeric sequence in terms of a complete set of four diad and eight triad frequencies. Any region with a strictly alternating M and G sequence was very short. The cation-specific vacuum ultraviolet c.d. spectra of alginate solutions, gels, and solid films have been recorded.266 A band at 185 nm has been assigned to carboxy groups while those at 169 and 149 nm were assigned to changes in the polymer backbone.

*

-


57

1-Carrageenan has shown substantial helix formation in the presence of Li', Na', and Me4N+ ions as measured by optical rotation, but the polymer does not gel. 2 6 7 Under identical Cs+,and NHq' ions. conditions, the polymer does gel with K+, Rb', Initially the individual The gel formation is in three stages. coils form double helices,then the double helices form domains, which finally form aggregates around the heavier Group I ions. A detailed assignment of the 13C chemical shifts of K and I-carrageenan in their Na' and K + forms has been given.268 Evidence for the conformational transition induced by temperature variation in the absence of any gel formation of K-carrageenan is also presented. The evidence is based on both n.m.r. and optical rotation experiments. Measurements of rigidity moduli and 39K and 23Na n.m.r. spectra have been used to investigate the roles played by Ca2+, K',and Na+ ions in 1-carrageenan gels. 2 6 9 Differences in binding were reflected in the rheological properties of the gels. Potassium 1-carrageenate gels have been prepared and studied by photon nm.270 The molecular and correlation spectroscopy at 633 vibrational motions of the gels were interpreted in terms of their elastic properties. Below the sol-gel transition temperature, oscillatory correlation functions were found with the frequency remaining constant for two weeks. Measurements of the shear modulus for Ca2+, K+, and Na+ 1-carrageenate gels as a function of biopolymer concentration and temperature showed that the Ca 2 + salt appears to possess a random glass-like structure whereas the K + and Na+ forms show a pseudocrystalline structure.271 The differences were tentatively attributed to differences in the solubility of the salt forms which, through controlling the gelation temperature, moderated the kinetics of the gelation process. The optical rotation and the conductivity of K-carrageenans in aqueous solutions have been investigated as functions of temperature in the presence of various electrolytes . 2 7 2 The activity coefficients of Na+ and K + ions have been determined and correlated with the conformation. The K + activity coefficient under ordered conformation is in agreement with a mechanism involving dimerization. The use of the carrageenan from the red alga Eucheuma striatum has been investigated as a possible substitute for agar in bacteriological tests. 2 7 3

58


Neocarratetraose 4-2-monosulphate B-hydrolase has been isolated from cell-free extracts of Pseudomonas carrageenovora.2 7 4 The hydrolysis products are neocarrabiose and neocarrabiose 4-2-sulphate. The pathway for the sequential degradation of the tetrasaccharide was proposed. A-Carrageenan has been shown to have a hypotensive effect in the rat that was dependent on platelet stimulation.275 Also in rats, intraperitoneal injection of ellagic acid and carrageenan The reduced paw oedema induced by carrageenan itself.276 relationship between the anticoagulant activity and the pro- and anti-inflammatory mechanism in A-carrageenan-induced inflammation has been investigated.277 The gelation behaviour of solutions of chitosan on reaction with acid anhydrides has been studied, and the effects of the time to onset of gelation and of varying the nature and concentration of the anhydride, the chitosan concentration, the temperature,and the nature of the cosolvent were all examined.278 Gelation occurred due to the decreased solubility of the polymer molecules brought on by g-acylation,and all other parameters influenced the rate of N-acylation. The syneresis of the gels was examined. The degree of N-acylation required to initiate gelation depended on the molecular weight of the acyl anhydride and the concentrations of c h i t ~ s a n . ~The ~ ~ energy of activation of N-acylation was determined for tj-acetylation and N-hexanoylation. A mechanism was proposed for the gelation process. An i.r. spectroscopic method has been used to determine the amide content of highly deacylated chitosans.280 A plot of the ratio of absorbance at the amide band ( 1 6 5 5 cm-1) to the C-H stretch frequency (2867 cm-l) against the degree of deacetylation was linear in the deacetylation range 90-100%. The degree of N-acetylation of chitosan was rapidly determined by periodate oxidation and i.r. spectroscopic methods. 281 The i.r. methods gave higher results but were thought to be more reliable. A new method of chitin determination, based on deacetylation and g.1.c. analysis of the liberated acetic acid,has been applied to a variety of chitosans.282 The results were in good agreement with other methods even though the rate of deacetylation of some chitosans is 10 times that of others. A highly crystalline form of a-chitin has been found in the grasping spines of the marine worm Sagitta and been examined by electron microscopy and electron diffraction.283


59

The chitin from carapace of Penaeus japonicus has been purified by a proteolytic enzyme from Pseudomonas malt~philia.'~~No deacetylation occurred and the protein content was reduced to virtually zero. The molecular weight of the enzymically prepared chitin was higher than that obtained by alkaline treatment. Polyelectrolytic complexes have been prepared from chitosan and sodium carboxymethylcellulose. 2 8 5 None were soluble in aprotic solvents but the series prepared at high pH values were more soluble in hot formic acid than those prepared at low pH values. The former were also different from the latter in the density of carboxy sites.286 A 'H n.m.r. method has been used to investigate the binding of methyl di-tj-acetyl-8-chitobioside to wheat-germ agglutinin. 2 8 7 The linewidth broadening of the methoxy protons allowed a calculation of kinetic association constants to be obtained. The bovine serum chitinase has been characterized as a true (l+4)-8-D-2-acetamido-2-deoxy-glucanase without any s - 8 - q - 2 acetamido-2-deoxy-glucosidase activity. 288 The purification and properties of the enzyme were reported. The chitin synthesizing system of insect tissue has been reported.289 A study on the adsorption of heavy-metal ions by chitin phosphate and chitosan phosphate has been made F g o The chemical composition of the porphyran from Porphyra columbina was in good agreement with data for similar polysaccharides. 29 One fraction had an optical rotation similar to that of the porphyran from P. capensis while the optical rotation of another fraction was similar to that of the phycocolloid of P. umbilicalis. The molar composition of the latter was D-galactose:6-~-methyl-D-galactose:3,6-anhydro-~ga1actose:sulphate in the ratio 1.00:~0.57:0.35:0.65. Several spectra could be assigned to various signals in the 'H n.m.r. anomeric protons. A biophotoreactor, using immobilized cells of Porphyridium cruentum in a polyurethane 'mousse', has been used to produce a capsular polysaccharide sulphate which is secreted into solution.29 Sulphated polysaccharides have been isolated from Caulerpa species and their function in wound healing has been assessed.293 Fucoidanase activities have been observed in fractions from the hepatopancreas of abalone. 2 9 4 The primary product from one enzyme

'


60

was Ti-fucose while another enzyme gave a series of neutral oligosaccharides. Laminarin, L-fucans, and alginic acid have all been isolated from Dictyopteris p l a g i o ~ r a m m a . The ~ ~ ~ laminarin contained chains terminating in a !-glucose residue and those terminating in a The &-fucans were present in Q-mannitol residue in the ratio 3 : l . a variety of extracts and each had a slightly different ratio of ~-fucose:~-xylose:~-galactose:~-mannose:~-glucuronic acid:sulphate. The structural features elucidated were similar to those fromother L-fucans. Polysaccharide material from Eucheuma spinosum appears to consist of two different components containing low and high sulphate contents. 2 9 6 The major component had the higher sulphate content while the molecular sizes of the two polysaccharides were d i fferent A series of acidic oligosaccharides (8) (20) has been obtained by graded acid hydrolysis of the methylated acidic polysaccharide associated with the coccoliths of the alga Emiliania huxleyi: the above structures were characterized. 2 9 7 The results along with methylation data from the native, carboxyreduced, and desulphated carboxy-reduced polysaccharides have been used to give a proposed structure for this complex polymer.298 Glycogen accumulation in vegetative cells of the blue-green alga Anabaena is a light-dependent process.299 The amount of glycogen produced was high during sporulation and this increased with the onset of maturation of the spores. The amino-acids L-methionine, k-tyrosine, b-glycine, and k-histidine gave the greatest enhancement of glycogen accumulation in supplemented cultures although all the amino-acids examined had a positive effect.3 0 0 The storage D-glucans in and isoenzyme distribution of Cyanidium caldarium have been in~estigated.~” The observations propose this organism to be a primitive Rhodophytan which is an algal-bridge linking the prokaryotic blue-green and the red algae. It seems to be a true transitional organism between the nucleated and anucleated algae and is unlikely to be an endosymbiotic association of more than two cells as speculated elsewhere. Two major arsenical constituents of the brown kelp Ecklonia radiata have been isolated and characterized as

.

-

2-hydroxy-~-sulphopropyl-5-deoxy-5-(dimethylarsenoso)-furanoside

and 2,3-dihydroxypropyl-5-deoxy-5(dimethylarsenoso)-furanoside

.302


61

a-E-GalpA-(1-+6)-a-D-Manp-(l+3)-E-Man (8)

E-GalpA- ( 1 -4)- p G a l p - (14 -Man ) (9)

~-GalpA-(1+4)-~-GalpA-(1-+2/6)-Manp-(1+3)-Man

(10)

g-GalpA- ( 1 +4 ) -g-GalpA- ( 1 +3 ) -E-Xyl (1 1 )

q-GalpA-( 1+2)-L=-Manp6Me-( 1+4)-!-GalpA-(

1+2)-&-Rha -

(12)

g-GalpA-( 1+2)-Manp-( l+&)-E-GalpA-( (13)

1+2)-L-Rha

62


g-GalpA( 1 +2 ) -Manp- ( 1 +4) -E-GalA (16)

g-GalpA- ( 1 +2) -4-Manp6Me- ( 1 +4) -E-GalpA- ( 1 +2 ) -L_-ManGMe (17)

~-GalpA-(1+2)-~-Manp6Me-(1+4)-g-GalpA-(l-+2)-Man

(18)


63

References 1

2 3 4 5 6 7 8

9 10 11

12 13

14 15 16 17

18

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

'Biochemistry of Plants. A Comprehensive Treatise', ed. J.Preiss, Academic Press, New York, 1980, Vol. 3 (Carbohydrates: Structure and Funct iod. 'Mechanisms of Saccharide Polymerization and Depolymerization', ed. J.J.Marshal1, Academic Press, New York, 1980. H.F.Mark, An ew. Chem. Int. Ed. En l., 1981, 20, 303. T.L.Bluhm Res., 1981, 89, 1. G.Rappenecker and P.Zugenmaier, Carbohydr. Res., 1981, 89, 11. J. Metzger, Fresenius' Z. Anal. Chem., 1981, 308, 29. R.G.Stone and J.A.Krasowski, Anal. Chem., 1981, 53, 736. K.Kainuma and T.Ogawa, J. Chromatogr., 1981, 212, 126. B.G.Osborne and S.Douglas, J. Sci. Food A ric., 1981, 3 l , 328. T.Handa, H.Yajima, and, sT.- K 1980, 2, 1723 F.W.Fales, Biopolymers, 1980, 2,1535. J.N.BeMiller and G.W.Pratt, Cereal Chem., 1981, 58, 517. M.Nedelcheva, E.Valcheva, S. Bencheva, and V.Valchev, Starg,1981, 195. K.Kulp and K.Lorenz, Cereal Chem., 1981, 58, 46. K.Lorenz and K.Kulp, Cereal Chem., 1981, 58, 49. R.J.Kartchner and B.Theurer, J. Agric. Food Chem., 1981, 29, 8. F.R.Dintzis and C.C.Harris, Cereal Chem., 1981, 58, 467. D.J.O'Donnel1, J.J.H.Ackerman, and G.E.Macie1, J. Agric. Food Chem., 1981, 29, 514. S.Hizukuri, Y.Takeda, M.Yasuda, and A.Suzuki, Carbohydr. Res., 1981, 94, 205. D.J.Manners and N.K.Matheson, Carbohydr. Res., 1981, 90, 99. K.Lorenz and K.Kulp, Starch, 1981, 33, 183. J.Torp, J. Sci. Food Agric., 1980, 30, 1354. P.Meredith, Starch, 1981, 33, 40. C.G.Biliaderis, D.R.Grant, and J.R.Vose, Cereal Chem., 1981, 58, 496. C.G.Biliaderis, D.R.Grant, and J.R.Vose, Cereal Chem., 1981, 58, 502. R.T.Tyler, C.G.Youngs, and F.W.Sosulski, Cereal Chem., 1981, 58, 144. M.E.O.Labaneiah and B.S.Luh, Cereal Chem., 1981, 58, 135. S.K.Sathe, Y.Iyer, and D.K.Salunkhe, J. Food Sci., 1981, 4 6 , 1914. S.Umadevi and D.B.Wankhede, Starch, 1981, 33, 23. K.Kayisu, L.F.Hood, and P.J.Van Soest, J. Food Sci., 1981, E, 1885. P.J.Loos, L.F.Hood, and H.D. Graham, Cereal Chem., 1981, 58, 282. B.A.Keating, A.R.Breen, and J.P.Evenson, J. Sci. Food Uric., 1981, 32, 997. I.Geilinger, R.Amado, and H.Neukom, Starch, 1981, 33, 76. A.Suzuki, A. Hizukuri, and Y.Takeda, Cereal Chem., 1981, 58, 286. T.T.Y.Llu and J.C.Shannon, Plant Physiol., 1981, 9, 518. Y.Ikawa, D.V.Glover, Y.Sugimoto, and H.Fuwa, Starch, 1981, 33, 9. C.D.Boyer, P.A.Damewood, and E.K.G.Simpson, Starch, 1981, 33, 125. K.Lorenz, Starch, 1981, 33, 73. Y.Sugimoto, K.Yamada, S.Sakamoto, and H.Fuwa, Starch, 1981, 33, 112. S.U.Sajjan and D.B.Wankhede, Starch, 1981, 33, 153. W.N.Bnmerling and B.PfannemSller, Starch, 1981, 33, 202. M.Wootton and M.A.Chaudhry, Starch, 1981, 33, 135. M.Wootton and M.A.Chaudhry, Starch, 1981, 33, 168. M.Wootton and M.A.Chaudhry, Starch, 1981, 33, 200. M.Eeh, t.Stropnik, V.DoleEekxS.Leskovar, Starch, 1981, 33, 45. A.R.Khan, J.A. Johnson, and R. J.Robinson, Cereal Chem., 1979, 56, 303. J. J.Raffi, J.-P.L. Agwel , C.M.Fre'javille, and L.R.Saint-L'ebe, J. Agric. Food Chem., 1981, 3,548. A.A.Ilin, L.S.Galbraikh, and B.P.Morin, Cellul. Chem. Technol., 1980, 14, 327. KC.Baur and R. J.Alexander, Cereal Chem., 1979, 56, 364.

.-

I.

-

64

50 51


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E.Perciva1, M.A.Rahman, and H.Weige1, Ph tochemistr 1981, 20, 1579. H.K.Tong, K.H.Lee, and H.A.Wong, Carbohyir. Res., 1;81, 88, %8. A.M.J.Fichtinger-Schepman, J.P.Kamerling, C.Versluis, and J.F.G.Vliegenthart, Carbohydr. Res., 1980, 86, 215. A.M.J.Fichtinger-Schepman, J.P.Kamerling, C.Versluis, and J.F.G.Vliegenthart, Carbohydr. Res., 1981, 93, 105. T.A.Sanna and S.Kanta, 2. ALlRem. Mikrobiol., 1979, l9, 571. T.A.Sarma and S.Kanta, Z. Ablgem. Mikrobiol., 1980, 20, 653. J.Seckbach and J.F.Frederick, Microbios, 1980, 29, 135. J.S.Edmonds and K.A.Francesconi, Nature (London), 1981, 28q, 602.

298 299 300 30 1 302

1980,

2,

323.

29

1979,

100, 455.

1980,

3, 132-

87.

Agric. Chem. Soc. Jpn.,

,

1979,

1,

2,

Micro bial Polysaccharides BY C.A. WHITE 1

T e i c h o i c Acids

A review h a s s u m m a r i z e d t h e b i o s y n t h e s i s , a s s e m b l y , a n d l o c a t i o n o f t e i c h o i c and t e i c h u r o n i c acids, which i n c l u d e s t h e b i o s y n t h e s i s o f l i p o t e i c h o i c a c i d s and t h e i r involvement i n t e i c h o i c a c i d biosytheses.’ M e m b r a n e s f r o m B a c i l l u s s u b t i l i s W23 s y n t h e s i z e d a l i p i d precursor of the linkage unit that attaches teichoic acid t o the cell wall I t c o n t a i n e d glycerophosphoryl-2-acetamido-2-deoxy-~g l u c o s e , l i n k e d t h r o u g h a n a c i d - l a b i l e bond t o a l f p i d . Glycerol-3-phosphate c y t i d y l y l t r a n s f e r a s e , Q-ribitol-5phosphate cytidylyltransferase, and p o l y ( r i b i t o 1 phosphate) s y n t h e t a s e a c t i v i t i e s have been measured i n c u l t u r e s o f B a c i l l u s s u b ti l i s W23 a s t h e y became p h o s p h a t e - s t a r v e d e i t h e r i n b a t c h c u l t u r e o r d u r i n g changeover from p o t a s s i u m l i m i t a t i o n t o p h o s p h a t e l i m i t a t i o n i n a ~ h e m o s t a t . ~The r e s u l t s i n d i c a t e d t h a t r e p r e s s i o n o f s y n t h e s i s o f a l l t h r e e enzymes o c c u r r e d a t t h e o n s e t o f p h o s p h a t e s t a r v a t i o n a n d t h a t t h i s was a c c o m p a n i e d by i n h i b i t i o n o r i n a c t i v a t i o n o f glycerol-3-phosphate c y t i d y l y l t r a n s f e r a s e and poly(ribito1 phosphate) synthetase. T h e s e r e s u l t s show t h a t t h e i n i t i a l response t o phosphate s t a r v a t i o n i n v o l v e s more than Syntheses of both i n h i b i t i o n o f o n e enzyme as p r e v i o u s l y proposed. linkage u n i t and p o l y ( r i b i t o 1 phosphate) are i n h i b i t e d independently. P r o t o p l a s t s o f B a c i l l u s s u b t i l i s W23 r e a d i l y s y n t h e s i z e d r i b i t o l t e i c h o i c acid from n u c l e o t i d e p r e c u r s o r s i n t h e s u r r o u n d i n g medium. W i t h C D P - r i b i t o l t h e y made p o l y ( r i b i t o 1 p h o s p h a t e ) , p r e s u m a b l y a t t a c h e d t o l i p o t e i c h o i c a c i d ~ a r r i e r :w~h e n C D P - g l y c e r o l were a l s o p r e s e n t , a 1 0 - f o l d a n d UDP-2-acetamido-2-deoxy-Q-glucose increase i n the r a t e of polymer s y n t h e s i s occurred, and t h e product c o n t a i n e d both t h e main c h a i n and t h e l i n k a g e u n i t . S y n t h e s i s was i n h i b i t e d by t r y p s i n o r 4 - c h l o r o m e r c u r i b e n z e n e s u l p h o n a t e i n t h e

.’

4: Microbial Polysaccharides

71

and i t was concluded t h a t i t occurred a t t h e o u t e r s u r f a c e During s y n t h e s i s , which was a l s o achieved r e a d i l y by whole c e l l s a f t e r a b r i e f p e r i o d o f w a l l l y s i s , t h e CMP p o r t i o n of t h e n u c l e o t i d e p r e c u r s o r s d i d n o t pass through t h e membrane. No evidence was o b t a i n e d f o r a t r a n s p h o s p h o r y l a t i o n mechanism f o r t h e I t was s u g g e s t e d t h a t r e a c t i o n w i t h translocation process. exogenous s u b s t r a t e s was due t o t e m p o r a r y e x p o s u r e of a p r o t e i n component of t h e enzyme complex a t t h e o u t e r s u r f a c e of t h e membrane d u r i n g t h e normal b i o s y n t h e t i c cycle. T o l u e n i t e d c e l l s of B a c i l l u s s u b t i l i s W23 s y n t h e s i z e d t h e t e i c h o i c a c i d , p o l y ( r i b i t o 1 phosphate), from exogenous precursor^.^ The s y n t h e s i s was dependent on concomitant s y n t h e s i s of t h e l i n k a g e u n i t t h a t j o i n s t e i c h o i c a c i d t o p e p t i d o g l y c a n . Under c o n d i t i o n s t h a t reduced c e l l a u t o l y t i c a c t i v i t y , a l a r g e p r o p o r t i o n of t h e t e i c h o i c a c i d became l i n k e d t o t h e c e l l w a l l , i n d e p e n d e n t l y o f p e p t i d o g l y c a n s y n t h e s i s . The s p e c i f i c a c t i v i t y of t h e s y s t e m was more t h a n 30 t i m e s t h a t o f i s o l a t e d membranes, s o t h a t a c t i v i t y c o u l d be measured r e a d i l y i n t h e c e l l s from 2 m l of an e x p o n e n t i a l c u l t u r e of b a c t e r i a . The i n f l u e n c e o f t h e l e c t i n s c o n c a n a v a l i n A a n d phytohaemagglutinin A and t h e p o l y e l e c t r o l y t e s d e x t r a n - s u l p h a t e ( a s i t s s o d i u m s a l t ) and d i e t h y l a m i n o e t h y l - d e x t r a n ( a s i t s hydrochlori d e ) o n b a c t e r i a l t r a n s f o r m a t i o n s i n B a c i l l u s s u b t i l i s h a s been s t u d i e d 6 i n o r d e r t o d i s c r i m i n a t e t h e t a r g e t o f l e c t i n s and p o l y e l e c t r o l y t e s i n competent c e l l s . B a c i l l u s s u b t i l i s var. n i g e r WM was grown i n continuous c u l t u r e under p h o s p h a t e - l i m i t e d and under m a g n e s i u m - l i m i t e d c o n d i t i o n s . Whole c e l l s , c e l l w a l l s , a n d t h e i s o l a t e d w a l l p o l y m e r s p e p t i d o g l y c a n , t e i c h o i c a c i d , and t e i c h u r o n i c a c i d were analysed by Curie-point p y r o l y s i s mass ~ p e c t r o m e t r y . ~ C h a r a c t e r i s t i c i o n peaks f o r t h e w a l l p o l y m e r s were e s t a b l i s h e d and f a c i l i t a t e d t h e i n t e r p r e t a t i o n o f the mass pyrograms of w a l l s and whole c e l l s . The mass pyrograms o f magnesium-limited c e l l s showed t h e c h a r a c t e r i s t i c peaks f o r p r o t e i n , peptidoglycan, and t e i c h o i c acid. A t u n i c a m y c i n - l i k e a n t i b i o t i c 2 4 0 1 0 a t a c o n c e n t r a t i o n of 1 pg/ml s e l e c t i v e l y inhibited the v i v o s y n t h e s i s of g l y c e r o l t e i c h o i c a c i d o f c e l l w a l l s i n B a c i l l u s c e r e u s AHU 1030.8 I n c u b a t i o n of membranes o f t h i s s t a i n w i t h 2-acetamido-Z-deoxy-Qglucopyranosyl p y r o p h o s p h o r y l u n d e c a p r e n o l and UDP-Z-acetamido-2deoxy-a-mannase l e d t o f o r m a t i o n o f a g l y c o l i p i d having a s a c c h a r i d e moiety i d e n t i c a l w i t h t h e c e l l - w a l l t e i c h o i c a c i d l i n k a g e u n i t (1). medium,

o f t h e membrane.


72

The membranes a l s o c a t a l y s e d t r a n s f e r o f g l y c e r o l p h o s p h a t e u n i t s The from CDP-glycerol t o t h i s disaccharide-linked l i p i d . b i o s y n t h e s i s o f t h e cell-wall g l y c e r o l t e i c h o i c acid i n t h i s s t r a i n seemed t o involve t h e disaccharide-linked l i p i d as an i n t e r m e d i a t e .

8-Q-Man~NAc-(1+4)-Q-GlcNAc (1)

The p r e s e n c e o f a c t i v e , or o f o n l y r e v e r s i b l y i n a c t i v a t e d , enzyme s y s t e m s f o r t h e s y n t h e s i s and i n c o r p o r a t i o n o f t e i c h o i c acid h a s b e e n d e t e r m i n e d 9 by w i t h d r a w i n g s a m p l e s o f b a c t e r i a f r o m a p h o s p h a t e - p u l s e d c h e m o s t a t c u l t u r e i n t o p h o s p h a t e - r i c h medium t h a t contained chloramphenicol. The i n c r e a s e d c o n t e n t o f w a l l t e i c h o i c a c i d f o l l o w i n g i n c u b a t i o n o f t h e s e s a m p l e s was d e t e r m i n e d by chemical analysis o f the recovered bacteria. Q - A l a n y l - l i p o t e i c h o i c a c i d from L a c t o b a c i l l u s casei c o n t a i n s a p o l y ( g l y c e r o 1 p h o s p h a t e ) m o i e t y t h a t i s s e l e c t i v e l y a c y l a t e d w i t h 0a l a n i n e ester residues." To c h a r a c t e r i z e f u r t h e r t h e m e c h a n i s m o f Q - a l a n i n e s u b s t i t u t i o n , i n t e r m e d i a t e s were s o u g h t t h a t p a r t i c i p a t e i n t h e assembly o f t h i s l i p o t e i c h o i c acid. From t h e i n c o r p o r a t i o n system u t i l i z i n g either t o l u e n e - t r e a t e d c e l l s or a combination o f membrane f r a g m e n t s a n d s u p e r n a t a n t f r a c t i o n , a series o f membranec o m p o u n d s was f o u n d . The a s s a y a s s o c i a t e d Q-{14C)alanyl-lipophilic o f t h e s e compounds depended on t h e i r e x t r a c t a b i l i t y i n t o m o n o p h a s i c c h l o r o f o r m -met h a n o 1-water (0.8 :3.2 :1 . 0 , vo l / v o l / v o 1) a n d s u b s e q u e n t p a r t i t i o n i n g i n t o chloroform. Four l i n e s o f evidence suggested t h a t t h e Q - a l a n y l - l i p o p h i l i c compounds are i n t e r m e d i a t e s i n t h e s y n t h e s i s o f Q - a l a n y l - l i p o t e i c h o i c acid. First, p a r t i a l d e g r a d a t i o n o f t h e p o l y ( g l y c e r o 1 p h o s p h a t e ) m o i e t y o f Q - a l a n y l - l i p o t e i c h o i c a c i d by phosphodiesterase II/phosphatase from Aspergillus n i g e r generated a series o f P - a l a n y l - l i p o p h i l i c compounds similar t o t h o s e e x t r a c t e d from t o l u e n e - t r e a t e d c e l l s d u r i n g t h e i n c o r p o r a t i o n o f Q - a l a n i n e . Second, e n z y m a t i c d e g r a d a t i o n o f t h e Q - a l a n y l - l i p o p h i l i c compounds by t h e a b o v e p r o c e d u r e g a v e Q - a l a n y l - g l y c e r o l , t h e same d e g r a d a t i o n p r o d u c t o b t a i n e d from a - a l a n y l - l i p o t e i c h o i c acid. Third, the i n c o r p o r a t i o n o f 9 - a l a n i n e i n t o t h e s e c o m p o u n d s r e q u i r e d t h e same components as t h e i n c o r p o r a t i o n o f Q - a l a n i n e i n t o membraneFourth, t h e phosphate-induced l o s s a s s o c i a t e d Q - l i p o t e i c h o i c acid. o f g- { C 1 a 1a n i n e - 1a be1 1e d 1i p o p h i 1i c c o m p o u n d s c o u 1d be c o r r e 1a t e d with t h e stimulation of phosphatidylglycerol synthesis i n the p r e s e n c e o f e x c e s s p h o s p h a t e . T h e s e e x p e r i m e n t s were i n t e r p r e t e d t o

73

4: Microbial Polysaccharides indicate that

the Q-alanyl-lipophilic

compounds w e r e Q - a l a n y l -

l i p o t e i c h o i c a c i d w i t h s h o r t p o l y m e r c h a i n s and were m o s t l i k e l y i n t e r m e d i a t e s i n t h e assembly o f t h e completed polymer,

a-alanyl-

l i p o t e i c h o i c acid. Teichuronic a c i d i s o l a t e d from the c e l l w a l l s o f Micrococcus

-----luteus

has

been

spectroscopy."

examined

by

natural-abundance

n.m.r.

13C

P r o t o n - d e c o u p l e d a n d p r o t o n - c o u p l e d s p e c t r a were

o b t a i n e d f o r n a t i v e t e i c h u r o n i c a c i d and a l s o a f t e r t h e t e i c h u r o n i c a c i d h a d been o x i d i z e d w i t h p e r i o d a t e and r e d u c e d w i t h b o r o h y d r i d e . The s p e c t r a a r e c o n s i s t e n t w i t h t h e s t r u c t u r e ( 2 ) .

Teichuronic a c i d

s y n t h e s i z e d i n v i t r o f r o m s u i t a b l e s u b s t r a t e s by t h e p a r t i c u l a t e enzyme f r a c t i o n o b t a i n e d f r o m M i c r o c o c c u s l u t e u s y i e l d e d a 13C n.m.r.

spectrum which i s i n d i s t i n g u i s h a b l e from t h a t o f the n a t i v e

teichuronic

acid,

indicating a

teichuronic a c i d synthesized

in

structural identity

o f

the

v i t r o w i t h t h a t i s o l a t e d from c e l l

walls.

+4)-B-Q-Man~ANAc-(l+6)-ct-Q-Glc~-(l+ (2) The

membrane-bound

enzymes

of

Micrococcus varians

which

p a r t i c i p a t e i n t h e syntheses o f t h e t e i c h o i c a c i d main c h a i n and linkage

unit

have

been

solubilized

f r a c t i o n a t e d by s u c r o s e d e n s i t y - g r a d i e n t f r a c t i o n s were obtained:

with

Triton

X-100

centrifugation.12

a heavy f r a c t i o n ,

and

Two m a i n

c o n t a i n i n g enzymes

e f f e c t i n g s y n t h e s i s o f t h e main chain attached t o t h e l i n k a g e u n i t , w h i c h was a s s o c i a t e d w i t h o n l y a s m a l l amount o f l i p i d ,

and a l i g h t

f r a c t i o n , w h i c h was r i c h i n p r e n y l p h o s p h a t e a n d c a t a l y s e d o n l y linkage-unit

synthesis.

The s e p a r a t i o n b y d e n s i t y w a s n o t b a s e d

e n t i r e l y on p o l y p e p t i d e c h a i n l e n g t h , as some o f t h e s h o r t e s t c h a i n s a p p e a r e d i n t h e d e n s e r f r a c t i o n s a n d some r e l a t i v e l y h i g h - m o l e c u l a r weight peptides occurred i n the l i g h t e s t fraction.

High a c t i v i t y

f o r l i n k a g e - u n i t s y n t h e s i s was o b s e r v e d i n a f r a c t i o n c o n t a i n i n g o n l y a few p e p t i d e s .

A d d i t i o n o f f i c a p r e n y l p h o s p h a t e t o t h e enzyme

p r e p a r a t i o n s h a d no s t i m u l a t o r y e f f e c t .

I t was c o n c l u d e d t h a t t h e

enzymes f o r m a i n - c h a i n and l i n k a g e - u n i t

s y n t h e s e s f o r m one o r m o r e

f a i r l y t i g h t l y a s s o c i a t e d c o m p l e x e s and t h a t p o l y p r e n y l p h o s p h a t e i s an i n t e g r a l ,

f i r m l y bound component o f t h e complex i n w h i c h t h e

linkage u n i t i s synthesized. The m a i n c h a i n o f t e i c h o i c a c i d s c a n be a s s e m b l e d i n c e l l - f r e e membrane p r e p a r a t i o n s by t h e t r a n s f e r o f r e s i d u e s f r o m t h e


74 appropriate nucleotide precursors t o a m p h i p h i l i c molecule, c e l l wall,

an i n c o m p l e t e l y c h a r a c t e r i z e d

lipoteichoic acid carrier.13

However,

linkage u n i t which i s synthesized independently. that,

i n the

the main chain i s attached t o peptidoglycan through a

i n these c e l l - f r e e

linkage units

are

systems,

also

able

to

It i s believed

l i p i d intermediates carrying

accept

residues

n u c l e o t i d e p r e c u r s o r s t o b u i l d up t h e m a i n c h a i n .

directly

from

I t was shown t h a t

t h e m a i n c h a i n a t t a c h e d t o l i p o t e i c h o i c a c i d c a r r i e r was t r a n s f e r r e d from l i p o t e i c h o i c acid c a r r i e r t o l i p i d s containing the linkage unit,

Thus,

i n t h e s e systems,

t h e r e a p p e a r t o be t w o r o u t e s t o t h e

biosynthesis o f teichoic acid/linkage-unit

complexes,

one by d i r e c t

a s s e m b l y o f t h e m a i n c h a i n o n l i n k a g e - u n i t l i p i d s a n d t h e o t h e r by t r a n s f e r o f the preassembled main chain from l i p o t e i c h o i c a c i d carrier t o the linkage unit.

I t was a l s o s h o w n t h a t l i n k a g e - u n i t

l i p i d s f r o m d i f f e r e n t o r g a n i s m s w e r e i n t e r c h a n g e a b l e and t h a t t h e s e w e r e u s e d f o r p o l y m e r s y n t h e s i s by B a c i l l u s s u b t i l i s 3610,

i n which

t h e t e i c h o i c a c i d i s a p o l y ( g l y c e r o 1 phosphate). The i n c r e a s i n g u s e o f S t a p h y l o c o c c u s a u r e u s r i b i t o l t e i c h o i c acid,

which possesses a 2-acetamido-2-deoxy-B-Q-glucopyranosyl

residue,

i n s e r o l o g i c a l d i a g n o s i s e s p e c i a l l y i n b a c t e r e m i a and

osteoarticular antigen.14

infections

I n the

course

prompted the of

the

a g g l u t i n i n immobilized on U l t r o g e l , acetamido-2-deoxy-Q-glucopyranosyl

separate

ribitol

This study

was

teichoic

purification of this

purification

a c i d from

the

wheat-germ

which has a f f i n i t y residues, other

was

for

2-

employed

to

contaminating antigens.

concerned w i t h the abnormal a f f i n i t y

acetamido-2-deoxy-B-~-glucosy~ r i b i t o l

S t a p h y l o c o c c u s a u r e u s f o r t h e wheat-germ

teichoic

of

t h e 2-

acid

o f

a g g l u t i n i n i m m o b i l i z e d on

U l t r o g e l w h i c h i s dependent on t h e i o n i c f o r c e s and t h e n a t u r e o f the contaminants. Native substitution w i t h the p-alanine ester o f lipoteichoic acids a f f e c t s t h e i r immunological properties, divalent cations,

the capacity to bind

and l i p o t e i c h o i c a c i d c a r r i e r a c t i v i t y . 1 5

i n f l u e n c e o f the Q-alanine e s t e r on a n t i - a u t o l y t i c

The

a c t i v i t y was

t e s t e d , u s i n g e x t r a c e l l u l a r a u t o l y s i n f r o m S t a p h y l o c o c c u s a u r e u s and n i n e l i p o t e i c h o i c acids w i t h a l a n i n e /phosphorus

molar ratios o f

b e t w e e n 0.23

h i g h e s t w i t h Q-

a n d 0.71.

The i n h i b i t o r y a c t i v i t y ,

a 1 a n i n e - f r e e 1i p o t e ic h o i c increasing alanine content, g r e a t e r t h a n 0.6. not

inhibitory,

a c id, e x p o n e n t ia 11y

Correspondingly, i n

de c r ea s e d

w it h

approaching zero a t s u b s t i t u t i o n s of

contrast

d i p o l a r i o n i c p h o s p h o l i p i d s were to

negatively

charged

ones.

75


G l y c o s y l a t i o n o f l i p o t e i c h o i c a c i d up t o a n e x t e n t o f 0.5 d i d n o t depress i n h i b i t o r y a c t i v i t y , was c o m p a r a t i v e l y s m a l l . various sources,

and e v e n a t a d e g r e e o f 0.8

the effect

On c o m p a r i s o n o f l i p o t e i c h o i c a c i d s f r o m

d i f f e r e n c e s i n l i p i d s t r u c t u r e s and c h a i n l e n g t h s

were w i t h o u t e f f e c t .

The i n h i b i t o r y a c t i v i t y d r a s t i c a l l y d e c r e a s e d

when t h e g l y c o l i p i d c a r r i e d a s i n g l e g l y c e r o p h o s p h a t e r e s i d u e or t h e

(3).

h y d r o p h i l i c c h a i n had t h e u n u s u a l s t r u c t u r e were o b t a i n e d w i t h t h e more complex

The same r e s u l t s

system o f

autolysis o f

I t was s u g g e s t e d t h a t t h e a n t i -

Staphylococcus a u r e u s c e l l s .

a u t o l y t i c a c t i v i t y o f l i p o t e i c h o i c a c i d r e s i d u e s i n a sequence o f glycerophosphate

units

and

that

the

negative

charges

a p p r o p r i a t e l y spaced p h o s p h o d i e s t e r groups p l a y a c r u c i a l r o l e . g-alanine

of The

e s t e r e f f e c t was d i s c u s s e d w i t h r e s p e c t t o t h e p u t a t i v e

vivo regulation

in

o f a u t o l y s i s by l i p o t e i c h o i c a c i d .

+6)-a-Q-Gale-(1+6)-a-g-Gale-O-CH2

I

a-g-Gale-0-CH

l

o

CH 2-0 -P+

OH (3) Streptococcus mutans

Ingbritt

was

grown i n a chemostat

d e s t i n e d d i l u t i o n r a t e s i n e i t h e r 0.5% ! - f r u c t o s e and a t d e s t i n e d pH v a l u e s i n 0.5% f r u c t o s e . a f f e c t e d by t h e c a r b o h y d r a t e s o u r c e , l o w e s t y i e l d b e i n g a t pH 5.5

at

o r 0.5% p - g l u c i t o l

The y i e l d o f c e l l s was

as w e l l as by t h e pH,

i n 0.5% p - f r u c t o s e . 1 6

with the

!-Fructose-grown

c e l l s showed g r e a t e r s u s c e p t i b i l i t y t o l y s i s by a m u r a m i d a s e t h a n t h e corresponding g-glucose-grown

cells,

b u t t h e r e w e r e no m a r k e d

differences i n the l y t i c s u s c e p t i b i l i t i e s o f the corresponding c e l l w a l l preparations or i n the serological r e a c t i v i t i e s o f w a l l lysates w i t h antiserum t o Streptococcus

mutans

Ingbritt.

The

greatest

amounts o f c e l l u l a r l i p o t e i c h o i c a c i d were o b t a i n e d a t h i g h d i l u t i o n r a t e s i n b o t h Q - f r u c t o s e and Q - g l u c i t o l , values

i n

!-fructose.

The

greatest

a s w e l l a s a t h i g h pH

amounts

of

l i p o t e i c h o i c a c i d were f o u n d a t l o w d i l u t i o n r a t e s ,

extracellular as e s t i m a t e d by

r o c k e t i m m u n o e l e c t r o p h o r e s i s and a l s o by h a e m a g g l u t i n a t i o n .

Three

m a j o r e x t r a c e l l u l a r p r o t e i n components w e r e s e p a r a t e d by s o d i u m dodecyl sulphate-polyacrylamide of

g e l e l e c t r o p h o r e s i s , and t h e e f f e c t s

g r o w t h c o n d i t i o n s on t h e s e components were d e t e r m i n e d .

Results

76


f o r batch-grown c u l t u r e s showed t h a t t h e r e was g e n o t y p i c v a r i a t i o n The i n t h e s u s c e p t i b i l i t y o f c e l l s t o l y s i s by a m u r a m i d a s e . e n h a n c e m e n t o f l i p o t e i c h o i c a c i d p r o d u c t i o n o f p - f r u c t o s e a n d ag l u c i t o l i n b a t c h c u l t u r e s was n o t i d e n t i c a l i n r e p r e s e n t a t i v e n o r was t h e e f f e c t o f s t r a i n s o f S t r e p t o c o c c u s m u t a n s s e r o t y p e 2, p-fructose found uniformly i n r e p r e s e n t a t i v e s t r a i n s of t h e d i f f e r e n t Streptococcus mutans serotypes. T h e p r o b l e m s c a u s e d by t h e n o n - s p e c i f i c b i n d i n g o f l i p o t e i c h o i c a c i d and glucan-binding p r o t e i n s o f Streptococcus mutans t o immunosorbent columns prepared from cyanogen bromide-activated S e p h a r o s e 48 h a v e b e e n d e s c r i b e d . 1 7 H u m a n p o l y m o r p h o n u c l e a r l e u k o c y t e s were s h o w n t o p o s s e s s s p e c i f i c binding sites f o r l i p o t e i c h o i c acid.18 L i p o t e i c h o i c acid b i n d i n g was r e v e r s i b l e a n d t i m e a n d t e m p e r a t u r e d e p e n d e n t . Scatchard p l o t analysis revealed an apparently single population o f 6.6 x l o 6 l i p o t e i c h o i c acid b i n d i n g s i t e s p e r human p o l y m o r p h o n u c l e a r l e u k o c y t e s w i t h a d i s s o c i a t i o n c o n s t a n t o f 5.6 ~ J M . Attachment of an a v i r u l e n t unencapsulated, M-negative of group A s t r e p t o c o c c i t o human p o l y m o r p h o n u c l e a r l e u k o c y t e s was i n h i b i t e d by l i p o t e i c h o i c a c i d b u t n o t by o t h e r b a c t e r i a l s o m a t i c a n t i g e n s tested. O c c u p a t i o n o f 30% o f t h e l i p o t e i c h o i c a c i d b i n d i n g s i t e s r e s u l t e d i n g r e a t e r t h a n 70% i n h i b i t i o n o f s t r e p t o c o c c a l a t t a c h m e n t o f human p o l y m o r p h o n u c l e a r l e u k o c y t e s . In contrast, lipoteichoic acid failed t o block attachment o f Escherichia c o l i o r antibodycoated streptococci, indicating that binding sites f o r E s c h e r i c h i a c o l i and t h e Fc p o r t i o n o f immunoglobulin G are d i s t i n c t from t h o s e f o r l i p o t e i c h o i c a c i d . Immunofluorescent studies demonstrated t h a t l i p o t e i c h o i c acid uniformly bound t o p o l y m o r p h o n u c l e a r l e u k o c y t e m e m b r a n e s f o r a s l o n g a s 2 h a t 37'C. C r o s s - l i n k i n g o f human p o l y m o r p h o n u c l e a r l e u k o c y t e s - b o u n d l i p o t e i c h o i c acid r e s u l t e d i n r a p i d capping o f l i p o t e i c h o i c r e c e p t o r sites. The r e s u l t s s u g g e s t t h a t l i p o t e i c h o i c a c i d i s a m o n o v a l e n t l i g a n d i n t e r a c t i n g w i t h m o b i l e r e c e p t o r s i n t h e p l a s m a membrane o f human p o l y m o r p h o n u c l e a r l e u k o c y t e s . 'H a n d 1 3 C n.m.r. has been shown t o d i s t i n g u i s h e a s i l y b e t w e e n c o v a l e n t l y and i o n i c a l l y a s s o c i a t e d a-alanine i n l i p o t e i c h o i c a c i d s , w i t h 13C n.m.r. showing regular 1,3-linkages in the polyglycerophosphate chain and a s s i g n a b l e resonances f o r t h o s e s u b u n i t s c a r r y i n g P - a l a n i n e and s u g a r r e s i d u e s . 1 9

4: Microbial Polysaccharides Peptidoglycans

2 The

77

immunological a c t i v i t i e s o f

b a c t e r i a l p e p t i d o g l y c a n s have

been r e v i e w e d and t h e s t r u c t u r e s o f p a r t i c u l a r p e p t i d o g l y c a n s and groups o f p e p t i d o g l y c a n s r e l a t e d t o a c t i v i t y . 2 0 Pep t i d o g l y c a n monomer (a-GlceNAc-Mur NAc-L-A l a - Q -i s o g l u t am i n e

-meso-diaminopimelic

acid-E-Ala-Q-Ala),

was i n c u b a t e d w i t h s l i c e s o f

d i s a c c h a r i d e and p e n t a p e p t i d e p o r t i o n s , mouse l i v e r ,

kidney,or

blood cells,

p l a s m a , a n d serum.21

unchanged a f t e r

-

l a b e l l e d w i t h 14C i n both the

s p l e e n as w e l l as w i t h mouse a n d human b l o o d , P e p t i d o g l y c a n monomer was i s o l a t e d

i n c u b a t i o n s w i t h mouse o r g a n s

and b l o o d c e l l s .

H o w e v e r , u p o n i n c u b a t i o n w i t h mouse o r human b l o o d , 1 0 - 5 0 % o f t h e p e p t i d o g l y c a n monomer u n d e r w e n t h y d r o l y s i s t o t h e c o r r e s p o n d i n g disaccharide

and p e n t a p e p t i d e .

serum

than

more

metabolized:

90% o f

the

After

i n c u b a t i o n s w i t h plasma

{14C)peptidoglycan

monomer

and was

a b o u t 5 0 % o f t h e a d m i n i s t e r e d r a d i o a c t i v e d o s e was

r e c o v e r e d i n t h e d i s a c c h a r i d e u n i t and a b o u t 35% i n t h e p e n t a p e p t i d e

I t was s u g g e s t e d t h a t i n b l o o d , p l a s m a , a n d

part.

man an N - a c e t y l m u r a m o y l - l - a l a n i n e

s e r u m o f mouse a n d

amidase e x i s t s

which s p l i t s the

a m i d e bond b e t w e e n t h e l a c t y l c a r b o x y l g r o u p o f t h e m u r a m y l r e s i d u e and t h e amino

mo l e c u l e

.

An

group o f

the peptide moiety

isotope-dilution

micro-analysis

using

procedure

for

quantitative N-terminal

3H- l a b e 1l e d 1- f l u o r o - 2 , 4 - d i n i t r o ben zene and a

m i x t u r e o f f r e e and a c e t y l a t e d l 4 C - 1 a b e l l e d s t a n d a r d s h a s been

described.22

The

derivatives

chromatography.

p r i m a r i l y intended f o r the estimation o f c e l l - w a l l peptidoglycan,

a m i n o a c i d s as i n t e r n a l

2,4-dinitrophenyl

a r e s e p a r a t e d by p o l y a m i d e t h i n - l a y e r

peptides.

i n the peptidoglycan

crosslinkage

Although

i n bacterial

t h e method i s c a p a b l e o f e x t e n s i o n t o o t h e r

When t e s t e d o n l y s o z y m e ,

t h e p r o c e d u r e gave r e s u l t s w h i c h

w e r e i n good a g r e e m e n t w i t h a c c e p t e d v a l u e s . The

conformational

energies

of

complexes

copolymers of 2-acetamido-2-deoxy-~-glucose w i t h hen e g g - w h i t e

of

alternating

and 2 - a c e t y l m u r a m i c a c i d

l y s o z y m e h a v e b e e n computed.23

This involved a

complete search o f t h e c o n f o r m a t i o n a l space a t t h e a c t i v e s i t e o f t h e enzyme a v a i l a b l e t o t h e s e s u b s t r a t e s and m i n i m i z a t i o n o f t h e conformational energies of ho m opo 1y m e r

( a -G 1ceNA c 16 ,

t h e n o n c o v a l e n t complexes.

G ~ c ~ N A cb )i n~d s p r e f e r e n t i a l l y on t h e l e f t s i d e o f cleft,

i n v o l v i n g r e s i d u e s s u c h as &-Arg-45,

The a l t e r n a t i n g c o p o l y m e r

As w i t h t h e

t h e hex as a c c h a r ide (Q -G lceNA c -M u r NAc ) 2I-Asn-46,

(Q-GlceNAc-MurNAc)j,

(g -

the active-site and i - T h r - 4 7 .

however,

binds with

78


i t s F-site r e s i d u e p r e f e r e n t i a l l y on t h e r i g h t s i d e o f t h e a c t i v e s i t e c l e f t , i n v o l v i n g r e s i d u e s s u c h a s L-Phe-34 a n d L-Arg-114. The l a c t i c a c i d s i d e c h a i n p r e v e n t s good b i n d i n g t o t h e F s i t e on t h e l e f t side. This r e s u l t can e x p l a i n t h e higher rate of c a t a l y s i s f o r t h e cell-wall s u b s t r a t e ( t h e a l t e r n a t i n g co-polymer). The r e l a t i v e a f f i n i t i e s o f t h e d i s a c c h a r i d e Q-GlcQNAc-MurNAc f o r a l l s e q u e n t i a l p a i r s o f s i t e s A-F ( i n c l u d i n g E a n d F s i t e s o n b o t h s i d e s o f t h e It is found t h a t t h e highest a f f i n i t y o f c l e f t ) are determined. t h i s d i s a c c h a r i d e i s f o r s i t e s C a n d D a n d r i g h t - s i d e s i t e s E a n d F. The e n e r g y o f t h e r e c e n t l y d e t e r m i n e d X-ray c r y s t a l l o g r a p h i c s t r u c t u r e o f MurNAc-Q-GlceNAc-MurNAc b o u n d t o t h e B, C, a n d D s i t e s o f hen egg-white lysozyme has been minimized and found t o l e a d a conformation q u i t e s i m i l a r t o one which has been predicted p r e v i o u s l y f o r t h e t r i s a c c h a r i d e ( Q - G ~ c ~ N A c ) ~ .T h e D r i n g i s u n d i s t o r t e d and binds c l o s e t o t h e s u r f a c e o f t h e a c t i v e - s i t e c l e f t . T h e s t r u c t u r e c a n b e e x t e n d e d i n t o s i t e s E a n d F by a d d i t i o n o f t w o g-GlceNAc r e s i d u e s , b u t o n l y o n t h e l e f t s i d e o f t h e a c t i v e - s i t e cleft. T h i s i n d i c a t e s t h a t p o l y m e r s b o u n d w i t h t h e i r D-site r e s i d u e s near t h e s u r f a c e o f t h e c l e f t must bind t o sites E and F on t h e l e f t s i d e of t h e c l e f t , as a l s o predicted previously. A series o f seven d i f f e r e n t UDP-N-acetylmuramyl peptide p r e c u r s o r s o f b a c t e r i a l c e l l - w a l l p e p t i d o g l y c a n h a s b e e n e x a m i n e d by r e v e r s e - p h a s e h i g h - p r e s s u r e l i q u i d c h r ~ m a t o g r a p h y . ~M~i x t u r e s o f t h e s e c o m p o u n d s were s u c c e s s f u l l y a n d r a p i d l y a n a l y s e d by u s i n g Waters U B o n d a p a k C 1 8 c o l u m n a s a s t a t i o n a r y p h a s e a n d i s o c r a t i c e l u t i o n s w i t h 0.05M a m m o n i u m p h o s p h a t e o r f o r m a t e b u f f e r s o f a p p r o p r i a t e pH. A c c u r a t e q u a n t i t a t i o n c o u l d a l s o be r e a d i l y a c h i e v e d by r e v e r s e - p h a s e h i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h y . A l l t h e s e t e c h n i q u e s are e x t r e m e l y u s e f u l f o r t h e p u r i f i c a t i o n o f these compounds and f o r a wide r a n g e o f b i o c h e m i c a l s t u d i e s c o n c e r n i n g t h e cytoplasmic s t e p s of t h e b i o s y n t h e s i s of peptidoglycan. T h e s o l i d - s t a t e c o n f o r m a t i o n a l a n a l y s i s o f Ac-Q-Ala-a-AlaOH.H20, c a r r i e d o u t by i n f r a r e d a b s o r p t i o n a n d X - r a y d i f f r a c t i o n , has i n d i c a t e d t h a t t h e molecules are not extended i n a r e g u l a r c o n f o r m a t i o n , b u t r a t h e r t h a t t h e y a r e p a r t i a l l y f o l d e d , 2 5 t h e +,$* torsional angles of the carboxyl-terminal reside i n p a r t i c u l a r being i n t h e r e g i o n o f t h e l e f t - h a n d e d a - h e l i x o f t h e R a m a c h a n d r a n map. The a c e t y l a m i n o a n d p e p t i d e g r o u p s are found i n t h e u s u a l t r a n s conformation, the latter e x h i b i t i n g a deviation from r i g i d p l a n a r i t y . Only i n t e r m o l e c u l a r hydrogen bonds o c c u r i n t h e c r y s t a l state. The s o l u t i o n c o n f o r m a t i o n a l a n a l y s i s , p e r f o r m e d by i n f r a r e d


79

a b s o r p t i o n a n d c.d., h a s r e v e a l e d t h a t t h e amount o f i n t r a m o l e c u l a r N-H. .O=C h y d r o g e n - b o n d e d f o l d e d f o r m s , i f a n y , s h o u l d be extremely small, even i n deuteriochloroform at high dilution. In water, solvated, unordered s p e c i e s l a r g e l y predominate. Incubation o f growing Bacillus s u b t i l i s with p e n i c i l l i n G led t o the s e c r e t i o n of a peptidoglycan-related polymer and a nonglycanb o u n d p e n t a p e p t i d e i n t o t h e c u l t u r e medium.26 T h e s e c r e t e d p o l y m e r was i s o l a t e d a n d c h a r a c t e r i z e d a s a l i n e a r c e l l - w a l l g l y c a n s t r a n d s u b s t i t u t e d p r e d o m i n a n t l y by u n c r o s s - l i n k e d p e n t a p e p t i d e s i d e c h a i n s . P o l y m e r f o r m a t i o n a n d s e c r e t i o n were m o s t l i k e l y t h e r e s u l t of continued s y n t h e s i s and elongation o t nascent glycan s t r a n d s i n t h e a b s e n c e o f s u b s e q u e n t p r o c e s s i n g by p e p t i d o g l y c a n t r a n s p e p t i d a s e o f Q - a 1a n i n e c a r b o x y p e p t i d a s e e n z y m e s . The nonglycan-bound L-Ala-n-iso-Glu-meso-diaminopimelic acid-P-Ala-D-Ala pentapeptide w a s p r o b a b l y f o r m e d by a n t j - a c e t y l m u r a m o y l - l - a l a n i n e amidase a c t i v e on t h e peptide side c h a i n s of t h e uncross-linked polymer. The u n c r o s s - l i n k e d p e p t i d o g l y c a n p o l y m e r was s h o w n t o be a g o o d s u b t r a t e for penicillin-sensitive P-alanine carboxypeptidase p u r i f i e d from mem b r a n e s o f B a c i l l u s s u b t i l i s , B a c i l l u s s t e a r o t h e r m o p h i l u s , a n d Escherichia c o l i . 9 - A l a n i n e r e l e a s e was n o t , h o w e v e r , c o u p l e d t o the c r o s s - l i n k i n g o f peptide side chains, suggesting t h a t t h e s e e n z y m e s do n o t f u n c t i o n a s p e p t i d o g l y c a n t r a n s p e p t i d a s e i n v i v o . No t r a n s p e p t i d a s e o r Q - a l a n i n e c a r b o x y p e p t i d a s e a c t i v i t y was d e t e c t e d i n m i x t u r e s o f h i gh-m o l e c u l a r-w e i g h t p e n i c i 11i n - b i n d i n g p r o t e i n s f r o m ga3L&s s u b t i l i s , Bacillus stearothermophilus, or Staphylococcus aureus. Possible reasons for the inability t o d e m o n s t r a t e these a c t i v i t i e s are discussed. I n a d d i t i o n , a n Nacetylmuramoyl-i-alanine amidase a c t i v i t y which c o p u r i f i e s w i t h penicillin-binding proteins with Bacillus s u b t i l i s , Staphylococcus a u r e u s , a n d E s c h e r i c h i a c o l i was p a r t i a l l y c h a r a c t e r i z e d . C e l l - w a l l p o l y m e r s were m e a s u r e d b o t h i n t h e c e l l s a n d i n t h e c e l l - f r e e medium o f s a m p l e s f r o m s t e a d y - s t a t e c h e m o s t a t c u l t u r e s o f B a c i l l u s s u b t i l i s , growing a t v a r i o u s rates under magnesium or phosphate limitation.” The p r e s e n c e o f b o t h p e p t i d o g l y c a n a n d a n i o n i c wall polymers i n t h e c u l t u r e s u p e r n a t a n t showed t h e occurrence of wall turnover i n t h e s e c u l t u r e s . Variable p r o p o r t i o n s o f t h e t o t a l p e p t i d o g l y c a n p r e s e n t i n t h e c u l t u r e s a m p l e s were f o u n d o u t s i d e t h e cells i n d u p l i c a t e c u l t u r e s , i n d i c a t i n g t h a t t h e rate of peptidoglycan turnover is variable i n Bacillus subtilis. Besides p e p t i d o g l y c a n , a n i o n i c w a l l p o l y m e r s were d e t e c t e d i n t h e c u l t u r e supernatant, teichoic acid i n magnesium-limited cultures, and


80 teichuronic acid i n phosphate-limited cultures. the

ratio

between

concentrations

was

than i n the walls.

the

peptidoglycan

significantly

lower

and

I n s e v e r a l samples,

the

anionic-polymer

i n the extracellular

fluid

T h i s d i v e r g e n c y was a t t r i b u t e d t o t h e o c c u r r e n c e

o f d i r e c t secretion o f anionic polymers a f t e r t h e i r synthesis. An enzyme w h i c h c a t a l y s e s t h e h y d r o l y s i s o f a c e t a m i d o g r o u p s o f 2-ace t am ido -2-deox y - g - g l u c o p y r anosy 1 r e s i d u e s i n c e l l - w a 11 pep t id o g l y c a n was f o u n d i n t h e s u p e r n a t a n t and 20,0009

pellet fractions o f

Autolysis of the l a t t e r fraction resulted i n

B a c i l l u s cereus.28

s o l u b i l i z a t i o n and a c t i v a t i o n o f t h e d e a c e t y l a s e . bacteria, of

2-amino-2-deoxy-P-glucopyranosyl

i n t h e deacetylase. able

from

the

basis

behaviour.

Among v a r i o u s

s t r a i n s o f B a c i l l u s cereus which c o n t a i n h i g h p r o p o r t i o n s residues are

particularly

2-acetamido-2-deoxy-~-glucose-6-phosphate

of

rich

The p e p t i d o g l y c a n d e a c e t y l a s e i s d i s t i n g u i s h -

their

cellular

d i s t r i b u t i o n and

deacetylase

on

chromatographic

The r a t e o f r e a c t i o n o f t h e d e a c e t y l a s e w i t h (P-GlceNAc-

MurNAcI3 i s l e s s t h a n 1/100 o f t h a t w i t h p e p t i d o g l y c a n , w h i l e t h e enzyme i s i n a c t i v e t o w a r d s (Q-GlceNAc-Mur N A ~ ) ~ - t j - G l c e N A c - M u r N A c , and mo n o m e r ic 2 - a c e t a m id o - 2 - d e o x y -g

- g 1u c o s e

d e r iv a t iv e s

.

The enzyme

also deacetylates p a r t i a l l y g-hydroxyethylated chitin,

for half-

m a x i m u m a c t i v i t i e s w e r e f o u n d t o b e 0 . 2 9 a n d 6.9 mg p e r m l ( 0 . 1 7 a n d 20mM w i t h r e s p e c t t o 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o p y r a n o s y l respectively.

residues),

The o c c u r r e n c e o f t h i s enzyme a c c o u n t s f o r t h e f o r m -

a t i o n o f c e l l - w a l l peptidoglycan containing non-acetylated

2-amino-

2-deoxy-~-glucopyranosyl residues. The c e l l w a l l s i s o l a t e d f r o m a x e n i c a l l y g r o w n l e p r o s y - d e r i v e d c o r y n e b a c t e r i a were s u b m i t t e d t o v a r i o u s c h e m i c a l and e n z y m a t i c degradations.

The g l y c a n s t r a n d s o f t h e w a l l p e p t i d o g l y c a n w e r e

e s s e n t i a l l y composed o f 2-acetamido-2-deoxy-~-glucopyranosyl-~acetylmuramic

acid disaccharide

units.29

S m a l l amounts

of

2-

acetamido-2-deoxy-~-glucopyranosyl-~-glycolylmuramic a c i d (less than 10%) were a l s o d e t e c t e d .

The m u r a m i c a c i d r e s i d u e s o f a d j a c e n t

g l y c a n s t r a n d s a r e s u b s t i t u t e d by a m i d a t e d t e t r a p e p t i d e u n i t s w h i c h , i n turn,

are c r o s s - l i n k e d through d i r e c t l i n k a g e s extending between

the C-terminal diaminopimelic

2-alanine acid

r e s i d u e o f one t e t r a p e p t i d e a n d t h e meso-

residue

of

another

tetrapeptide.

Such

a

s t r u c t u r e i s very s i m i l a r t o t h a t o f the w a l l peptidoglycan found i n the

taxonomically

related

micro-organisms

of

Corynebacteriurn,

Mycobacterium, and N o r c a r d i a groups. Two

different

cell-wall

peptidoglycan synthetase

c a r r i e d by p e n i c i l l i n - b i n d i n g

systems a r e

p r o t e i n s 1 A a n d 1B p u r i f i e d f r o m


-E--s-c h e r i c h i a

-

81

c o l i . Both s y s t e m s c o n s i s t of two enzyme a c t i v i t i e s c a r r y i n g o u t s u c c e s s i v e r e a c t i o n s of peptidoglycan s y n t h e s i s from t h e 1 i p i d - 1i n k e d p r e c u r s o r 2 - ace t a m i do - 2 - d e o x y -B - g l u c o p y r a n o s y 1-!acetylmuramyl-(pentapeptide)-diphosphate-undecaprenol, namely t h o s e o f p e p t i d o g l y c a n t r a n s g l y c o s y l a s e a n d B-lactam a n t i b i o t i c - s e n s i t i v e transpeptidase. The a c t i v i t i e s o f t h e two enzyme s y s t e m s d i f f e r i n o p t i m a l c o n d i t i o n s a n d s e n s i t i v i t i e s o f B-lactam a n t i b i o t i c s . The p r o p e r t i e s of the p u r i f i e d p e n i c i l l i n - b i n d i n g p r o t e i n 1 A have been r e p o r t e d . 30 A p u r i f i e d p r e p a r a t i o n o f N-acetylmuramoyl-L-alanine a m i d a s e , a m u r e i n h y d r o l a s e f r o m E s c h e r i c h i a c o l i , was f o u n d t o l o s e i t s a c t i v i t y during incubation i n the presence of b a c t e r i a l phospholipid suspension^.^' W h e t h e r i t was c o - d i s p e r s e d w i t h t h e p h o s p h o l i p i d s o r a d d e d t o s o n i c a t e d p h o s p h o l i p i d s u s p e n s i o n , t h e e n z y m e was i n h i b i t e d (or i n a c t i v a t e d ) from t h e f i r s t m i n u t e s o f i n c u b a t i o n a t 37OC. As t h e p h o s p h a t i d y l g l y c e r o l / c a r d i o l i p i n r a t i o o f t h e p h o s p h o l i p i d s u s p e n s i o n was i n c r e a s e d ( a l l o t h e r t h i n g s b e i n g e q u a l ) , a f u r t h e r d e c r e a s e o f a m i d a s e a c t i v i t y was o b s e r v e d . T h e h i g h e s t l o s s e s o f a c t i v i t y were f o u n d a f t e r c o - d i s p e r s i o n o f t h e enzyme and t h e s u b s t r a t e t o g e t h e r w i t h t h e p h o s p h o l i p i d s , t h e r e s u l t i n g suspension being formed of l a r g e r multilayered vesicles, a s r e v e a l e d by e l e c t r o n m i c r o s c o p y . I n t h e s e c o n d i t i o n s , t h e e f f e c t o n e n z y m e a c t i v i t y w a s o n l y p a r t i a l l y a c c o u n t e d f o r by t h e p r o p o r t i o n o f t h e e n z y m e t h a t was e n t r a p p e d i n t h e v e s i c l e s . T h e entrapment c a p a c i t y of t h e enzyme ( u s i n g a 35S-labelled enzyme p r e p a r a t i o n ) a n d o f t h e s u b s t r a t e ( 3 H - l a b e l l e d ) by t h e m u l t i l a m e l l a r p h o s p h o l i p i d i c vesicles d i d n o t s i g n i f i c a n t l y change a s a f u n c t i o n o f t h e i r r e l a t i v e c o n t e n t o f p h o s p h a t i d y l g l y c e r o l and c a r d i o l i p i n . T h e m e c h a n i s m by w h i c h a m i n o a c i d s t a r v a t i o n o f E s c h e r i c h i a c o l i induces r e s i s t a n c e against t h e l y t i c and b a c t e r i c i d a l e f f e c t s o f p e n i c i l l i n has been studied.32 Starvation of Escherichia coli s t r a i n W7 o f t h e a m i n o a c i d s I - l y s i n e o r I - m e t h i o n i n e r e s u l t e d i n t h e r a p i d development of r e s i s t a n c e t o a u t o l y t i c cell-wall d e g r a d a t i o n , w h i c h may be e f f e c t i v e l y t r i g g e r e d i n g r o w i n g b a c t e r i a by a n u m b e r o f c h e m i c a l o r p h y s i c a l t r e a t m e n t s . T h e m e c h a n i s m o f t h i s e f f e c t i n t h e amino acid-starved cells involved t h e production of a murein r e l a t i v e l y r e s i s t a n t t o the hydrolytic a c t i o n of crude m u r e i n h y d r o l a s e e x t r a c t s prepared from n o r m a l l y g r o w i n g E s c h e r i c h i a c o l i . R e s i s t a n c e t o t h e a u t o l y s i n s was n o t d u e t o t h e c o v a l e n t l y linked lipoprotein. Resistance t o murein hydrolase developed most r a p i d y a n d most e x c l u s i v e l y i n t h e p o r t i o n of c e l l wall s y n t h e s i z e d

82


a f t e r the onset of

amino a c i d s t a r v a t i o n .

I-lysine

Lysozyme d i g e s t s o f t h e

m u r e i n s y n t h e s i z e d d u r i n g t h e f i r s t 10 m i n o f

autolysin-resistant

starvation yielded

( i n addition t o the characteristic

degradation products) a high-molecular-weight

m a t e r i a l t h a t was

absent from the lysozyme d i g e s t s o f c o n t r o l c e l l - w a l l

preparations.

I t has been p r o p o s e d t h a t i n h i b i t i o n o f p r o t e i n s y n t h e s i s c a u s e s a r a p i d m o d i f i c a t i o n o f murein s t r u c t u r e a t the c e l l - w a l l

g r o w t h zone

i n s u c h a manner t h a t a t t a c h m e n t o f m u r e i n h y d r o l a s e m o l e c u l e s i s inhibited.

The m e c h a n i s m may i n v o l v e some a s p e c t s o f t h e r e l a x e d

c o n t r o l system since p r o t e c t i o n a g a i n s t p e n i c i l l i n - i n d u c e d l y s i s d e v e l o p e d much s l o w e r

i n amino

acid-starved

relaxed controlled

c e l l s t h a n i n i s o g e n i c s t r i n g e n t l y c o n t r o l l e d (*A+)

(=A)

Physiological concentrations

of

bacteria.

guanosine 5’-diphosphate

i n h i b i t e d t h e s y n t h e s i s o f l i p i d i n t e r m e d i a t e s and p e p t i d o g l y c a n c a t a l y s e d by coli.33

a p a r t i c u l a t e enzyme p r e p a r a t i o n f r o m

concentration i n the

Escherichia

The i n h i b i t i o n o f t h e s e r e a c t i o n s was d e p e n d e n t o n t h e of

assay.

guanosine The

5’-diphosphate

degree o f

3’-diphosphate

inhibition of

and

MgC12

lipid-intermediate

s y n t h e s i s d e c r e a s e d a s t h e m o l a r r a t i o o f MgC12 : g u a n o s i n e 5 ’ diphosphate 3’-diphosphate

was i n c r e a s e d ,

a n d no i n h i b i t i o n was

o b s e r v e d a b o v e a MgC12 : g u a n o s i n e 5 ’ - d i s p h o s p h a t e ratio

o f 2.5.

i n h i b i t i o n by significant

3’-diphosphate

The s y n t h e s i s o f p e p t i d o g l y c a n was m o r e s e n s i t i v e t o guanosine 5’-disphosphate

inhibition

occurred

under

3’-diphosphate,

conditions

where

and lipid

i n t e r m e d i a t e s y n t h e s i s was u n a f f e c t e d ( i . e .

a t MgC12 : g u a n o s i n e 5’-

diphosphate 3’-diphosphate

o r more).

other

nucleotides

did

r a t i o s o f 2.5

not

inhibit

the

A

synthesis

variety o f of

l i p i d

i n t e r m e d i a t e s and p e p t i d o g l y c a n . Azureomycin B (10 pg/ml), a z u r e a nov.

sp.,

a new a n t i b i o t i c f r o m P s e u d o n o c a r d i a

caused t h e a c c u m u l a t i o n o f

i n h i b i t i o n o f p e p t i d o g l y c a n s y n t h e s i s i n an particulate fraction {3H)pentapeptide

l i p i d i n t e r m e d i a t e and

2

v i t r o system u s i n g a

f r o m B a c i l l u s meqaterium KM

a n d c o l d UDP-Q-GlcgNAc

p e n t a p e p t i d e and U D P - { 3 H ) - Q - G l c ~ N A c as

substrate^.^^

c o n c e n t r a t i o n s o f a zu re o mycin B ( o v e r 100ug/ml), a c c u m u l a t i o n was a l s o i n h i b i t e d . E s c h e r i c h i a c o l i Y-10 p e n t a p e p t i d e were

UDP-M urNAcA t higher

lipid-intermediate

When p a r t i c u l a t e f r a c t i o n f r o m

and UDP-{14C}-p-Glc~NAc

used,

with

o r c o l d UDP-MurNAc-

accumulation o f

and c o l d UDP-MurNAc-

l i p i d i n t e r m e d i a t e and

i n h i b i t i o n o f p e p t i d o g l y c a n s y n t h e s i s were a l s o observed.

This

i n d i c a t e d t h a t t h e p r i m a r y t a r g e t o f azureomycin B i s t h e t r a n s f e r o f t h e d i s a c c h a r i d e p e p t i d e u n i t (a-GlcgNAc-M urNAc-pen t a p e p t i d e

1


83

from lipid-bound p r e c u r s o r t o acceptor. ---m e s o - L a n t h i o n i n e was i d e n t i f i e d i n t h e h y d r o l y s a t e o f -_----------F u s o b a c t e r i u m --------n u c l e a t u m p e p t i d o g l y c a n by g . c . m . s . and t h e d i s t r i b u t i o n o f t h i s unusual sulphur-containing dibasic amino acid among p e p t i d o g l y c a n s of v a r i o u s s p e c i e s a n d s t r a i n s of g e n u s F u s o b a c t e r i u m i n v e s t i g a t e d . 35 The p e p t i do g l y c a n s o f F u s o b a c t e r i u rn b a c t e r i a c o u l d be s u b d i v i d e d a c c o r d i n g t o c o n s t i t u e n t d i b a s i c a m i n o acid into: a lanthionine type, a diaminopimelic acid type,and a mixed type. The a m i n o a c i d s i n t h e p e p t i d o g l y c a n o f F u s o b a c t e r i u m n u c l e a t u m F e v 1 h a v e b e e n r e p o r t e d t o be Q - g l u t a m i c a c i d , m e s o - l a n t h i o n i n e , a n d p - ( 4 2 % ) a n d L _ - a l a n i n e (58%).36 A b o u t 7 0 % o f t h e l a n t h i o n i n e r e s i d u e s were n o t s u s c e p t i b l e t o d i n i t r o p h e n y l a t i o n , e v i d e n t l y because they a r e involved i n cross-linkages. Consequently, lysozyme d i g e s t i o n o f t h e p e p t i d o g l y c a n y i e l d e d 20 t o 2 5 % u n c r o s s - l i n k e d A chemical analysis o f d i s a c c h a r i d e t r i - and t e t r a - p e p t i d e s . i s o l a t e d g l y c o p e p t i d e s i n d i c a t e d t h a t the s t r u c t u r e of t h e b u i l d i n g b l o c k o f t h i s p e p t i d o g l y ca n i s 2 - a c e t am i d o - 2 - d e o x y -p - g l u c o s e -tja c e t y l m u r a m i c acid-L-alanine-p-glut am i c a c i d - m e s o - l a n t h i o n i n e ( -p a l a n i n e 1. E v i d e n c e h a s b e e n p r e s e n t e d t o s u p p o r t t h e c l a s s i f i c a t i o n o f t h e F u s o b a c t e r i u m n u c l e a t u m F e v 1 p e p t i d o g l y c a n a s a new A16, d i r e c t l y c r o s s - l i n ked, m e s o - l a n t h i o nine-co n t a i n i n g pep t i d 0 g l y can. The d e n s i t y p r o p e r t i e s o f b a c t e r i a l c e l l walls, when c e n t r i f u g e d under c o n d i t i o n s o f low o s m o l a l i t y , s u p p o r t t h e e v i d e n c e f o r an open s t r u c t u r e f o r c e l l walls from Micrococcus l y s o d e i k t i c u s , B a c i l l u s lichenifgymlp, C a c t o b a c i l l u s fermenturn, and P r o p r i o n i b a c t e r i u m t h e o n i i .37 The C - m y c o s i d i c g l y c o p e p t i d o l i p i d t y p i n g a n t i g e n s f r o m a l l serovars i n the t4ycgbacterium "'ig~/Mycobacterium intracellulare/Mycobacterium s c r o f u l a c e u m c o m p l e x h a v e b e e n e x a m i n e d t o varying extents.38 D e t a i l e d a n a l y s i s o f t h o s e from s e r o v a r s 8, 9 , 1 6 , a n d 25 s h o w t h a t t h e a n t i g e n s c o n s i s t o f s h o r t a c e t y l a t e d o l i g o s a c c h a r i d e s l i n k e d t o a common f a t t y acyl-peptidyl-g-(3,4-di-gmethyl-I=-rhamnose) c o r e (4). The o l i g o s a c c h a r i d e u n i t s , i n a f o r m s u i t a b l e f o r c h e m i c a l s t u d i e s , were l i b e r a t e d a s o l i g o s a c c h a r i d e a l d i t o l s on t r e a t m e n t o f the g l y c o p e p t i d o l i p i d s w i t h a l k a l i n e borohydride solution. The a l d i t o l i n t h e reduced o l i g o s a c c h a r i d e s f r o m a l l s o u r c e s was 6 - d e o x y - l - t a l i t o l . M o r e o v e r I - r h a m n o s e was a l s o a l w a y s p r e s e n t , i n d i c a t i n g t h a t a b a s a l d i s a c c h a r i d e , L-rhamnosyl-6deoxy-l-talosyl, is always l i n k e d t o t h e a l l o - t h r e o n i n e i n t h e acylpeptide. I n a d d i t i o n t h e o l i g o s a c c h a r i d e s from the


84

g l y c o p e p t i d o l i p i d s of each s e r o v a r were d i s t i n g u i s h e d by t h e i r own i n d i v i d u a l i s t i c sugars: 3-g-methyl-~-glucose i n serovar 8 2,3-diO-methyl-l-fucose i n s e r o v a r 9 , 2-2-methyl-&-fucose i n s e r o v a r 25, 4-g-methyl-_L-rhamnose i n t h e o l i g o s a c c h a r i d e from o n e of t h e two g l y c o p e p t i d o l i p i d s i n s e r o v a r 16, and a p p a r e n t l y a n o t h e r I-rhamnose substituent i n the other oligosaccharide. The g l y c o p e p t i d o l i p i d a n t i g e n s i n t h e i r s t r u c t u r a l p r i n c i p a l s , c e l l u l a r l o c a t i o n , and physiological r o l e bear a s t r i k i n g miniscular resemblance to c e l l w a l l c o m p o n e n t s o f o t h e r b a c t e r i a s u c h a s O - a n t i g e n i c and R a n t i g e n i c 1i po p o l y sa ccha r i de s. {Fatty A c y l } - ~ - P h e - ~ - a T h r - ~ - A l a - ~ - A l a n i n o l - O - ~ - R h a ~ 3 , 4 M e 2

1

T

{ O l i g o s a c c h a r i de } -0-Ac (4)

The s p e c i f i c o l i g o s a c c h a r i d e u n i t s o f t h e C - m y c i s i d i c g l y c o p e p t i d o l i p i d a n t i g e n s from s e r o v a r i e t i e s i n t h e Mycobacterium -avium/Mycobacterium L n t r a c e l l u l G / M y c o b a c t e r i u m scrofulaceum complex were l i b e r a t e d a s o l i g o s a c c h a r i d e a l d i t o l s by t r e a t m e n t of t h e glycopeptidolipids w i t h a l k a l i n e borohydride. The c o m p l e t e s t r u c t u r e s o f t h e o l i g o s a c c h a r i d e a l d i t o l s have been d e r i v e d f r o m t h e 'H n . m . r . spectra or those of t h e i r permethylated (or p e r t r i d e u t e r i o m e t h y l a t e d ) d e r i v a t i v e s , t h e m a s s s p e c t r a of t h e m e t h y l a t e d d e r i v a t i v e s , and from m e t h y l a t i o n f r a g m e n t a t i o n analysis.39 P e r i o d a t e o x i d a t i o n was a l s o used t o c o n f i r m t h e p o s i t i o n o f t h e l i n k b e t w e e n t h e u l t i m a t e and p e n u l t i m a t e s u g a r s . S t r u c t u r e s (5)-(7) w e r e p r o p o s e d ( w i t h some e x t r a p o l a t i o n o f e n a n t i o m e r i c c o n f i g u r a t i o n s where e v i d e n c e f o r a s s i g n m e n t i s not y e t c o m p l e t e ) f o r t h e o l i g o s a c c h a r i d e a l d i t o l s from s e r o v a r s 8 , 9, and 25, r e s p e c t i ve 1y 6 -Deox y -I - t a 1i t o 1 ( 6-de ox y -_h - t a 1ose i n t h e o r i g i na 1 glycopeptidolipid) invariably occupies the reducing terminus. LRhamnose i s i n v a r i a b l y t h e p e n u l t i m a t e s u g a r , and t h e l i n k between I-rhamnose and 6-deoxy-L-talose i s i n v a r i a b l y (1+2). Moreover, t h e r e s u l t s p o i n t t o t h e o u t e r o n e o r two a p p e n d a g e s f o r t h e p r o v i s i o n of individually distinctive features required for antigen speci f i c it y The s u r f a c e p r o p e r t i e s o f c e l l s o f M y c o b a c t e r i u m B C G , Mycobac ----t e r i u m p h 1e i , My co b a c t e r i u m smgm a t i s, an d My c o b a c t e r i u m

.

.


85

-m-i c r o t i

have been shown t o be i d e n t i c a l ,

medium,

t h e age o f t h e c e l l s ,

various

v i g o r o u s t r e a t r n e n t ~ . ~ ' The n e g a t i v e s u r f a c e c h a r g e f o r

i r r e s p e c t i v e o f the growth

and t h e c o l o n i a l m o r p h o l o g y , and a f t e r

a l l these species a r i s e s from the phosphate groups o f

cells of

phosphodiester

linkages

between

the

a r a b i n o g a l a c t a n of the b a s i c c e l l - w a l l

peptidoglycan

and

the

s t r u c t u r e , w h i c h i s common t o

a l l species o f Mycobacteria. The p e p t i d o g l y c a n o f N e i s s e r i a g o n o r r h o e a g h a s b e e n f o u n d t o contain g-acetyl

groups,

by

use

of

the

hydroxamate

formation

and f o u n d t o be o n l y p a r t l y s e n s i t i v e t o l y ~ o z y m e . ~ ~

reaction,

B-~-Glc~3Me-(1+3)-a-~-Rhae-(1+2)-6-deoxy-~-Talitol 0

6

'\/C

/\

Me

CO,

a-L-Fuce2,3Me2-[

1 + 4 ) - u - L - F u c ~ 2 , 3 M e ~ - [ 1+3)-a-L-Rhae-[

1+2)-

6-deoxy-k-tali to (6)

Low c o n c e n t r a t i o n s o f B - l a c t a m

a n t i b i o t i c s caused an i n c r e a s e d

uptake o f r a d i o a c t i v e 2-amino-2-deoxy-Q-glucose dodecyl

sulphate-insoluble

gonorrhoeae.42

T h e r e was

peptidoglycan of no a p p r e c i a b l e

i n t o the sodium

growing

Neisseria

change i n t h e

[small)

amount o f sodium d o d e c y l s u l p h a t e - s o l u b l e p o l y m e r p r e s e n t i n t h e cultures.

The sodium d o d e c y l s u l p h a t e - i n s o l u b l e

product i n control

c e l l s was o n l y p a r t i a l l y d i s s o l v e d by e g g - w h i t e

lysozyme (about

40%), b u t c o u l d a l l be r e l e a s e d by t h e C h a l a r o p s i s B muramidase. c e l l s exposed t o B - l a c t a m s

s u s c e p t i b l e t o l y s o z y m e i n c r e a s e d t o 60%. C h a l a r o p s i s B d i g e s t s by t h i n - l a y e r contained

disaccharide-peptide

Examination o f

the

c h r o m a t o g r a p h y showed t h a t t h e y

monomers

a c e t y l a t i o n and b i s - d i s a c c h a r i d e - p e p t i d e a c e t y l g r o u p s , o r w i t h none.

I n

the proportion o f labelled peptidoglycan

with

and

without

d i m e r s w i t h one o r t w o

g0-

B-Lactam a n t i b i o t i c s caused a decrease

i n t h e d e g r e e o f 2 - a c e t y l a t i o n b u t d i d n o t g r e a t l y a f f e c t t h e amount

86


of peptidoglycan cross-linking.

They a l s o e n l a r g e d t h e b a c t e r i a and

c o n s e r v e d a n d t h i c k e n e d t h e s e p t a t h a t c o u l d be o b s e r v e d i n t h i n s e c t i o n s by e l e c t r o n m i c r o s c o p y . r e s u l t s and

e f f e c t s o f B-lactams

The r e l a t i o n s h i p b e t w e e n t h e s e

2

on

v i t r o synthesis o f peptido-

g l y c a n by e t h e r - t r e a t e d N e i s s e r i a g o n o r r h o e a e has been d i s c u s s e d .

-m i r a b i l i s

The m u r e i n o f

i s r e p o r t e d t o be u n i q u e w i t h

respect t o the g-acetylation

o f p a r t o f i t s N-acetylmuramic a c i d

residues.43

synchronized

Working

with

r a d i o a c t i v e 2-acetam ido-2-deoxy-Q-glucose

c e l l s and p r o v i d i n g w i t h t h e medium,

the

m u r e i n was p u l s e - l a b e l l e d i n v i v o f o r 1 0 m i n p e r i o d s a t d i f f e r e n t times of the c e l l cycle.

A f t e r i s o l a t i o n o f m u r e i n and e n z y m a t i c

cleavage w i t h endo-N,g-diacetylmuramidase d i s t r i b u t i o n of radio-activity

from Chalaropsis,

the

between u n c r o s s l i n k e d m u r e i n s u b u n i t s

(monomers) and t h e p e p t i d e - c r o s s l i n k e d

I t was

d i m e r s was a n a l y s e d .

found t h a t t h e m u r e i n s y n t h e s i z e d a t d i f f e r e n t t i m e s d u r i n g t h e c e l l c y c l e d i d n o t show s i g n i f i c a n t v a r i a t i o n r e g a r d i n g t h e d e g r e e o f c r o s s l i n k a g e and t h e d e g r e e o f g - a c e t y l a t i o n .

of g-acetylation sevenfold

of

l o w e r than t h a t o f murein from

asynchronous c u l t u r e s . non-g-acetylated

A pulse-chase

chase,

generation later, became e v i d e n t .

degree

continuously

labelled

experiment revealed t h a t only

p a r t o f w h i c h became c - a c e t y l a t e d s u b s e q u e n t l y .

S i n c e a c e r t a i n amount o f the

the

m u r e i n s u b u n i t s were i n c o r p o r a t e d i n t o t h e g r o w i n g

murein sacculus, during

However,

p u l s e - l a b e l l e d m u r e i n was f o u n d t o be a b o u t

which

newly

incorporated subunits

reappeared

i n

the

sacculus

was

lost

about

one

l i m i t e d murein turnover during the c e l l cycle

The d e g r e e o f c r o s s l i n k a g e o f b o t h t h e g - a c e t y l a t e d

as w e l l as t h e n o n - ) - a c e t y l a t e d d u r i n g subsequent growth.

newly

synthesized murein increased

The l a b e l l e d p e p t i d e - c r o s s l i n k e d

dimers

w e r e c l e a v e d by an e n d o p e p t i d a s e i s o l a t e d P r o t e u s m i r a b i l i s .

The

d i s t r ib u t i o n o f r a d i o a c t i v i t y be t w een t h e r e s u 1t i n g no n - 2 - ace t y 1a t e d and 2 - a c e t y l a t e d a c e t y l a t e d dimer

monomers

n o n - g - a c e t y 1a t e d s u b u n it subsequent b i o s y n t h e s i s .

m ono-)-ace subunits,

indicated that

most

fraction carried radioactivity

t y l at e d dimer

w h i ch

w as

of

the

mono-2-

exclusively

n o n - 2 - a c e t y 1a t e d

Since o n l y a s m a l l p o r t i o n o f t h e l a b e l l e d f r a c t i o n c a r r i e d r a d i o a c t iv i t y

i n

represented

an i n t e r m e d i a t e

i n the g-acetylation

both

i t was

and o n l y a t s p e c i f i c t i m e s o f m u r e i n b i o s y n t h e s i s ,

concluded t h a t t h i s p o r t i o n o f t h e l a b e l l e d mono-)-acetylated fraction

i n the during

dimer

process.

A d e f i n e d sequence o f s t e p s o f m u r e i n b i o s y n t h e s i s has been p r o p o s e d as a r e s u l t o f t h e r e s u l t s d e scrib e d . A n o v e l mur e i n b u i l d i n g b l o c k ,

2-ace tam i d o - 2 - d e o x y - Q - g 1 ycosy 1-

a7


-N - a c e t y l m u r a m o y l - d i p e p t i . d e , P r ot e u s m i r a b i l i s ,

formed d u r i n g t h e c e l l - d i v i s i o n c y c l e o f

has been r e p o r t e d . 4 4

g l u c o s e r e s i d u e was f o u n d t o c o n t a i n ,

The 2 - a c e t a m i do-2-deoxy-gi n some i n s t a n c e s , 2 - a c e t y l

groups. The Pseudomonas a e r u g i n o s a o u t e r membrane was i s o l a t e d w i t h attached

peptidoglycan

and

ethylenediaminetetraacetate, major

outer

membrane

fractioned

and lysozyme.

proteins

with

X-100,

I are noncovalently

H2, a n d

F,

Triton

The d a t a s u g g e s t e d t h a t

associated with the p e p t i d ~ g l y c a n . ~ ’ The u n i q u e p r e s e n c e o f

a polyamine covalently

p e p t i d o g l y c a n h a s b e e n r e p ~ r t e d . ~ The ~ , ~p o~l y a m i n e was f o u n d t o e x i s t a s a c o m p o n e n t o f c e l l - w a l l Selenomonas

ruminantium,

a

strictly

linked to a cadaverine

peptidoglycan o f

anaerobic

bacterium.

{ l 4 C } C a d a v e r i n e added t o t h e g r o w t h medium was i n c o r p o r a t e d i n t o t h e cells,

a n d a b o u t 70% o f t h e t o t a l r a d i o a c t i v i t y i n c o r p o r a t e d was

found i n the peptidoglycan fraction.

When t h e { 1 4 C ) c a d a v e r i n e -

l a b e l l e d p e p t i d o g l y c a n p r e p a r a t i o n was a c i d h y d r o l y s e d , 14C

counts were recovered as cadaverine.

l a b e l l e d p e p t i d o g l y c a n p r e p a r a t i o n was t h r e e s m a l l f r a g m e n t s w h i c h were ninhydrin reaction. was composed o f

a l l o f the

{14C)cadaverine-

digested w i t h lysozyme i n t o

r a d i o a c t i v e and were p o s i t i v e i n

One m a j o r s p o t ,

QL-alanine,

The

a compound o f t h e f r a g m e n t s ,

Q-glutamic

acid,

diaminopimelic acid,

c a d a v e r i n e , m u r a m i c a c i d , and 2 - a m i n o - 2 - d e o x y - Q - g l u c o s e . two

amino

groups

peptidoglycan,

of

cadaverine

was

a n d t h e o t h e r was f r e e .

covalently

One o f t h e

linked

to

the

The c h e m i c a l c o m p o s i t i o n o f

t h e p e p t i d o g l y c a n p r e p a r a t i o n o f t h i s s t r a i n was d e t e r m i n e d t o be a s f o l 1ow s:

-

I-a 1a n i n e -Q-

a 1a n i ne-Q- g l u t am ic a c i d - mes o d i am in o p im e 1ic

acid-cadaverine-muramic (1.O:l

.O:l.O:l

.0:1.1:0.9:1

acid-2-amino-2-deoxy-~-glucose

.O).

T h e { I 4 C ) c a da ve r i n e - l a b e 1 l e d p e p t ido g l y c a n was d e g r a d e d w i t h t h e l y t i c enzymes p r e p a r e d f r o m S t r e p t o m y c e s a l b u s G i n t o t h r e e s m a l l f r a g m e n t s i n c l u d i n g a m a j o r f r a g m e n t (band A compound).48 B a n d A c o m p o u n d was c o m p o s e d o f I - a l a n i n e , d i a m i n o p i m e l i c acid,

Q - g l u t a m i c a c i d , meso-

Q-alanine, and c a d a v e r i n e i n t h e m o l a r r a t i o

0.98:1.0:1.0:0.98:0.97.

Diaminopimelic

acid,

&-alanine, and

c a d a v e r i n e w e r e N - t e r m i n a l r e s i d u e s i n b a n d A compound.

{14Clcadaverine-labelled acid hydrolysis,

band A

compound was

t w o p e p t i d e f r a g m e n t s were o b t a i n e d .

c o n s i s t e d o f d i a m i n o p i m e l i c a c i d and Q - a l a n i n e , was t h e N - t e r m i n a l of I-alanine,

subjected

amino a c i d ,

When t h e to

partial

One o f them

diaminopimelic acid

and t h e o t h e r f r a g m e n t was composed

Cj-glutamic acid, and cadaverine, o f which I - a l a n i n e

88


and c a d a v e r i n e w e r e N - t e r m i n a l . p e p t i d e s t r u c t u r e of

I t was c o n c l u d e d t h a t t h e p r i m a r y

b a n d A compound i s L - a l a n y l - E - g l u t a m o y l - m e s o -

d i a m i n o p i m e l y l - g - a l a n i n e and t h a t c a d a v e r i n e l i n k s c o v a l e n t l y t o t h e g-glutamic a c i d residue. S t r e p t o c o c c u s m u t a n s BHT was g r o w n i n T o d d - H e w i t t d i a l y s a t e medium c o n t a i n i n g 2-acetamido-2-deoxy-E-{

1 4 C ) g l u c o s e f o r 6 t o 11

generations.

A f t e r t r e a t m e n t w i t h c o l d and h o t t r i c h l o r o a c e t i c a c i d

and t r y p s i n ,

5 2 t o 65% o f t h e r a d i o a c t i v i t y r e m a i n e d p r e s e n t i n

insoluble peptidoglycan-containing residues.

Hen e g g - w h i t e l y s o z y m e

or rnutanolysin t r e a t m e n t of t h e peptidoglycan residues r e s u l t e d i n t h e r e l e a s e o f 80 a n d 97%, r e s p e c t i v e l y , o f t h e 1 4 C l a b e l t o t h e supernatant f r a ~ t i o n . ~ ’ H y d r o c h l o r i c a c i d h y d r o l y s a t e s o f such supernatants present

in

showed t h a t insoluble

compounds t h a t

essentially

peptidoglycan

a l l of

the

fractions

c o m i g r a t e d on p a p e r c h r o m a t o g r a p h y

deoxy-!-glucose

( 6 0 % ) o r m u r a m i c a c i d (30%).

radioactivity

was

present

i n

w i t h 2-amino-2-

Treatment o f whole

c e l l s w i t h l o w and h i g h c o n c e n t r a t i o n s o f lysozyme a l o n e r e s u l t e d i n l o s s e s o f 45 and 70% o f t h e i n s o l u b l e p e p t i d o g l y c a n ,

respectively,

y e t r e l e a s e o f d e o x y r i b o n u c l e i c a c i d f r o m c e l l s was n o t d e t e c t e d . Sequential addition inorganic

salts

of

after

appropriate

concentrations

lysozyme treatment

l i b e r a t i o n o f deoxyribonucleic acid.

of

selected

did result i n the

Deoxyribonucleic acid release

was c o r r e l a t e d w i t h a f u r t h e r r e l e a s e o f p e p t i d o g l y c a n f r o m t h e insoluble fraction.

However, t h e t o t a l amount o f p e p t i d o g l y c a n l o s t

e f f e c t e d by t h e l o w c o n c e n t r a t i o n o f l y s o z y m e and NaSCN ( l y s i s ) was s i g n i f i c a n t l y l e s s t h a n t h e amount o f p e p t i d o g l y c a n h y d r o l y s e d by h i g h c o n c e n t r a t i o n s o f l y s o z y m e a l o n e (no l y s i s ) ,

which suggested

t h a t t h e o v e r a l l amount o f p e p t i d o g l y c a n l o s t d i d n o t c o r r e l a t e w e l l with cellular lysis.

The t o t a l a m o u n t o f i n s o l u b l e p e p t i d o g l y c a n

l o s t a t the highest s a l t concentrations greater

t e s t e d was f o u n d t o b e

t h a n c o u l d b e a c c o u n t e d f o r by l y s o z y m e - s e n s i t i v e

o f the peptidoglycan, obtained

suggested

possibly implicating autolysins. that

topologically localized,

hydrolysis

of

linkages

The r e s u l t s

peptidoglycan

bonds

in

b u t s t r a t e g i c a l l y i m p o r t a n t s i t e s was a

more s i g n i f i c a n t f a c t o r i n t h e sequence t h a t r e s u l t s i n loss o f cellular

integrity (lysis).

A p e p t i d o g l y c a n l a y e r o f Treponema p a l l i d u m k a z a n was i s o l a t e d

by

solubilization of

whole

cells

with

1% w a r m

sodium

dodecyl

s u l p h a t e and s u b s e q u e n t d i g e s t i o n o f an i n s o l u b l e r e s i d u e w i t h proteases.

E l e c t r o n m i c r o s c o p y r e v e a l e d t h a t t h e p e p t i d o g l y c a n was

i s o l a t e d as a s i n g l e - l a y e r e d s a c c u l u s o f l e s s t h a n 5 nm t h i c k n e s s ,

89

4: Microbial Polysaccharides f r e e d f r o m a x i a l f i l a m e n t s and an e n v e l o p e sheath.50

An i s o l a t e d

p e p t i d o g l y c a n f r a c t i o n was m a i n l y c o m p o s e d o f 2 - a m i n o - 2 - d e o x y - Q glucose,

muramic acid,

alanine,

g l y c i n e i n molar r a t i o s of

0-glutamic

acid,

L-orthithine,

and

Amino-

0.65:0.68:1.63:1.00:0.75:1.03.

and c a r b o x y l - t e r m i n a l a m i n o a c i d a n a l y s e s s u g g e s t e d t h e i n v o l v e m e n t

o f a t l e a s t a p a r t o f the g l y c i n e r e s i d u e i n c r o s s - l i n k i n g between t h e a m i n o g r o u p o f i - o r n i t h i n e r e s i d u e a t one s t r a n d o f t h e s t e m peptide

subunit

neighbouring

and

the

strand.

carboxyl

The

group

treponemal

of

alanine

peptidoglycan

o f

the

lacked the

i m m u n o a d j u v a n t a c t i v i t y b o t h t o s t i m u l a t e a n t i b o d y p r o d u c t i o n and t o induce delayed-type

hypersensitivity

the properties necessary t o splenocytes

and g u i n e a - p i g

against

stimulate

peritoneal

ovalbumin,

as w e l l as

guinea-pig

macrophages,

and

mouse

unlike the c e l l

w a l l s or p e p t i d o g l y c a n s ( g r o u p A t y p e o f S c h l e i f e r and K e n d l e r s ’ classification) the

i s o l a t e d from

mammal.

However,

many b a c t e r i a l s p e c i e s p a r a s i t i c t o

the

peptidoglycan

activated

the

human

complement s y s t e m t h r o u g h t h e a l t e r n a t i v e pathway, as w e l l as t h e c l a s s i c a l one, rabbit

blood

and caused a l i b e r a t i o n o f 5 - h y d r o x y t r y p t a m i n e platelets

i n

a

similar

manner

to

the

i n

cell-wall

p e p t i d o g l y c a n s o f b o t h g r o u p A and B t y p e s .

Lipopolysaccharides

3

The c h e m i s t r y and b i o l o g i c a l s i g n i f i c a n c e o f 3-deoxy-g-manno-2o c t u l o s o n i c a c i d have been r e v i e w e d Y 5 l elucidation

and

polysaccharides, and

enzymology

i t s

location

chemical

of

an

and

including i t s structure linkages

i n

b a c t e r i a l

s y n t h e s i s and m o n o s a c c h a r i d e c h e m i s t r y , inhibitor

to

metabolism.

Bacterial

l i p o p o l y s a c c h a r i d e and i t s l i p i d A component has been r e v i e w e d briefly.52

C o n f i r m a t i o n o f t h e s t r u c t u r e o f 3-deoxy-P-manno-2-

o c t u l o s o n i c a c i d by X - r a y t h e r e p o r t of

t h e X-ray

c r y s t a l l o g r a p h y h a s been b r o u g h t n e a r e r by s t r u c t u r e o f m e t h y l ( m e t h y l 4,5,7,6-tetra-g-

a c e t y 1-3-deoxy-a-Q-manno-2-octulopyranosid I o n a t e (8).53 The

l i p o p o l y s a c c h a r i d e

f r o m

A c t i n o b a c L l l u s

a c t i n o m y c e t e m c o m i t a n s s t r a i n s Y4 and N27 was i s o l a t e d by t h e p h e n o l water procedure.

Morphologically,

and branched f i l a m e n t s weight.54

Chemical

lipopolysaccharide carbohydrate,

of

which

analysis both

the molecule consisted o f ribbon

c o m p r i s e d 3% o f of

strains

the

the cellular

isolated

showed

them

l i p i d , 3-deoxy-Q-manno-2-octulosonic

and to

consist

acid,

dry

purified of

heptose,

90


amino-deoxyhexose, and p h o s p h a t e .

The m a j o r f a t t y a c i d s o f t h e l i p i d

A m o i e t y were m y r i s t i c and B - h y d r o x y m y r i s t i c a c i d s . f uco se,

p- g a l a c t o se,

p - g l u c o se,

h e p t o se,

I-Rhamnose,

2-amino-2-deoxy-Q-gluco

Ise,

a n d 2 -am i n o -2-deox y -Q-ga 1a c t o se c o m p r i s e d t h e mono sa c c h a r ide pa r t i o n o f the lipopolysaccharide.

Biological-activity

both lipopolysaccharide molecules t o

be a c t i v e

r e a c t i o n a n d i n v i t r o 45Ca bone r e s o r p t i o n ,

studies revealed

i n t h e Schwartzman

a s w e l l as i n macrophage

a c t i v a t i o n a n d l e t h a l i t y and i n p l a t e l e t a g g r e g a t i o n .

The c o r e o l i g o s a c c h a r i d e o f Aeromonas h y d r o p h i l a (Chemotype

111) l i p o p o l y s a c c h a r i d e has

been

i n ~ e s t i g a t e d . ~ The ~

i n v o l v e d t h e use o f m e t h y l a t i o n a n a l y s i s , trioxide,

p a r t i a l hydrolysis with acid,

periodate oxidation,

degradation,

and t a g g i n g o f t h e r e d u c i n g end group.

unusual

having

i n

constituent.

3 - a c e t am id o - 3 , 6 - d i

Structure

studies

o x i d a t i o n and chromium

(9) was

deoxy-L- g l ucose

proposed

for

Smith

The c o r e i s the

as

a

core

o l i g osaccharide. a -Q - G 1 c g

1

I

6 dd-B-L-GlcgNAc-( -

1+3) -a-e-Galg-(

1+3)-a-LQ-Hepp(

1+2) -a-LQ-Hepe

4

I

1 a -Q - G 1 c e

LQ -H e p e

=

L-g 1y c e r o -Q - m a nno - h e p t o p y r a no s e (91

The sugar c o m p o s i t i o n o f t h e O - a n t i g e n i c lipopolysaccharides i s o l a t e d f r o m Group F (once c l a s s i f i e d a s Aeromonas) v i b r i o s was

91


analysed. 56 3-Deoxy-Q-manno-2-oct uloso n i c a c i d was t o t a l l y absent from t h e l i p o p o l y s a c c h a r i d e s . As common component s u g a r s , p g l u c o s e , a - g a l a c t o s e , i - q l y c e r o - Q - m a n n o - h e p t o s e , and 2-amino-2d e o x y - P - g l u c o s e w e r e p r e s e n t . The G r o u p F v i b r i o s e x a m i n e d w e r e f o u n d t o be d i v i d e d i n t o t w o g r o u p s , d e s i g n a t e d t e n t a t i v e l y a s g r o u p s 1 a n d 11, o n t h e b a s i s o f t h e p a t t e r n of t h e s u g a r composition of t h e i r l i p o p o l y s a c c h a r i d e s . As a d d i t i o n a l s u g a r c o m po ne n t s , 2 - am i no - 2 d e ox y -Q - m a n no s e , 2 - am i no - 2,6 - d i d e ox y -Q - g 1u co s e, and t w o u n i d e n t i f i e d amino s u g a r s were p r e s e n t i n group I , while Irhamnose, Z - a m i n o - 2 - d e o x y - Q - g a l a c t o s e , an u n i d e n t i f i e d amino sugar, and a r e l a t i v e l y h i g h c o n t e n t o f a - g l y c e r o - Q - m a n n o - h e p t o s e were found i n g r o u p 11. The main f a t t y a c i d s p r e s e n t i n l i p o p o l y s a c c h a r i d e s from B a c t e r o i d e s f r a g i l i s NCTC 9343 were i d e n t i f i e d a s 13-methylt e t r a d e c a n o i c , B-3-h~d r ox ypent ade c a n o i c , p-3- hy drox y hex ade canoi c, p3 - h y d r o x y - 1 5 - m e t h y l - h e x a d e c a n o i c , and 0 - 3 - h y d r o x y h e p t a d e c a n o i c acids.57 Of t h e s e 1 3 - m e t h y l - t e t r a d e c a n o i c a c i d i s e x c l u s i v e l y e s t e r b o u n d , and 3 - h y d r o x y - 1 5 - m e t h y l - h e x a d e c a n o i c acid i s exclusively i n v o l v e d i n amide l i n k a g e . The o t h e r 3-hydroxy f a t t y a c i d s a r e b o t h e s t e r and amide b o u n d . A l l 3-hydroxy f a t t y a c i d s p o s s e s s t h e c o n f i g u r a t i o n , and t h e 3 - h y d r o x y l group o f e s t e r - l i n k e d 3-hydroxy f a t t y a c i d s i s n o t s u b s t i t u t e d . L i p o p o l y s a c c h a r i d e s of r e l a t e d Bacteroides species (Bacteroides thetaiotaomicron, Bacteroides o v a t u s , B a c t e r o i d e s d i s t a s o n i s , a n d B a c t e r o i d e s v u l g a t u s 1 showed a f a t t y - a c i d spectrum w i t h s i m i l a r and d i s t i n c t f e a t u r e s compared t o t h a t o f Bacteroides f r a g i l i s lipopolysaccharides. Pur i f i e d 1i p o po 1y s a cch a r i de e x t r a c t e d w i t h pheno 1-water from smooth B r u c e l l a a b o r t u s was h y d r o l y s e d w i t h 1% a c e t i c a c i d a t 1 0 0 ° C . 5 8 The d e g r a d e d p o l y s a c c h a r i d e r e l e a s e d gave r e a c t i o n s o f i d e n t i t y w i t h t h e n a t i v e p o l y s a c c h a r i d e hapten i n phenol-water- o r t r i c h l o r o a c e t i c a c i d - e x t r a c t e d e n d o t o x i n preparations o f Brucella a b o r t u s w i t h t h e p o l y s a c c h a r i d e e x t r a c t e d by t r i c h l o r o a c e t i c a c i d from rough B r u c e l l a m e l i t e n s i s s t r a i n 8115. The l a t t e r p o l y s a c c h a r i d e was p r e s e n t i n t h e s o l u b l e c y t o p l a s m i c f r a c t i o n , b u t n o t i n t h e membrane f r a c t i o n , of d i s r u p t e d 8115 c e l l s . I t could n o t be e x t r a c t e d from t h r e e rough m u t a n t s o f B r u c e l l a a b o r t u s o r from -------B r u c e l l a ----canis or Brucella o v i s cells. B o t h the degraded p o l y s a c c h a r i de and na t i ve p o l y saccha r i de hap t e n shared de t e rm i nan t s p r e s e n t o n s m o o t h l i p o p o l y s a c c h a r i d e and m i s s i n g from t h e rough B r ucella g e l i t e n s i s polysaccharide. S u g a r s found i n p u r i f i e d l i p o p o l y s a c c h a r i d e , t h e n a t i v e h a p t e n , and degraded p o l y s a c c h a r i d e

-

a-

92


a - -~ - G l c ~ N A c - ( 1 + 2 ) - a-- ~ - G l c ~ - ( l + 2 ) --a - ~ - G a l ~ - ( l + 3 ) - a - ~ - G l c ~ - ( l + 6

I

1

a-Q-Gale (10)

a-~-Gal~-(1+2)-a-~-Gal~-(l+2)-a-~-Glc~-(l+3)-a-~-Glc~-(l+ 3

I

1

B-P-GlcQ (11)

i 1

a-Q-Gale (12)

a - -~ - G l c ~ - ( 1 + 2 ) - a - ~ - G l c ~ - ( l + 2 ) - a - ~ - G a l ~ - ( l + 3 ) - a - ~ - G l c ~ ( l + 3

I

1 a-9-GlceNAc (13)

a-Q-Gal~-(1+2)-a-~-Gal~-(l+2)-a-q-Glc~-(l+3)-a-~-Glc~-(l+ -

i 1

B-P-Galg (14)

~-~-Glc~NAc-(1+6)-a-~-Glc~-(l+2)-a-g-Glc~-(l+3)-a-~-Glc~-(l+ 6

I

1

a-P -Ga le

93


-

-

i n c l u d e d Q-manno s e , Q g l u c o s e , 2 -am ino -2,6 - d i de ox y -Q g l u c o s e , 2 -

-

am ino-2-deox y -tj- g l u c o se,

a n d 3 - d e o x y -Q m a n n 0 - 2 - 0 c t u l o so n i c a c i d.

The B r u c e l l a m e l i t e n s i s p o l y s a c c h a r i d e c o n t a i n e d o n l y a t r a c e amount o f 2-amino-2,6-dideoxy-Ba c i d d e t e c t a b l e by

g l uco se a n d n o 3-deoxy-B- m a n n o - o c t u l o s o n i c

t h e t h i o b a r b i t u r a t e assay.

Sera

from

some

r a b b i t s i m m u n i z e d w i t h p u r e s m o o t h l i p o p o l y s a c c h a r i d e a n d some, not

all,

cows

infected

with

field strain of

but

Brucella abortus

recognized the determinants missing from the Brucella melitensis polysaccharide.

A subclass-specific enzyme-linked

immunoassay

showed t h a t most o f t h e a n t i b o d y i n s e r a f r o m i n f e c t e d cows w h i c h b i n d s t o smooth l i p o p o l y s a c c h a r i d e and t o t h e n a t i v e h a p t e n i s o f t h e immunoglobulin G 1 subclass. The c h e m i c a l s t r u c t u r e o f t h e 0 - s p e c i f i c

----------Citrobacter

036 l i p o p o l y s a c c h a r i d e has

methylation analysis s p e ~ t r o m e t r y . ~T~h i s

using gas-liquid polysaccharide

polysaccharide o f

been e s t a b l i s h e d

chromatography i s

a

linear

by

and mass

homopolymer

composed o f ( 1+2) - 1 i n k e d 4-deox y-6 - Q - a r a b i n o - hexopy r a n o s y 1 r e s i d u e s . cores

from

E n t e r o b a c t e r a ce ae 1ipo po 1y s a c c h a r i de s h a v e be e n i n ve s t iga t e d,

The

structures

for

the

us in g

s p e c i f i c d e g r a d a t i o n a n d 'H methods.60

hexose

n.m.r.

regions

of

studies as the p r i n c i p a l

Complete s t r u c t u r e s f o r these r e g i o n s i n t h e Salmonella,

t h e E s c h e r i c h i a c o l i R1,

R2,

R3,

R4,

and E s c h e r i c i a c o l i K12 and

E s c h e r i c h i a c o l i B c o r e s a r e p r o p o s e d ((10)-(16), r e s p e c t i v e l y ) .

The

apparent s i m i l a r i t i e s between these s t r u c t u r e s i n d i c a t e a c l o s e relationship.

They a r e a l l composed o f f i v e h e x o s e r e s i d u e s . t h e E s c h e r i c h i a c o l i R2 c o r e s d i f f e r

The S a l m o n e l l a a n d

o n l y i n t h e n a t u r e o f one

h e x o s e r e s i d u e a n d t h e E s c h e r i c h i a c o l i R I , R2, R4 a n d E h e r i c h i a

c o l i K12 c o r e s a l l c o n t a i n c o l i core. There a r e o n l y a n d t h e R4 c o r e s .

t h e disaccharide u n i t of

The S a l m o n e l l a a n d t h e R3 c o r e ,

t h e same P-Glc-Q-Gal-Q-Glc

the Escherichia

minor s t r u c t u r a l d i f f e r e n c e s between R 1 trisaccharide unit.

finally,

contain

Some c o m p l e m e n t a r y

i n f o r m a t i o n o n t h e s t r u c t u r e o f t h e h e p t o s e r e g i o n has a l s o been obtained.

T h i s r e g i o n i s more u n i f o r m , a n d i t w o u l d n o t be s u r p r i s i n g

ifa l l c o m p l e t e h e p t o s e r e g i o n s i n S a l m o n e l l a , A r i z o n a ,

Citrobacter,

E s c h e r i c h i a c o l i , S h i g e l l a , K l e b s i e l l a , Enterobacter,and p r o v e d t o h a v e e s s e n t i a l l y t h e same s t r u c t u r e ( 1 7 ) .

Serratia

One r e s u l t o f

94


t

N

u I

0

n Y

n I

b N t U

I

0

0 Y I

N

n

z

m t

I

U

I

N

u

Y

N

I

u N r 0 z

I

e

0 0

a

I

2

N

I

u a .t a-

e-N

4

0 1 1 All

I

N

a

I

I

n

M

t

4 v

I

a-al

2 X

b-4

I

n u

2 I nil

A1I

A1I I

a I

n

M

t

4 u

I

a 4

U

w-4

I 0 1 1 I

a

a

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0 .

M 4

L)

w

4 u I

al m

4

U

I nit I

a

n I

N

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.-I

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o

95

4: Microbial Polysaccharides this

study6'

was

the

finding

that

a l l

&-glycero-!-manno-

h e p t o p y r a n o s y l r e s i d u e s seem t o be u - l i n k e d . The h e p t o s e r e g i o n o f t h e l i p o p o l y s a c c h a r i d e o f E s c h e r i c h i a The Q - g l u c o s y l r e s i d u e l i n k e d t o c o l i K12 CR34 h a s been s t u d i e d . 6 1 t h e h e p t o s e I 1 w a s f o u n d t o b e s u b s t i t u t e d by a P - g a l a c t o s y l g r o u p a n d t h e l i n e a r c h a i n o f t h e c o r e p o l y s a c c h a r i d e h a s t w o (1+3) heptoses.

linked

The h e p t o s e I 1 i s s u b s t i t u t e d by a l a t e r a l (1+7) l i n k e d

h e p t o s e I11 and t h e h e p t o s e I i s l i n k e d i n (1+5) t o 2-deoxy-Q-mannoo c t u l o s o n i c acid.

h e p t o s e I,

The t h r e e s u g a r s o f t h e l i n e a r c h a i n ,

h e p t o s e 11, and Q-glucose, a r e s u b s t i t u t e d by p h o s p h a t e ,

pyrophosphate,

o r p y r o p h o s p h o r y l e t h a n o l a m i n e g r o u p s l i n k e d t o C-4 h y d r o x y l g r o u p s . i n some p o l y s a c c h a r i d e c h a i n s , one o r t w o s u b s t i t u t i n g

However,

g r o u p s may b e a b s e n t , w h i c h

may e x p l a i n t h e h e t e r o g e n e i t y i n t h e

l e n g t h o f the core polysaccharide chains. Structural studies o f

lipopolysaccharides o f Escherichia c o l i

K 1 2 have d e m o n s t r a t e d t h a t s m o o t h s t r a i n s p r o d u c e l i p o p o l y s a c c h a r i d e

w i t h c o n s i d e r a b l e h e t e r o g e n e i t y w i t h r e s p e c t t o t h e l e n g t h o f 0a n t i g e n i c c h a i n s w h e n "P

n.m.r.

methods

average

only

p r o v i d e d an

heterogeneity.62

was u s e d , a l t h o u g h t r a d i t i o n a l composition

over

S t a n d a r d i s o l a t i o n p r o c e d u r e s were

a

range

of

shown t o f a i l

t o e x t r a c t some 30% o f t h e t o t a l l i p o p o l y s a c c h a r i d e p r e s e n t i n t h e cells,

t h e s i g n i f i c a n c e o f w h i c h was d i s c u s s e d i n r e l a t i o n t o o u t e r

membrane s t r u c t u r e . When l i p o p o l y s a c c h a r i d e s f r o m E s c h e r i c h i a c o l i 8 w e r e s o n i c a t e d together w i t h pure spin-labelled phospholipids without the a d d i t i o n o f unlabelled phospholipids,

e x t e n s i v e l i n e b r o a d e n i n g was o b s e r v e d

due t o t h e c l o s e p r o x i m i t y o f s p i n - l a b e l l e d m o l e c u l e s t o e a c h o t h e r , a result suggesting that spin-labelled phospholipids existed i n s e g r e g a t e d domains c o n t a i n i n g few Such

mixed

bilayers

were

l i p o p o l y s a c c h a r i d e molecules.63

incubated

under

various

conditions,

i n c l u d i n g t h e a d d i t i o n o f N a C l a n d MgC12 t o t h e m e d i u m a n d t h e i n c o r p o r a t i o n o f t h e m a j o r outer-mem b r a n e p r o t e i n , bilayer,

porin,

i n t o the

a n d t h e i n t e r m i x i n g o f t h e d o m a i n s w a s f o l l o w e d by t h e

decrease i n l i n e width.

The d i f f u s i o n o f t h e l a b e l l e d p h o s p h o l i p i d s

i n t o l i p o p o l y s a c c h a r i d e d o m a i n s was h a r d l y d e t e c t a b l e when t h e m i x e d b i 1ay e r co n t a ine d s p i n - l a be 1l e d p h osp h o l ip i ds a n d 1ipo po 1y s a c c h a r i de i n approximately equimolar was o b s e r v e d when a 1 7 - f o l d present,

ratios.

Although progressive d i f f u s i o n

m o l a r e x c e s s o f l i p o p o l y s a c c h a r i d e was

i t i s v e r y slow even under t h e o p t i m a l c o n d i t i o n s ,

r e q u i r i n g s e v e r a l days f o r a n e a r l y complete mixing. s e r i e s o f experiments,

usually

I n another

s p i n - l a b e l l e d p h o s p h o l i p i d s were d i l u t e d w i t h

96


a 1 0 0 - f o l d e x c e s s of u n l a b e l l e d p h o s p h o l i p i d s and t h e n mixed w i t h lipopolysaccharides. I n t h e s e e x p e r i m e n t s , t h e f l u i d i t y of t h e d o m a i n s c o n t a i n i n g s p i n - l a b e l l e d p h o s p h o l i p i d s was shown t o be i d e n t i c a l , e v e n a f t e r 3 d a y s of i n c u b a t i o n , w i t h t h e f l u i d i t y of b i l a y e r s containing only phospholipids, i n c o n t r a s t t o t h e e x p e c t a t i o n of t h e d i m i n i s h e d f l u i d i t y i f p h o s p h o l i p i d m o l e c u l e s became f i n e l y i n t e r s p e r s e d w i t h l i p o p o l y s a c c h a r i d e m o l e c u l e s . These two d i f f e r e n t l i n e s o f a p p r o a c h t h e r e f o r e s u p p o r t e d t h e i d e a t h a t p h o s p h o l i p i d (and most probably l i p o p o l y s a c c h a r i d e ) domains i n m i x e d b i l a y e r s t e n d t o be r a t h e r s t a b l e and p e r s i s t f o r l o n g p e r i o d s o f time. The n a t u r a l a f f i n i t y of v a r i o u s b a c t e r i a l g l y c o p e p t i d e s and l i p o p o l y s a c c h a r i d e s f o r mammalian c e l l membranes has been e s t i m a t e d q u a n t i t a t i v e l y by c o m p a r i s o n w i t h t h e a d s o r p t i o n of l i p o p o l y s a c c h a r i d e from E s c h e r i c h i a c o l i N C T C 8623 t o e r y t h r o c y t e s , t h y m o c y t e s , bone-marrow c e l l s , s p l e e n c e l l s , p e r i t o n e a l lymphocytes, and macro phage^.^^ I m m u n o p o t e n t i a t i n g a c t i v i t y was e s t i m a t e d b y m e a s u r i n g t h e a b i l i t y of t h e b a c t e r i a l f r a c t i o n s t o s t i m u l a t e a h u m o r a l r e s p o n s e t o o v a l b u m i n i n H A M / l C R m i c e . When t h e a f f i n i t y f o r mammalian c e l l membranes was compared w i t h t h e s t i m u l a t i o n of t h e a n t i b o d y r e s p o n s e , i t was found t h a t a n e g a t i v e c o r r e l a t i o n f o r P 0 . 0 0 0 5 ) and a p o s i t i v e p e r i t o n e a l m a c r o p h a g e s ( g s = -0.94, c o r r e l a t i o n f o r p e r i t o n e a l l y m p h o c y t e s ( z s = + 0 . 9 7 , P 0 . 0 0 0 5 ) and s p l e e n c e l l s (rs = +0.76, 0.005) e x i s t e d . A heptose-deficient l i p o p o l y s a c c h a r i d e s t r a i n of E c h e r i c h i a c o l i 0 8 , s t r a i n F151, was f o u n d t o r e l e a s e p o r t i o n s of i t s o u t e r membrane when c e l l s w e r e e x p o s e d t o l O m M c i t r a t e b u f f e r ( p H 2.75) f o r 30 m i n and s u b s e q u e n t l y e x p o s e d t o l O O m M t r i s ( h y d r o x y m e t h y 1 ) The o u t e r - m e m b r a n e c o m p o n e n t aminomethane b u f f e r (pH 8 . 0 p 5 r e l e a s e d was f o u n d t o be composed of p r o t e i n , l i p o p o l y s a c c h a r i d e , phospholipid (card i o l i p i n , phospha t i d y l e t h a n o lam ine, and p h o s p h a t i d y l g l y c e r o l ) , and a l k a l i n e p h o s p h a t a s e . T h e outer-membrane component was r e l e a s e d from t h e c e l l envelope i n t h e a b s e n c e of c e l l l y s i s , a s no n - g l u c o s e - 6 - p h o s p h a t e dehydrogenase a c t i v i t y or s u c c i n a t e dehydrogenase a c t i v i t y was d e t e c t e d . M o r p h o l o g i c a l l y , t h e o u t e r - membrane component appeared t o c o n s i s t of l a m i n a r f r a g m e n t s and v e s i c l e s which had an a s s o c i a t e d a l k a l i n e p h o s p h a t a s e a c t i v i t y . I n t h e c h r o m a t i n or" s p l e e n c e l l s of m i c e and r a t s i m m u n i z e d w i t h l i p o p o l y s a c c h a r i d e s f r o m E s c h e r i c h i a c o l i , a new s p e c i e s , and a n t i g e n n o n s p e c i f i c f r a c t i o n , o f n o n - h i s t o n e c h r o m a t i n p r o t e i n s has been d e s c r i b e d . 6 6 The p o s s i b l e r o l e of t h i s f r a c t i o n i n t h e

97


r e g u l a t o r y p r o c e s s o f gene a c t i v a t i o n d u r i n g t h e immune r e s p o n s e as e x p r e s s e d by

the synthesis of

t h e IgM c l a s s o f

antibodies

was

discussed. E x p e r i m e n t a l e v i d e n c e h a s been p r e s e n t e d w h i c h i n d i c a t e s t h a t

1,2-dihydro-l-hydroxy-6-methyl-2-(propanesulphonyl)-thieno(3,2-~-~ (1,2,3)-diazaborine

(18),

a heterocyclic,

boron-containing

sub-

s t a n c e , p r e v e n t s b a c t e r i a l p r o l i f e r a t i o n by i n h i b i t i n g l i p o p o l y s a c c h a r i d e b i o ~ y n t h e s i s . ~T h~e r e w a s a r e d u c t i o n i n Q - g a l a c t o s e i n c o r p o r a t i o n i n t o the lipopolysaccharide o f whole c e l l s , n e w l y f o r m e d l i p o p o l y s a c c h a r i d e c h a i n s appear t h e p r e s e n c e o f t h e d i a z a b o r i n e compound, t r o n microscopy.

and no

i n whole b a c t e r i a i n

as d e m o n s t r a t e d by e l e c -

The 3 - d e o x y - ~ - m a n n o - 2 - o c t u l o s o n i c

acid metabolism

seemed t o be t h e t a r g e t o f t h e d i a z a b o r i n e compound as L - a r a b i n o s e 5 - p h o s p h a t e i n c o r p o r a t i o n i n t o m a c r o m o l e c u l a r m a t e r i a l was a f f e c t e d .

OH

The p u r i f i e d l i p o p o l y s a c c h a r i d e e n d o x i n - l i k e c o m p o n e n t f r o m L i s t e r i a monocytogenes has been shown t o a c t as b o t h an i n v i t r o mitogen

for

lymphocytes

and

i n

in

vivo

adjuvant

i n

antibody

production. A

novel peptidoglycan-associated

l i p o p r o t e i n was

found i n t h e

c e l l e n v e l o p e o f P r o t e u s ~ i r a b i l i s . T~h ~i s p r o t e i n was s h o w n t o h a v e an a p p a r e n t polyacrylamide gel,

molecular of

18,000.

weight,

i n sodium dodecyl

sulphate

The p r o t e i n was p r e s e n t i n t h e c e l l

envelope i n a form very c l o s e l y , but n o t c o v a l e n t l y , associated w i t h t h e p e p t i d o g l y c a n l a y e r a n d was r e c o v e r e d p r e d o m i n a n t l y f r o m t h e o u t e r membrane a f t e r s e p a r a t i o n o f t h e c e l l e n v e l o p e s . 1-14C)P a l m i t i c a c i d and I 2 - 3 H I g l y c e r o l were i n c o r p o r a t e d i n t o t h e p r o t e i n . A

fatty-acid-containing

polypeptide

(lipopolypeptide)

by d i g e s t i o n o f t h e p u r i f i e d p e p t i d o g l y c a n - a s s o c i a t e d

was

isolated

lipoprotein

It w i t h t r y p s i n i n t h e p r e s e n c e o f 0.05% s o d i u m d o d e c y l s u l p h a t e . was c o m p o s e d o f 3 1 a m i n o a c i d r e s i d u e s , an u n i d e n t i f i e d compound {XI,

and ca. 3 f a t t y - a c i d

residue^.^' A

l i p o - o l i g o p e p t i d e was a l s o

98


isolated after

further

t h e absence o f composed o f

4

compound { X I ,

digestion of

sodium amino

a n d ca.

dodecyl

lipopolypeptide with t r y p s i n i n

sulphate.

acid residues

3 fatty-acid

Lipo-oligopeptide

(L-Asx,

2 I-Ser,

residues.

was

L-Lys),

The C - t e r m i n a l

a

amino

a c i d s o f l i p o p o l y p e p t i d e and l i p m l i g o p e p t i d e were d e t e r m i n e d as

I-

arginine

N-terminus

o f

lipopolypeptide,

or

and

L-lysine,

respectively.

peptidoglycan-associated

lipoprotein,

The

l i p o a l i g o p e p t i d e c o u l d n o t be i d e n t i f i e d by c o n v e n t i o n a l N - t e r m i n a l a n a l y s i s , i n d i c a t i n g t h a t t h e N - t e r m i n u s i s p r o b a b l y masked.

The

a m i n o a c i d s e q u e n c e o f t h e l i p w l i g o p e p t i d e d e r i v e d f r o m t h e Nterminal

--Proteus

region o f

the peptidoglycan-associated

lipoprotein o f

m i r a b i l i s was d e t e r m i n e d t o be { X I - k - S e r - I = - S e r - L - A s n - i -

L Y S . ~ The ~ u n i d e n t i f i e d compound { X I p r e s e n t a t t h e N - t e r m i n u s was i d e n t i f i e d a s g l y c e r y l - l - c y s t e i n e {S-(p r o p a n e -2’,3’- d i o 1) -3- t h i o - 2 amino-propanoic

acid).

lipopolypeptide,

The p a r t i a l a m i n o a c i d s e q u e n c e o f t h e

w h i c h c o n t a i n e d t h e l i p o a l i g o p e p t i d e a t i t s N-

t e r m i n a l p a r t , w a s a l s o d e t e r m i n e d , m a i n l y by Edman d e g r a d a t i o n . The s t r u c t u r e o f t h e N - t e r m i n a l p a r t o f p e p t i d o g l y c a n - a s s o c i a t e d lipoprotein

was d e t e r m i n e d t o

be

(3 f a t t y

acids

glyceryl-L-Cys-I-

-

Ser-L-Ser-L-Asn-I-Lys-L-Asn-L-Asn-l-Asp-I-Asp-~-Glu-I-Thr-l-Asp-L-Thr-L-

Ser.....}.

These p r o p e r t i e s , e x c e p t f o r m o l e c u l a r w e i g h t a n d n o n -

covalent association w i t h the peptidoglycan, t h o s e o f Braun’s l i p o p r o t e i n . Braun’s

showed r e s e m b l a n c e t o

However, t h e p r o t e i n was d i s t i n c t f r o m

l i p o p r o t e i n i n r e g a r d t o amino a c i d c o m p o s i t i o n .

A similar

p e p t i d o g l y c a n - a s s o c i a t e d l i p o p r o t e i n was p r e s e n t w i d e l y i n t h e c e l l envelopes o f various Gram-negative contains

about

twelve

times

as

l i p o p r o t e i n as E s c h e r i c h i a c o l i . associated lipoprotein o f

bacteria. much

Proteus m i r a b i l i s

peptidoglycan-associated

Antiserum against peptidoglycan-

-m i r a b i l i s

was c r o s s - r e a c t i v e

a g a i n s t p e p t i do g l y c a n - a s s o c i a t e d l i p o p r o t e i n o f

E s c h e r ic h ia c o 1i,

b u t n o t a g a i n s t Braun’s l i p o p r o t e i n o f E s c h e r i c h i a c o l i . The

stimulation

of

incorporation o f

{3HIg-galactose

into

membrane g l y c o c o n j u g a t e s , m e a s u r e d i n a p r e c i p i t a t i o n t e s t ,

was u s e d

as a c r i t e r i o n f o r a c t i v a t i o n o f

I n this

assay,

bone-marrow

p u r i f i e d bacterial lipopolysaccharide,

cells.72

lipoprotein,

and

m u r e i n monomer and d i m e r f r a g m e n t s a l l a c t i v a t e d r a t bone-marrow cells i n vitro. t i m e course,

The r e s p o n s e was dose d e p e n d e n t ,

a n d was n o t s e r u m d e p e n d e n t .

followed a defined

c-Acetylated murein dimer

f r a g m e n t s f r o m P r o t e u s m i r a b i l i s wtere much l e s s a c t i v e t h a n t h e i r

u n s u b s t it u t e d c o u n t e r p a r t s , i n d i c a t i n g a s t r u c t u r a l spe c i f ic i t y f o r

murein activation.

Removal o f a d h e r e n t a n d phagocy t i z i n g c e l l s f r o m


99

t h e marrow suspensions d i d n o t a l t e r t h e s e r e s u l t s . The l a b e l l e d , a c t i v a t e d c e l l s c o n s t i t u t e d a d i s t i n c t p o p u l a t i o n of buoyant d e n s i t y 1 . 0 6 4 t o 1 . 0 6 9 g/cm3 when c e n t r i f u g e d o n a c o n t i n u o u s g r a d i e n t of P e r c o l l . Enrichment of t h e t a r g e t - c e l l p o p u l a t i o n was achieved by a c o m b i n a t i o n o f a d h e r e n t - c e l l r e m o v a l and d i s c o n t i n u o u s d e n s i t y g r a d i e n t c e n t r i f u g a t i o n t o remove g r a n u l o c y t e s and e r y t h r o p o i e t i c cells. I t was c o n c l u d e d t h a t a p o p u l a t i o n o f m y e l o p o i e t i c p r e c u r s o r s could be a c t i v a t e d by d i r e c t c o n t a c t w i t h b a c t e r i a l c e l l w a l l c o n s t i t u e n t s . The s t i m u l a t i o n o f Q - g a l a c t o s e i n c o r p o r a t i o n was n o t coupled t o a c t i v e d e o x y r i b o n u c l e i c a c i d s y n t h e s i s i n t h e marrow cells. T h u s , t h e a c t i v a t i o n was i n t e r p r e t e d a s an i n d u c t i o n o f d i f f e r e n t i a t i o n r a t h e r than a m i t o t i c e v e n t . To o b t a i n i n f o r m a t i o n about t h e n a t u r e of t h e immunogens i n t h e r i b o s o m a l v a c c i n e ( f r a c t i o n 11) o f Pseudomonas a e r u g i n o s a , t h e s p e c i f i c i t y of r a b b i t a n t i b o d i e s t o F r a c t i o n I 1 has been ~ t u d i e d . ’ ~ Crossed i m m u n o e l e c t r o p h o r e s i s d e m o n s t r a t e d t h e p r e s e n c e of a n t i b o d i e s which p r e c i p i t a t e d w i t h ribosomal a n t i g e n s , b u t n o t w i t h lipopolysaccharide. B y means of an e n z y m e - l i n k e d irnmunosorbent a s s a y , a n t i b o d i e s t o l i p o p o l y s a c c h a r i d e were d e t e c t e d i n a n t i b o d i e s t o f r a c t i o n 11. A n t i b o d i e s t o f r a c t i o n I1 c o u l d p r o t e c t mice a g a i n s t a l e t h a l c h a l l e n g e w i t h Pseudomonas a e r u g i n o s a . Absorption experiments demonstrated t h a t t h e p r o t e c t i v e a b i l i t y of a n t i b o d i e s t o f r a c t i o n I1 was due t o cell-envelope a n t i g e n s , whereas a n t i b o d i e s t o ribosomal antigens d i d n o t contribute t o the protection. A n t i b o d i e s t o l i p o p o l y s a c c h a r i d e c o u l d be d e t e c t e d i n mice 1 week a f t e r a s i n g l e v a c c i n a t i o n w i t h f r a c t i o n 11. I t was concluded t h a t t h e p r o t e c t i v e a c t i v i t y of f r a c t i o n I1 was due, a t l e a s t i n p a r t , t o t h e p r e s e n c e of l i p o p o l y s a c c h a r i d e i n t h e r i b o s o m a l v a c c i n e . Treatment o f f r a c t i o n I1 w i t h r i b o n u c l e a s e decreased t h e p r o t e c t i v e a c t i v i t y of t h e r i b o s o m a l v a c c i n e . A d d i t i o n of s y n t h e t i c polyadenylic acid-polyuridylic acid restor ed t h e p ro t e c t i v e a c t i v i t y o f r i b o n u c l e a s e - t r e a t e d f r a c t i o n 11, i n d i c a t i n g t h a t R N A i n t h e r i b o s o m a l v a c c i n e m i g h t a c t a s an a d j u v a n t o r a c a r r i e r i n t h e p r e s e n t a t i o n of t h e c o n t a m i n a t i n g c e l l - e n v e l o p e a n t i g e n s . The p r o t e c t i v e a c t i v i t y and t h e t o x i c i t y of f r a c t i o n I1 were compared w i t h t h e p r o t e c t i v e a c t i v i t y and t h e t o x i c i t y of f r a c t i o n I , which co n t a i ne d c e 1l-en ve 1ope co m P O ne n t s , i n c 1u d i n g 1ipopol y s a c c h a r i de , and o f p u r i f i e d l i p o p o l y s a c c h a r i d e . The r e s u l t s i n d i c a t e d t h a t p r o t e c t i o n by t h e ribosomal vaccine was a s s o c i a t e d w i t h a s l i g h t l y h i g h e r t o x i c i t y than was p r o t e c t i o n by f r a c t i o n I , whereas p u r i f i e d l i p o p o l y s a c c h a r i d e was t h e most t o x i c vaccine.

100


A k a l i n e t r e a t m e n t o f P s e u d o m o n a s gg~g~ifiisg t y p e 5 l i p o p o l y s a c c h a r i d e r e s u l t e d i n r e d u c e d t o x i c i t y a s m e a s u r e d by b o t h t h e L i m u l u s amoebocyte assay and t h e r a b b i t p y r o g e n i c i t y test.74 Chemical a n a l y s i s o f t h e d e a c y l a t e d l i p o p o l y s a c c h a r i d e r e v e a l e d t h a t e s t e r - l i n k e d f a t t y a c i d s were r e m o v e d w h i l e t h e a m i d e - l i n k e d f a t t y acids remained i n t a c t . The n e u t r a l and amino s u g a r c o m p o s i t i o n s f o r n a t i v e l i p o p 0 l y s a c c h a r i de a n d d e a c y 1a t e d l i p o p o l y s a c c h a r i d e were identical within experimental error. Antigenic determinants f o r c o m p l e m e n t - d e p e n d e n t h u m a n o p s o n i c a n t i b o d y were r e t a i n e d u n d e r these d e a c y l a t i o n c o n d i t i o n s . To e n h a n c e i t s i m m u n o g e n i c i t y , d e a c y l a t e d l i p o p o l y s a c c h a r i d e was c o v a l e n t l y c o u p l e d t o P s e u d o m o n a s p i l i and t h e 1,4-diaminobutyl d e r i v a t i v e s o f Pseudomonas exotoxin A and t e t a n u s t o x o i d . Q u a n t i t a t i v e amino s u g a r a n a l y s e s r e v e a l e d t h a t 2.6 a n d 3 . 2 m o l o f d e a c y l a t e d l i p o p o l y s a c c h a r i d e s were c o v a l e n t l y bound t o a m i n o b u t y l Pseudomonas e x o t o x i n A and a m i n o b u t y l t e t a n u s toxoid, r e s p e c t i v e l y . Gel-electrophoresis data i n d i c a t e d a t least 1 mol of d e a c y l a t e d l i p o p o l y s a c c h a r i d e c o v a l e n t l y bound p e r p i l u s subunit protein. I n i t i a l immunologic data i n d i c a t e d that antibody a g a i n s t d e a c y l a t e d 1 i p o p o l y s a c c h a r i . d e c o u l d be i n d u c e d w h e n t h e deacylated l i p o p o l y s a c c h a r i d e i s c o v a l e n t l y attached t o p r o t e i n carriers. The p o l y s a c c h a r i d e m o i e t y was i s o l a t e d by m i l d a c i d h y d r o l y s i s f r o m t h e slime g l y c o p r o t e i n o f P s e u d o m o n a s a e r u g i n o s a s t r a i n BI. After gel f i l t r a t i o n , the polysaccharide obtained from the c a r b o h y d r a t e p e a k f r a c t i o n s was f o u n d t o b e l i p i d - a n d p r o t e i n free.75 A n a l y s e s i n d i c a t e d t h a t t h e p o l y s a c c h a r i d e c o n t a i n e d t h e c a r b o h y d r a t e components o f the p a r e n t g l y c o l i p o p r o t e i n . Molecular s i z e o f t h e p o l y s a c c h a r i d e was e s t i m a t e d by g e l f i l t r a t i o n a s 70,000 t o 10,000. The p o l y s a c c h a r i d e showed no i n d i c a t i o n s o f t o x i c i t y i n mice a t d o s e s f a r i n e x c e s s o f t h e l e t h a l d o s e f o r t h e p a t e n t g l y c o l i p o p r o t e i n , n o r d i d t h e mice d e v e l o p t h e l e u k o p e n i a t h a t characteristically follows intraperitoneal injection of glycolipoprotein. The p o l y s a c c h a r i d e acted a s a n i n h i b i t o r o f i n d i re c t haem a g g l u t i n a t i o n o f g l y c o 1i po p r o t e i n- c o a t e d e r t h r o cy t e s i n t h e p r e s e n c e o f a n t i - g l y c o l i p o p r o t e i n s e r u m . H o w e v e r , i t was n o t a n t i g e n i c i t s e l f i n rabbits. Coupled w i t h methylated bovine serum albumin, t h e p o l y s a c c h a r i d e c o n t i n u e d t o lack t h e l e u k o p e n i c and t o x i c p r o p e r t i e s o f the p a r e n t g l y c o l i p o p r o t e i n , but the coupled p o l y s a c c h a r i d e was c a p a b l e o f s t i m u l a t i n g i n d i r e c t h a e m a g g l u t i n a t i n g antibody a g a i n s t both the polysaccharide and the g l y c o l i p o p r o t e i n coating erythrocytes. Moreover, t h e antibody t o the coupled

101


p o l y s a c c h a r i d e p r o t e c t e d m i c e a g a i n s t c h a l l e n g e w i t h l e t h a l doses o f v i a b l e Pseudomonas a e r u g i n o s a w i t h t h e same e f f e c t i v e n e s s a s a n t i g l y c o l i p o p r o t e i n serum. The i d e n t i f i c a t i o n o f t h e new a m i n o s u g a r

d i d e o x y - Q - g l u c o p r a n u r o n i c a c i d (19)

2,3-diacetamido-2,3-

a s a c o n s t i t u e n t o f t h e 0-

s p e c i f i c p o l y s a c c h a r i d e s o f Pseudomonas a e r u g i n o s a s t r a i n 1 7 0 0 1 4 h a s been b r o u g h t a b o u t by use o f 1 3 C n.m.r.

AcHN

a n d c h e m i c a l methods.76

AcHN

OH

The p r o c e d u r e u s e d f o r t h e i s o l a t i o n o f from

strains

o f

Pseudomonas

the lipopolysaccharide

aeruginosa appears

t o

c o n f l i c t i n g r e s u l t s i n t h e i n t e r p r e t a t i o n o f 31P n.m.r. The

use

of

the

Westphal

lipopolysaccharide with

extraction

procedure7'

to

gives

a h i g h e r phosphorus content

o b t a i n e d by u s e d o f t h e B ~ v i pnr o~c e ~d u r e .

lead

spectra. a

than that

The h i g h e r l e v e l s o f

n.m.r.

p h o s p h o r u s c o n t e n t h a v e b e e n a t t r i b u t e d 7 7 b y u s e o f "P

to

the presence o f t r i p h o s p h a t e residues, probably i n t h e l i p i d A and inner-core

regions.

The

presence

of

triphosphate residues i n

-P-s-e u d o m o n a s a e r u g i n o s a i s i n c o n t r a s t t o

results obtained

for

S a l m o n e l l a s p e c i e s and E s c h e r i c h i a c o l i . Among t h e a e r o b i c p s e u d o m o n a d s ,

the closely related species

Pseudomonas d i m i n u t a and Pseudomonas v e s i c u l a r i s f o r m subgroup,

the

inclusion of

which i n

becoming i n c r e a s i n g l y suspect.

the

an i s o l a t e d

genus Pseudomonas

i s

Chemotaxonomic grounds f o r t h e

s e p a r a t e s t a t u s o f t h e s e s p e c i e s i n c l u d e t h e p o s s e s s i o n by t h e i r type

strains

o f

a

unique

range

o f

polar

l i p i d s

and

l i p o p o l y s a c c h a r i d e s i n w h i c h l i p i d A i s based o n 2,3-diamino-2,3d i de ox y -Q- g l uco se r a t h e r t h a n 2- am ino -2 -de ox y -Q - g l

UCG se

.

The 1i po

-

p o l y s a c c h a r i d e f r o m Pseudomonas d i m i n u t a NCTC 8545 h a s a n u m b e r o f o t h e r unusual f e a t u r e s i n c l u d i n g t h e presence o f Q-threo-2-pentu l o s e ( i - x y l u l o s e ) .79 B a c t e r i o p h a g e 12P caused l y s i s o f not

other

plant

Specificity of

pathogenic

or

Pseudomonas p h a s e o l i c o l a b u t

saprophytic

pseudonomads.80

t h e b a c t e r i o p h a g e may depend o n r e c o g n i t i o n o f some


102 unique

feature

of

polysaccharides.

the

Pseudomonas

phaseolicola

cell-surface

Exopolysaccharide and l i p o p o l y s a c c h a r i d e from

----------P s e u d o m o n a s e ----------haseolicola

were

more e f f i c i e n t a t

inhibiting

bacteriophage attachment than were i d e n t i c a l l y prepared e x t r a c t s from

five

other

phaseolicola

pseudomonad s p e c i e s .

w i t h

Mutants

bacteriophage

of

Pseudomonas

r e s i s t a n c e

produced

e x o p o l y s a c c h a r i d e and l i p o p o l y s a c c h a r i d e d i f f e r e n t i n amount and composition from those o f the parental strain. the

e x o p o l y s a c c h a r i d e and

P-s-e u d o m o n a s e h aseolicola -

Thus i n some manner

lipopolysaccharide

structures

o f

are d i s t i n c t from those o f other p l a n t

p a t h o g e n i c and s a p r o p h y t i c s p e c i e s .

The d i f f e r e n c e may p r e v e n t

r e c o g n i t i o n a n d i n i t i a t i o n o f r e s i s t a n c e e v e n t s when Pseudomonas phaseolicola challenges i t s susceptible host plant, The l i p o p o l y s a c c h a r i d e o f

bean.

R h o d o m i c r o b i u m v a n n i e l i i A T C C 17100

h a s been s t u d i e d a n d f o u n d t o c o n t a i n a - g l u c o s e a n d Q-mannose ( m o l a r r a t i o 2:l)

3-2-

w i t h t r a c e amounts o f p - g l u c o s e and p - x y l o s e and

m e t h y l - Q - x y l o s e . 81

2-Ace t a m i d o - 2 - d e o x y - Q - g 1 ucose,

3-deoxy-~-manno-2-octulosonic

u r o n i c acids, and

a c i d were a l s o found

t o be m a j o r

co m po ne n t s t o g e t h e r w it h 6-hy d r ox y p a 1m i t ic a c i d (ex c l u s ive 1y am ide bound),

6-hydroxymyristic

bound).

H e p t o s e s a n d p h o s p h a t e w e r e f o u n d t o be a b s e n t .

A f r a c t i o n (ca.

acid, and m y r i s t i c a c i d ( m a i n l y e s t e r The l i p i d

4 0 % o f t h e m a t e r i a l d r y w e i g h t ) l i b e r a t e d by m i l d

a c i d h y d r o l y s i s c o n t a i n s Q-mannose ( i n t h e n o n r e d u c i n g t e r m i n a l p o s i t i o n s ) i n a d d i t i o n t o 2-amino-2-deoxy-~-glucose

a n d an unknown

n i n h y d r i n - p o s i t i v e compound, b u t t h e p r e s e n c e o f a-mannose a s a t r u e c o n s t i t u e n t o f l i p i d A (as opposed t o i t b e l o n g i n g t o an a d d i t i o n a l , c o n t a m i n a t i n g l i p i d a s may be t h e c a s e i n l i p o p o l y s a c c h a r i d e s f r o m

---Chromatium

vinosum

I___-

determined. f r a c t io n a r e

and T h i o c a p s a r o s e o p e r s i c i n a ) has y e t

The m a i n c o n s t i t u e n t s o f

p - g l uc o s e ,

acid, and u r o n i c a c i d s .

a -m a nno s e ,

I t should

the

to

be

degraded p o l y s a c c h a r i d e

3 -de o x y -Q - m a n n o - 2 - o c t u l o s o n i c be

noted that

uronic acids

co n t r i b u t e ne ga t ive c h a r g e s i n a p h o s p h a t e - f r e e 1i p o po 1y sa c c h a r ide Interestingly,

9-xylose

and 3 - 2 - m e t h y l - a - x y l o s e

were

.

enriched

r e l a t i v e t o t h e o t h e r s u g a r s when R h o d o m i c r o b i u m v a n n i e l i i A T C C 1 7 1 0 0 was g r o w n f o r s e v e r a l p a s s a g e s i n t h e same R8AH medium. There

is

a

common

structure

(core

l i p o p o l y s a c c h a r i d e s o f R h o d o s p i r i l l u m tenue.”

region)

i n

the

I t i s composed o f a

b r a n c h e d t r i s a c c h a r i d e o f L - g l y c e r o - p - m a n n o - h e p t o s e ( a n d o f 3-deox y ~-manno-2-octulosonic acid),

a s r e v e a l e d by m e t h y l a t i o n a n a l y s e s o f

degraded polysaccharides o f f o u r d i f f e r e n t R h o d o s p i r i l l u m tenue strains.

The s t r u c t u r e i s s i m i l a r o r m i g h t e v e n be i d e n t i c a l t o t h e

103

4: Microbial Polysaccharides i n n e r core o f e n t e r o b a c t e r i a l 0 antigens.

I n addition,

each o f t h e

four R h o d o s p i r i l l u m tenue lipopolysaccharides contains a s t r a i n s p e c if ic r e g io n t h a t c o n s is t s o f h e p t o s e ( s ) ( & -9 1y c e r o - Q - m a n n o h e p t o s e o r a-glycero-p-manno-heptose o r b o t h ) o r hexoses. There i s a p a r t i a l s u b s t i t u t i o n o f t h e core r e g i o n and t h e s t r a i n - s p e c i f i c r e g i o n by p h o s p h o r u s , The

showing m i c r o h e t e r o geneit y .

lipopolysaccharide

from

polymixin-resistant pmrA

the

m u t a n t s o f S a l m o n e l l a t y p h i m u r i u m has been c h a r a c t e r i z e d a n d f o u n d t o be a n u n u s u a l t y p e o f p h o s p h o l i p i d ( 2 0 ) b u i l t up o f a c e n t r a l d i s a c c h a r i de o f t w o 2-am ino -2-deox y g l uco se r e s i d ue s. 83 Pho sp h a t e

-n-

e s t e r s a r e bound i n t h e 4-

and l - p o s i t i o n s

r e s p e c t i v e l y , t o g i v e t h e p h o s p h o l i p i d backbone.

o f r e s i d u e s I 1 a n d I, The l i p i d c h a r a c t e r

i s g i v e n by s u b s t i t u t i o n o f t h e b a c k b o n e w i t h 7 m o l o f l o n g - c h a i n f a t t y acids:

4 mol o f 3-hydroxymyristic

nonhydroxylated f a t t y acids,

2. 1

a c i d and 3 m o l o f

m o l each o f l a u r i c ,

myristic,and

p a l m i t i c acids, r e s p e c t i v e l y , w i t h s m a l l amounts o f 2 - h y d r o x y m y r i t i c acid.

The e x a c t p o s i t i o n s o f t h e s e g r o u p s h a v e o n l y p a r t l y b e e n

identified.52,83

The backbone c o n t a i n s t w o a m i n o g r o u p s s u b s t i t u t e d

by 3 - h y d r o x y m y r i s t i c h y d r o x y l groups,

a c i d and,

besides t h e two phosphate-substituted

another f o u r h y d r o x y l groups o f which p o s i t i o n 3 o f

r e s i d u e I 1 forms t h e l i n k t o t h e s p e c i f i c p o l y s a c c h a r i d e v i a 3-

deoxy-9-manno-2-octulosonic ester-bound backbone,

acid.

3-hydroxymyristic

There i s evidence t h a t

the

two

acids are linked d i r e c t l y t o the

s u b s t i t u t i n g t w o o f t h e t h r e e a v a i l a b l e h y d r o x y l groups.

The n o r m a l f a t t y a c i d s ,

i n c o n t r a s t , a p p e a r t o be l i n k e d a t t h e 3 -

hydroxy groups o f t h e 3 - h y d r o x y m y r i s t i c a c i d residues:

m y r i s t i c and

2- hy d r ox ym y r i s t ic a c i d s s u b s t it u t e t h e e s t e r - b o un d 3- hy d r ox ym y r i s t ic

acid, w h i l e p r e l i m i n a r y acids acid.

to

evidence i n d i c a t e s

be e s t e r - l i n k e d

to

t h e l a u r i c and m y r i s t i c

t h e t w o amide-bound

3-hydroxymyristic

I n t h i s way l i p i d A i s c h a r a c t e r i z e d by a h i g h c o n t e n t o f a

r a t h e r unusual s t r u c t u r e o f long-chain hydroxymyristate.

Thus o n l y

the

s u b s t i t u t e t h e backbone d i r e c t l y , linkage,

fatty-acid

e s t e r s o f 3-

3-hydroxylated

acids would

t w o i n amide and t w o i n e s t e r

w h i l e t h e n o n - h y d r o x y l a t e d a c i d s (and 2 - h y d r o x y m y r i s t i c

a c i d ) w o u l d be e s t e r - l i n k e d t o t h e 3 - h y d r o x y m y r i s t i c

a c i d groups.

M u t a t i o n s i n gene r f a H o f S a l m o n e l l a t y p h i m u r i u m a t 8 4 u n i t s o n t h e l i n k a g e map make l i p o p o l y s a c c h a r i d e o f c h e m o t y p e s Ra, a n d R c . ~ F~ - F a c t o r

e x p r e s s i o n i n RfaH'

f o l l o w i n g p r o p e r t i e s when c o m p a r e d w i t h RfaH'

Flac,

Rb2,

Rb3,

s t r a i n s was r e d u c e d i n t h e

number o f phage f 2 i n f e c t i v e c e n t r e s ,

strains:

transfer o f

l y s i s by a n d p r o p a g a t i o n

o f phages f 2 and M13, p r o p o r t i o n o f c e l l s w i t h v i s i b l e F - p i l i ,

and


1 04

where F

= -C=O

I

( 2 m o l e s p e r 3 hydroxyl groups)

HC-O(R )

I

(7H2) 10 CH 3

and R = l a u r i c a c i d ( 1 m o l e ) m y r i s t i c a c i d ( 1 mole) p a l m i t i c a c i d ( 1 mole) 2-hydroxymyristic a c i d ( t r a c e )

(201

105


f o r m a t i o n o f m a t i n g a g g r e g a t e s w i t h F- c e l l s . Inhibition o f m u l t i p l i c a t i o n o f Br60, a f e m a l e - s p e c i f i c phage, was n o t r e d u c e d i n Rfah' F l a c s t r a i n s . P l a s m i d t r a n s f e r f r o m RfaH' s t r a i n s was r e d u c e d u n a f f e c t e d f o r I n c g r o u p s B, Ia, L, N,

f o r I n c g r o u p s F I , F I I , a n d T, P,and W ,

a n d i n c r e a s e d f o r I n c g r o u p M when c o m p a r e d w i t h p l a s m i d

t r a n s f e r f r o m RfaH'

strains.

R e d u c e d F - f a c t o r f u n c t i o n i n RfaH'

s t r a i n s was n o t due t o d e f e c t i v e l i p o p o l y s a c c h a r i d e s i n c e s t r a i n s with

mutations

rfa

i n other

genes

were

unaffected i n plasmid

Gene r f a H a p p e a r s t o be h o m o l o g o u s w i t h gene s f r B i n

transfer.

E s c h e r i c h i a c o l i K-12,

which maps a t t h e same l o c a t i o n ,

influences

F-factor function,

and a f f e c t s s y n t h e s i s o f l i p o p o l y s a c c h a r i d e .

gene

s f r B has

product

an t i t e r m i na t o r The

.

of

been p r o p o s e d t o

mobility

of

membrane

lipopolysaccharide,

i s

essential

components,

for

particularly

biogenesis

membrane and i s a p r i m a r y e v e n t i n phage i n f e c t i o n . f l u i d dynamic p r o p e r t i e s o f

the outer

The

be t r a n s c r i p t i o n

of

the outer To d e f i n e t h e

membrane a s

related to

f u n c t i o n more a c c u r a t e l y t h e c a p a b i l i t y t o measure l a t e r a l d i f f u s i o n coefficients

vivo

pf

r h o d a m i n a t e d G30 l i p o p o l y s a c c h a r i d e f u s e d

i n t o S a l m o n e l l a t y p h i m u r i u m GPOA f i l a m e n t o u s b a c t e r i a h a s been developed.85 ( f 1uo r e s c e nce

The

method

used

r e d i s t r ib u t io n a f t e r

extends

the

FRAP

p h o t obl e a c h i n g) t o

procedure

b a c t e r i a and

the r e s u l t s demonstrate r a p i d d i f f u s i o n o f lipopolysaccharide

(2.0

2 0.9) A

cm2s-l)

x

=

f a m i l y o f mutants o f Salmonella tyhimurium w i t h a l t e r e d

lipopolysaccharide to

(D

over micrometre distances.

freeze-thaw

c o r e c h a i n l e n g t h s were assessed f o r s e n s i t i v i t y

and o t h e r

stresses.86

Deep r o u g h s t r a i n s w i t h

decreased c h a i n l e n g t h i n t h e l i p o p o l y s a c c h a r i d e c o r e were more s u s c e p t i b l e t o novobiocin, l a u r y l sulphate

p o l y m y x i n B,

d u r i n g growth,

to

bacitracin,

and sodium

ethylenediaminetetracetic

acid

and sodium l a u r y l s u l p h a t e i n r e s t i n g suspension, and t o s l o w and r a p i d freeze-thaw

i n w a t e r and s a l i n e ,

more outer-membrane Variations

i n

the

dramatically affect neomycin,

lipopolysaccharide the s e n s i t i v i t y of

chain

length

did not

the s t r a i n s t o t e t r a c y c l i n e ,

o r NaCl i n g r o w t h c o n d i t i o n s o r t h e degree o f f r e e z e - t h a w -

induced cytoplasmic-membrane strains

and t h e s e s t r a i n s e x h i b i t e d

damage t h a n t h e w i l d t y p e o r l e s s r o u g h s t r a i n s .

incorporated

damage.

larger

The d e e p e r r o u g h i s o g e n i c

quantities

o f

less-stable

l i p o p o l y s a c c h a r i d e and l e s s p r o t e i n i n t o t h e o u t e r membrane t h a n d i d t h e w i l d t y p e o r l e s s rough mutants, a f f e c t e d outer-membrane

i n d i c a t i n g t h a t the mutations

synthesis o r organization or

both.

106


Nikaido’s

model of

t h e r o l e o f l i p o p o l y s a c c h a r i d e and p r o t e i n i n

determining the resistance o f Gram-negative b a c t e r i a o f lowm o l e c u l a r - w e i g h t h y d r o p h o b i c a n t i b i o t i c s was d i s c u s s e d i n r e l a t i o n t o t h e s t r e s s of freeze-thaw. The s p e c i f i c a n t i - l i p o p o l y s a c c h a r i d e s e r u m a n t i b o d y r e s p o n s e i n BALB/c

mice, b e f o r e and a f t e r an o r a l b o o s t e r w i t h d i f f e r e n t non-

pathogenic s t r a i n s o f Salmonella typhimurium, ELISA t e c h n i q u e . 8 7

was m e a s u r e d b y a n

Serum r e s p o n s e t o l i p o p o l y s a c c h a r i d e was

found

t o depend on t h e l e n g t h o f t h e l i p o p o l y s a c c h a r i d e p o l y s a c c h a r i d e moiety i n rough mutants.

T h e M206 s m o o t h m u t a n t i n d u c e d a l e s s

marked a n t i - l i p o p o l y s a c c h a r i d e response.

The i m m u n o g e n i c i t y o f t h e

p o l y s a c c h a r i d i c c o r e a p p e a r s t o be m o d i f i e d by t h e 0 a n t i g e n .

The

s p e c i f i c r e s p o n s e i n t h e g u t o n l y becomes marked f o l l o w i n g a f t e r b o o s t e r o f Ra, Rc,and The 2 - h a p t e n i c reported

to

contain

However, 2 - h a p t e n products of

M206 m u t a n t s . p o l y s a c c h a r i d e o f S a l m o n e l l a m o n t e v i d e o h a s been glyceraldehyde

partial hydrolysis o f

separate experiments perchloric

acid

at

i t s

preparatlons from a

terminus.

lipopolysaccharide,

gave { 3 H ) g l y c e r o l

a n d {3H)NaBH4.

reducing

galE mutant contained

Further

which

upon t r e a t m e n t study

of

i n

with

the g-hapten

r e d u c i n g t e r m i n u s s u g g e s t e d t h a t i t was a c t u a l l y P-mannose.88 The c e l l - w a l l

polymers o f the thermophilic cyanobacterium

S y n e c h o c o c c u s PCC6716 h a v e b e e n i s o l a t e d a n d a n a l y s e d , t h e m a j o r compounds o f t h e t h e r m o p h i l i c s t r a i n b e i n g s i m i l a r b u t n o t i d e n t i c a l t o those o f the mesophilic strains.89

The l i p o p o l y s a c c h a r i d e was

f o u n d t o c o n t a i n B - h y d r o x y p a l m i t i c a c i d as t h e o n l y h y d r o x y l a t e d f a t t y acid together

with myristic,

p a l m i t i c , and p a l m i t o l e i c a c i d s .

The c a r b o h y d r a t e s p r e s e n t w e r e Q - g l u c o s e , I - r h a m n o s e , 2 - a m i n o - 2 deoxy-a-glucose,and w i t h traces of

an u n i d e n t i f i e d n i n h y d r i n - p o s i t i v e component

Q-mannose,

Q-galactose,

H e p t o s e s and 3 - d e o x y - a - m a n n o - 2 - o c t u l o s o n i c

L-fucose,

and P - x y l o s e .

a c i d w e r e f o u n d n o t t o be

present. The

ability

of

various

bacterial

l i p o p o l y s a c c h a r i d e s and

mycoplasmal l i p o p o l y s a c c h a r i d e s ( l i p o g l y c a n s ) t o i n d u c e macrophagemediated t u m o u r - c e l l was d e t e r m i n e d . 9 0

a-x a n t u m less

o r A c h o l e p l a s m a q r a n u l a r u m had no a c t i v i t y o r

activity

0128:812,

k i l l i n g and L i m u l u s a m e b o c y t e l y s a t e c l o t t i n g L i p o g l y c a n s f r o m t h e mycoplasma Acholeplasma

than

lipopolysaccharides

from

lo4

to

Escherichia

lo5 coli

E s c h e r i c h i a c o l i K235, o r S a l m o n e l l a m i n n e s o t a R595 i n

c a u s i n g L i m u l u s l y s a t e c l o t t i n g and t u m o u r - c e l l

k i l l i n g by

p e r i t o n e a l macrophages f r o m n o r m a l or b a c i l l u s C a l m e t t e - G u e r i n -


107

i n f e c t e d mice. Previous s t u d i e s have s h o w n t h a t t h e l i p i d A p o r t i o n o f b a c t e r i a l l i p o p o l y s a c c h a r i d e i s r e s p o n s i b l e f o r t h e e f f e c t s on macrophage-mediated tumour-cell k i l l i n g and L i m u l u s l y s a t e c l o t t i n g . The k n o w n d i f f e r e n c e s i n t h e l i p i d s t r u c t u r e s o f b a c t e r i a l l i p o p o l y s a c c h a r i d e s and mycoplasmal lipopolysaccharides ( l i p o g l y c a n s ) may account f o r t h e noted d i f f e r e n c e s i n t h e b i o l o g i c p o t e n c i e s observed.

4

Capsular Polysaccharfdes

The i n t e r a c t i o n o f l e c t i n s and l e c t i n - l i k e s u b s t a n c e s w i t h b a c t e r i a h a s been r e v i e w e d 9 ' and t h e i n t e r a c t i o n d i s c u s s e d a s a means o f d i s t i n g u i s h i n g between m i c r o b i a l s p e c i e s , i d e n t i f y i n g t h e p r e s e n c e of a p a r t i c u l a r s u g a r r e s i d u e and f r a c t i o n a t i n g t h e po 1y s a c c h a r i des from b a c t e r i a . 91 S o l u b l e a n t i g e n s were o b t a i n e d by e x t r a c t i n g f i v e s e r o t y p e s t r a i n s o f C l o s t r i d i u m p e r f r i n g e n s t y p e A w i t h w a t e r a t 100°C.92 The t y p e - s p e c i f i c p o l y s a c c h a r i d e s w e r e p r e c i p i t a t e d w i t h e t h a n o l , and t h e common a n t i g e n s were recovered from t h e e t h a n o l s u p e r n a t a n t s b y c o n c e n t r a t i o n , d i a l y s i s , and l y o p h i l i z a t i o n . Refluxing the w a t e r - e x t r a c t e d c e l l r e s i d u e s w i t h 1 %a c e t i c a c i d f o l l o w e d b y c o n c e n t r a t i o n , d i a l y s i s , and l y p o p h i l i z a t i o n gave a d d i t i o n a l common antigen fractions. A comprehensive, s i d e - b y - s i d e comparison of t h e antigen fractions, the ethanol precipitate, the ethanol supernatant, and t h e a c e t i c a c i d s u p e r n a t a n t , r e v e a l e d t h a t common a n t i g e n s were r e c o v e r e d i n a l l t h r e e f r a c t i o n s and t h a t t h r e e d i s t i n c t e n t i t i e s w e r e r e s p o n s i b l e f o r t h e f o r m a t i o n o f t h e o b s e r v e d common immunoprecipitin l i n e s . Whereas many f r a c t i o n s possessed a l l t h r e e i m m u n o p r e c i p i t i n l i n e s , o t h e r s c o n t a i n e d o n l y one o r t w o . The s e r o l o g i c a l h o m o l o g y observed between t h e v a r i o u s a n t i g e n f r a c t i o n s was a p p a r e n t l y a consequence of 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e and 2acetamido-2-deoxy-P-mannose c o n t a i n i n g polymers. The common a n t i g e n s were presumably a s s o c i a t e d w i t h t h e c e l l envelope and may be t h e t y p e o f m a r k e r s s o u g h t p r e v i o u s l y b y o t h e r s f o r t h e serological identification of C l o s t r i d i u m perfringens. The bacterium formerly named Fusobacterium p o l y s a c c h a r o l y t i c u m and now r e c l a s s i f i e d a s a C l o s t r i d i u m s p e c i e s h a s been s h o w n t o produce s p o r e s and have a c e l l - w a l l s t r u c t u r e o f t h e Gram-positive type d e s i t e i t s appearance a s Gram-negative when s t a i n e d . 9 3 The c a p s u l a r p o l y s a c c h a r i d e s f r o m C r y t o c o c c u s n e o f o r m a n s

108


s e r o t y p e A and from a p o s s i b l e , mutant s t r a i n t h e r e o f have been s t r u c t u r a l l y a n a l y ~ e d . ~T h~e l a t t e r m a t e r i a l i s s i m i l a r t o t h e c a p s u l a r a n t i g e n o f C r y t o c o c c u s n e o f o r m a n s s e r o t y p e D. Epidemiological and immunological evidence i n d i c a t e s t h a t t h e K 1 c a p s u l a r p o l y s a c c h a r i d e c o n f e r s the p r o p e r t y o f v i r u l e n c e on Escherichia coli. One a p p r o a c h t o s t u d y i n g w h e t h e r t h e K 1 a n t i g e n is s u f f i c i e n t t o confer virulence or i f other Escherichia c o l i s t r u c t u r e s are necessary i s t o i s o l a t e t h e K 1 genes f o r g e n e t i c and biochemical analysis. R e c o m b i n a n t DNA m e t h o d o l o g y p r o v i d e d a p o w e r f u l t o o l f o r s u c h an a p p r o a c h . The m o l e c u l a r c l o n i n g o f t h e E s c h e r i c h i a c o l i K 1 a n t i g e n genes h a s been reported.95 The c l o n e d K 1 g e n e s s y n t h e s i z e d a c a p s u l e i n Escherjchja c o l i K12 i n d i s t i n g u i s h a b l e c h e m i c a l l y and i m m u n o l o g i c a l l y from t h a t o f w i l d type K 1 strains. T h e c a p s u l a r p o l y s a c c h a r i d e was i s o l a t e d f r o m E s c h e r i c h i a c o l i 010:K5:H4: i t c o u l d n o t be o b t a i n e d f r o m a n u n c a p s u l a t e d ( K 5 - ) m u t a n t . I t c o n t a i n s 2 - a c e tam i do - 2 - d e o x y - Q - g l u c o se a n d E - g l u c u r o n i c a c i d i n a m o l a r r a t i o o f 1:1.96 A c i d h y d r o l y s i s o f t h e a c i d i c p o l y s a c c h a r i d e a s w e l l a s S m i t h d e g r a d a t i o n a n d d e g r a d a t i o n by deamination of the carboxyl-reduced p o l y s a c c h a r i d e s u g g e s t e d t h a t t h e p o l y s a c c h a r i d e is composed o f a d i s a c c h a r i d e r e p e a t i n g u n i t . T h e d a t a o b t a i n e d by m e t h y l a t i o n a n a l y s i s a n d n.m.r. s p e c t r o s c o p y indicated t h a t the repeating sequence of the capsular polysaccharide i s similar t o t h a t o f desulpho-heparin w i t h the s t r u c t u r e (21). + 4 ) -B-Q-GlcQA-( 1+4) - a - Q - G l C Q N A C - (

1+

(21) T h e l i n k a g e p a t t e r n o f t h e K6 a n t i g e n was i n v e s t i g a t e d u s i n g m a t e r i a l f r o m t h e u r i n a r y p a t h o g e n E s c h e r i c h i a c o l i LP 1092.97 The p o l y s a c c h a r i d e c o n s i s t s o f 9 - r i b o s e a n d 3-deoxy-Q-manno-2Colorimetric procedures, Smith o c t u l o s o n a t e i n a r a t i o o f 2:l. d e g r a d a t i o n , m e t h y l a t i o n a n a l y s i s , a n d n.m.r. s p e c t r o s c o p y were a p p l i e d t o t h e whole polysaccharide and t o a t r i s a c c h a r i d e r e p e a t i n g u n i t o b t a i n e d by m i l d - a c i d - c a t a l y s e d h y d r o l y s i s . T o g e t h e r , t h e d a t a were c o m p a t i b l e o n l y w i t h a b r a n c h e d - c h a i n s t r u c t u r e ( 2 2 ) . A method has been developed t o s e p a r a t e t h e c e l l e n v e l o p e o f e n c a p s u l a t e d ( t y p e b) Haemophilus i n f l u e n z a e i n t o i t s o u t e r - and inner-membrane components w i t h p r o c e d u r e s t h a t a v o i d e d two p r o b l e m s encountered i n f r a c t i o n a t i o n o f t h i s envelope: (i) t h e tendency o f t h e o u t e r and i n n e r membranes t o h y b r i d i z e and ( i i ) t h e tendency o f


109

the apparently

f r a g i l e i n n e r membrane t o f r a g m e n t i n t o d i f f i c u l t l y

sedimentable

units.98

Log

radioactively labelled, press.

phase

cells,

whose

lipids

were

were l y s e d by passage t h r o u g h a F r e n c h

The l y s a t e was a p p l i e d t o a d i s c o n t i n u o u s s u c r o s e g r a d i e n t ,

a n d e n v e l o p e - r i c h m a t e r i a l was c o l l e c t e d b y c e n t r i f u g a t i o n o n t o a c u s h i o n o f dense s u c r o s e under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s . This

material

centrifugation

was i n

a

then

further

sucrose

fractionated

gradient

to

f r a c t i o n s w h i c h were p a r t i a l l y c h a r a c t e r i z e d . density,

by

yield

isopycnic

four

membrane

On t h e b a s i s o f t h e i r

radioactivity,

bouyant

composition,and

content o f phospholipid, protein, lipopolysaccharide,

and s u c c i n i c dehydrogenase, follows:

fraction 1

inner-membrane vesicles

these f r a c t i o n s

outer-membrane

were

entrapped

inner

f r a c t i o n o f i n n e r membrane,

p o o r f r a c t i o n o f i n n e r membrane.

polypeptide i d e n t i f i e d as

vesicles with very l i t t l e

c o n t a m i n a t i o n (10 m M . B o t h enzymes were i n h i b i t e d by C u 2 + (>29mM), Z n 2 + (>l.OmM), and e t h y l e n e g l y c o l - b i s ( B - a m i n o e t h y l e t h e r ) - l l , N - t e t r a - a c e t i c a c i d ( > l o mM). T r i c h o d e r m a r e e s e i b u t n o t C l o s t r i d i u m t h e r m o c e l l u m c e l l u l o s e - s o l u b i l i z i n g a c t i v i t y was 20% i n h i b i t e d by Q - g l u c o s e ( 7 3 m M ) and c e l l o b i o s e ( 2 9 m M ) . Both c e l l u l a s e s p r e f e r e n t i a l l y cleaved t h e i n t e r n a l g l y c o s i d i c bonds of cello-oligosaccharides. The o v e r a l l r a t e s o f c e l l o - o l i g o s a c c h a r i d e d e g r a d a t i o n were higher f o r Trichoderma r e e s e i than f o r C l o s t r i d i u m -t-h--e r m o c e l l u m c e l l u l a s e , e x c e p t t h a t t h e r a t e s of c o n v e r s i o n o f c e l l o h e x a o s e t o c e l l o t r i o s e were e q u i v a l e n t . The f o r m a t i o n and e x c r e t i o n o f a c e t y l m a l t o s e and a c e t y l a t e d m a l t o d e x t r i n s a f t e r accumulation o f maltose i n E s c h e r i c h i a c o l i have been s t u d i e d . 1 3 4 As a c e t y l m a l t o s e was n o t a s u b s t r a t e f o r t h e m a l t o s e t r a n s p o r t s y s t e m i t was p r o p o s e d t h a t t h e a c e t y l a t i o n


122

p r o c e s s was an e f f e c t i v e d e t o x i f i c a t i o n mechanism. When E s c h e r i c h i a c o l i s t r a i n CP78 (&+) w a s s t a r v e d f o r ii s o l e u c i n e by t h e a d d i t i o n o f L - v a l i n e , t h e amount o f Q - g l u c o s e i n p o l y m e r i c f o r m i n t h e c e l l s i n c r e a s e d m a r k e d l y compared t o t h a t o f t h e c o n t r o l cells.135

I n contrast,

(*-I.

s t r a i n CP79

increase of

t h i s phenomenon was n o t s e e n i n

The i n c r e a s e i n CP78 was shown t o be due t o t h e

glycogen.

These r e s u l t s i n d i c a t e t h a t

=’

s y n t h e s i s was a u g m e n t e d u n d e r s t r i n g e n t c o n t r o l . using other isogenic pairs o f o t h e r amino acids. valyl-tRNA

strains starved for

When t h e c u l t i v a t i o n t e m p e r a t u r e o f s t r a i n s

and 1 0 8 6 0 2 (&-)

1 0 8 6 0 1 (*+)

&+ and

glycogen

T h i s was c o n f i r m e d

possessing temperature-sensitive

s y n t h e t a s e was s h i f t e d f r o m 3OoC t o 4OoC,

L-

no d i f f e r e n c e

was o b s e r v e d i n t h e r e s p o n s e o f g l y c o g e n s y n t h e s i s b e t w e e n t h e t w o strains.

These

necessary f o r

results

the

guanosine 5’-diphosphate through

indicate

augmentation o f

stimulation

o f

that

3,-diphosphate the activity

i n v o l v e d i n glycogen synthesis.

protein

synthesis

was

glycogen s y n t h e s i s and t h a t did not exert i t s effect

of

pre-existing

enzyme(s)

These c o n c l u s i o n s w e r e s u p p o r t e d by

t h e r e s u l t s o f experiments u s i n g chloramphenicol and r i f a m p i c i n . The r a t e s o f Q - g l u c o s e u t i l i z a t i o n o f CP78 and CP79 w e r e d e c r e a s e d t o n e a r l y t h e same e x t e n t by I - v a l i n e a d d i t i o n .

This suggests t h a t

t h e r e g u l a t i o n s i t e o f glycogen s y n t h e s i s under s t r i n g e n t c o n t r o l resides i n a

step

after

the

transport

of

P-glucose

by

the

p h o p h o t r a n s f e r a s e system. ADP-Q-glucose

pyrophosphorylase from E s c h e r i c h i a c o l i B mutant

CL1136-504 h a s been p u r i f i e d t o h o m o g e n e i t y . t e t r a m e r o f 200,000 subunits.136 enzymes

of

The n a t i v e enzyme i s a

d a l t o n s composed o f f o u r i d e n t i c a l 50,000

Mr

The s t r u c t u r a l g e n e s f o r t h e g l y c o g e n b i o s y n t h e t i c Escherichia coli,

glycogen synthase,

ADP-Q-glucose

p y r o p h o s p h o r y l a s e , a n d b r a n c h i n g enzyme w e r e c l o n e d f r o m c h r o m o s o m a l DNA b a c t e r i a l p l a s m i d , A

pBR322. 137

b a c t e r i a l a g g l u t i n i n was e x t r a c t e d f r o m g r o u n d c o r n ( W I

h y b r i d 64A x W117) seed w i t h p h o s p h a t e - b u f f e r e d s a l i n e (pH 6.0) p r e c i p i t a t e d w i t h (NH4)2S04 a t 7 0 % s a t u r a t i o n .

and

The a c t i v i t i e s o f

t h i s a g g l u t i n i n a g a i n s t 22 s t r a i n s o f E r w i n i a s t e w a r t i i (agent o f b a c t e r i a l w i l t o f c o r n ) t h a t v a r i e d i n v i r u l e n c e were determined.138 Specific agglutination (agglutination t i t e r per m i l l i g r a m o f p r o t e i n p e r m i l l i l i t e r ) values were c o r r e l a t e d n e g a t i v e l y w i t h v i r u l e n c e ratings.

S t r a i n s w i t h h i g h s p e c i f i c a g g J u t i n a t i o n values (15 o r

h i g h e r ) were a v i r u l e n t o r w e a k l y v i r u l e n t ,

s t r a i n s with low s p e c i f i c

a g g l u t i n a t i o n values (10 o r l o w e r ) were h i g h l y v i r u l e n t ,

with two

123

4: Microbial Polysaccharides exceptions.

A

virulent

s t r a i n produced butyrous

c o l o n i e s and

r e l e a s e d o n l y s m a l l amounts o f e x t r a c e l l u l a r p o l y s a c c h a r i d e i n t o t h e medium,

and t h e

fluidal

colonies

cells and

l a c k e d capsules. released

V i r u l e n t s t r a i n s produced

large

amounts

of

extracellular

p o l y s a c c h a r i d e p r o d u c e d by each s t r a i n ( a s d e t e r m i n e d by i n c r e a s e i n viscosity

of

contrast

lipopolysaccharide compositions

strains.

t h e medium) and t h e s p e c i f i c a g g l u t i n a t i o n When c e l l s o f

six

were

fluidal strains

value.

similar were

I n

i n

a l l

washed by

r e p e a t e d l y c e n t i f u g i n g and r e s u s p e n d i n g them i n b u f f e r , t h e y were a g g l u t i n a t e d more s t r o n g l y by c o r n a g g l u t i n i n t h a n were unwashed cells.

When

agglutination

avirulent

cells

were

washed,

their

values d i d not increase s i g n i f i c a n t l y .

c e l l u l a r polysaccharide-deficient

specific

Eight extra-

mutants o f E r w i n i a s t e w a r t i i ,

s e l e c t e d f o r r e s i s t a n c e t o t h e capsule-dependent

b a c t e r i o p h a g e K9,

h a d l o w e r v i r u l e n c e by h i g h e r s p e c i f i c a g g l u t i n a t i o n t h a n d i d t h e i r of

extracellular

p o l y s a c c h a r i d e appears t o be e s s e n t i a l f o r v i r u l e n c e .

corresponding

wild-type

parents.

Production

Extracellular

may p r e v e n t a g g l u t i n a t i o n o f b a c t e r i a i n t h e h o s t , t h u s a l l o w i n g their multiplication, The

dextran

mesenteroides described.13'

produced

obtained

from

Branch

points

by

a

new

strain

contaminated at

0-3

and

of

case 0-2

Leuconostoc

juice

has

been

2:l)

(ratio

were

d e t e r m i n e d by m e t h y l a t i o n w h i l s t t h e o x i d i z e d d e x t r a n was f o u n d t o liberate,

on p a r t i a l a c i d h y d r o l y s i s ,

- - g 1u c o s e ,

a c id ) Q

-

i c a c i d ) ( 1+6)

-0-Q -

6-g-(a-Q-glucopyranosyluronic

glucopyranosy1)-(1+3)-Q-glucose,and acid)-Q-glucose

2-g-(a-a-glucopyranosyluronic

0 - ( a -Q - g 1u c o p y r a n o s y 1u r o n

( w h i c h may h a v e b e e n d e r i v e d f r o m t h e t r i o u r o n i c

a c i d 1. R a b b i t a n t i - d e x t r a n 81355 s e r a p r e p a r e d by i n j e c t i n g r a b b i t s w i t h L e u c o n o s t o c m e s e n t e r o i d e s NRRL 8 1 3 5 5 w e r e s e p a r a t e d o n a S e p h a d e x G75 c o l u m n i n t o t w o f r a c t i o n s , o n e b i n d i n g a n d t h e o t h e r not

binding to

the

column.140

Oligosaccharide

inhibition

of

p r e c i p i t a t i o n o f t h e t w o f r a c t i o n s w i t h d e x t r a n 81355 i n d i c a t e d t h a t b o t h f r a c t i o n s had (1+3)-a-Q-specificity.

However, a n t i b o d i e s i n

t h e n o n - b i n d i n g f r a c t i o n were shown t o be d i r e c t e d a g a i n s t

g l u c o p y r a n o s y l - ( 1 + 3 ) - a - e - g-l u c o p y r a n o s y l - ( l + 6 ) - g l u c o s e ,

g-a-Q-

w h i l s t those

i n t h e b i n d i n g f r a c t i o n were d i r e c t e d a g a i n s t g - a - a - g l u c o p y r a n o s y l -

(1+6)-a-~-glucopyranosyl-(l+3)-~-glucose. (1+6)-a-P-Specific by

anti-dextran

a n t i b o d y was

i n j e c t i n g N4 d e x t r a n - c o n c a n a v a l i n

interactions o f f i v e synthetic linear

A

raised i n rabbits

conjugate,

and

the

dextrans w i t h r a b b i t anti-N4

124


d e x t r a n were s t u d i e d . l 4 l antibody

The a b i l i t y o f Q - g l u c a n s t o p r e c i p a t e

depended on t h e i r a v e r a g e m o l e c u l a r

t h e same

conditions.

solubility

of

the

This

p h e n o m e n o n was

antigen-antibody

i n h i b i t i o n assay i n d i c a t e d t h a t specific

antibody

samples with

weight,

h i g h e r m o l e c u l a r w e i g h t p r e c i p i t a t i n g more a n t i b o d y ,

n i t r o g e n under

shown t o

complex.

be due

to

Oligosaccharide

t h e maximum s i z e o f t h e (l+6)-a-Q-

combining s i t e corresponded t o

isomaltose.

The

p r e c i p i t a t e d a n t i b o d y c l a s s was shown t o be I g G i m m u n o g l o b u l i n , a n d it

was

mostly

directed

to

linear

non-terminal

a-Q-glucosidic

D e t e r m i n a t i o n o f a n t i b o d y n i t r o g e n and Q-glucan i n t h e

linkages.

precipitates numbers o f

indicated that

e-glucose

the

ratio of

r e s i d u e s was

antibody

molecule t o

1:16 i n t h e e x t r e m e a n t i b o d y

excess r e g i o n . The

concentration

viscosity

(0)

(GI

and

shear-rate

has been s t u d i e d f o r

(y)

dependence

p o l y s a c c h a r i d e s o l u t i o n s ( i n c l u d i n g dextran), and t h e s t r i k i n g g e n e r a l i t i e s a r e observed:142 dilute-to

concentrated-solution

concentration 10

c,* =:

(nsp v a r i e s a t

c1*4

for

viscosity

and

frequency

v i s c o s i t y are c l o s e l y superimposable, p l o t s of

n/n0

a g a i n s t y/yOe1 ( w h e r e

o0

a critical

and a t

shear-rate

dependence

at

s p e c i f i c v i s c o s i t y (nsp)

dilute solutions,

(ii) the

following

(i) the t r a n s i t i o n from

behaviour occurs

4 / { n } , when z e r o - s h e a r

concentrated solutions),

of

a wide range o f random-coil

of

c3*3 f o r

dependence

dynamic

of

(oscillatory)

(iii) double l o g a r i t h m i c i s zero-shear

viscosity,

and

yoe1 i s t h e shear r a t e a t w h i c h y = yo/lO) a r e e s s e n t i a l l y i d e n t i c a l f o r a l l c o n c e n t r a t e d s o l u t i o n s s t u d i e d , and t h u s t h e t w o p a r a m e t e r s

no

and yoe1 c o m p l e t e l y d e f i n e t h e v i s c o s i t y a t a l l shear r a t e s o f

p r a c t i c a l importance. Five strains of

Microccus,

accumulated a,a-trehalose glucose.143

a,a-Trehalose

representing three species,

when g r o w n i n t h e p r e s e n c e o f

a-

was n o t f o u n d i n t w o S t a p h y l o c o c c u s

species or i n Planococcus c i t r e u s . S u d a n o p h i l i c and i o d i n e - s t a i n i n g c y t o p l a s m i c g r a n u l e s were i s o l a t e d a t various developmental stages during growth o f Nocardia asteroides (strain

55)

and g l y c o g e n g r a n u l e s ,

and

i d e n t i f i e d as p o l y - b e t a - h y d r o x y b u t y r a t e

r e ~ p e c t i v e 1 y . l ~O~u r i n g g r o w t h i n n u t r i e n t

b r o t h c o n t a i n i n g 1% D - g l u c o s e , hydroxybutyrate respectively,

and

maximal accumulation o f

glycogen

granules,

o f i t s dry weight,was

up

t o

10

poly-betaand

20%,

obtained i n the filamentous

c e l l s a t 16 h j u s t p r i o r t o the onset o f c e l l fragmentation during t h e s t a t i o n a r y phase.

The d e c r e a s e o f t h e c y t o p l a s m i c g r a n u l e s was

125

4: Microbial Polysaccharides concomitant

with

fragmentation

of

the

cells

to

rod-like

and

s p h e r i c a l c e l l s , s u g g e s t i n g t h a t t h e p o l y m e r s may s e r v e a s c a r b o n and energy

source

during

macrophogenesis.

detected i n t h e t h r e e c e l l forms.

Both g r a n u l e s were

A t higher concentrations o f

a-

g l u c o s e ( 4 % ) more g l y c o g e n g r a n u l e s ( 9 t i m e s ) a c c u m u l a t e d t h a n p o l y b e t a - h y d r o x y b u t y r a t e ( 4 t i m e s ) , a n d g l y c o g e n h y d r o l y s i s was a l s o faster

than

that

of

suggesting

poly-beta-hydroxybutyrate,

preferences o f glycogen over poly-beta-hydroxybutyrate c a r b o n s o u r c e under t h i s g r o w t h c o n d i t i o n . of

granules

were s i g n i f i c a n t l y

concentration

( l o % ) , and

as e n e r g y and

Growth and b i o s y n t h e s i s

r e d u c e d by

very

h i g h !-glucose

the usual pleomorphic developmental stages

were r e d u c e d t o a d i m o r p h i c l i f e c y c l e .

Thus,

b i o s y n t h e s i s of b o t h

g r a n u l e s and morphogenesis a r e under c a t a b o l i t e r e p r e s s i o n .

Both

p o l y m e r s were a l s o f o u n d i n N o c a r d i a b r a s i l i e n s i s and N o c a r d i a otitidis-caviarurn,

indicating that

cytoplasmic accumulation o f

m u l t i p l e g r a n u l e s i s common i n t h e genus. The m e t h o d u s e d t o i s o l a t e a h i g h - m o l e c u l a r - w e i g h t

immunogenic

p o l y s a c c h a r i d e f r o m Pseudomonas a e r u g i n o s a i m m u n o t y p e 1 ( I T - 1 )

was

m o d i f i e d t o p e r m i t t h e i s o l a t i o n o f a s i m i l a r polysaccharide from Pseudomonas a e r u g i n o s a IT-2.145

T h i s a n t i g e n was composed p r i m a r i l y

o f c a r b o h y d r a t e , had a complex monosaccharide c o m p o s i t i o n ,

including

sugars n o t found i n t h e lipopolysaccharide,

a n d was n o n p y r o g e n i c i n

r a b b i t s a n d n o n t o x i c i n m i c e a t h i g h doses.

This m a t e r i a l protected

mice from

challenges

w i t h l i v e homologous

cells.

Pseudomonas

a e r u g i n o s a I T - 2 p o l y s a c c h a r i d e gave a l i n e o f i d e n t i t y w i t h t h e 0 side

chain

of

the

lipopolysaccharide,

polysaccharide i n molecular ability

to

immunize

mice

weight,

but

differed

from

chemical composition,

actively.

Lipopolysaccharide

this and from

Pseudomonas a e r u g i n o s a I T - 2 c o n t a i n e d an i m m u n o l o g i c a l d e t e r m i n a n t n o t found

o n Pseudomonas a e r u g i n o s a I T - 2 p o l y s a c c h a r i d e ,

d e t e c t e d due t o i t s s t a b i l i t y d u r i n g t r e a t m e n t Thus,

a

high-molecular-weight

polysaccharide

Pseudomonas a e r u q i n o s a I T - 2 p o l y s a c c h a r i d e ,

w h i c h was

with dilute alkali. antigen

from

w h i c h was s e r o l o g i c a l l y

i d e n t i c a l t o t h e l i p o p o l y s a c c h a r i d e 0 s i d e c h a i n b u t was c h e m i c a l l y and p h y s i c a l l y d i s t i n c t ,

was r e c o v e r e d .

Also,

l i k e Pseudomonas

a e r u q i n o s a I T - 1 s t r a i n s , Pseudomonas a e r u g i n o s a I T - 2 c o n t a i n s an alkali-stable

i m m u n o d e t e r m i n a n t on t h e l i p o p o l y s a c c h a r i d e t h a t may

represent a c o r e - l i k e antigen. Spontaneous a l g i n a t e - p r o d u c i n g

( m ~ v] a r i a n t s

f r o m s t r a i n s o f Pseudomonas f l u o r e s e n c e s ,

-P-s-e u d o m o n a s

were i s o l a t e d

Pseudomonas p u t i d a , and

mendocina a t a frequency o f 1 i n

lo8

by s e l e c t i n g f o r

126


c a r b e n i c i l l i n resistance.146

The i n f r a r e d s p e c t r u m o f t h e b a c t e r i a l

e x o p o l y s a c c h a r i d e was t y p i c a l o f

an a c e t y l a t e d a l g i n a t e s i m i l a r

to

t h a t p r e v i o u s l y d e s c r i b e d i n Azotobacter v i n e l a n d i i and i n mucoid v a r i a n t s o f Pseudomonas a e r u g i n o s a . i s o l a t e d f r o m Pseudomonas s t u t z e r i ,

-P-s-e u d o m o n a s

testostergnL,

Mucoid v a r i a n t s

Pseudgmmas

a c i d o v o r a n s , Pseudomonas c e p a c i a , o r

were

not

Pseudomonas p s e u d o a l c a l i g e n e s ,

GLgLn~tg, P s e u d o m o n a s

Pseudomonas m a l t o p h i l i a .

G r a m - n e g a t i v e h y d r o g e n b a c t e r i u m Pseudomonas h y d r o g e n o v o r a was found

t o

excrete

an

anthrone-H2S04

p 0 1 y s a c c h a r i d e . l ~ ~A b o u t

12 g / l i t r e

of

p o s i t i v e

viscous

the polysaccharide

was

p r o d u c e d a u t o t r o p h i c a l l y o n gaseous h y d r o g e n a t t h e s t a t i o n a r y p h a s e of

growth.

Biosynthesis of

the

nitrogen-deficient condition. 39.29%,

H

6.23%,

polysaccharide

49.67%,

0

occurred under

I t s e l e m e n t a r y c o m p o s i t i o n was C 0.21%,

N

p o l y s a c c h a r i d e c o n t a i n e d !-galactose,

and

Q-glucose,

rhamnose as i t s m a i n components.

ash

4.6%.

The

Q-mannose, and

L-

The p o l y s a c c h a r i d e h a d a n t i -

t o b a c c o m o s a i c v i r u s and a n t i - t u m o u r a c t i v i t i e s . The p o s s i b i l i t y o f s e p a r a t i n g p y r u v u l a t e d p o l y s a c c h a r i d e s i n t o pyruvate-rich

and p y r u v a t e - p o o r

u s i n g an a f f i n i t y matrix.148 strand types exists.

f r a c t i o n s has been d e m o n s t r a t e d

T h u s i n some p o l y m e r s , a m i x t u r e o f

The a f f i n i t y m a t r i x was p r e p a r e d by c o u p l i n g

a n t i b o d i e s t o a R h i z o b i u m p o l y s a c c h a r i d e t o Sepharose g e l .

Elution

was a c c o m p l i s h e d by t h e a d d i t i o n o f p y r u v a t e t o t h e e l u t i n g b u f f e r . N o n - p y r u v y l a t e d p o l y s a c c h a r i d e s were n o t a d s o r b e d . Rhizobia

are

Gram-negative

n o d u l a t i n g t h e r o o t s of

bacteria

leguminous p l a n t s ,

e x t r a c e l l u l a r polysaccharide-deficient

normally

capable

of

and t h e f a i l u r e o f f i v e

mutants t o nodulate suggested

that the polysaccharide i s required f o r t h i s nodulation.

I t has

b e e n r e p o r t e d t h a t among a l a r g e r s a m p l e o f m u t a n t s w i t h a l t e r e d polysaccharide production,

i s o l a t e d from two species

( i n c l u d i n g one o f t h e o r i g i n a l s e t p l u s 34 new o n e s ) ,

o f Rhizobium production of

t h e e x t r a c e l l u l a r p o l y s a c c h a r i d e does n o t c o r r e l a t e w i t h a b i l i t y t o n o d u l a t e a p p r o p r i a t e hosts.149

Therefore,

although involvement o f a

m i n o r component c a n n o t be r u l e d o u t , t h e r e i s no e v i d e n c e t h a t t h e whole p o l y s a c c h a r i d e i s r e q u i r e d f o r n o d u l a t i o n . I m m u n o e l e c t r o n m i c r o s c o p y was c o m b i n e d w i t h p a r t i a l characterization o f i s o l a t e d exopolysaccharide t o study binding o f s o y b e a n l e c t i n by R h i z o b i u m j a p o n i c u m s t r a i n USDA 138.150

Lectin-

b i n d i n g a c t i v i t y r e s i d e d i n two forms o f exopolysaccharide produced d u r i n g growth:

an a p p a r e n t l y

very

f o r m and a l o w e r - m o l e c u l a r - w e i g h t

high-molecular-weight d i f f u s i b l e form.

capsular

A t low-speed


127

c e n t r i f u g a t i o n , t h e c a p s u l a r form cosedimented w i t h c e l l s t o form a v i s c o u s , w h i t e , c e l l - g e l c o m p l e x which was n o t d i f f u s i b l e i n 1% a g a r , and t h e d i f f u s i b l e form remained i n t h e c e l l - f r e e s u p e r n a t a n t . E l e c t r o n - m i c r o s c o p i c o b s e r v a t i o n of t h e c e l l - g e l c o m p l e x a f t e r l a b e l l i n g w i t h soybean l e c t i n - f e r r i t i n c o n j u g a t e r e v e a l e d t h a t c a p s u l a r p o l y s a c c h a r i d e s , f r e q u e n t l y a t t a c h e d t o o n e end o f t h e c e l l s , w e r e r e c e p t o r s f o r l e c t i n . The o u t e r membrane of t h e c e l l bound no l e c t i n . Various p r e p a r a t i o n s of e x o p o l y s a c c h a r i d e i s o l a t e d from t h e c u l t u r e s u p e r n a t a n t w e r e t e s t e d f o r l e c t i n b i n d i n g , i n t e r a c t i o n w i t h homologous s o m a t i c a n t i g e n , and t h e p r e s e n c e of 3deoxy-a-manno-2-octulosonic a c i d and w e r e c h r o m a t o g r a p h e d i n S e p h a r o s e 48 and 6 8 g e l b e d s . L e c t i n b i n d i n g was r e s t r i c t e d t o a p o l y s a c c h a r i d e component designated a s l e c t i n - b i n d i n g polysaccharide. T h i s polysaccharide, as present i n the cell-free c u l t u r e s u p e r n a t a n t , was a d i f f u s i b l e a c i d i c p o l y s a c c h a r i d e devoid of 3 - d e o x y - ~ - m a n n o - 2 - o c t u l o s o n i c a c i d , w i t h m o l e c u l a r weight of 2 x l o 6 t o 5 x l o 6 . I t was c o n c l u d e d t h a t s o y b e a n l e c t i n - b i n d i n g component o f Rhizobium japonicum i s an e x t r a c e l l u l a r p o l y s a c c h a r i d e and not a l i p o p o l y s a c c h a r i d e and t h a t t h e d i f f u s i b l e l e c t i n - b i n d i n g p o l y s a c c h a r i d e probably d i f f e r s from t h e very h i g h - m o l e c u l a r - w e i g h t l e c t i n - b i n d i n g p o l y s a c c h a r i d e of t h e loose c a p s u l e ( s l i m e ) only i n t h e d e g r e e of p o l y m e r i z a t i o n . The e x t r a c e l l u l a r p o l y s a c c h a r i d e o f R h i z o b i u m m e l i l o t i 201 c o n s i s t s of two a c i d i c p o l y s a c c h a r i d e s , APS-I and APS-I1 .151 APS-I is composed o f D-glucose, D-mannose,and Q - g l u c u r o n i c a c i d i n a molar w h e r e a s APS-I1 i s composed of E - g l u c o s e , r a t i o of 3:3:2, g a l a c t o s e , a - m a n n o s e , a n d p y r u v i c a c i d i n a m o l a r r a t i o of 4:3:2:1. APS-I1 w a s s e p a r a t e d f r o m t h e e x t r a c e l l u l a r p o l y s a c c h a r i d e p r e p a r a t i o n b y h y d r o l y s i n g APS-I t o i t s o c t a s a c c h a r i d e r e p e a t i n g u n i t w i t h a s p e c i f i c enzyme. APS-I and APS-I1 were a l s o s e p a r a t e d by t r e a t m e n t w i t h c e t y l p y r i d i n i u m c h l o r i d e and by p a p e r e l e c t r o p h o r e s i s of t h e d e p y r u v y l a t e d p o l y s a c c h a r i d e . An i m p r o v e d , g l y c o s y l - s e q u e n c i n g method h a s been u s e d f o r e l u c i d a t i n g t h e s t r u c t u r e of t h e a c i d i c p o l y s a c c h a r i d e s e c r e t e d b y R h i z o b i u m -------m e l i l o t i s t r a i n 1021.152 T h e p o l y s a c c h a r i d e was --------methylated, t h e e t h e r p a r t i a l l y hydrolysed, t h e p r o d u c t s were r e d u c e d , and t h e a l d i t o l s e t h y l a t e d . The r e s u l t i n g m i x t u r e of p e r a l k y l a t e d o l i g o s a c c h a r i d e - a l d i t o l s was a n a l y s e d by h.p.1.c. m.s. C h e m i c a l - i o n i z a t i o n mass s p e c t r a were o b t a i n e d a t 2 s i n t e r v a l s , a s t h e v a r i o u s , p e r a l k y l a t e d oligosaccharide-alditols were e l u t e d from t h e h.p.1.c. column. P e r a l k y l a t e d mono-, d i - , t r i - , and t e t r a -

e-

128

carbohydrate Chemistry

s a c c h a r i d e - a l d i t o l s c o u l d be r e a d i l y d e t e c t e d , and a t l e a s t p a r t i a l l y i d e n t i f i e d , by t h e i r M + 1 i o n s , i n conjunction w i t h o t h e r c h a r a c t e r i s t i c i o n s p r e s e n t i n t h e i r c h e m i c a l - i o n i z a t i o n mass s p e c t r a . T h e p e r a l k y l a t e d d i - and t r i - s a c c h a r i d e - a l d i t o l s w e r e f u r t h e r a n a l y s e d b y g.c. m.s. i n t h e e l e c t r o n - i m p a c t mode. The s t r u c t u r e of t h e a c i d i c p o l y s a c c h a r i d e s e c r e t e d by Rhizobium -------m e l i l o t i s t r a i n 1021 i s t h e same a s t h a t of t h e p r e v i o u s l y c h a r a c t e r i z e d p o l y s a c c h a r i d e s e c r e t e d b y Rhizobium m e l i l o t i s t r a i n U-27.

The s t r u c t u r e of an e x t r a c e l l u l a r , a c i d i c p o l y s a c c h a r i d e from R h i z o b i u m m e l i l o t i I F 0 1 3 3 3 6 was s t u d i e d b y a method i n v o l v i n g of successive fragmentation w i t h s p e c i f i c B-e-glycanases F l a v o b a c t e r i u m M64.153 The p o l y s a c c h a r i d e i s composed of r e p e a t i n g u n i t s of t h e o c t a s a c c h a r i d e ( 3 3 ) . An a c i d i c c o m p o n e n t was i d e n t i f i e d a s q-riburonic acid. F i v e c u l t u r e s of Rhizobium m e l i l o t i (57017, 202, 204, 207, 209) and o n e o f R h i z o b i u m t r i f o l i i ( 5 6 0 ) p r o d u c e d w a t e r - s o l u b l e p o l y s a c c h a r i d e s c o n t a i n i n g P-glucose, Q - g a l a c t o s e , a n d p y r u v i c a c i d i n a m o l a r r a t i o of 7 : l : l and some s u c c i n i c and a c e t i c a c i d s . 1 5 4 These were i d e n t i f i e d a s s u c c i n o g l y c a n - l i k e p o l y s a c c h a r i d e s on t h e b a s i s of t h e i r components, m e t h y l a t i o n a n a l y s i s , a n d f r a g m e n t a t i o n w i t h two s p e c i f i c B - Q - g l y c a n a s e s . One c u l t u r e o f R h i z o b i u m m e l i l o t i ( I F 0 13336) produced w a t e r - s o l u b l e p o l y s a c c h a r i d e c o n t a i n i n g ! - g l u c o s e , Q - g a l a c t o s e , 0 - g l u c u r o n i c a c i d , a n d a c e t i c a c i d i n a molar r a t i o o f 5:1:1:2 and an u n i d e n t i f i e d component. Two c u l t u r e s of Rhitobium -------m e l i l o t i (201, 206) produced w a t e r - s o l u b l e p o l y s a c c h a r i d e s c o n t a i n i n g g - g l u c o s e , ; - g a l a c t o s e , g-mannose, and g - g l u c u r o n i c a c i d i n a m o l a r r a t i o o f 4:2:3:1 and 4:1:2:1, r e s p e c t i v e l y , and some p y r u v i c a c i d . Rhizobium t r i f o l i i I F 0 13337 and Rhizobium japonicum I F 0 13338 p r o d u c e d w a t e r - s o l u b l e p o l y s a c c h a r i d e s c o n t a i n i n g Q g l u c o s e , Q - g l a c t a c t o s e , Q - g l u c u r o n i c a c i d , p y r u v i c a c i d , and a c e t i c Two i s o l a t e s f r o m t h e s t o c k a c i d i n a m o l a r r a t i o o f 6:1:1:2:1. c u l t u r e of R h i z o b i u m t r i f o l i i 560 p r o d u c e d l a r g e a m o u n t s o f t h e water-insoluble polysaccharide curdlan. T h i s is t h e f i r s t report i n Rhizobium of t h e o c c u r r e n c e of c u r d l a n and of s p o n t a n e o u s m u t a t i o n s i n a b i l i t y t o produce s u c c i n o g l y c a n - l i k e p o l y s a c c h a r i d e and c u r d l a n . Carbohydrate m u t a n t s were i s o l a t e d from Rhizobium m e l i l o t i L530 u s i n g t h e t r a n s l o c a t a b l e d r u g - r e s i s t a n c e e l e m e n t Tn5.155 Enzyme a s s a y s w i t h c e l l - f r e e e x t r a c t s of f o u r m u t a n t s showed t h a t t h e y lacked p-mannitol dehydrogenase, ; - r i b o s e k i n a s e , ;-xylose isomerase, and !-fructose k i n a s e , r e s p e c t i v e l y . An I - a r a b i n o s e mutant was a l s o

129

4: Microbial Polysaccharides 4

4 U

b

rl

lu

(3

I

4'

m I

n

M 4

+

4

v

.--I (3

I

4' m I

i

4

u

I

4' m I

I

4' m

I

I

2

4

(3 I

4'

m I

n

+

U

4

u

(3

I

4' 0

n

+

U

4 v

I

4'

u

130


isolated.

U p t a k e s t u d i e s showed t h a t t h e Q - r i b o s e ,

a - x y l o s e , and

p-

f r u c t o s e m u t a n t s s t i l l u t i l i z e d t h e s u g a r s on w h i c h t h e y were u n a b l e t o grow,

p o s s i b l y i n d i c a t i n g t h a t Rhizobium m e l i l o t i possesses

a l t e r n a t i v e m e t a b o l i c r o u t e s w h i c h do n o t r e s u l t i n g r o w t h . mutants were a b l e t o n o d u l a t e a l f a l f a k i n a s e m u t a n t was u n a b l e t o f i x

A l l the

plants, but the n-fructose

n i t r o g e n and t h e L - a r a b i n o s e m u t a n t

showed e i t h e r l a t e or no n i t r o g e n - f i x i n g a b i l i t y . The fulvum,

ADP-!-glucose

pyrophosphorylases

R h o d o s p i r i l l u m molischianum,

from

Rhodospirillum

and R h o d o s p i r i l l u m t e n u e w e r e

p a r t i a l l y p u r i f i e d , and t h e i r k i n e t i c p r o p e r t i e s were studied.156 The e n z y m e f r o m t h e t h r e e o r g a n i s m s was f o u n d t o b e a c t i v a t e d b y p y r u v a t e and t h u s was s i m i l a r

t o t h e R h o d o s p i r i l l u m r u b r u m enzyme

t h a t h a d been p r e v i o u S l y s t u d i e d . fulvum,

The enzymes f r o m R h o d o s p i r i l l u m

R h o d o s p i r i l l u m molischianum,

a l s o a c t i v a t e d by oxamate,

and R h o d o s p i r i l l u m t e n u e were

an a n a l o g o f p y r u v a t e .

Other a-keto

a c i d s , a - k e t o b u t y r a t e and h y d r o x y p y r u v a t e , a c t i v a t e d t o a s m a l l e r extent.

The p r e s e n c e o f p y r u v a t e i n c r e a s e d t h e a p p a r e n t a f f i n i t y

f o r adenosine 5’-triphosphate

a n d M g C 1 2 f o r a l l t h r e e enzymes.

R h o d o s p i r i l l u m m o l i s c h i a n u m enzyme h a s v e r y i n h i b i t i o n by adenosine 5’-monophosphate, phosphate.

However,

pyrophosphorylase monophosphate, ADP.

Rhodospirillum

i s very

sensitive

to

The

l i t t l e sensitivity tenue

to

or inorganic

ADP,

ADP-Q-glucose

i n h i b i t i o n by a d e n o s i n e 5’-

and t h e R h o d o s p i r i l l u m f u l v u m enzyme i s i n h i b i t e d by

Increasing pyruvate concentration reversed the i n h i b i t i o n

o r ADP.

c a u s e d by a d e n o s i n e 5’-monophosphate t h e g l y c o s y l donor f o r s y n t h e s i s of

vivo glycogen pyruvate

and,

synthesis i n

the

Rhodospirillum tenue,

i s

S i n c e ADP-Q-glucose

is

glycogen, i t i s p o s s i b l e t h a t

in

regulated

case

of

by

the

concentration

Rhodozejrillum

fulvum

of and

by t h e r a t i o o f p y r u v a t e c o n c e n t r a t i o n t o

i n h i b i t o r concentration. ADP-Q-glucose

synthetase from

Rhodospirillum tenue

the photosynthetic bacterium

has been p u r i f i e d g r e a t e r

than

95%.

m o l e c u l a r w e i g h t o f t h e e n z y m e i s a p p r o x i m a t e l y 215,000, s u b u n i t m o l e c u l a r w e i g h t o f a b o u t 51,000. composed o f

The enzyme a p p e a r s t o be

f o u r s i m i l a r ifn o t i d e n t i c a l s u b u n i t s . 1 5 7

amino a c i d composition o f

The

with a

t h e enzyme i s s i m i l a r

E s c h e r i c h i a c o l i and S a l m o n e l l a t y p h i m u r i u m ,

Although the t o that

o f

no a p p a r e n t h o m o l o g y

has been o b s e r v e d between t h e i r N - t e r m i n a l amino a c i d sequences. Antisera

prepared against

the

Rhodospirillzm tenue

p a r t i a l l y i n h i b i t the activities o f other photosynthetic bacteria.

ADP-a-glucose

enzyme

synthetases

can from

131

4: Microbial Polysaccharides Q-Mannitol, component

of

extracellular

not

previously

reported

as

an

intracellular

i t has been f o u n d as an end p r o d u c t o f a n a e r o b i c c a r b o h y d r a t e m e t a b o l i s m , bacteria,

although

accumulated w i t h i n s t r a i n s of

a l l 10 s t a p h y l o c o c c a l species t e s t e d

a f t e r a e r o b i c i n c u b a t i o n o f washed c e l l s u s p e n s i o n s i n p h o s p h a t e buffered

1%! - g l u c o s e

f o r 2 h.lS8

b e f o r e and a f t e r i n c u b a t i o n ,

Phenol e x t r a c t s of the c e l l s ,

were a n a l y s e d f o r e - m a n n i t o l c o n t e n t by

p e r i o d a t e u t i l i z a t i o n and p a p e r c h r o m a t o g r a p h y phosphate

content,

with

and f o r q - m a n n i t o l -

Q-mannitol l-phosphate

Staphylococcus aureus Towler,

the

content

of

dehydrogenase.

Q-mannitol

In

increased

f r o m a 0 h v a l u e o f a n d p r o t e i n c o d e d by E 3 o f a d e n o v i r u s t y p e 2 . H e p a t i t i s B s u r f a c e a n t i g e n c o n t a i n s a p r o t e i n (rnol. w t . 2.5 x l o 4 ) a n d a g l y c o p r o t e i n ( m o l . w t . 3.0 x l o 4 ) o f i d e n t i c a l a m i n o a c i d I t i s p r o p o s e d t h a t t h e t w o a n t i g e n c o m p o n e n t s may composition.26 d i f f e r only i n t h e presence of carbohydrate i n t h e glycoprotein and Some p h y s i c o c h e m i c a l t h a t t h e y a r e i n f a c t t h e same g e n e p r o d u c t . and immunological properties o f a glycopeptide obtained a f t e r p r o t e l y t i c cleavage of h e p a t i t i s B s u r f a c e a n t i g e n have been r e p o r t e d .27 A method f o r determining s p e c i f i c r e a c t i v i t y t o herpes simplex v i r u s t y p e s 1 a n d 2 has been d e v e l o p e d , u s i n g human s e r a t o i m m uno p r e c i p i t a t e s p e c i f i c g l y c o p r o t e i n a n t i g e n s f r o m v i rus - i n f e c t e d c e l l e x t r a c t s . 2 8 The v i r a l g l y c o p r o t e i n s p r e c i p i t a t e d from t h e s e e x t r a c t s w e r e t h e n a n a l y s e d by S D S - p o l y a c r y l a m i d e g e l electrophoresis. Mono c l o n a l a n t i bo d i e s d i r e c t e d a g a i n s t h e r p e s s i m p l e x v i r u s g l y c o p r o t e i n s have been u s e d t o p r o t e c t mice a g , a i n s t a c u t e v i r u s induced neurological disease.29 Evidence t h a t t h e virus-induced g l y c o p r o t e i n gC e x p r e s s e s t y p e - s p e c i f i c a n t i g e n i c d e t e r m i n a n t s , w h e r e a s g l y c o p r o t e i n gD e x p r e s s e s t y p e - c o m m o n d e t e r m i n a n t s , i s reported. By u s i n g a t e m p e r a t u r e - s e n s i t i v e m u t a n t o f h e r p e s s i m p l e x v i r u s t y p e 1, a n d i n h i b i t o r s o f g l y c o s y l a t i o n , s p e c i f i c g l y c o p r o t e i n s o f t h e v i r u s h a v e b e e n s h o w n t o be i n v o l v e d i n T c e l l - m e d i a t e d c y t o l y s i s o f v i r u s - i n f e c t e d cells.30 Two-dimensional gel e l e c t r o p h o r e s i s h a s been used t o i d e n t i f y p o l y p e p t i d e s and g l y c o p r o t e i n s o f h e r p e s s i m p l e x v i r u s t y p e 1.31 Pulse chase e x p e r i m e n t s and t r e a t m e n t o f i n f e c t e d cells w i t h neuraminidase s u g g e s t s t h a t t h r e e g l y c o p r o t e i n s c o n t a i n neuraminic acid and t h a t s y n t h e s i s o f t w o o f t h e t h r e e o c c u r s by a t l e a s t t e n d i s c r e t e s t e p s . The h e r p e s s i m p l e x v i r u s t y p e l - s p e c i f i c g l y c o p r o t e i n has been p u r i f i e d by a f f i n i t y c h r o m a t o g r a p h y o n i m m o b i l i z e d s o y a b e a n agglutinin and Helix pomatia lectin.32 The b i n d i n g o f t h e glycoprotein t o these l e c t i n s indicates the presence of a terminal 2-ace t am i d o - 2 - d e o x y-or -p -ga 1a c t 0 s y 1 r e s i d u e i n a n o l i go sa c c h a r i de , a finding not previously reported f o r glycoproteins of enveloped viruses. Two m o n o c l o n a l a n t i b o d y p r e p a r a t i o n s w h i c h a r e h e r p e s s i m p l e x v i r u s t y p e 2 - s p e c i f i c have been i s o l a t e d . 3 3 One o f t h e a n t i b o d i e s

172


precipitated

a

glycoprotein with

g l y c o p r o t e i n E of Drecipitated a

the

v i r u s whereas

characteristics similar to

the other antibody preparation

glycoprotein not

previously

described.

This

g l y c o p r o t e i n i s t e n t a t i v e l y d e s i g n a t e d g l y c o p r o t e i n F. E n e r g y d e p l e t i o n o f v i r a l c e l l s c a u s e d by t h e a d d i t i o n t o t h e c u l t u r e medium o f

carbonyl

cyanide

formation o f N,N’-diacetylchitobiosyl the pool size o f affects the

p-glucosyl

pool

size

of

(CCCP),

3-chlorophenylhydrazone

an uncoupler o f o x i d a t i v e phosphorylation,

does n o t a f f e c t

pyrophosphoryl dolichol,

phosphoryl dolichol,

the

or

and o n l y s l i g h t l y

GDP-Q-manno~e.~~ Using t h i s

system,

i n f l u e n z a v i r u s h a e m a g g l u t i n i n c a n be g l y c o s y l a t e d i n c h i c k e m b r y o fibroblasts

o r a nona-a-mannosyl

e i t h e r a penta-a-mannosyl

derivative.

The

construction o f

a

recombinant

c o n s i s t i n g o f an SV40 v e c t o r and a c l o n e d f u l l - l e n g t h

viral

genome

DNA c o d i n g f o r

t h e h a e m a g g l u t i n i n s u r f a c e g l y c o p r o t e i n o f i n f l u e n z a v i r u s h a s been reported.35 produced

a

Infection of glycoprotein

primate cells similar

i n

with

the hybrid

molecular

size

virus

to

the

haemagglutinin o f influenza virus. Some virus,

b i o c h e m i c a l and b i o l o g i c a l p r o p e r t i e s o f i n f l u e n z a C

a s w e l l a s some m o r p h o l o g i c a l a s p e c t s o f t h e o r g a n i z a t i o n o f

t h e g l y c o p r o t e i n s o n t h e v i r a l e n v e l o p e a n d t h e p l a s m a membranes o f infected cells,

have been r e p o r t e d . 3 6

The c a r b o h y d r a t e p o r t i o n o n

t h e h a e m a g g l u t i n i n o f i n f l u e n z a v i r u s does n o t a p p e a r t o be o f m a j o r importance i n d e f i n i n g the a n t i g e n i c i t y o f the h a e m a g g l ~ t i n i n . ~ ~ Although t h e a b i l i t y o f u n t r e a t e d and g l y c o s i d a s e - t r e a t e d v i r u s i n h i b i t

the

binding

haemagglutinin i s almost

o f

antibodies

directed

indistinguishable,

against 50% o f

release o f

carbohydrate from i n t a c t v i r u s p a r t i c l e s s i g n i f i c a n t l y haemaggluti n at i n g

activity.

The

haem a g g l u t i n i n

to the the

affected membrane

g l y c o p r o t e i n o f i n f l u e n z a v i r u s i s composed o f a t r i p l e - s t r a n d e d c o i l e d c o i l o f a - h e l i c e s e x t e n d i n g 76A f r o m t h e membrane, g l o b u l i n r e g i o n o f a n t i p a r a l l e l B-sheet,

which

and a

contains the

r e c e p t o r - b i n d i n g s i t e and the v a r i a b l e a n t i g e n i c determinants, i s p o s i t i o n e d on t o p of l i k e topology,

t h i s stem.38

Each s u b u n i t h a s a n u n u s u a l l o o p -

s t a r t i n g a t t h e membrane, e x t e n d i n g l 3 5 A d i s t a l l y ,

a n d f o l d i n g b a c k t o e n t e r t h e membrane.

Four antigenic s i t e s on t h e

t h r e e - d i m e n s i o n a l s t r u c t u r e have been i d e n t i f i e d . 3 9

A t l e a s t one

a m i n o a c i d s u b s t i t u t i o n i n e a c h s i t e s e e m s t o be r e q u i r e d f o r t h e p r o d u c t i o n o f new e p i d e m i c s t r a i n s . Glycosylated t r y p t i c peptides o f

the haemagglutinin o f

i n f l u e n z a A v i r u s have been s e p a r a t e d u s i n g a c o m b i n a t i o n o f i o n -

173

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides exchange

chromatography

and g e l

f i l t r a t i ~ n . ~ ' Seven d i f f e r e n t

g l y c o s y l a t e d t r y p t i c p e p t i d e classes were o b t a i n e d from t h e HA1 p o l y p e p t i d e , w h i c h i s d e r i v e d f r o m t h e N - t e r m i n a l segment o f t h e h a e m a g g l u t i n i n , a n d o n l y one g l y c o s y l a t e d p e p t i d e f r o m t h e C - t e r m i n a l p o r t i o n (HA2).

Three t y p e s o f c a r b o h y d r a t e c h a i n s ,

complex,

high

a-

mannose, a n d h y b r i d t y p e , a r e p r e s e n t i n t h e H A 1 p o l y p e p t i d e whereas HA2 c o n t a i n s

the

oligosaccharide

complex-type

side

chains

oligosaccharide

of

the

major

chains.

glycoprotein

The

HA1

of

i n f l u e n z a v i r u s h a e m a g g l u t i n i n have been e x a m i n e d f o r a n t i g e n i c activity

using a

qualitative

solid-phase

and

radioimmunoassay.41

quantitative

differences

o l i g o s a c c h a r i d e s i d e c h a i n s were d e t e c t e d ,

the

exception o f

terminus.42 complex

a

highly

c h a i n has been e l u c i d a t e d ,

aggregated

r e g i o n near

A single glycosylated I-asparagine

oligosaccharide

these

the antigenically active

The amino a c i d sequence o f a Hong

side chains a r e cross-reactive.

Kong i n f l u e n z a h a e m a g g l u t i n i n l i g h t (HA2) with

Although

between

structure

was

the

C-

residue bearing a

noted.

The

amino

acid

sequence and o l i g o s a c c h a r i d e d i s t r i b u t i o n f o r t h e h a e m a g g l u t i n i n f r o m t h e e a r l y Hong Kong i n f l u e n z a v i r u s A/Aichi/2/68(X-31) reported.43 variant

Although the

was

found

to

oligosaccharide

be

identical

h a e r n a g g l u t i n i n f r o m A/Memphis/72, specific I-asparaginyl the individual sequence

residues,

carbohydrate

analysis

shows

the

to

that

i n

the

t h e monosaccharide compositions o f

u n i t s were

different.

existence o f

structural

b e t w e e n t h e Hav 7 a n d t h e Hong K o n g (H3) Two o f

heavy

haemagglutinin from

the

found

w i t h sugar u n i t s a t t a c h e d t o s i x

i n f l u e n z a virus.44 chain o f

have been

distribution i n this

Amino

acid

relationships

haemagglutinins of

the six oligosaccharide the

u n i t s on t h e

Hong K o n g

variant

A/Memphis/102/72

have been shown t o be a n t i g e n i c a l l y r e l a t e d t o t h e

h o s t antigen.45

They

attached t o

C-Asn-8

c o n t a i n t h e N-acetyl-lactosamine-type and &-Asn-22.

residues

The a n t i g e n i c s t r u c t u r e o f

i n f l u e n z a v i r u s h a e m a g g l u t i n i n has been d e f i n e d u s i n g h y b r i d o r n a a n t i b o d i e s . 46 The

uncleaved

(HNO)

and

the

cleaved

(HN)

forms

h a e r n a g g l u t i n i n n e u r a m i n i d a s e o f New c a s t l e d i s e a s e v i r u s , Ulster,

the

strain

have been s u b j e c t e d t o e l e c t r o p h o r e s i s u n d e r r e d u c i n g and

non-reducing

conditions.47

The c o n v e r s i o n o f o n e f o r m t o t h e o t h e r

i n v o l v e s t h e r e l e a s e by p r o t e o l y s i s o f a g l y c o p e p t i d e (mol. x lo3).

of

wt.

8.0

T h e i s o e l e c t r i c p o i n t s h a v e b e e n d e t e r m i n e d a n d t h e c.d.

spectra analysed f o r both p o s t - t r a n s l a t i o n a l and uncleaved Newcastle

disease

virus

p r o t e o l y t i c a l l y cleaved

g l y ~ o p r o t e i n s . ~ I~t i s


174

d e m o n s t r a t e d t h a t c l e a v a g e i s p a r a l l e l e d by a c o n f o r m a t i o n a l c h a n g e of t h e g l y coprot e i n s The d i s s o c i a t i o n a n d r e c o n s t i t u t i o n o f t h e S e m l i k i F o r e s t v i r u s membrane u s i n g n o n - i o n i c d e t e r g e n t have been s t u d i e d . 4 9 Different mechanisms of r e c o n s t i t u t i o n give rise t o symmetric and a s y m m e t r i c vesicles. Glycoprotein E l of the virus i n g - 3 - i n f e c t e d cells is g l y c o s y l a t e d a n d i n s e r t e d i n t o the e n d o p l a s m i c r e t i c u l u m membrane w i t h o u t s i m u l t a n e o u s s y n t h e s i s o f t h e e n v e l o p e p r o t e i n ( m o l . w t. 6.2 x lo4), w h i c h i s a p r e c u r s o r o f t w o o t h e r e n v e l o p e g l y c ~ p r o t e i n s . ~ ' A r e v e r s i b l e defect i n t h e g l y c o s y l a t i o n of t h e membrane p r o t e i n s o f S e m l i k i F o r e s t v i r u s G-1 m u t a n t h a s b e e n d e s c r i b e d . 5 1 At a r e s t r i c t i v e t e m p e r a t u r e t h e v i r a l membrane g l y c o p r o t e i n s a r e a r r e s t e d i n t h e rough e n d o p l a s m i c r e t i c u l u m , but a r e t r a n s p o r t e d t o t h e Golgi complex once t h e c u l t u r e s are s h i f t e d t o a p e r m i s s i v e temperature. An e f f i c i e n t m e t h o d f o r t h e i s o l a t i o n o f l a r g e q u a n t i t i e s o f S e n d a i v i r u s F - g l y c o p r o t e i n by r e d u c t i o n o f t h e i n t a c t v i r u s p a r t i c l e s w i t h d i t h i o t h r e i t o l and s o l u b i l i z a t i o n o f t h e r e d u c e d v i r u s w i t h n o n - i o n i c d e t e r g e n t has b e e n r e p o r t e d . 5 2 Two m e m b r a n e g l y c o p r o t e i n s ( H A N A p r o t e i n a n d F - p r o t e i n ) o f HVJ ( S e n d a i ) v i r u s c o n t a i n r e s i d u e s o f I - f u c o s e , Q - g a l a c t o s e , Q-mannose, a n d 2-ace tam i d o - 2 - d e o x y - & - g l u c o s e , b u t no 2 - a c e t a m i d o - 2 - d e o x y - Q galactose or neuraminic acid.53 Both g l y c o p r o t e i n s c o n t a i n o l i g o s a c c h a r i d e c h a i n s o f t h e h i g h g-mannose t y p e a n d o f t h e complex type. T h e com p l e x - t y p e o l i go s a c c h a r i des c o n t a i n 2 - a c e t a m i d o - 2 deox y - 3 -&a -1- f uco s y 1 - 4 - 2 4 -Q-ga 1a c t o s y 1-Q-g1 uco s y 1 g r o u p s i n t h e i r outer- chain moieties. P a r a m y x o v i r u s f u s i o n (F) p r o t e i n s a r e a c t i v e l y i n v o l v e d i n t h e i n d u c t i o n o f membrane fusion.54 The F p r o t e i n o f S e n d a i v i r u s i s a c t i v a t e d by p r o t e o l y t i c c l e a v a g e t o y i e l d t w o d i s u l p h i d e - l i n k e d p o l y p e p t i d e s (F1 a n d F2). C o n f o r m a t i o n s of t h e i n a c t i v e uncleaved p r e c u r s o r g l y c o p r o t e i n and t h e a c t i v e c l e a v e d form have been D i r e c t e v i d e n c e was o b t a i n e d f o r t h e p r e s e n c e o f compared. hydrophobic binding sites o n t h e active form of t h e F p r o t e i n , but n o t o n t h e i n a c t i v e form. Grow t h - d e p e n d e n t c h a n g e s i n a - m a n n o s y l o l i g o s a c c h a r i d e s d e r i v e d from a s i n g l e s p e c i e s o f membrane g l y c o p r o t e i n have been s t u d i e d i n a v i r a l system.55 E l a n d E 2 g l y c o p r o t e i n s o f S i n d b i s v i r u s e x p r e s s d i f f e r e n t r e l a t i v e q u a n t i t i e s o f f o u r o l i g o s a c c h a r i d e s. Metabolically labelled Sindbis virion and cell-associated v i r a l g l y c o p e p t i d e s h a v e b e e n a n a l y s e d by a c o m b i n a t i o n o f g e l f i l t r a t i o n and specific glycosidase digestion.56 T h e g e n e r a l m o d e l o f I-

.

5: Glycoproteins, Glycopeptides, Proteoglycans, m d Animal Polysaccharides asparaginyl oligosaccharide

p r o c e s s i n g f o r t h e n e u t r a l and a c i d i c -

t y p e s t r u c t u r e s i n normal and l e c t i n - r e s i s t a n t cells

was

confirmed.

175

The

presence o f

Chinese hamster o v a r y

unusual

small

neutral

s t r u c t u r e s i n t h e m a t u r e v i r a l g l y c o p r o t e i n f r o m t h e c e l l l i n e s was a l s o noted. The r o l e o f c a r b o h y d r a t e i n t h e m o r p h o g e n e s i s o f v e s i c u l a r s t o m a t i t i s v i r u s has been s t u d i e d u s i n g t u n i c a m y c i n t o i n h i b i t g l y c o ~ y l a t i o n . ~I ~t i s c o n f i r m e d t h a t d i f f e r e n t g l y c o p r o t e i n s h a v e d i f f e r e n t carbohydrate requirements f o r Simple

mutational

requirement.

changes

Micelles

i n

a

formed

spontaneously p a r t i t i o n i n t o vesicles.58

plasma-mem b r a n e i n s e r t i o n .

protein by

can

also

glycoproteins

affect

of

this

this virus

sonicated phosphatidyl-choline

The h y d r o p h o b i c t a i l f r a g m e n t o f t h e g l y c o p r o t e i n i s

r e s i s t a n t t o p r o t e o l y s i s when t h e g l y c o p r o t e i n is i n s e r t e d i n t o t h e vesicle bilayer.

The i n t r a c e l l u l a r l o c a t i o n o f a n i n t e g r a l membrane

g l y c o p r o t e i n o f v e s i c u l a r s t o m a t i t i s v i r u s h a s been e ~ a m i n e d . ~ ’ I t s p a s s a g e t o t h e c e l l s u r f a c e has been s y n c h r o n i z e d a n d t h u s s t a g e s i n t h e i n t r a c e l l u l a r p a t h w a y t h a t i t f o l l o w s w e r e mapped.

Glycoprotein

o f v e s i c u l a r s t o m a t i t i s v i r u s has b e e n i s o l a t e d a n d i t s b i n d i n g t o c e l l s u r f a c e s studied.60

The s t r u c t u r e s o f t h e o l i g o s a c c h a r i d e s o f

the v i r u s grown i n d i f f e r e n t teratocarcinoma c e l l l i n e s a r e n o t identical

for

sensitivity glycosidases.61 stomatitis sy n t h e s i z i n g

a l l to

cell

lines,

digestion Baby-hamster

virus

and

by

as a

evidenced mixture

by

o f

differences i n endo-

and

KO-

kidney c e l l s i n f e c t e d with v e s i c u l a r

starved

of

Q-glucose

are

capable

o f

-m an no sy 1a t e d 1ip i d -1i n ke d o 1igo sa c c h a r i de s b u t ca nno t

e f f i c i e n t l y c o m p l e t e t h e a s s e m b l y o f Q-Glce3-Q-Maneg-Q-G1cpNAc2pyrophosphoryl dolichol,

r e s u l t i n g i n an a b n o r m a l l y g l y c o s y l a t e d G

p r o t e in .62 Polyoma

virus

requires

oligosaccharide receptors f o r

specific

cell-surface

infection of

sialo-

3T6 c e l l s . 6 3

A

t r i s a c c h a r i d e s e q u e n c e (2) c a n s e r v e a s a s p e c i f i c c e l l - s u r f a c e receptor

both for

polyma

virus-mediated

haemagglutination and f o r

polyoma v i r u s i n f e c t i o n o f h o s t c e l l s . a-Neue5Ac-( 2 + 3 ) - B - Q - G a l p (

1+4) -8-GalpNAc

(2) The a c t i o n o f

toyocamycin on t h e biosynthesis o f

v i r a l

glycoproteins i n c e l l s chronically infected w i t h a murine r e t r o v i r u s i s i n r e d u c i n g t h e content o f envelope g l y c o p r o t e i n (mol.

wt.

7.0 x

176


104).64

However, t h e

precursor

m o l e c u l a r w e i g h t o f 8.5

x

of

this

glycoprotein,

having a

i s synthesized n o r m a l l y and i s

lo4,

processed i n t o i t s f i n a l products, which accumulate i n t h e c e l l s . from

The p u r i f i e d m a j o r e n v e l o p e g l y c o p r o t e i n ( m o l . w t . 8.5 x l o 4 ) avian m y e l o b l a s t o s i s v i r u s contains 45%carbohydrate i n c l u d i n g

25 % 2 - a c e t am ido - 2 distributed backbone.

- d e ox y -Q - g l u co s e .65

between

seven

to

nine

One s t r u c t u r a l m o d e l ,

p r o p e r t i e s of

the

0 1igo sa c c h a r ide c h a in s a r e

points

on

the

polypeptide

which r e c o n c i l e s t h e hydrodynamic

glycoprotein with

nearly

spherical

architecture

o b s e r v e d by e l e c t r o n m i c r o s c o p y , r e q u i r e s t h e o r g a n i z a t i o n o f t h e p o l y p e p t i d e c h a i n and a p p r o x i m a t e l y h a l f o f t h e c a r b o h y d r a t e i n t o a g l o b u l a r form.

The r e m a i n i n g c o v a l e n t l y l i n k e d o l i g o s a c c h a r i d e s

would extend outwardly

fcom

the globular s t r u c t u r e as randomly

o r i e n t e d chains. I n

order

bunyavirus, c o n t r o l of

to

characterize

Belmont

virus

as

a

possible

e v i d e n c e has been p r e s e n t e d o f s i m i l a r t r a n s l a t i o n a l t h e p r o t e i n s a n d g l y c o p r o t e i n s s p e c i f i e d by

the two

v i r u s e s i n mammalian cells.'' Cauliflower

mosaic

virus

capsid

f 1uo r e s c e i n -de r i va t i z e d co n c a n a v a l i n A ,

glycoproteinaceous nature of The

synthesis,

polypeptide t h us

binds

to

demo n s t r a t i n g t h e

the r n ~ l e c u l e . ' ~

glycosylation, and i d e n t i f i c a t i o n

p r o t e i n s i n d u c e d by E p s t e i n - B a r r

of

v i r u s have b e e n r e p o r t e d . 6 8

s p o n t a n e o u s a n d i n d u c e d s y n t h e s e s of

the

early The

virus antigens i n Raji

c e l l s immobilized on surfaces coated w i t h anti-lymphocy t e g l o b u l i n have been s t u d i e d . "

A m a j o r g l y c o p r o t e i n ( g p 350/220)

B a r r v i r u s h a s been d e t e c t e d by i m m u n o f l u o r e s c e n c e

o f Epstein-

with monoclonal

a n t i b o d i e s o n b o t h t h e p l a s m a membrane a n d i n t h e c y t o p l a s m . 7 0 F o u r s t r u c t u r a l p o l y p e p t i d e s o f H a z a r a v i r u s h a v e been r e s o l v e d

.

by so d i um do de c y 1 s u l p h a t e p o 1y a c r y 1am ide g e l e l e c t r o p h o r e s i s T h r e e g l y c o p r o t e i n s ( m o l . w t s . 8.4 x l o 3 , 4.5 x lo3, a n d 3.0 x l o 3 ) are associated with

the

v i r i o n envelope, and a

nonglycosylated

polypeptide i s associated w i t h t h e nucleocapsid. The GIX

a n t i g e n o f m u r i n e r e t r o v i r u s has b e e n a n a l y s e d u s i n g

m o n o c l o n a l a n t i b ~ d i e s , ' ~r e a c t i v i t y w i t h w h i c h a p p e a r s t o r e q u i r e a stable

configurational

change i n t h e

coinciding w i t h glycosylation,

precursor

protein

rather than d i r e c t p a r t i c i p a t i o n of

carbohydrate i n the antigenic site.

Thus l a t e r e n z y m i c r e m o v a l o f

c a r b o h y d r a t e c h a i n s does n o t a l t e r r e a c t i v i t y w i t h t h e a n t i b o d i e s . Two m e a s l e s v i r u s g l y c o p r o t e i n s ,

a haemagglutinin and a f u s i o n

p r o t e i n , have been i n c o r p o r a t e d i n t o a r t i f i c i a l l i p i d b i l a y e r s . 7 3

177

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides The

resultant

'virosomes'

haemagglutinating

had

activity,

visible

and

should

'spikes'

and

provide a

s t u d y i n g immune responses t o m e a s l e s v i r u s ,

possessed

reagent

for

independent o f the

immunosuppressive e f f e c t s o f t h e whole v i r u s . M o n o s p e c i f i c a n t i b o d i e s have been p r e p a r e d t o each o f e n v e l o p e p a r a m y x o v i r u s g l y c o p r o t e i n s (HN a n d F ) . 7 4

two

Antibodies t o

t h e HN g l y c o p r o t e i n i n h i b i t e d haem a g g l u t i n a t i n g a n d neuram i n i d a s e activities.

A n t i - F a n t i b o d i e s i n h i b i t e d haemolysis.

The c o m p l e t e n u c l e o t i d e s e q u e n c e o f t h e g l y c o p r o t e i n g e n e i n r a b i e s v i r u s has been d e t e r m i n e d . 7 5 to

be!

made o f

several

T h i s has a l l o w e d a p r e d i c t i o n

features o f

the glycoprotein,

from

the

deduced a m i n o a c i d sequence. The e f f e c t s o f c e l l t r a n s f o r m a t i o n o n t h e g l y c o s y l a t i o n a n d p r o c e s s i n g o f P r a g u e C Rous s a r c o m a v i r u s e n v e l o p e p o l y p e p t i d e s have been r e p o r t e d . 7 6 the

The m a j o r d e t e c t a b l e d i f f e r e n c e i s a n i n c r e a s e i n

r e l a t i v e amount

o f

larger acidic-type

containing residues o f neuraminic acid, 2-deoxy-~-glucose,

oligosaccharides

a-galactose, and 2-acetamido-

compared w i t h t h e n e u t r a l - t y p e o l i g o s a c c h a r i d e s .

G l y c o p e p t i d e s d e r i v e d from

a g l y c o p r o t e i n (mol.

been e x a m i n e d a f t e r s e q u e n t i a l

glycosidase

wt.

8.5

digestions

x lo4) h a v e followed

by

g e l f i l t r a t i ~ n . ~The ~ major h y b r i d species has an oligo-Q-mannosyl c o r e o f f i v e Q-mannosyl- and t w o 2-acetamido-2-deoxy-Q-glucosyl residues together

with

substitution

residues w i t h an a c i d i c s i d e a c e t y l n e u r a mi n i c a c i d ,

of

one

of

c h a i n composed o f

the

p-mannosyl

residues o f

N-

Q - g a l a c t o p y r a n o s e , a n d 2-acetam i d o -2-deox y-Q-

glucopyranose. Immunological s t u d i e s on t h e r e l a t i o n s h i p o f t h e major envelope g l y c o p r o t e i n o f HEL-12

v i r u s and t h e analogous g l y c o p r o t e i n s o f

S i m i a n sarcoma-Simian a s s o c i a t e d v i r u s have

been reported.78

A

a n d baboon endogenous v i r u s

glycopeptide

(mol.

w t .

2.0

x

lo5)

a s s o c i a t e d w i t h t h e t r a n s f o r m a t i o n o f S i m i a n sarcoma v i r u s h a s been

.

iden t i f i e d 79 A g l y c o p r o t e i n s p e c i f i e d by s p l e e n f o c u s - f o r m i n g v i r u s i n t h r e e

c e l l l i n e s v a r i e s i n m o l e c u l a r s i z e and p e p t i d e c o m p o s i t i o n b u t retains

both x e n o t r o p i c and e c t o t r o p i c murine leukaemia v i r u s

antigenicity.80

Both q u a l i t a t i v e and q u a n t i t a t i v e d i f f e r e n c e s i n

the post-translational

processing o f t h e envelope g l y c o p r o t e i n s o f

p o l y c y t h a e m i a - and a n a e m i a - i n d u c i n g s t r a i n s o f s p l e e n f o c u s - f o r m i n g v i r u s have been r e p o r t e d . 8 1 The i n v i t r o t r a n s l a t i o n o f U n k u n i e m i v i r u s - s p e c i f i c RNA h a s led to

the identification o f a non-structural

p r o t e i n and a


178 p r e c u r s o r t o membrane g l y co p r o t e i n s . 82 Vaccinia

wt.

(mol.

virus-induced haemagglutinin i s a single glycoprotein

lo4>

x

8.5

v i r us - i n d u c e d

w h i c h a p p e a r s a t t h e p l a s m a membrane a s a

function. 83

l-lin k e d

A 1 t h ough

predominate i n the molecule,

o 1i g o s a c c h a r i de s

a b o u t 25% o f t h e o l i g o s a c c h a r i d e c h a i n s

which a r e g - g l y c o s i d i c a l l y l i n k e d t o t h e p r o t e i n a r e r e s p o n s i b l e f o r the

biological

activity

of

the

molecule.

Glycoproteins

and

p h o s p h o p r o t e i n s have been i d e n t i f i e d a s s t r u c t u r a l e l e m e n t s o f t h i s

.

v i r u s B4 wt.

Immunogenic g l y c o p r o t e i n s (mol. 1.18

x l o 5 ) i s o l a t e d from

varicella-zoster

lo4,

x 104,and

9.8

vaccine

strains

of

v i r u s have been i d e n t i f i e d . 8 5

I n r e c o n s t i t u t i o n experiments E l and E 2 o f

x

6.5

l a b o r a t o r y and

u s i n g t h e envelope

western equine e n c e p h a l i t i s virus,

glycoproteins

the haemolytic

a c t i v i t y i s associated w i t h the E l glycoprotein which a c t s as a haemaggl u t i n i n . 86

Plant Glycoproteins

2 A

l a r g e number o f l e g u m e - s e e d

glyc~proteins.~’ A glycoproteins functional

high

and t h e i r

significance

p o l y p e p t i d e s have been i d e n t i f i e d a s

of

degree

corresponding

binding lectins

between

might

i n m a i n t a i n i n g l a r g e aggregates

have of

seed some

protein

i n compact i n s o l u b l e f o r m . Twelve N - g l y c o s y l a t e d p o l y p e p t i d e s have been i d e n t i f i e d i n glyoxysomal

membranes

p o l y a c r y lam i d e w t s . 9.0

x

of

castor

electrophoresis

lo4,

7.1

x

lo4,

A glycoprotein,

5.6 x

beans.88

correspond

lo4,

4.7

to

Major

bands o n SDS-

g l yco p r o t e i n s o f m 01.

x 1 0 4 , a n d 4.3

x

lo4.

involved w i t h the control of i n t e r c e l l u l a r

r e c o g n i t i o n and o f p o l l e n i n c o m p a t i b i l i t y has been i s o l a t e d f r o m stigmas o f glycoprotein,

S-allele

g e n o t y p e S2S2

of

Brassica oleracea.89

w h i c h i s n o t p r e c i p i t a t e d by c o n c a n a v a l i n A,

r e s i d u e s o f L-arabinose,

!-galactose,

Evidence t h a t l i p i d - l i n k e d a s e q u e n t i a l manner f r o m

!-glucose,

The

contains

a n d P-mannose.

o l i g o s a c c h a r i d e s can be a s s e m b l e d i n

UDP - 2 - a c e t a m i d o -2-deox y - B - g l uco se a n d GDP -

P-mannose by membrane p r e p a r a t i o n s f r o m d e v e l o p i n g s o y b e a n c o y l e d o n s has

been p r e s e n t e d . ”

Tissue

slices

synthesize products which

resemble t h e completed 7 s storage g l y c o p r o t e i n s . Concanavalin

A

has

been shown

to

bind to

the

endoplasmic

r e t i c u l u m and t h e s t a r c h g r a i n s u r f a c e o f r o o t s t a t o c y t e s ( L e p i d i u m s a t i v u m L9).91

of

cress

The r e c e p t o r f o r t h e l e c t i n a p p e a r s t o be a

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides

179

high-molecular- weight glycoprotein which is r e s i s t a n t t o s o l u b i l i t a t i o n a f t e r g l u t a r a l d e h y d e f i x a t i o n of t h e cells. Two s o l u b l e g l y c o p r o t e i n c o m p o n e n t s f r o m r y e g r a s s ( L o l i u m p e r e n n e ) have been i s o l a t e d a n d p a r t i a l l y c h a r a ~ t e r i z e d . ~ ’ They a r e c h e m i c a l l y and i m m u n o l o g i c a l l y d i s t i n c t . One o f t h e g l y c o p r o t e i n s i s t h e m a j o r a l l e r g e n o f r y e - g r a s s p o l l e n and b i n d s s p e c i f i c a l l y t o i m m u n o g l o b u l i n E o f s e n s i t i z e d human s u b j e c t s . 9 3 Immunological c r o s s - r e a c t i v i t y t h a t i s d u e , a t l e a s t i n p a r t , t o common a n t i g e n i c carbohydrate components i s reported. Two t y p e s o f p r o t e i n g l y c o s y l a t i o n i n c e l l - f r e e p r e p a r a t i o n s o f d e v e l o p i n g c o t y l e d o n s o f P h a s e o l u s v u l g a r i s have been p o ~ t u l a t e d . ~ ~ I n a n i n v i t r o s y s t e m , t h e a t t a c h m e n t o f 2-acetamido-2-deoxy-Qglucosyl r e s i d u e s t o phytohaemagglutinin and phaseolin does n o t involve lipid-linked intermediates w h i l s t the glycosylation of a class of p r o t e i n s of heterogeneous molecular weights involves the l i p i d pathway. A q u e o u s e x t r a c t s o f l e a v e s , r o o t s , a n d stems o f g r e e n b e a n , a n d l e a v e s o f mung b e a n , s o y b e a n , a n d l i m a b e a n , c a u s e a g g l u t i n a t i o n o f c e l l s o f a s a p r o p h y t i c b a c t e r i u m P s e u d o m o n a s p ~ t i d a . P~u ~r i f i e d p r e p a r a t i o n s of t h e a g g l u t i n i n are composed of g l y c o p r o t e i n s , containing k-arabinose, e-galactose, and e-galacturonic a c i d as t h e p r e d o m i n a n t c a r b o h y d r a t e c o m p o n e n t s . The p r o p e r t i e s o f l a c c a s e , i s o l a t e d from S c h i n u s m o l l e trees, i n c l u d i n g m o l e c u l a r w e i g h t , amino a c i d and c a r b o h y d r a t e c o m p o s i t i o n , have been d e ~ c r i b e d . ’ ~ The presence o r absence o f an inducing hormonal glycoprotein i n t h e g r o w t h medium o f Volvox l a r t e r i d e t e r m i n e s w h e t h e r t h e algaegrow sexually or asexually.97 The s i t e and time o f f o r m a t i o n o f t h i s g l y c o prot e i n h a v e been est a b l i shed.

3

Lectins

Review a r t i c l e s d e a l i n g w i t h l e c t i n s i n c l u d e a s t u d y of l e c t i n s i n The endogenous b i o l o g i c a l f u n c t i o n s o f l e c t i n s higher plants.98 have b e e n r e ~ i e w e d . ’ ~ E x a m p l e s f r o m p l a n t s , c e l l u l a r slime moulds, The a n d a n i m a l t i s s u e s are u s e d i n t h e a n a l y s i s of t h i s problem. c a r b o h y d r a t e b i n d i n g s p e c i f i c i t y and c e l l - membrane i n t e r a c t i o n o f Vicia s a t i v a l e c t i n have b e e n r e p 0 r t e d . l ” Affinity chromatography f o r t h e p u r i f i c a t i o n o f l e c t i n s h a s been described.lo1 Stable, high c a p a c i t y a f f i n i t y adsorbents used i n t h e i s o l a t i o n o f l e c t i n s have b e e n p r e p a r e d by d e r i v a t i z a t i o n o f e p o x y - a g a r o s e w i t h v a r i o u s

--


180 carbohydrates .Io2 Isoelectric

focusing

i n polyacrylarnide

gels

containing

immobilized sugars r e s u l t s i n a s h i f t i n the p o s i t i o n o f p r o t e i n bands c a p a b l e o f

i n t e r a c t i o n w i t h these ligands.lo3

These a f f i n i t y

g e l s have been u s e d t o d e m o n s t r a t e i n t e r a c t i o n s w i t h l e c t i n s . s e n s i t i v e assay

A

has been d e v e l o p e d f o r m e a s u r i n g t h e b i n d i n g o f

l e c t i n s t o g l y c o c o n j u g a t e s i n w e l l s of p o l y v i n y l c h l o r i d e ~ 1 a t e s . l ' ~ A chromophore-labelled

Q - g a l a c t o - Q - m a n n a n h a s b e e n p r e p a r e d by

c o u p l i n g a m o n o c h l o r o t r i a z i n y l dye t o guaran.lo5

The p r o d u c t h a s

been u s e d f o r t h e q u a n t i t a t i v e e s t i m a t i o n o f l e c t i n s h a v i n g a f f i n i t y f o r Q - g a l a c t o s y l o r 2-acetam ido-2-deox y - Q - g a l a c t o s y l r e s i d u e s . The f o l l o w i n g l e c t i n s h a v e b e e n p u r i f i e d a n d some o f t h e i r p h y s i c a l a n d c h e m i c a l c h a r a c t e r i s t i c s r e c o r d e d ( T a b l e 1) . l o 6 - l 1 4 I o d i n a t e d l e c t i n s have

been u s e d f o r

the determination o f the

d e g r e e of d e g l y c o s y l a t i o n o f h i g h n - m a n n o s y l g l y c o p r o t e i n s f o l l o w i n g digesti o n w i t h e

- B -2-ace tam i d o -2-deox y - E - g l ucanase H. '15 I n t e r m o l e c u l a r f o r c e s i n l e c t i n- c a r boh y d r a t e in t e r a c t i o n h a v e

been analysed on features.'l6

the

basis o f

their

s t r u c t u r e and chemical

The r o l e o f w a t e r , a s w e l l a s t h a t o f o t h e r p h y s i c o -

c h e m i c a l p a r a m e t e r s i n f l u e n c i n g t h e f o r m a t i o n o f such c o m p l e x e s i n aqueous m e d i a ,

i s a l s o discussed.

A model d e p i c t i n g t h e importance

o f hydrogen bonding and charge-transfer

i n t e r a c t i o n s as t h e main

s o u r c e s o f complex s t a b i l i t y i n t h e a s s o c i a t i o n between l e c t i n s and carbohydrates i s proposed. By p e r f o r m i n g e l e c t r o p h o r e s i s p e r p e n d i c u l a r t o a s t a t i o n a r y pH gradient i n polyacrylamide gels containing a s p e c i f i c l i g a n d e i t h e r covalently f i x e d o r entrapped i n the gel matrix, i t i s possible t o measure d i s s o c i a t i o n c o n s t a n t s o f l e c t i n s a n d t h e i r pH dependence i n a pH r a n g e . l l 7

The m e t h o d h a s b e e n u s e d t o s t u d y t h e l e c t i n s f r o m

R i c i n u s communis a n d L e n s c u l i n a r i s . Aqueous e x t r a c t s f r o m l e a v e s , l e a v e s o f mung bean, saprophytic

r o o t s , a n d s t e m s o f g r e e n bean a n d

soybean,and l i m a bean a g g l u t i n a t e c e l l s o f a putida."

Purified

p r e p a r a t i o n s c o n t a i n b o t h c a r b o h y d r a t e and p r o t e i n ,

r e q u i r e Mg2+ f o r

activity,

bacteria,

Pseudomonas

and a r e h e a t s t a b l e .

pathogenic bacteria

No a g g l u t i n a t i o n o f

A s u r v e y o f t w e n t y - t w o commonly i n g e s t e d f r u i t s , seeds has i n d i c a t e d the presence, interact cells.l19

with

other

plant

or nonpseudomonad s a p r o p h y t e s was o b s e r v e d . i n many,

vegetables, and

o f l e c t i n s w h i c h can

c o m p o n e n t s o f human s a l i v a a n d S t r e p t o c o c c u s

I n t e r a c t i o n s o f soybean and p e a n u t l e c t i n s w i t h

t h a t n o d u l a t e soybean,

peanut, o r

mutans

rhizobia

b o t h p l a n t s have b e e n studied."'


0

0

0

0

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U

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0

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M

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182

Carbohydrate Chemisrry

No r e l a t i o n s h i p b e t w e e n t h e a b i l i t y o f p e a n u t o r soybean r h i z o b i a t o n o d u l a t e t h e r e c i p r o c a l h o s t p l a n t and t h e i r a b i l i t y

to bind to

the

l e c t i n o f t h a t p l a n t c o u l d be d e m o n s t r a t e d . The seeds o f P i s u m s a t i v u m ,

-V.

Canavalia ensiformis,

-s a t i v a , a n d R i c i n u s c o m m u n i s c o n t a i n p r o t e i n s ,

Vicia

faba,

termed l e c t i n

binders, which a r e associated w i t h t h e i r r e s p e c t i v e lectins.lZ1

The

p e a l e c t i n b i n d e r i s a t e t r a m e r i c g l y c o p r o t e i n composed o f i d e n t i c a l

w t. 5.1

s u b u n i t s (mol.

x

lo4),

and i t s i n t e r a c t i o n w i t h t h e l e c t i n i s

a b o l i s h e d by l o w pH o r by g - g l u c o s e . w t .

p r o t e i n (mol.

3.5

x

lo4)

The c o n c a n a v a l i n A b i n d e r i s a

c o n t a i n i n g no

covalently

bound

carbohydrate.

A s t u d y o f t h e t i s s u e s o f s o y b e a n a n d j a c k bean s e e d s

demonstrates

t h a t t h e l e c t i n o f a p a r t i c u l a r s p e c i e s may e x h i b i t a

very

narrow

range

glycoconjugates.’**

o f

s p e c i f i c i t y

towards

endogenous

This suggests t h a t n o t o n l y i s t h e r e one m a j o r

l e c t i n i n the cotyledons o f a given plant

b u t a l s o one s o l u b l e

g l y c o p r o t e i n i n t h e c o t y l e d o n s t h a t t h e l e c t i n can r e c o g n i z e .

The

s t r i k i n g s p e c i f i c i t y shown b e t w e e n h o m o l o g o u s l e c t i n a n d r e c e p t o r i s n o t m a i n t a i n e d when t h e l e c t i n i s u s e d t o . i s o l a t e g l y c o p r o t e i n s f r o m t h e same t i s s u e i n o t h e r p l a n t s p e c i e s .

G 1 y co p e p t ide s a n d o l igo s a c c h a r id e s de r ive d f r o m proteins

have

been

different lectins.lZ3

used

g l ycoaspa r a g i ne s ,

define

the

1-g l y co s y 1

specificity

of

twelve

Lectins considered i d e n t i c a l i n terms of

monosaccharide s p e c i f i c i t y d i f f e r e n c e s i n more

to

possess an a b i l i t y

complex

structures.

to recognize fine

The

observation

that

g l yco pe p t i d e s, a n d g l yco p r o t e i ns p o s s e s s a h i g h e r

a f f i n i t y f o r l e c t i n s t h a n t h e r e l a t e d o l i g o s a c c h a r i d e s has been e x p l a i n e d by t h e f a c t t h a t t h e g l y c a n - a m i n o a c i d l i n k a g e l e a d s t o s t r u c t u r e s more r i g i d t h a n t h o s e o f t h e o l i g o s a c c h a r i d e s themselves. Many o f

t h e l e c t i n s u s e d i n t h i s s t u d y seem t o p o s s e s s i n

o r near

t h e i r carbohydrate-binding s i t e a hydrophobic area such as t h a t d e s c r i b e d f o r c o n c a n a v a l i n A,

a n d t h i s c o u l d be t h e c a u s e o f n o n -

s p e c i f i c hydrophobic i n t e r a c t i o n s o f glycopeptides

Fluorescence b i nd i ng o f

between t h e l e c t i n s and r e s i d u e s

o r g l y c o p r o t e i n s , a s d e s c r i b e d f o r c o n c a n a v a l i n A. p o l a r i z a t i o n has been u s e d t o i n v e s t i g a t e t h e

4-m e t h y 1um be 11if e r y 1 - 6 -Q -ga 1a c t o p y r a n 0 s i de

p r e c at o r i u s a g g l ut i n i n

. 24

to

A b r us

L e c t i n a c t i v i t y has been d e t e c t e d i n seeds o f t w e n t y A c a c i a species o f A u s t r a l i a n origin.125

Fourteen of

the

species

had been

r e p o r t e d by o t h e r w o r k e r s t o c o n t a i n no l e c t i n . P e a n u t l e c t i n has been p u r i f i e d by a d s o r p t i o n o n g l u t a r a l d e h y d e cross-linked

h e m a t i c d e s i a l y l a t e d a n d r e t i c u l a t e d human s t r o m a . l Z 6

183

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides The

lectin

(mol.

1.25

wt.

lo5),

x

although

homogeneous on

e l e c t r o p h o r e s i s , y i e l d s e i g h t m a j o r bands o n i s o e l e c t r i c f o c u s i n g . The s c r e e n i n g o f p e a n u t ( A r a c h i s ) a n d i t s w i l d r e l a t i v e s f o r m u l t i p l e m o l e c u l a r f o r m s o f p e a n u t l e c t i n and f o r g e n o t y p e s d e v o i d of

the

b e e n r e ~ 0 r t e d . l ~A ~ p e a n u t

l e c t i n has

lectin-negative

p h e n o t y p e was d e t e c t e d i n f o u r g e n o t y p e s ( i s o l a t e d from

h a s been a n a 1 ~ s e d . l ~The ~ amount o f e a c h

i s o l e c t i n v a r i e s f r o m seed t o seed, each seed d i s p l a y i n g a u n i q u e distribution of the five isolectins. intact

s imp 1i c i f o 1i a -B. --

cleavage

by

an endo-

It i s postulated that the

I B4 i s o l e c t i n u n d e r goes p r o t e o l y t i c

or =-protease

t o

form

f i r s t t h e AB3

i s o l e c t i n , w h i c h i n t u r n i s t h e n p r o c e s s e d f u r t h e r t o y i e l d t h e A2B2

184


isolectin,

and so o n u n t i l A4

i s produced.

4-Methylumbelliferyl

g l y c o s i d e s have been used i n b i n d i n g s t u d i e s w i t h t h e l e c t i n s A4,

BSI-B4,

and BS I 1

from

d i a l y s i s,

Equilibrium

Bandeiraea ( G r i f f o n i a )

fluorescent

BSI-

simplicif01ia.l~~

enhancement,

quantitative

p r e c i p i t a t i o n , and h a p t e n i n h i b i t i o n t e c h n i q u e s have been used t o investigate

the

simplicifolia

binding

characteristics

of

four

of

the

2.

I i s 0 1 e c t i n s . l ~ ~One o f t h e m o s t u n u s u a l f e a t u r e s i s

that,although

different,

b o t h t h e A a n d B s u b u n i t s h a v e t h e same

f o r a-Q-galactosyl residues. Although both s u b u n i t s a l s o b i n d 2 - a c e t a m i d o - 2 - d e o x y - ~ - g a l a c t o s y l r e s i d u e s , t h e A s u b u n i t has a n

affinity

association constant

f o r t h e amino sugar more t h a n 1 0 0 0 - f o l d g r e a t e r

t h a n the B s u b u n i t . Affinity

chromatography

i m m o b i l i z e d 4-aminophenyl means of

equations

of

divalent

B-P-glucopyranoside

concanavalin

A

on

h a s been a n a l y s e d by

based o n t h e s i m p l e s t m o d e l ,

i n which t h e l e c t i n

has e q u i v a l e n t a n d i n d e p e n d e n t b i n d i n g s i t e s a n d one l e c t i n m o l e c u l e b i n d s o n l y o n e i m m o b i l i z e d l i g a n d a t a time.135 P o l y a c r y 1am i d e

e l e c t rophore s i s o f

gra d i ent

r e v e a l s the presence o f

various minor

co n c a n a V a l i n A

bands w h i c h a r e n o t

detected

by p o l y a c r y l a m i d e d i s c e l e c t r o p h ~ r e s i s . ~The ~ ~ m i n o r components a r e suggested t o the

combined

s iev i ng

.

be d i f f e r e n t m o l e c u l a r s p e c i e s s e p a r a t i n g by v i r t u e o f effect

of

electrophoretic

mobility

Over w i d e r a n g e s o f t e m p e r a t u r e a n d pH, of only subunits

and m o l e c u l a r

concanavalin A consists

d i m e r s a n d t e t r a m e r ~ . ' ~ ~The l a r g e f r a c t i o n i n commercial preparations

o f hydrolysed

causes s i g n i f i c a n t

populations

o f d i m e r i c species t h a t a s s o c i a t e o n l y weakly or n o t a t a l l .

The

e f f e c t o f t h e b i n d i n g o f saccharide l i g a n d s on t h e r e v e r s i b l e dimert e t r a m e r e q u i l i b r i u m o f t h e l e c t i n has b e e n s t u d i e d by h i g h - s p e e d s e d i m e n t a t i o n e q ~ i 1 i b r i u m . l ~C~o n t r a r y

to

e a r l i e r p u b l i s h e d work,

s a c c h a r i d e b i n d i n g does n o t a p p e a r t o a f f e c t e q u i l i b r i u m o f t h e n a t i v e c o n c a n a v a l i n A, irreversible

behavio ur

i n

preparations

proportions o f hydrolysed subunits.

the

dimer-tetramer

a l t h o u g h i t does i n t r o d u c e containing

sign1f icant

The d i m e r - t e t r a m e r e q u i l i b r i u m

i s l i n k e d t o t h e weak b i n d i n g o f c a l c i u m a t a s i t e p r o b a b l y i n t h e dimer-dimer

interface.

interaction

of

Studies

concanavalin

A

have

with

g l u c o py r a no s y 1- a - e - m a n n o p y r a n o s i de ga l a c t o p y r a n 0 s y l - a - D = - m a n n o p y m a 1 t o ~ i d e . l ~T h ~e disaccharides

i s

existence

proposed.

,

been

Some o f

two the

the

2-2-a-P-

4 - n i t r o p h e n y 1- 2 - 2 - a - Q -

r a n 0 s i de, of

reported of

4-nitrophenyl and

4-ni trophenyl

b i n d i n g modes specificity

for

the

requirements

185


f o r t h e i n t e r a c t i o n o f concanavalin A w i t h t h e non-reducing g l y c o s y l group are characterized, Temperature-jump

and a r e s u m m a r i z e d i n Scheme 1.

relaxation studies using 4-methylumbelliferyl

a-Q-mannopyranoside as a f l u o r e s c e n c e i n d i c a t o r l i g a n d have been used i n an e x a m i n a t i o n o f

the

binding kinetics o f

m e t h y l a-Q-

m a n n o p y r a n o s i d e t o c o n c a n a v a l i n A. 140

Scheme 1 An

i.r.-attenuated

total-reflectance

s p e c t r o s c o p i c method has

been u s e d t o s t u d y t h e i n t e r a c t i o n o f m o n o l a y e r s o f c o n c a n a v a l i n A The e f f e c t s o f f i l m

w i t h mono- and p o l y - s a c c h a r i d e s . 1 4 1 pH,

urea,

Mn2+, a n d Ca2+ o n t h e b i n d i n g o f d e x t r a n ,

pressure,

m e t h y l a-Q-

mannopyranoside, and Q - g a l a c t o s e t o t h e l e c t i n w e r e s t u d i e d . thermodynamic parameters o f t h e Mn2+-binding i n the

concanavalin A

dimer

c a l o r i m e t r i c technique.142

state

have

been

The

reaction with

determined

The r e s u l t s i n d i c a t e t h a t

by

a

the free

e n e r g y c h a n g e f o r t h e p r o c e s s i s d o m i n a t e d by a p o s i t i v e e n t r o p y change

and

that

independent. the

binding of

process

i s

effects.

the

two

i d e n t i c a l S1 s i t e s

of

the

dimer

ar,e

C a l o r i m e t r i c s t u d i e s have a l s o been used t o examine saccharides

induced

by

Kinetic

activation of

both

and

to

concanavalin

favourable

equilibrium

Ca2+ i n d i c a t e t h a t

The

A.143

enthalpic

studies

of

and

binding entropic

concanavalin

ion binding at

A

S2 produces a

general ordering o f the ligands a t t h i s s i t e which i n t u r n orders those side-chain residues involved i n saccharide r e ~ o g n i t i 0 n . l ~ ~ The c a r b o h y d r a t e - b i n d i n g

activity

v a r i o u s l e v e l s o f Ca2+ a n d Mn2+

of

concanavalin

has

A containing

b e e n r e ~ 0 r t e d . l ~T h~e

p r e c i p i t a t i o n a c t i v i t y o f t h e l e c t i n w i t h g l y c o g e n as w e l l as t h e binding of affected

lectin to

4-nitrophenyl

a-p-mannopyranoside

by t h e n u m b e r o f b o u n d C a 2 + - i o n s .

are strongly

The k i n e t i c s o f t h e

i n t e r a c t i o n s o f Ca2+ w i t h c o n c a n a v a l i n A i n t h e p r e s e n c e o f C o 2 + , Mn2+, and/or of

Zn2+ h a v e been f o l l o w e d by m e a s u r e m e n t o f t h e q u e n c h i n g

fluorescence of

4-methylumbelliferyl

bound t o ~ r 0 t e i n . l ~Some ~

observations

a-a-mannopyranoside on

metal-ion

when

requirements

a n d s u g a r b i n d i n g by B a n d e i r a e a s i z p l i c i f o l i a I l e c t i n a r e a l s o reported. The c o - o p e r a t i v e

binding properties o f concanavalin A with

186


g l y c o c o n j u g a t e s a r e dependent o n t h e p r e s e n c e o f h y d r o p h o b i c b i n d i n g sites.14'

The d e g r e e o f c o - o p e r a t i v i t y

can be s u b s t a n t i a l l y a l t e r e d

by c o n f o r m a t i o n a l changes o f t h e l i g a n d . Irradiation of

concanavalin A

tetrameric

with

a high-pressure

me r c u ry lamp i n t h e presence o f c h l o r a c e t a m i d e r e s u l t s i n t h e l e c t i n showing a monomeric m o l e c u l a r w e i g h t w i t h r e t e n t i o n o f b i n d i n g capacity.148 Fluorescein-concanavalin

conjugates

A

are

capable

of

d i s t i n g u i s h i n g b e t w e e n n o r m a l and m a l i g n a n t human ~ e 1 l s . l ~ ' The f o u r

-V i c i a faba

l e c t i n s c o n c a n a v a l i n A,

agglutinin,

Lens c u l i n a r i s a g g l u t i n i n ,

and P i s u m s a t i v u m

be i d e n t i c a l i n t e r m s o f s p e c i f i c i t y

agglutinin,

considered t o

or a-;-glucose,

f o r a-;-mannose

p o s s e s s t h e a b i l i t y t o r e c o g n i z e f i n e d i f f e r e n c e s i n more c o m p l e x carbohydrate structures.150 A

lectin

from

the

a g g l u t i n a t i n g sheep haemolysis

of

seeds

and o t h e r

rabbit

of

Croton

tiglium

erythrocytes

erythrocyte^.'^^

i s

as w e l l

These e f f e c t s

capable as

of

inducing

are i n h i b i t e d

by sheep e r y t h r o c y t e g l y c o p e p t i d e s . D a t u r a s t r a m o n i u m l e c t i n has been p u r i f i e d on c o l u m n s o f diacetylchitobiose

i m m o b i l i z e d on a g a r o s e . l o 6

37% c a r b o h y d r a t e ,

sugar,

of

which I-arabinose

and a h i g h c o n t e n t o f

hydroxy-C-proline i d e n t i c a l with,

residues. the

lectin

i s t h e most

C-cysteine,

glycine,

potato.152

predominant I - s e r i n e , and but not

Although they

chito-oligosaccharides,

N,"-

contains

This l e c t i n i s s i m i l a r to,

l e c t i n from

similar specificities for

The

have

the Datura l e c t i n

shows i t s g r e a t e s t a f f i n i t y f o r g l y c o p e p t i d e s and t h e p o t a t o l e c t i n

for chito-oligosaccharides.

I n both lectins,

the hydroxy-l-proline

residues are substituted w i t h 6-i-arabinofuranosides,

and t h e r e a r e

L-serine residues i n the glycosylated region that are substituted with

a-4-galactopyranosyl

residues.

The

structure

of

the

glycosylated r e g i o n o f the l e c t i n s i s very s i m i l a r t o t h a t o f the hydroxy-&-proline-rich

glycopeptides of plant c e l l walls,

and t h e s e

l e c t i n s c o u l d be p r e c u r s o r s o f s u c h m a t e r i a l . Metal-chelate

affinity

chromatography

p u r i f i c a t i o n o f the Dolichos b i f l o r u s

has been used i n t h e

s e e d 1 e ~ t i n . l ~E q ~u i l i b r i u m

d i a l y s i s s t u d i e s show t h a t t h e l e c t i n h a s t w o c o m b i n i n g s i t e s p e r ma l e c u l e f o r me t h y 1 2-ace t am i d o -2-deoxy - a - g a l a c t 0 s i d e

. 54

Subunit I

i s p r i m a r i l y r e s p o n s i b l e f o r t h e c a r b o h y d r a t e - b i n d i n g p r o p e r t i e s of the lectin.

Monoclonal antibodies s p e c i f i c f o r subunit I of

the

D o l i c h o s b i f l o r u s l e c t i n combine w i t h t h e C - t e r m i n a l p o r t i o n o f t h e subunit

a n d may

be i n t e r a c t i n g w i t h

the

active

site.155

This

187

5: Glycoproteins, Glycopeptides, Proteoglytans, and Animal Polysaccharides

a n t i b o d y does n o t r e a c t w i t h a n o t h e r l e c t i n - l i k e p r o t e i n f r o m t h e s t e m s and l e a v e s o f t h e p l a n t . 1 5 6

The m o n o c l o n a l a n t i b o d y i s o f

i m p o r t a n c e i n t h a t i t can d i s t i n g u i s h s u b u n i t I f r o m s u b u n i t I 1 o f t h e seed l e c t i n . one a n o t h e r

A l t h o u g h t h e s e t w o s u b u n i t s appear t o d i f f e r f r o m

only at

their

C-terminal

I 1 i s not.157

whereas s u b u n i t

Atomic

ends,

subunit

I i s active

absorption spectrophotometry

h a s e s t a b l i s h e d t h e p r e s e n c e o f Ca2+, Mg2+, Mn2+, Zn2+, a n d Cu2+ i n native golichos biflorus subsequent constant

addition of

1 e ~ t i n . l ~ The ~ e f f e c t s o f r e m o v a l and

different

o f the l e c t i n for

m e t a l i o n s on t h e a s s o c i a t i o n are

2-acetamido-2-deoxy-q-galactose

reported. The l e c t i n o f E r y t h r i n a c o r a l l o d e n d r o n s e e d s i s s i m i l a r t o soybean l e c t i n ,

being a glycoprotein

(mol.

1.1 x

wt.

lo5) and

2-acetamido-2-deoxy-qbinding t o 2-amino-2-deoxy-Q-galactose, g a l a c t o s e , a- and B - p - g a l a c t o s i d e s , a n d g - g a l a c t o s e . 1 5 9

-

l e c t i n i s o l a t e d from

A

the roots o f

soybean ( G l y c i n e

!ax)

s e e d l i n g s i s s i m i l a r b u t n o t i d e n t i c a l t o t h e c o r r e s p o n d i n g seed The l e c t i n i s a s s o c i a t e d w i t h t h e o u t e r s u r f a c e s o f t h e

lectin.16'

r o o t and i s c o n c e n t r a t e d i n t h e segments o f t h e r o o t a t w h i c h r o o t h a i r and e a r l y secondary r o o t s a r e observed. and

s o l u b l e soybean l e c t i n , i s of

From e x a m i n a t i o n o f

&

g e n o t y p e s o f G l y c i n e max f o r m e m b r a n e - b o u n d a n d b u f f e r i t seems u n l i k e l y t h a t t h e membrane l e c t i n

c y t o p l a s m i c origin.161

5gJa)

(slycine

I n contrast

l e c t i n

has

erythroagglutinating activity

been

after

t o o t h e r r e p o r t s soybean shown

complete

t o

r e t a i n

removal o f

i t s

Mn2+ f r o m

t h e w h o l e molecule.162 The l e n t i l

subunit

(Lens

s t r u c t u r e and c o m p l e t e a m i n o a c i d s e q u e n c e

cglinaris)

l e c t i n have

been

determined.163

of A

c o m p a r i s o n b e t w e e n t h e s e c o n d a r y s t r u c t u r e o f c o n c a n a v a l i n A and t h e p r o b a b l e s e c o n d a r y s t r u c t u r e o f l e n t i l l e c t i n a s p r e d i c t e d by t w o different

methods

indicates

that

the

folding

of

these

two

p o l y p e p t i d e s has been w e l l c o n s e r v e d d u r i n g e v o l u t i o n . I n a re-examination specifities

of

of

the h i g h - a f f i n i t y

pea and l e n t i l l e c t i n s ,

the

carbohydrate-binding ability

ofvarious

g l y c o p e p t i d e s t o b i n d t o c o l u m n s o f t h e i m m o b i l i z e d l e c t i n s was studied.164

I-Fucose

r e s i d u e s a r e i n d e e d an i m p o r t a n t

determinant

i n t i g h t b i n d i n g t o pea and l e n t i l l e c t i n b u t n o t t o c o n c a n a v a l i n A. The l e c t i n f r o m M a c l u r a p o m i f e r a , a f t e r p u r i f i c a t i o n by i o n exchange and a f f i n i t y

c h r o m a t o g r a p h y , has been r e s o l v e d i n t o f i v e

s t r u c t u r a l l y r e l a t e d p r o t e i n components o f s i m i l a r h a e m a g g l u t i n a t i n g and c a r b o h y d r a t e - b i n d i n g

activity.165

P u r i f i c a t i o n o f the l e c t i n


188

h a s a l s o been a c h i e v e d by a d s o r p t i o n o n t o p o l y l e u c y l h o g A+H b l o o d f o l l o w e d by e l u t i o n w i t h m e l i b i o s e o r 2 - a c e t a m i d o -

group substances,

2-deoxy-;-galactose.166 104,and 1.2

Three s u b u n i t s (mol.

wt.

1.45

interaction i s important combining

site

of

lo4,

x

x lo4) are present i n the i n t a c t lectin.

1.35

f o r b i n d i n g o f t h e l e c t i n t o sugars.

the

lectin

appears

to

be

x

Hydrophobic

as

large

The as

a

disaccharide. The i n t e r a c t i o n o f been s t u d i e d . 1 6 7 g r o u p s a t t h e C-2,

I w i t h c a r b o h y d r a t e has

mistletoe lectin

I n h i b i t i o n data suggest

that

unmodified hydroxyl

C-3, a n d C-4 p o s i t i o n s o f t h e Q - g a l a c t o p y r a n o s y l

r i n g are essential f o r binding t o the active s i t e o f the l e c t i n . The ligands

association with

the

c h a r a n t i a have of

protein

constants

for

the

P-galactose-specific

been d e t e r m i n e d t h r o u g h

fluorescence.168

binding

of

lectin

from

the

Analysis

of

a

series

light-induced the

iodide

quenching quenching

s u g g e s t s t h a t t h e r e is a s l i g h t i n c r e a s e i n t h e a c c e s s i b i l i t y o f t r y p t o p h a n r e s i d u e s o f t h e l e c t i n on b i n d i n g l a c t o s e . the binding characteristics of sugar

by

fluorescence

i.

of

Momordica

L-

Studies o f

charantia l e c t i n t o i t s specific

spectroscopy

using 4-methylumbelliferyl

f3-g-

g a l a c t o p y r a n o s i d e show t h e q u e n c h i n g o f t h e s u g a r u p o n b i n d i n g t o the

lectin.169

The

binding i s carbohydrate-specific

and i s

i n h i b i t e d by l a c t o s e . Considerable

homology

i n structures

and b i o l o g i c a l a c t i v i t i e s

has been r e p o r t e d f o r 62 c u l t i v a r s o f P h a s e o l u s v u l g a r i s , basis

of

isolectin

A

patterns.’”

number

of

on t h e

cultivars

are

s u f f i c i e n t l y d i f f e r e n t t o w a r r a n t s u b c l a s s i f i c a t i o n and f u r t h e r characterization. Inclusion o f diets

of

rats

nitrogen.171

p u r e l e c t i n s f r o m t h e seeds o f

increases

both

faecal

and

p.

urinary

vulqaris i n losses

of

The a n i m a l s d e v e l o p a n t i b o d i e s o f l o w a v i d i t y f o r t h e

dietary lectins.

L e c t i n t o x i c i t y i s t e n t a t i v e l y suggested t o r e s u l t

f r o m t h e combined e f f e c t s o f i n t e r f e r e n c e w i t h n o r m a l i n t e s t i n a l d i g e s t i o n and/or

absorption o f

p r o t e i n through

damaged e n t e r o c y t e s

and o f s y s t e m i c r e s p o n s e s o f t h e r a t t o t h e i n t e r n a l i s e d l e c t i n . The

subunit

compositions

of

individual

tetrameric

p h y t o h a e m a g g l u t i n i n i s o l e c t i n s f r o m P h a s e o l u s v u l g a r i s have been e x a m i n e d by i s o e l e c t r i c

focusing

and sodium

dodecylsulphate

e l e c t r o p h ~ r e s i s . ~The ~ ~ p r o p o r t i o n o f s u b u n i t s r e s u l t i n g f r o m each i s o l e c t i n was d e t e r m i n e d f o r a d i r e c t

physical conformation o f the

i s o l e c t i n subunit structures. Early

events

i n

the

synthesis

of

polypeptides

of

Ricinis

5: Glycoproteins, Glycopeptides, Proteoglycans, .and Animal Polysaccharides

--communis

189

a g g l u t i n i n t y p e 1 have been r e ~ 0 r t e d . l ~C~o - t r a n s l a t i o n a l

t r a n s l o c a t i o n a c r o s s t h e e n d o p l a s m i c r e t i c u l u m membrane i s a c c o m p a n i e d by p r o t e o l y s i s and, w h e r e a p p r o p r i a t e , core glycosylation. The

agglutinins

from

Solanum

tuberosum

and wheat-germ

a g g l u t i n i n i n t e r a c t w i t h k e r a t a n s u l p h a t e and c h i t i n ~ u 1 p h a t e . l ~ ~ D i f f e r e n c e s i n t h e r e a c t i v i t y between t h e polysaccharides and t h e two

l e c t i n s are

reported.

Chemical m o d i f i c a t i o n

of

amino

or

ca r bo x y 1 gro up s , I - a r g i n i ne , I - m e t h io n i ne, and L - h i s t id i ne r e s i due s does n o t a l t e r s i g n i f i c a n t l y t h e h a e m a g g l u t i n a t i n g a c t i v i t y o f p o t a t o 1 e ~ t i n . l ~I - ~ Tyrosine involved i n the activity,

and I - t r y p t o p h a n findings

residues are closely

which are

similar

to

those

r e p o r t e d f o r o t h e r p r o t e i n s t h a t b i n d o l i g o m e r s o f 2-acetamido-2de ox y -p- g l uco s e. The dependence o f t h e Sophora j a p o n i c a l e c t i n o n b i v a l e n t m e t a l i o n s a s w e l l a s o n t h e pH r a n g e f o r o p t i m a l h a e m a g g l u t i n a t i o n a n d precipitation

i n the

presence

o f

bivalent

e ~ t a b 1 i s h e d . l ~F~u r t h e r c h a r a c t e r i z a t i o n o f

c a t i o n s h a s been

the

combining s i t e o f

t h i s l e c t i n i s reported.

-Ulex

europaeus h a e m a g g l u t i n i n I1 ( C y t i s u s - t y p e

inhibited affinity

by

di-N-acetylchitobiose,

chromatography.l'l

g l uco s e

.

anti-H(O))

i s

been p u r i f i e d

by

sugar w i t h s m a l l e r

@-xylose, !-galactose,

Ribitol

has

The l e c t i n i s a g l y c o p r o t e i n i n w h i c h

Q-mannose i s t h e p r e d o m i n a n t glucose,

and

amounts o f

Q-

L-fucose, and 2-amino-2-deoxy-Q-

t e i c h o i c a c i d o f Staphylococcus

aureus, w h i c h

contains

t e r m i n a 1 2 - ace t am i d o - 2 - d e o x y-B - E - g l uco sy 1 r e s i d u e s , po s s e s s e s a n a b n o r m a l a f f i n i t y f o r i m m o b i l i z e d wheat-germ a g g l u t i n i n i n b i n d i n g a t h i g h i o n i c strength.177 Interactions

of

monomeric

amino

sugars

with

wheat-germ

~pectroscopy.'~~ a g g l u t i n i n h a v e b e e n s t u d i e d b y 'H a n d 19F n.m.r. -N - A c e t y l n e u r a m i n i c a c i d a n d 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e have a common b i n d i n g s i t e o n t h e l e c t i n .

D e u t e r i u m n.m.r.

resonance has

been used t o d e l i n e a t e t h e m o l e c u l a r d y n a m i c s o f s u g a r s bound t o 1 e ~ t i n s . l ~2H~ r e l a x a t i o n t i m e s and r o t a t i o n a l for

2- { 2H 1 -ace tam i d o -2-deox y-a-

3 - { 2H 1 - g l uco se

absence o f wheat-germ a g g l u t i n i n were s t u d i e d . a p p e a r s t o have n e g l i g i b l e m o t i o n a l freedom on binding.

correlation

times

i n t h e p r e se nce and The p y r a n o s e r i n g

r e l a t i v e t o the protein

The a c e t a m i d o s i d e c h a i n i s a l s o i m m o b i l i z e d i n t h e

b i n d i n g s i t e , t h e o n l y m o t i o n a v a i l a b l e b e i n g r o t a t i o n o f t h e C2H3 group about i t s t h r e e f o l d axis.


190

T h e e l u t i o n p r o f i l e s o f v a r i o u s g l y c o p e p t i d e s m o d i f i e d by glycosidase treatment, Smith degradation, acetolysis, and h y d r a z i n o l y s i s show t h a t t h e s t r u c t u r e (3) i s e s s e n t i a l f o r t h e b i n d i n g o f g l y c o p e p t i de s t o immo b i l i z e d w h e a t - g e rm a g g l “ t i n i n 8o Both t h e N,”-diacetylchitobiose m o i e t y and t h e B-Z-acetamido-2d e o x y - a - g l u c o s y l r e s i d u e l i n k e d t o C-4 o f t h e B - l i n k e d Q - m a n n o s y l r e s i d u e c o n t r i b u t e t o t h e i n t e r a c t i o n of t h e g l y c o p e p t i d e w i t h t h e lectin. The s u b s t i t u t i o n a t C-6 o f t h e i n n e r m o s t B-2-acetamido-Zd e o x y - Q - g l u c o s y l r e s i d u e by a n a - I = - f u c o s y l r e s i d u e o r a t C-6 o f t h e B - l i n k e d g - m a n n o s y l r e s i d u e by a n o t h e r g - m a n n o s e r e s i d u e r e d u c e s t h e a f f i n i t y of g l y c o p e p t i d e s f o r the column. In contrast to results f r o m o t h e r w o r k e r s , no i n t e r a c t i o n o f g l y c o p e p t i d e s c o n t a i n i n g 3a c e t y l n e u r a m i n i c acid r e s i d u e s was o b s e r v e d w i t h t h e l e c t i n . Some p r o p e r t i e s o f V i c i a g r a m i n e a l e c t i n p u r i f i e d by a f f i n i t y chromatography have been d e s c r i b e d , a n d t h e b i n d i n g of t h e l e c t i n t o v a r i o u s e r y t h r o c y t e s has been c h a r a c t e r i z e d . l a 1 The c o m p l e t e amino acid s e q u e n c e o f t h e a - s u b u n i t o f V i c i a s-a-t i v a l e c t i n h a s b e e n e s t a b l i s h e d a n d c o m p a r e d w i t h t h e k n o w n s e q u e n c e s o f l e c t i n s f r o m P i s u m s a t i v u m , L e n s c u l i n a r i s , 1. f a b a , a n d C a n a v a 1i a e n s i f o r m is ( co n ca n a v a l i n A );’ 82 A p a r t i a l l y p u r i f i e d h a e m a g g l u t i n i n a s s o c i a t e d w i t h c e l l walls from t h e h y p o c o t y l s o f Vigna r a d i a t a e x h i b i t s b o t h h a e m a g g l u t i n a t i n g activity and a - Q - g a l a c t o s i d a s e activity.183 It is suggested that, s i m i l a r t o t h e s e e d l e c t i n o f t h e same p l a n t , b o t h a c t i v i t i e s m i g h t The n a t i v e m o l e c u l e c o u l d be p r e s e n t i n t h e same m o l e c u l e . d i s s o c i a t e i n t o smaller u n i t s e x h i b i t i n g either of t h e two a c t i v i t i es

.

B-Q-GlcpNAc-( 1+4)-B-Q-Mang-(

1+4) -B-g-GlceNAc-( 1+4) -

B -Q -G1 ceNA c- 1 -A -Asn

A tetrameric Q-galactose-binding

p r o t e i n o f mung b e a n ( V i g n a t h a t d i s p l a y s b o t h h a e m a g g l u t i n i n a c t i v i t y a n d aa - g a l a c t o s i d a s e a c t i v i t y c a n be r e v e r s i b l y d i s s o c i a t e d i n t o a l o w m o 1e c u l a r- w e i gh t m o no m e r i c f o r m w h i c h po s s e s se s o n l y e n z ym i c activity.184 T h e p o s s i b i l i t y o f ffi v i v o c h a n g e s i n s u b u n i t e q u i l i b r i a , when c o m b i n e d w i t h t h e a c c o m p a n y i n g a l t e r a t i o n s i n activity, is suggested a s a possible physiological role of phy t o h a e m a g g l u t i n i n s .

_-----radiata)


191

Abrin A , p u r i f i e d from t h e s e e d s of Abrus p r e c a t o r i u s , h a s p h y s i c a l and b i o l o g i c a l p r o p e r t i e s s i m i l a r t o the b e t t e r known a b r i n C.185 P o s s i b l e s t r u c t u r a l r e l a t i o n s h i p s between t h e t w o g l y c o p r o t e i n s a n d a r e l a t e d a g g l u t i n i n from t h e same s o u r c e w e r e examined. A l t h o u g h c o v a l e n t l y a t t a c h e d c a r b o h y d r a t e i s a r e l a t i v e l y minor p a r t of t h e o v e r a l l s t r u c t u r e of r i c i n , o x i d a t i o n w i t h s o d i u m p e r i o d a t e r e s u l t s i n t h e d e s t r u c t i o n of _p-mannosyl r e s i d u e s and l o s s of t o x i c i t y o n t h e HeLa c e l l , w h i l e A-chain and 5 - c h a i n ( c e l l binding) a c t i v i t i e s a r e n o t s i g n i f i c a n t l y altered.186 A hybrid molecule c o n s i s t i n g of t h e 6 chain o f r i c i n l i n k e d t o i n s u l i n v i a a d i s u l p h i d e b o n d b i n d s t o t a r g e t c e l l s and m a i n t a i n s se ve r a l i n s u l i n - l i ke b i o l o g i c a l a c t i v i t i e s .l S e v e r a l human and m u r i n e tumour c e l l l i n e s undergo e x t e n s i v e a g g r e g a t i o n i n t h e p r e s e n c e of f e t u i n and a s i a l o f e t u i n . 1 8 8 This a c t i v i t y can be i n h i b i t e d by l a c t o s e and t o a l e s s e r e x t e n t b y g a l a c t o s e , 2- am i no-2-deox y -a- g a 1 a c t o s e , an d 2 - a c e t am i d o - 2 - de ox y -Q galactose. The i m p l i c a t i o n s o f t h e e x i s t e n c e o f a c a r b o h y d r a t e b i n d i n g p r o t e i n ( s ) o n t h e s u r f a c e o f m a l i g n a n t c e l l s o n t h e i r fi v i v o behaviour a r e d i s c u s s e d . Clq Serum a m y l o i d P c o m p o n e n t ( 9 . 5 s a l - g l y c o p r o t e i n ) , ( s u b c o m p o n e n t of t h e C 1 component o f complement), and C - r e a c t i v e p r o t e i n have l e c t i n - l i k e com b i n i n g s i t e s f o r c e r t a i n Q - g a 1 a ~ t a n s . l ~ ~ Two new a d d i t i o n a l c o m b i n i n g s i t e s o f t h e C - r e a c t i v e p r o t e i n have been d e s c r i b e d . F o e t a l - c a l f s k e l e t a l muscle c o n t a i n s t w o l e c t i n s having b i n d i n g a c t i v i t i e s i n h i b i t e d b y l a c t o s e and a s e p a r a t e h a e m a g g l u t i n a t i n g a c t i v i t y which i s n o t i n h i b i t e d by 1 a c t 0 s e . l ~ ~ The b i n d i n g of a s i a l o o r o s o m u c o i d t o t h e i s o l a t e d r a b b i t h e p a t i c l e c t i n a p p e a r s t o be a s a t u r a b l e and r e v e r s i b l e p r o c e s s a s s h o w n by s t e a d y - s t a t e and k i n e t i c a n a 1 y ~ i s . l ~The ~ minimal m o l e c u l a r w e i g h t (1.04 x lo5) of the intact rat hepatic receptor for a s i a l o g l y c o p r o t e i ns has been measured d i r e c t l y i n plasma rnembranes.lg2 The v a l u e o b t a i n e d i s s h o w n t o be s i g n i f i c a n t l y a f f e c t e d by the presence of d e t e r g e n t employed i n s o l u b i l i z a t i o n and is0 1a t i o n pro ce d u r e s. P r o t e i n h a e m a g g l u t i n i n s have been d e t e c t e d i n h o m o g e n a t e s o f e a c h o f t h e s i x c o x a l d e p r e s s o r m u s c l e s i n t h e l e g of t h e co c kr o a ch 93 The q ua n t i t a t i ve a b i 1i t y of so m e gl y co sa m i no g 1y ca n s t o i n h i b i t these haemagglutinins c o r r e l a t e s w i t h t h e muscles' i n n e r v a t i o n by i d e n t i f i e d motor neurons. The complete amino a c i d sequence of chicken h e p a t i c l e c t i n has

a-

.

192


been d 0 ~ u m e n t e d . l ~The ~ B-Q-galactoside-specific

l e c t i n present i n

e m b r y o n i c - c h i c k s k e l e t a l m u s c l e does n o t appear t o be i n v o l v e d i n myotube f o r m a t i o n

2

from chick-embryo

k i d n e y b i n d s s t r o n g l y t o asialoglycoconjugates.196

~ i t r 0 . l ~ '

A

developmentally

I t s a f f i n i t y towards 2-amino-2-deoxy-~-glucosyl, and 2-amino-2-deoxy-Q-mannosyl

galactosyl,

regulated l e c t i n

2-amino-2-deoxy-g-

residues i s low.

The

i n h i b i t o r y e f f e c t o f t h e amino s u g a r s i n h a e m a g g l u t i n a t i o n assays i s n o t due t o s i m p l e e l e c t r o s t a t i c i n t e r a ~ t i 0 n . l ~A ~ n e u r a m i n i c a c i d b i n d i n g l e c t i n i s o l a t e d from

r otunda cauda has

t h e horseshoe crab Carcinoscorpius

been p u r i f i e d by

affinity

chromatography

on

i m m o b i l i z e d f e t ~ i n . ' ~The ~ l e c t i n , which i s a g l y c o p r o t e i n (mol.

wt.

lo5)

4.2 x

lo4),

and c o n t a i n s s u b u n i t s (mol.

wts.

2.7

i s antigenically unrelated t o the other

binding lectin, Limulin

limulin,

binds

x

lo4

a n d 2.8 x

neuraminic acid-

from t h e horseshoe c r a b L i m u l u s polyphemus.

specifically

to

glycolylneuraminic acid residue

gangliosides

. 199

bearing

1-

an

The b i n d i n g r e q u i r e s a f r e e

c a r b o x y l g r o u p and a f r e e h y d r o x y l g r o u p a t C-4 o f t h e n e u r a m i n i c acid but the side chain o f the N-glycolylneuraminic required.

Wheat-germ

gangliosides acetamido

agglutinin

binds

b e a r i n g an N - a c e t y l n e u r a m i n i c

group

but

not

the

acid i s not

preferentially acid

c a r b o x y l group

i s

to The

residue.

involved i n the

binding. L e c t i n s o f d i f f e r i n g s p e c i f i c i t i e s have been d e t e c t e d i n t h e crop, midgut, and haemolymph o f t h e i n s e c t Rhodinus p r o l i x u s . 2 0 0 These a r e s p e c i f i c f o r 2 - a c e t a m i d o - 2 - d e o x y - t J - m a n n o s e , 2-acetamido-2deoxy-Q-galactose,

a n d a-

Receptors for

and B-!-galactose.

the

l e c t i n s were d e t e c t e d i n e p i m a s t i g o t e f o r m s o f Trypanosoma c r u z i ,

a

p r o t o z o a n p a r a s i t e o f t h e i n s e c t a n d o f humans. A

possible

----Sarcophaga described.201 smaller

mechanism o f

peregrina

on

induction o f the

injury

of

the

body

insect wall

lectin of has

been

The l a r g e r s u b u n i t o f t h e l e c t i n i s c o n v e r t e d t o t h e

subunit

by

proteolysis,

i n a process essential

for

c o n s t r u c t i n g a c t i v e l e c t i n h a v i n g a f f i n i t y f o r !-galactose. The sponge H a l i c h o n d r i a p a n i c e a c o n t a i n s a l e c t i n t h a t h a s been i s o l a t e d and p u r i f i e d . 2 0 2

F r o m t h e same s p o n g e s p e c i e s , b a c t e r i a

have been i s o l a t e d and i d e n t i f i e d as Pseudomonas i n s o l i t a .

The

l e c t i n , d e t e c t e d on t h e s u r f a c e o f m u c o i d c e l l s f r o m H.panicea,

may

be i n v o l v e d i n a s y m b i o t i c r e l a t i o n s h i p b e t w e e n t h e sponge a n d t h e bacterium. A

mycobacterial haemagglutinin,

o f ~ y x o c o c c u sx a n t h u s ,

a development-specific

lectin

h a s b e e n p ~ r i f i e d . ~ ' ~W, h ~i l e~ ~s i m p l e

193


sugars f a i l t o i n h i b i t t h e h a e m a g g l u t i n a t i n g a c t i v i t y o f the l e c t i n , a g l y c o p e p t i d e c o n t a i n i n g t h e t r i s a c c h a r i d e ( 4 ) , common t o many glycoproteins, i s a potent i n h i b i t o r . Two l e c t i n s have

been p u r i f i e d f r o m

A g a r i c u s edulis.’05

Both

a r e c a p a b l e o f a g g l u t i n a t i n g e r y t h r o c y t e s a n d i m m u n o g l o b u l i n s A, and M ,

but

no

monosaccharide

was

capable o f

G,

inhibiting the

interaction. NeupSAc-( 2 + 3 ) - B - Q - G a l p ( 1+3I-!-GalNAc

A chitin-binding

h a e m a g g l u t i n i n h a s been i s o l a t e d f r o m c u l t u r e

C o n id io b o l u s

of

f i1t r a t e s

chromatography

Haemagglutination

i s s t r o n g l y i n h i b i t e d by c h i t o - o l i g o s a c c h a r i d e s . protease, lectin,

p r o d u c e d by

g.

lampranges,

n it y a f fi

f o 11ow in g

lamerrnags

on f o r m a l i n i z e d e r y t h r o c y tes.*06 acts,

A pronase-like

i n company

with the

on erythrocytes t o accelerate the a c t i v i t y of the l e ~ t i n . ~ ’ ’

The l e c t i n h a s c h i t i n - b i n d i n g

p r o p e r t i e s s i m i l a r t o t h o s e o f wheat

germ a g g l u t i n i n . Purpurin,

t h e l e c t i n from

o f seven t e t r a m e r i c forms, The i s o l e c t i n s ,

D i c t y o s t e l i u m purpureum,

i s made up

assembled from f o u r d i s t i n c t subunits.208

although related,

can be f u n c t i o n a l l y d i s c r i m i n a t e d

o n t h e b a s i s o f t h e i r r e l a t i v e a f f i n i t i e s f o r columns d e r i v a t i z e d w i t h complementary s a c c h a r i d e s . Oleic

acid

concentrations d ioleolyl

and

dioleolyl

agglutinate

phosphatidic

rabbit

phospha t i d y 1 c h o l i n e

and r a t i s not

acid

a t

erythrocytes,

low while

haem a g g l ~ t i n a t i n g . ~ ~ ’

E r y t h r o c y t e a g g l u t i n a t i o n by l i p i d s i s i n d i s t i n g u i s h a b l e i n some respects

from

that

haemagglutination

caused

reactions

by

lectins,

exhibit

cell

i n

particular

specificity

and

both are

i n h i b i t e d by c e r t a i n g l y c o p r o t e i n s .

4 A review

Fibronectin

d e a l i n g w i t h c u r r e n t c o n c e p t s c o n c e r n i n g t h e s t r u c t u r e and

f u n c t i o n o f f i b r o n e c t i n has been p u b l i s h e d . 2 1 0 R a t p l a s m a f i b r o n e c t i n h a s been i s o l a t e d and c h a r a c t e r i z e d and

m o n o s p e c i f i c a n t i b o d i e s p r e p a r e d t o it.211

The a n t i b o d i e s o n l y

w e a k l y c r o s s - r e a c t w i t h p l a s m a f i b r o n e c t i n s o f c h i c k e n , horse, and


194 human.

A

wt.

second g l y c o p r o t e i n (mol.

collagen-binding

activity,

was

Immunologically pure f i b r o n e c t i n cannot p l a s m a by o n e - s t a g e

affinity

x lo4),

7.0

isolated

from

also having rat

plasma.

be p r e p a r e d f r o m

human

chromatography on i m m o b i l i z e d g e l a t i n

o r i m m o b i l i z e d f i b r o n e c t i n due t o n o n - s p e c i f i c a b s o r p t i o n o f o t h e r p r o t e i n s o n t h e support.212

P u r i f i e d f i b r o n e c t i n can be o b t a i n e d

u s i n g a s e c o n d s e p a r a t i o n by g e l p e r m e a t i o n c h r o m a t o g r a p h y . Although

human

amniotic

f l u i d

fibronectin

and

plasma

f i b r o n e c t i n a r e i m m u n o l o g i c a l l y i n d i s t i n g u i s h a b l e and a r e e q u a l l y a c t i v e promoters of c e l l attachment,

they d i f f e r i n carbohydrate

composi t i o n . 2 1 3 solid-phase

A

radioimmunoassay

has been d e v e l o p e d f o r t h e

d e t e r m i n a t i o n o f f i b r o n e c t i n l e v e l s i n plasma.214

An i m m u n o l o g i c a l

a s s a y has been u s e d t o m e a s u r e t h e u p t a k e o f endogenous a n d exogenous fibronectin

by c e l l s i n c u l t u r e . 2 1 5

f i b r o n e c t i n from

The

t h e medium i s i n f l u e n c e d by

cellular

uptake

of

c e l l shape a n d by t h e

presence o f collagen. C u l t u r e d human-mammary n o r m a l t i s s u e s and p r i m a r y fibronectin.*16

epithelial carcinomas

c e l l s derived from

produce

The p r e s e n c e o f c e l l - a s s o c i a t e d

both

large quantities

of

f i b r o n e c t i n as w e l l

a s t h e s y n t h e s i s a n d s e c r e t i o n o f f i b r o n e c t i n i n t o t h e medium by these

c e l l s a n d by

studied

fibroblasts

.

structural

A

comparison

derived from of

these t i s s u e s were

fibronectins

from

t r a n s f o r m e d h a m s t e r c e l l l i n e s has been r e p o r t e d . 2 1 7 major s i t e s of types o f cells,

normal

and

Although

the

p h o s p h o r y l a t i o n i n f i b r o n e c t i n a r e t h e same i n b o t h f i b r o n e c t i n f r o m t r a n s f o r m e d c e l l s a p p e a r s t o be

p h o s p h o r y l a t e d t o a much h i g h e r e x t e n t t h a n t h a t f r o m n o r m a l c e l l s . Human g e r m - c e l l t u m o u r s p r o d u c e f i b r o n e c t i n r e s e m b l i n g i t s a m n i o t i c fluid

variant,

but

differ

from

f ib r o ne c t i n s have been s ugge s t e d t o

plasma

fibronectin.218

Such

be p o t e n t ia 1 o n co de v e l o pm e n t a 1

ma r k e rs. The s p e c i f i c c a l c i u m - d e p e n d e n t serum a m y l o i d P component

for

b i n d i n g r e a c t i v i t y f o r human

f i b r o n e c t i n and a C4- binding

protein

i s reported.219 Human p e r i p h e r a l b l o o d m o n o c y t e s p o s s e s s a t r y p s i n - s e n s i t i v e p l a s m a membrane r e c e p t o r f o r s u r f a c e - b o u n d

fibronectin.220

Binding

o f monocytes t o f i b r o n e c t i n l e a d s t o enhanced f u n c t i o n a l e x p r e s s i o n of

their

plasma-membrane

receptors

for

the

Fc

portion

i m m u n o g l o b u l i n G a n d f o r t h e t h i r d component o f c o m p l e m e n t .

of

It i s

proposed t h a t plasma f i b r o n e c t i n a c t s as a c i r c u l a t i n g probe f o r

195


f i b r i n o r f o r e x p o s e d o r a l t e r e d c o l l a g e n a t s i t e s o f t i s s u e damage. Treatment o f f i b r o n e c t i n w i t h e i t h e r m e t h y l m e r c u r i c bromide o r iodoacetamide

as

sulphydryl

blocking

agents

protects

the

g l y c o p r o t e i n a g a i n s t p r e c i p i t a t i o n a l l o s s e s d u r i n g p r e p a r a t i o n . 221 A

combination

o f

electron

microscopy,

c.d.

i.r.

and

spectroscopy, a n d s c a n n i n g m i c r o c a l o r i m e t r y has been u s e d i n a s t u d y o f t h e s t r u c t u r a l o r g a n i z a t i o n o f human p l a s m a f i b r o n e c t i n . 2 2 2 Four

functionally

fibronectin

have

distinct

domains

i n

hamster

plasma

been i s o l a t e d and c h a r a c t e r i z e d f o l l o w i n g

p r o t e o l y t i c d i g e s t i o n o f t h e g l y ~ o p r o t e i n . ~I n~t a ~c t fibronectin i s considered t o contain four carbohydrate chains per subunit, b e i n g a t t a c h e d t o a d o m a i n o f m o l . w t . 4.0 o f mol.

wt.

1.4

lo4

x

three

and one t o a domain

lo5.

x

H e a t d e n a t u r a t i o n o f human p l a s m a f i b r o n e c t i n p r o c e e d s t h r o u g h a t l e a s t three stages, o b s e r v e d a t 6 8 , 82,and

w i t h endothermal denaturing t r a n s i t i o n s 119°C.224

A three-domain s t r u c t u r e f o r t h e

g l y c o p r o t e i n i s p r o p o s e d i n w h i c h t h e domain w h i c h u n f o l d s a t 68' associated

gelatin

with 82'

unfolding at immunological

b i n d i n g and

appears t o

activity.

The

cell

binding,

while

be a s s o c i a t e d w i t h m u c h o f 119'

domain

has h e p a r i n

a c t i v i t y a s w e l l a s some i m m u n o l o g i c a l a c t i v i t y . b i n d i n g domain o f f i b r o n e c t i n i s immunogenic,

i s

that the

binding

The g e l a t i n -

and a n t i s e r a a g a i n s t

t h i s domain r e c o g n i z e c e l l u l a r f i b r o n e c t i n g e l a t i n - b i n d i n g

sites.225

I n h i b i t i o n o f g e l a t i n b i n d i n g b u t n o t c e l l s p r e a d i n g by a n t i - g e l a t i n binding

d o m a i n Fab'

fragments

confirms

the hypothesis

that

f i b r o n e c t i n has separate s i t e s m e d i a t i n g these a c t i v i t i e s . on d i g e s t i o n w i t h c a t h e p s i n D, r e l e a s e s t w o

Plasma f i b r i n o g e n ,

p o o l s o f l o n g - c h a i n p o l y p e p t i d e s o f d i f f e r e n t m o l e c u l a r w e i g h t s and w i t h graded a f f i n i t y

having

weaker

fibronectin;

f o r i m m o b i l i z e d heparin.226

affinity

contains

the

The f r a c t i o n

N-terminal

region

t h a t having the stronger a f f i n i t y consists o f

d i s u l p h i d e - l i n k e d peptide chains o f related structure.

different

o f two

l e n g t h and s h a r i n g a

The h i g h e r - m o l e c u l a r - w e i g h t p o l y p e p t i d e c o n t a i n s

an a d d i t i o n a l t r a n s a m i d a s e - s e n s i t i v e s i t e .

Human p l a s m a f i b r o n e c t i n

h a s b e e n t r e a t e d w i t h ~ h y m o t r y p s i n . ~C~h a ~racterization o f the i s o l a t e d f r a g m e n t s h a s shown t h a t t h e o r d e r o f t h e f u n c t i o n a l domains from t h e N-terminus i s s t a p h y l o c o c c a l binding, l i n k i n g and b i n d i n g s i t e s , area, and h e p a r i n - b i n d i n g Horse serum

gelatin-binding site,

f i b r i n cross-

c e l l attachment

site.

fibronectin

h a s been r e d u c e d a n d a l k y l a t e d t o

p r o d u c e a m o d i f i e d g l y c o p r o t e i n w h i c h no l o n g e r b i n d s t o g e l a t i n ,

196


b u t s t i l l p r o v i d e s a s u b s t r a t e f o r myoblast attachment.228 was

to

obtained

fibronectin

demonstrate

wt.

(mol.

x

6 .0

chymotryptic

a

fragment

lo4)

was u n a b l e t o p r o m o t e c e l l

a t t a c h m e n t u n l e s s f i b r o n e c t i n was p r e s e n t i n t h e medium, that

the gelatin-binding

fragment

f i b r o ne c t i n- f i b r o ne c t i n b i n d i n g. f i b r o n e c t i n from affinity

also

chromatography

The a c t i n - b i n d i n g

of

suggesting

spe c i f i c a c t i n - b i n d i n g s i t e i n

A

fragments

r e l e a s e d from

wt.

a fragment (mol.

s i t e i s close to,

collagen-binding

site.

2.7

but not i d e n t i c a l with, The

binding o f

human

the

plasma

microtitre

The b i n d i n g i s

b u t n o t by h e p a r i n o r b o v i n e

i n h i b i t e d by b o t h a c t i n a n d g e l a t i n , n o t take

by

x l o 4 ) was i s o l a t e d .

f i b r o n e c t i n t o a c t i n has been s t u d i e d u s i n g a c t i n - c o a t e d

not

After

fibronectin

w e l l s i n e n z y m e - l i n k e d immunosorbent assays.230 serum a l b u m i n .

of

contains a s i t e involved i n

c h i c k e n f i b r o b l a s t s h a s been i d e n t i f i e d . 2 2 9

l i m i t e d proteolysis, reported

Evidence

that

The b i n d i n g o f c o l l a g e n a n d a c t i n t o f i b r o n e c t i n may

place simultaneously.

interfere

with

cell

Since the

attachment,

binding of

the

cell

collagen

does

binding s i t e of

f i b r o n e c t i n i s probably d i f f e r e n t from t h e a c t i n - b i n d i n g

site.

S t r u c t u r a l d i f f e r e n c e s be t w een p l asm a s a n d f i b r o b l a s t c e l l u l a r fibronectins

have

regions.231

One s t r u c t u r a l d i f f e r e n c e o c c u r s n e a r t h e g e l a t i n -

been i d e n t i f i e d i n a t

least

three

different

b i n d i n g s i t e and t h e o t h e r s a r e n e a r h e p a r i n - b i n d i n g s i t e s . A

number

of

cell

types

(HeLa,

Ehrlich

ascites,

and

t r a n s f o r m e d r a t k i d n e y ) show a l o w r a t e o f C a 2 + u p t a k e ,

viral

which i s

stimulated a f t e r incubation with f i b r ~ n e c t i n . ~ ~ ~ The i n t e r a c t i o n o f i s o l a t e d p l a s m a f i b r o n e c t i n w i t h s t i m u l a t e d a n d n o n s t i m u l a t e d human p l a t e l e t s h a s

been ~ h a r a c t e r i z e d . ~ The ~~

s p e c i f i c b i n d i n g o f a l a r g e number o f

fibronectin molecules to

throm b i n - s t i m u l a t e d p l a t e l e t s suggests t h a t

f i b r o n e c t i n may p l a y a

r o l e i n modulating p l a t e l e t

a g g r e g a t i o n i n d u c e d by

adhesion and/or

t h i s stimulus. The i n c o r p o r a t i o n o f C - p r o l i n e and g l y c i n e i n t o f i b r o n e c t i n and c o l l a g e n by c u l t u r e s o f s k i n f i b r o b l a s t s o b t a i n e d f r o m p a t i e n t s w i t h insulin-dependent

diabetes

mellitus

has

been r e p o r t e d . 2 3 4

Changes

i n t h e r a t i o o f f i b r o n e c t i n t o c o l l a g e n between s k i n f i b r o b l a s t s o f n o r m a l and d i a b e t i c s u b j e c t s d u r i n g The XIII,

covalent binding of

h a s been e x a m i n e d i n a s y s t e m

o n macroporous agarose, presence o f beads i s

in

v i t r o a g i n g were not ed.

various s t r u c t u r a l

p r o t e i n s by

i n which p r o t e i n s ,

factor

immobilized

a r e i n c u b a t e d w i t h l a b e l l e d p r o t e i n s i n the

factor X I I I a ,

determined.235

and t h e r a d i o a c t i v i t y Coupling occurred

of

t h e urea-washed

between

fibrin

and


197

f i b r o n e c t i n and b e t w e e n m y o s i n and f i b r o n e c t i n . The a m i n o a c i d sequence i n b o v i n e f i b r o n e c t i n w h i c h c o n t a i n s L - g l u t a m i n e r e s i d u e s labelled with

1, 4 - { 1 4 C ) - p u t r e s c i n e

determined.236

--glutamine This L

by

factor

X I 1 1

has

been

residue i s located a t position 3

from t h e N-terminus o f f i b r o n e c t i n . F i b r o n e c t i n and p r o c o l l a g e n a r e r e l e a s e d f r o m t h e m a t r i x o f h u m a n f i b r o b l a s t s by t h r o m b i n . 2 3 7

Collagenase alone i s unable t o

b r i n g about t h e release o f f i b r o n e c t i n .

The r e l e a s e o f t h e s e t w o

g l y c o p r o t e i n s by t h r o m b i n may be i n v o l v e d i n wound h e a l i n g i n v i v o . Hum a n

f i b r one c t in

p l asm a

at

physio logic a l

concentrations

s t i m u l a t e s t h e l e v e l o f t o t a l p l a t e l e t a d h e s ion and t h e i r on s u r f a c e s c o a t e d w i t h f i b r i l l a r collagen.238

spreading

The s t i m u l a t i n g

e f f e c t o f f i b r o n e c t i n i s due t o i t s i n t e r a c t i o n w i t h c o l l a g e n s i n c e f r e e f i b r o n e c t i n does n o t i n f l u e n c e t h e a d h e s i o n

o r spreading o f

platelets. Plasma f i b r o n e c t i n mediates c o l l a g e n t y p e I11

the

b i n d i n g o.f s o l u b l e n a t i v e

macro phage^.^^^

to trypsinized

The a s s o c i a t i o n

i s a u g m e n t e d by h e p a r i n o r h e p a r a n s u l p h a t e , b u t h y a l u r o n i c a c i d reverses the effect. F i b r o n e c t i n h a s been shown t o b i n d t o a g g r e g a t i n g c o l l a g e n fibres.240

Since fibronectin i n h i b i t s the

i t

fibrillogenesis

may

regulate

the

size

rate of

of

collagen

collagen

fibres.

C a r t i l a g e p r o t eo g l y c a n s i n h i b i t fi bro nectin-me d i a t e d adhesion t o collagen.241

The p r e s e n c e o f f i b r o n e c t i n i n c o l l a g e n f i b r i l s o f

human f i b r o b l a s t s has been shown, u s i n g i m m u n o l o g i c a l t e c h n i q u e s . 2 4 2

A g e l a t i n - b i n d i n g p r o t e i n (mol.

wt.

7.0

x

lo4),

which i s q u i t e

d i s t i n c t f r o m f i b r o n e c t i n , i s s y n t h e s i z e d by n o r m a l a n d m a l i g n a n t adherent

cells.243

The g l y c o p r o t e i n seems n o t t o

be a f r a g m e n t

of

f i b r o n e c t i n a n d shows no i m m u n o l o g i c a l c r o s s r e a c t i v i t y w i t h i t .

5

The

Collagen

biochemical

properties

i n c l u d i n g collagen, connective-tissue-

of

basement

have been reviewed.244

r e s e a r c h symposium

membrane

components,

The p r o c e e d i n g s o f a

include chapters dealing w i t h

r e c e n t advances i n methods f o r i n v e s t i g a t i n g t h e m o l e c u l a r s t r u c t u r e of

collagens,

purification collagen.245 c o l l a g e n .246

c h r o m a t o g r a p h i c and e l e c t r o p h o r e t i c of

collagen, A symposium

and

tissue-specific

has been d e v o t e d t o

methods

for

the

differences

i n

the biology

of

Carbohydrate Chemistry I n a novel s t r u c t u r a l model f o r c o l l a g e n , i t i s proposed t h a t t h e r e a r e no i n t e r - c h a i n h y d r o g e n b o n d s a n d v a n d e r Waals' contacts.247 I n s t e a d , a t r i p l e - h e l i c a l s t r u c t u r e i s s t a b i l i z e d by water m o l e c u l e s , w h i c h f o r m i n t e r c h a i n b r i d g e s t h r o u g h h y d r o g e n b o n d s w i t h carbonyl groups. Collagen has been s e l e c t i v e l y s o l u b i l i z e d from m i x t u r e s o f c o l l a g e n and e l a s t i n u s i n g phenol, acetic acid, and water mixtures,248 and a l s o u s i n g c y c l e s i n v o l v i n g pepsin, p a n c r e a t i c e l a s t a s e , and d i t h i ot h r e i P r e p a r a t i o n s f r o m norm a 1 h a m s t e r a n d b a b o o n l u n g s c o n t a i n p r i n c i p a l l y t y p e s I a n d I11 c o l l a g e n . A c h r o m a t o g r a p h i c p r o c e d u r e c o n s i s t i n g of g e l p e r m e a t i o n a n d a n i o n - e x c h a n g e c h r o m a t o g r a p h y f o r p u r i f y i n g n a t i v e t y p e s I , 11, a n d I11 c o l l a g e n s f r o m a v a r i e t y o f s o u r c e s h a s b e e n r e p o r t e d . 2 5 0 The n a t i v e c o l l a g e n p r e p a r e d by t h i s m e t h o d i s m o r e s t a b l e t h a n t h a t p r e p a r e d by s a l t f r a c t i o n a t i o n , a s r e f l e c t e d by t h e h i g h e r a n d s h a r p e r thermal t r a n s i t i o n t e m p e r a t u r e . Radioimmunoassay p r o c e d u r e s f o r 7s c o l l a g e n a n d l a m i n i n h a v e been u s e d t o a n a l y s e t h e s e p r o t e i n s i n s e r u m , c e l l c u l t u r e s , a n d t i s s u e s .251 T h e r e l a t i v e a m o u n t s o f t y p e s I a n d I11 c o l l a g e n s h a v e b e e n e s t i m a t e d a f t e r e x t r a c t i o n of non-collagenous p r o t e i n from r a b b i t The m e t h o d h a s b e e n lung t i s s u e i n sodium dodecyl sulphate.252 a p p l i e d t o s m a l l t i s s u e s a m p l e s s u c h a s w o u l d b e o b t a i n e d by l u n g biopsy. The s k i n c o l l a g e n s f r o m t h e l a m p r e y , E n t o s p h e n u s j a p o n i c u s , a n d t h e g r e a t b l u e s h a r k , P r i o n a c e g l a u c a , c o n t a i n t w o d i s t i n c t acomponents and are classified a s t y p e I-like collagen.253 The h e t e r o g e n e i t y of cyanogen bromide-cleavage p e p t i d e s o f h u m a n c o l l a g e n s , t y p e s I , 11, I I 1 , a n d V , h a v e b e e n d e m o n s t r a t e d by t w o-dim e n s i o n a l p o l y a c r y lam i d e ge 1 e l e c t r o p h o r e s i s. 25 The amino a c i d s e q u e n c e o f t h e o l i g o s a c c h a r i d e a t t a c h m e n t s i t e w i t h i n t h e proa-2 chain of chick type I collagen has been I n a d d i t i o n 0 - m a n n o s e- r i c h g l y c o p e p t i d e s o b t a i n e d i d e n t i f i e d.255 from b o t h p r o a - 1 ( I ) a n d proa-2 c a r b o x y l p r o p e p t i d e s have been i s o l a t e d and characterized. Evidence f o r a s t r u c t u r a l mutation o f procollagen type I i n a p a t i e n t w i t h E h l e r s - D a n l o s s y n d r o m e , t y p e VII, has b e e n The f i b r o b l a s t s o f t h e p a t i e n t s y n t h e s i z e b o t h a n reported.256 abnormal proa-2 chain and a normal proa chain. Human t y p e I1 c o l l a g e n d i f f e r s f r o m t y p e I c o l l a g e n i n m o l e c u l a r p a c k i n g , a s d e m o n s t r a t e d by X - r a y d i f f r a c t i o n s t u d i e s . 2 5 7


199

U n d e r p h y s i o l o g i c a l c o n d i t i o n s t h e t y p e I1 c o l l a g e n i s s h o w n t o c o n t a i n 5 0 - 1 0 0 % m o r e water t h a n t h e t y p e I c o l l a g e n . The c o n t e n t o f g l y c o s y l a t e d h y d r o x y - L - l y s i n e r e s i d u e s i s t h o u g h t t o be t h e v a r i a b l e m o s t l i k e l y t o be m o d u l a t i n g f i b r i l l a r h y d r a t i o n . C o l l a g e n s s y n t h e s i z e d by c u l t u r e d b o v i n e c o r n e a l e n d o t h e l i a l cells have been characterized.258 T y p e I11 c o l l a g e n i s t h e m a j o r component both d e p o s i t e d i n t h e e x t r a c e l l u l a r m a t r i x and secreted i n t o t h e media. B a s e m e n t m e m b r a n e c o l l a g e n s , t y p e I V a n d V, a r e a l s o found i n each compartment. The a 2 ( v ) c h a i n o f human c o l l a g e n when c l e a v e d w i t h c y a n o g e n b r o m i d e c o n t a i n s t e n p e p t i d e s , a c c o u n t i n g f o r 956 a m i n o a c i d residues.259 Possible homologies between t h e a2(v) peptides and p e p t i d e s d e r i v e d f r o m o t h e r c o l l a g e n c h a i n s were n o t e d . A n t i b o d i e s have been p r e p a r e d t o c o l l a g e n a s e - r e s i s t a n t t e r m i n a l Competition radioimmunoassay r e g i o n s of pro-type I V collagen.260 e x p e r i m e n t s show r e c o g n i t i o n o f t h e a n t i b o d i e s f o r r e d u c e d a n d a l k y l a t e d 7s c o l l a g e n , s u p p o r t i n g t h e p o s s i b i l i t y t h a t t h i s c o l l a g e n i s a c r o s s l i n k e d domain o f t y p e I V c o l l a g e n . A high-molecular-weight c o l l a g e n o u s p r o t e i n has been i s o l a t e d , a f t e r l i m i t e d p e p s i n d i g e s t i o n , from t h e e n d o m e t r i a l v i l l i o f b o v i n e placenta.261 I t r e s e m b l e s t h e p r o t e i n i s o l a t e d from human a o r t a s i n c o n t a i n i n g t h r e e polypeptide c h a i n s w i t h similar amino acid com po s i t io n 262 The p o l y p e p t i de cha i n s s h ow c h a ra c t e r i s t i cs t y p i c a l o f basement membrane c o l l a g e n s , h a v i n g a high c o n t e n t o f hydroxy-kl y s i n e , w h i c h i s a l m o s t a l l g l y c o s y l a t e d . The o c c u r r e n c e of s i m i l a r a m o u n t s o f L - c y s t e i n e , L-ty r o s i n e , a n d 2 - a m i n o - 2 - d e o x y - Q - g l u c o s e i s not observed i n o t h e r collagen types. An u n u s u a l c o l l a g e n f r a c t i o n h a s b e e n i s o l a t e d a n d c h a r a c t e r i z e d from bovine h y a l i n e c a r t i l a g e and i n t e r v e r t e b r a l disc.263 Evidence s u p p o r t s a model c o n t a i n i n g t h r e e i d e n t i c a l c h a i n s ( m o l . w t . 3 . 3 x l o 4 > l i n k e d by i n t e r c h a i n d i s u l p h i d e b o n d s t o f o r m a s h o r t t r i p l e-he1 i ca 1 p s e u d o a-si z e d com po n e n t The c o l l a g e n o u s d o m a i n o f b o v i n e g l o m e r u l a r b a s e m e n t m e m b r a n e h a s been i s o l a t e d i n s o l u b l e form and shown t o c o n t a i n t h e previously designated polypeptide XIV.264 The amino a c i d a n d carbohydrate compositions and cyanogen bromide p a t t e r n s i n d i c a t e t h a t polypeptide X I V has a s t r u c t u r e similar to t h a t of C chain i s o l a t e d f r o m o t h e r v a s c u l a r t i s s u e s a n d t h a t i t may r e p r e s e n t a m a j o r s t r u c t u r a l segment o f t h e e n t i r e c o l l a g e n o u s domain. The c o l l a g e n o u s domain o f r a b b i t r e n a l t u b u l a r basement membrane i s c o m p o s e d o f a m u l t i p l i c i t y o f c o m p o n e n t s r a n g i n g f r o m m o l . wts. 3 . 4

.

.

200


lo4

t o 1 0 6 . 265 As a s e l f - a s s e m b l y

system,

renature as t r i p l e - s t r a n d e d

guinea-pig

d e t e r m i n e d by t h e r a t i o s o f t h e a - c h a i n s The c o l l a g e n s f r o m

-e l e g a n s

collagen chains tend t o

m o l e c u l e s whose

chain compositions are

i n the mixture.266

the c u t i c l e o f

t h e nematode C a e n o r h a b d i t i s

h a v e b e e n s e p a r a t e d by m o l e c u l a r - s i e v e c h r o m a t o g r a p h y a n d

shown t o c o n t a i n no h y d r o x y - l - l y s i n e The

collagen

-c--e l l u l o s a e

isolated

from

residues.267 the

platyhelminth

I,-pr o 1i n e .26

ve r t e b r a t e co 11age n ex ce p t t ha t it co n t a i n s no h y d r ox y An e x t r a - e m b r o n i c and

rat

Cysticerus

c o n t a i n s an amino a c i d c o m p o s i t i o n c h a r a c t e r i s t i c o f

contains

base membrane,

collagen

together

R e i c h e r t ' s membrane, with

four

wt.

g l y c o p r ~ t e i n s . ~One ~ ~ o f t h e g l y c o p r o t e i n s (mol. unrelated t o laminin,

o f mouse

non-collagenous

w h i l s t t h e o t h e r t h r e e (mol.

5.0

wts.

x lo4) i s 4.15

x

lo5,

2.45 x lo5, a n d 1.70 x lo5, r e s p e c t i v e l y ) s h a r e some i m m u n o c h e m i c a l ch a r a c t e r i s t ics o f 1am i n i n. A 7 s c o l l a g e n h a s been i d e n t i f i e d a s a c r o s s l i n k i n g domain o f

mouse t u m o u r m a t r i x t y p e I V c o l l a g e n . 2 7 0 Transformed e p i t h e l i a l b o v i n e l e n s c e l l s s y n t h e s i z e and s e c r e t e i n t h e c u l t u r e medium o n l y type

I V

collagen.271

Like

the

interstitial

molecule i s a disulphide-bonded collagenous and a non-collagenous

procollagen,

the

glycoprotein containing both a domain.

The absence o f m a t u r a t i o n o f c o l l a g e n c r o s s - l i n k s

i n fish skin

has been r e p o r t e d . 2 7 2 A c l o s e s i m i l a r i t y e x i s t s between o c t o p u s s k i n c o l l a g e n and

c a l f s k i n t y p e I collagen.273

B o t h c o l l a g e n s r e s e m b l e each o t h e r ,

n o t o n l y i n c h a i n c o m p o s i t i o n b u t a l s o i n chemical composition. The m o l e c u l a r o r g a n i z a t i o n of t h e c o l l a g e n o u s components o f t h e i n t e s t i n a l basement membrane o f A s c a r i s suum has been i n v e s t i g a t e d by c h a r a c t e r i z a t i o n o f t h e i r p h y s i c a l p r o p e r t i e s i n b o t h t h e n a t i v e and denatured states.274

The c o l l a g e n o u s domain c o n s i s t s m a i n l y o f

a component composed o f t w o e s s e n t i a l l y i d e n t i c a l t r i p l e - h e l i c a l s u b u n i t s j o i n e d e n d t o end by d i s u l p h i d e b o n d s w i t h e a c h s u b u n i t being cross-linked

by

disulphide

bonds b e t w e e n a l l

constituent

chains. A

study

of

collagen-sulphated

glycosaminoglycuronan

i n t e r a c t i o n s i n d e v e l o p i n g r a t t a i l t e n d o n has been r e p o r t e d i n 75

t e r m s o f f ib r i11oge ne s i s and f ib r e mat u r a t i on.

Bovine corneal e n d o t h e l i a l c e l l s synthesize predominantly type I11 collagen, V

i n culture

w i t h l e s s e r amounts o f t y p e s I a n d

and a p p a r e n t l y l i t t l e i f any o f t y p e I

V

.

~

~

~

,

~

~

~


201

M3A, a d e r i v a t i v e o f the c l o n a l s k e l e t a l m u s c l e myoblast l i n e secretes i n t o t h e c u l t u r e medium a n a b n o r m a l f o r m of c o l l a g e n ( m o l . w t . 5.6 x l o 4 ) a n d a g r e a t l y r e d u c e d a m o u n t o f h i g h - m o l e c u l a r w e i g h t c o l l a g e n a-chains.278 I n a d d i t i o n M 3 A does n o t secrete u n s u l p h a t e d c h o n d r o i t i n w h i l e L6 d o e s . Comparison of the behaviour o f 1e u c i n e am i n o p e p t i d a s e a n d ca r bo x y p e p t i d a s e - m o d i f i e d c o 11 a g e n s suggests that the carboxyl telopeptide has a major r o l e i n the grow t h s t a g e s o f s e l f -assem b l y 2 7 9 M a t r i x - f r e e cells f r o m c h i c k embryo t e n d o n s s y n t h e s i z e pro-y c o l l a g e n c h a i n s which are either not triple-helical o r are i n an u n s t a b l e t r i p l e-he1 i c a l c o n f o r m a t i on.28o These c h a i n s c o n t a i n l e s s h y d r o x y - & - p r o l i n e t h a n t h e t r i p l e - h e l i c a l p r o c o l l a g e n s e c r e t e d by t y e cells. Their content o f glycosylated hydroxy-L-lysine is, h o w e v e r , g r e a t e r t h a n t h a t o f t h e p r o c o l l a g e n s e c r e t e d by t h e c e l l s . The b i o s y n t h e s i s a n d p r o c e s s i n g o f t y p e V p r o c o l l a g e n s i n s e v e r a l c h i c k t i s s u e s p r o d u c e p r o c o l l a g e n s ( p r ~ a l V ) (~p r, o a 2 V I 2 , a n d (proalV)3.281 A comparative b i o s y n t h e t i c s t u d y o f these g l y c o p r o t e i n s by c h i c k t e n d o n f i b r o b l a s t s a n d by h a m s t e r l u n g c e l l c u l t u r e s h a s b e e n r e po r t e d . 82 The N - g l y c o s y l a t i o n o f I - l y s i n e a n d h y d r o x y - l - l y s i n e r e s i d u e s i n t y p e I c o l l a g e n from s t r e p t o z o t o c i n - d i a b e t i c r a t s has been c o n f i r m e d , a n d t h e s t a b i l i t y o f t h e c o m p l e x h a s b e e n s h o w n t o be d u e t o a n Amadori rearrangement.283 This type of glycosylation does not represent an acceleration of the normal maturation process i n collagen involving the reducible cross-links. Post-translational modifications i n the biosynthesis of type I V c o l l a g e n by a h u m a n t u m o u r c e l l l i n e h a v e b e e n s t u d i e d . 2 8 4 The d i f f e r i n g e x t e n t s o f m o d i f i c a t i o n s s h o w n by t h i s c e l l l i n e a n d n o r m a l h u m a n s k i n f i b r o b l a s t s a r e e x p l a i n e d by d i f f e r e n c e s i n t h e a c t i v i t i e s o f L-lysy l h y d r o x y l a s e and hydroxy-k-lysyl-g1 ucosy 1 t r a n s f e r a s e s between t h e two types. Collagen hydroxylases and glycosyltransferases o f cultured human-foetal l u n g f i b r o b l a s t s m i g h t n o t be c o - o r d i n a t e l y regulated.285 Regardless of hydroxylation events, glycosylation of t h e p e p t i d e might be l i m i t e d t o a s p e c i f i c f r a c t i o n of t h e hydroxyI-lysine residues during the post-translational modification of collagen. L6,

.


202 Glycogen

6

A review d e a l i n g w i t h t h e comparative biochemistry o f s t a r c h and g l y c o g e n has been p u b l i s h e d . 2 8 6

A h i g h l y b r a n c h e d , p o l y d i s p e r s e , h i g h - m o l e c u l a r - w e i gh t g l yco gen from

rat

liver

has

been i s o l a t e d

using centrifugation,

gentle

h e a t i n g , a n d g e l c h r ~ m a t o g r a p h y . ~U~s ~ ing a d u l t fasted rats, t h i s g l y c o g e n was shown t o water-ethanol

be b e t t e r t h a n h i g h - m o l e c u l a r - w e i g h t

extracted

glycogen

for

the

binding of

cold

glycogen

m e t a b o l i z i n g e nz ymes. The

simultaneous

demonstration o f

glycogen and p r o t e i n i n

c a r d i a c t i s s ue g l y co some s has been a c h i e ve d h i s t o chem i c a l l y . 2 8 8 An a b n o r m a l u r i n a r y o l i g o s a c c h a r i d e i n patients with

glycogen storage

p a t t e r n h a s been o b s e r v e d

disease type

III.289 No

ch a r a c t e r i z a t i o n o f t h e o l i g o sa c c h a r i de s was a t t e m p t e d . S t r u c t u r a l aspects o f the c a t a l y t i c and r e g u l a t o r y f u n c t i o n o f g l y c o g e n p h o s p h o r y l a s e have been reviewed.290

The r e g u l a t i o n o f

g l y c o g e n p h o s p h o r y l a s e a n d g l y c o g e n s y n t h a s e by a d r e n a l i n i n S o l e u s

m us c l e in ph o sp h o r y 1as e k i na se - de f ic i ent m ice h a s be e n r e pa r t e d

.

D a t a have been p r o d u c e d w h i c h a r e n o t c o n s i s t e n t w i t h t h e t h a t i n s u l i n a c t i v a t e s g l y c o g e n s y n t h a s e by

91

view

producing an i n h i b i t o r

o f 3’5’ c y c l i c a d e n o s i n e m o n o p h o s p h a t e - d e p e n d e n t

protein

kinase i n

Nor do t h e y s u p p o r t t h e h y p o t h e s i s t h a t

r a t s k e l e t a l muscle.292

i n s u l i n a c t s by d e c r e a s i n g t h e a c t i v i t y o f a n i n h i b i t o r o f a m u l t i s u b s t r a t e p h o s p h o p r o t e i n k i nase. Bovine

heart

glycogen

with

heart.293

combination o f

The

a

synthase

phosphorylated

glycogen

has

synthase

the

been

kinase

specifically isolated

e n z y m e a n d 3’5’

from

c y c l i c AMP-

dependent p r o t e i n k i n a s e l e a d s t o s p e c i f i c p h o s p h o r y l a t i o n i n t h r e e enzyme s i t e s .

Each f o r m o f t h e g l y c o g e n s y n t h a s e , p h o s p h o r y l a t e d i n

s p e c i f i c enzyme s i t e s , A

protein

has b e e n i s o l a t e d a n d s t u d i e d k i n e t i c a l l y .

kinase

from

phosphorylate t h e 8-subunit

rabbit

reticulocytes,

able

i f not

and glycogen synthase k i n a s e from r a b b i t s k e l e t a l muscle are,

.

i d e n t i c a l , c l o s e l y r e 1a t e d p r o t e i n s 2 9 4 Yea s t 1,4 -a -Q - g l u c o s i da s e

, hom o l o go u s

against isolated r a t hepatocytes

have

a 1bum i n , a n d

I gG r a is e d

been c r o s s - l i n k e d

glutaraldehyde t o produce a stable, a c t i v e enryme-polymer (mol.

wt.

1.0

x 106).295

t o

o f e u k a r y o t i c i n i t i a t i o n f a c t o r 2 (IF2),

using

complex

After intravenous i n j e c t i o n i n t o rats,

the

complex

i s p r e f e r e n t i a l l y associated w i t h the Kupffer c e l l s o f

liver.

The p r o c e d u r e h a s b e e n u s e d a s a m o d e l f o r e n z y m e t h e r a p y

the

203


f o r t h e p o s s i b l e l o w e r i n g o f h e p a t o c y t e g l y c o g e n c o n t e n t i n t y p e I1 glycogenesis

(Pompe’s

Glycosaminoglycans and P r o t e o g l y c a n s

7

-

Analysis. of

free

disease).

A semi-quantitative micromethod f o r the determination

glycosaminoglycans

i n

serum

has

been

developed as

a

s c r e e n i n g m e t h o d f o r m u c o p o l y s a c c h a r i do s i s . ~ ’ ~ G l y c o s a m i n o g l y c a n s a r e adsorbed i n DEAE-cellusose paper b e f o r e s t a i n i n g w i t h A l c i a n B l u e a n d m e a s u r e m e n t o f t h e o p t i c a l d e n s i t y o f t h e r e s u l t i n g dye complex. G l y c o s a m i n o g l y c a n s have been i d e n t i f i e d and measured a f t e r a s e l e c t i v e and stepwise

sequence

of

treatments w i t h hyaluronidase,

c h o n d r o i t i n s u l p h a t e l y a s e A C a n d ABC, nitrous procedure

To the

determine

remaining

the

@-B-&-galactanase,

result of

each

and

degradative

glycosaminoglycans are subjected t o

cellulose acetate electrophoresis. H y a l u r o n i c a c i d and t h e i s o m e r i c c h o n d r o i t i n s u l p h a t e s have been s i m u l t a n e o u s l y d e t e r m i n e d a f t e r h y d r o l y s i s w i t h c h o n d r o i t i n a s e ABC

( E C 4.2.2.4.).298

( d e t e c t i o n l i m i t 0.02

The m e t h o d i s r e p r o d u c i b l e a n d s e n s i t i v e p m o l d i s a c c h a r i d e 1.

The s p e c i f i c i t y o f a r a d i o i m m u n o a s s a y m e t h o d f o r m e a s u r i n g h u m a n a r t i c u l a r c a r t i l a g i n o u s p r o t e o g l y c a n s h a s been reported.”’ T h e a n t i b o d i e s u s e d show a s p e c i e s s p e c i f i c i t y s i n c e t h e r e i s n o c r o s s - r e a c t i o n w i t h t h e c o r r e s p o n d i n g r a t o r dog p r o t e o g l y c a n s . A n t i g e n i c s i t e s a p p e a r t o be l o c a t e d i n t h e p r o t e i n r e g i o n a n d n o t i n t h e glycosaminoglycan r e g i o n o f t h e molecule. C o m p l e x e s f o r m e d b e t w e e n serum p o l y - a n i o n s a n d c e t y l p y r i d i n i u m c h l o r i d e h a v e been q u a n t i f i e d by l a s e r n e p h e l ~ r n e t r y . ~ ~ ’Measurement o f t h e c o m p l e x e s i n serum b e f o r e a n d a f t e r d i g e s t i o n w i t h s p e c i f i c enzymes e n a b l e s t h e m e a s u r e m e n t o f h y a l u r o n i c a c i d a n d c h o n d r c d t i n s u l p h a t e i n s m a l l v o l u m e s o f serum. c h o n d r o i t i n 4-

and 6 - s u l p h a t e s

The

uronic acid levels i n

and d e r m a t a n s u l p h a t e have been

determined a f t e r electrophoresis o f the l i b e r a t e d uronic acids on T i t a n 111 c e l l u l o s e a c e t a t e An h.p.1.c.

plate^.^"

m e t h o d has

been d e s c r i b e d f o r

the analysis o f

r e d u c e d u n s a t u r a t e d d i s a c c h a r i d e s d e r i v e d f r o m e n z y m i c d i g e s t i o n and sodium borohydride r e d u c t i o n o f c h o n d r o i t i n sulphates, sulphate,

heparan sulphate,and heparin.302

possibility

of

obtaining

anomeric

dermatan

The m e t h o d a v o i d s t h e

forms

of

unsaturated

204


d i sa ccha r i d e s

.

A modification of measure

the

an amino

sulphaminohexose

s u g a r a n a l y s i s h a s been u s e d t o content

of

heparin

and

heparan

~ u l p h a t e . ~ 'P ~o l y m e r - b o u n d N - s u l p h a t e d 2-amino-2-deoxyhexoses s p e c i f i c a l l y d e a m i n a t e d t o y i e l d f r e e 2,5-anhydro-g-mannose, t h e n measured u s i n g t h e 3 - m e t h y l - 2 - b e n z o t h i a z o l i n o n e The culture

degradation of may

be

m o n i t o r i n g the

proteoglycan

and

release of

soluble

reagent.

collagen

measured simultaneously

are

which i s cells

i n

i n a p l a t e assay

by

by

r a d i o a c t i v e degradation

products

from a d i s h c o a t e d w i t h a g e l c o n t a i n i n g 3 H - l a b e l l e d p r o t e o g l y c a n and 1 4 C - l a b e l l e d c o l l a g e n . 3 0 4

S y n o v i a l c e l l s a r e a b l e t o degrade

both substrates, owing t o the release o f a proteoglycan-degrading n e u t r a l p r o t e i n a s e and o f co lla g e n a se . A

monodimensional c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s

capable of r e s o l v i n g k e r a t a n sulphate, sulphate,

heparin,

t h e b a s i s of chloride acid,

c h o n d r o i t i n 4-sulphate,and

The e l e c t r o p h o r e t i c b e h a v i o u r

desulphated chondroitin,

sulphate w i t h two d i f f e r e n t buffer concentration, examined.306

A

is

dermatan

h y a l u r o n i c a c i d , on

t h e i r d i f f e r e n t i a l m i g r a t i o n i n H4-e.d.t.a.

so 1u t ions. 305

system

heparan sulphate,

and l i t h i u m o f h y a luronic

heparan sulphate, and c h o n d r o i t i n

b u f f e r s under v a r y i n g c o n d i t i o n s o f

electrophoresis

two-buffer

t i m e , and v o l t a g e has been

monodirectional

electrophoresis

t e c h n i q u e , w h i c h o f f e r s some o f t h e a d v a n t a g e s o f r e s o l u t i o n s e e n w i t h two-dimensional

methods

y e t r e t a i n s t h e a d v a n t a g e s o f band

comparison and q u a n t i t a t i v e dimensional electrophoresis, Intact

chick

flexible.308

limb

analysis

characteristic

of

one-

h a s been p r o p o s e d .

bud

Restrictions

proteoglycan

on

chain

monomer

flexibility

i s

occur

highly i n

the

n e i g h bo u r h o o d o f t h e l i n k a g e b e t w e e n p r o t e i n a n d p o l y s a c c h a r i d e c h a i n s where h i g h l o c a l c h a i n c o n c e n t r a t i o n s cause c o l l i s i o n s and entanglements.

Proteoglycans

from

bovine

nasal,

bovine a r t i c u l a r ,

a n d r a t c h o n d r o s a r c o m a c a r t i l a g e have been a n a l y s e d by h.p.1.c.

or a

s i l i c a - b a s e d m a t e r i a l bonded w i t h a n amide phase.307 Cation binding t o

m u l t i - c h a i n and s i n g l e - c h a i n

g l y c a n p e p t i d e s has been s t u d i e d b y 23Na+ n.m. r. Hyperlipidemic

rabbit

serum

impairs

the

glycosamino-

s p e c t r o s c o p y .309 precipitation

of

g l y c o samino g l y c u r o n a n s f r o m t i s s u e - c u l t u r e medium by c e t y l p y r i d i nium chloride.310 of

samples

derivatives

The i n t e r f e r i n g compounds c a n be r e m o v e d by e x t r a c t i o n i n l i p i d solvents. thereof

on low-density

have

Various

g l y c o s a m i n o g l y c a n s and

been s u b j e c t e d t o a f f i n i t y

l i p o p r o t e i n - s u b s t i t u t e d agarose.311

chromatography By u s e o f

a

205


v a r i e t y o f m o d i f i c a t i o n and degradation procedures, t h e chemical c h a r a c t e r i s t i c s t h a t maximize the a f f i n i t y o f a glycan f o r the l i p o p r o t e i n have been assessed.

The i n t e r a c t i o n b e t w e e n t h e t w o

c l a s s e s o f m a c r o m o l e c u l e s i s dependent o n t h e p r e s e n c e o f s u l p h a t e groups and i n c r e a s e s w i t h charge d e n s i t y .

The i n t e r a c t i o n o f serum

l i p o p r o t e i n s and a proteoglycan from

bovine aorta

reported.312

has

been

P o s i t i v e charges o n t h e p r o t e i n moiety o f low-densi t y

l i p o p r o t e i n s are r e q u i r e d f o r the i n t e r a c t i o n , as are presence o f the

protein

core

.

p r o t eo g l y ca n

and

the

glycosaminoglycan

chains

o f

the

P u r i f i e d p r o t e o g l y c a n s f r o m human a r t i c u l a r c a r t i l a g e have b e e n used

i n

the

development

of

a

radioimmunoassay

procedure.313

C o n d i t i o n s f o r l a b e l l i n g and subsequently p u r i f y i n g t h e p r o t e o g l y c a n are described,

Occurrence,

and o p t i m a l c o n d i t i o n s f o r t h e assay a r e d e t a i l e d .

I s o l a t i o n , and S t r u c t u r e .

-

The

f r a c t i o n a t i o n and

c h a r a c t e r i z a t i o n o f p r o t e o g l y cans i s o l a t e d from chondro c y t e c e l l cultures

have

been d e s c r i b e d . 3 1 4

The

proteoglycans

were

compared

with the proteoglycans extracted from bovine nasal o r t r a c h e a l cartilage.

P r o t e o g l y c a n s h a v e been p r e p a r e d f r o m human f e m o r a l - h e a d

a r t i c u l a r cartilage.315 scattering,and proteoglycan

As a r e s u l t o f g e l c h r o m a t o g r a p h y ,

ultracentrifugal studies, molecule

i n cartilage

light

a p h y s i c a l model o f t h e

has

been

proposed.

Three

p r o t e o g l y c a n f r a c t i o n s p r o d u c e d by human-em b r y o l u n g f i b r o b l a s t s h a v e b e e n i s o l a t e d a n d ~ h a r a c t e r i z e d . ~T h~e~ f r a c t i o n s c o n t a i n w t . 4.0 x l o 4 ) , c h o n d r o i t i n

m a i n l y heparan s u l p h a t e c h a i n s (mol. s u l p h a t e c h a i n s (mol. (mol.

2.5

wt.

x

lo4),

lo4),

w t. 4.0 x

respectively.

and dermatan sulphate chains

Glycosaminoglycans i s o l a t e d from

t r y p t i c d i g e s t s o f a t r i a o f t h e human h e a r t have been i d e n t i f i e d a s containing hyaluronic acid, sulphates,

heparan sulphate,

and dermatan sulphate.317

human m e n i s c u s a p p e a r t o be o f t w o types.318 a t

high

buoyant

density

on

caesium

c h o n d r o i t i n 4- and 6 -

The p r o t e o g l y c a n s o f a d u l t Preparations i s o l a t e d

chloride

density-gradient

c e n t r i f u g a t i o n c o n t a i n molecules o f l a r g e hydrodynamic s i z e t h a t a r e composed o f p r o t e i n ,

chondroitin sulphate,

k e r a t a n sulphate, and

neuraminic acid

possess

aggregate

comparable Preparations

and

with from

de rma t a n s u l pha t e,

those low

o f

subunit

and

hy a l i ne- c a r t i l a g e

buoyant

density,

which

are

co n t a i n sm a l l e r- m o l e c u l a r- w e i g h t

w h i c h do n o t i n t e r a c t w i t h h y a l u r o n i c a c i d .

structures

p r o t e o g l y cans. enriched i n p r o t e o g l y cans

The g l y c o s a m i n o g l y c a n

c o m p o s i t i o n s o f c a n i n e m e n i s c i have been reported.319

Although the


206

c o m p o s i t i o n i s t h e same i n d i f f e r e n t r e g i o n s o f t h e m e n i s c i , t o t a l amounts vary baboons

considerably.

( P a ~ i gp a p i o )

electrophoretic

Articular

contains

mobility

on

a

the

c a r t i l a g e o f young

proteoglycan

large-porosity

gels

c h a r a c t e r i z e d as h a v i n g a high p r o t e i n content.320

of

high

and

i s

Proteoglycans

f r o m p i g a o r t a have b e e n e x t r a c t e d s e q u e n t i a l l y w i t h i n o r g a n i c s a l t s o l u t i o n s under a s s o c i a t i v e and d i s s o c i a t i v e c o n d i t i o n s , fractionation

by

f o l l o w e d by

g e l permeation chromatography i n order

t o

c h a r a c t e r i z e and compare t h e i r c h e m i c a l p r o p e r t i e s and h y d r o d y n a m i c sizes.321 fraction

Low-buoyant-density proteoglycans found i n the l i n k from, a v i a n c a r t i l a g e c o n t a i n some o f t h e a n t i g e n i c However, t h e r e i s

d e t e r m i n a n t s f o u n d o n t h e p r o t e o g l y c a n monomer.322

a t l e a s t one monomer d e t e r m i n a n t w h i c h i s n o t p r e s e n t i n t h e l i n k fraction. Proteoglycan

subunits

of

bovine

nasal

cartilage

contain

phosphate e s t e r groups t h a t a r e s e n s i t i v e t o a c i d phosphatase and are considered t o molecule.323

be a r e g u l a r c o n s t i t u e n t o f

the proteoglycan

S t r u c t u r a l studies on the E-xylosyl-L-serine

linkage

i n p r o t e o g l y c a n s o f human, p o r c i n e , a n d s h a r k c a r t i l a g e s h a v e b e e n reported.324

The f i n d i n g t h a t human a n d p o r c i n e p r o t e o g l y c a n s have

t h e same a m i n o a c i d sequence i n t h e l i n k a g e r e g i o n a s t h a t r e p o r t e d p r e v i o u s l y f o r b o v i n e p r o t e o g l y c a n s u g g e s t s t h a t t h e r e i s a common s t r u c t u r a l f e a t u r e i n t h e core p r o t e i n s o f these mammalian c a r t i l a g e p r o t eo g l y cans. Z o n a l - r a t e c e n t r i f u g a t i o n i n s u c r o s e g r a d i e n t s h a s been u s e d t o study i n t e r a c t i o n s i n proteoglycan aggregation.325

The l i n k p r o t e i n

was shown t o i n t e r a c t w i t h t h e i s o l a t e d h y a l u r o n i c a c i d - b i n d i n g r e g i o n o f t h e p r o t e o g l y c a n monomer. isolated hyaluronic

acid,

the

Since i t also i n t e r a c t s with binding o f

proteoglycans

hyaluronate i n the presence o f l i n k p r o t e i n s i s t r i f u n c t i o n a l .

to The

b i n d i n g p r o p e r t i e s o f c a r t i l a g e l i n k p r o t e i n and t h e hyaluronatebinding region of

cartilage

proteoglycan t o

hyaluronic acid

i m m o b i l i z e d o n a g a r o s e h a v e been c o m p a r e d a n d shown t o h a v e s i m i l a r properties.326

A

gross physical characterization o f the hyaluronic

acid-binding r e g i o n o f proteoglycan from p i g l a r y n g e a l c a r t i l a g e has been a c h i e v e d

using

densitometric

and

small-angle

neutron

s t u d i e s . 327 P l a t e l e t f a c t o r 4 i s a basic t e t r a m e r i c p r o t e i n which i n t e r a c t s w i t h sulphated polymers.328

The t r a n s f e r o f t h i s f a c t o r f r o m i t s

p r o t e o g l y c a n c a r r i e r t o n a t u r a l and s y n t h e t i c p o l y m e r s h a s been reported.

The r e s u l t s o f e l e c t r i c

b i r e f r i n g e n c e s t u d i e s show

that


207

c h o n d r o i t i n 4 - s u l p h a t e m o l e c u l e s have a very high d e g r e e o f m o b i l i t y

.

w i t h i n t h e p r o t eo g l y c a n str u c t u r e 329 A novel low-molecular-weight

chondroitin sulphate proteoglycan ( m o l . w t . 7.6 x l o 4 ) h a s b e e n i s o l a t e d f r o m b o v i n e n a s a l c a r t i l a g e , and i n d i f f e r e n t l a y e r s o f bovine h i p a r t i c u l a r cartilage.330 The p r o t e o g l y c a n c o n t a i n s two o r t h r e e c h o n d r o i t i n s u l p h a t e c h a i n s a n d some o l i g o s a c c h a r i d e s , and h i g h c o n t e n t s o f L - l e u c i n e and I - c y s t e i n e as compared w i t h o t h e r proteoglycans. The i n h i b i t o r o f Clq, a s u b u n i t o f t h e f i r s t component o f complement, has been i d e n t i f i e d a s a c h o n d r o i t i n 4-sulphate proteoglycan.331 Unlike the major species o f c a r t i l a g e p r o t e o g l y c a n , the serum p r o t e o g l y c a n does n o t form a complex w i t h h y a l u r o n i c acid. Rat b r a i n c o n t a i n s a s i n g l e p o l y d i s p e r s e m a c r o m o l e c u l e i n w h i c h c h o n d r o i t i n s u l p h a t e c h a i n s and o l i g o s a c c h a r i d e s are both covalently l i n k e d t o a common p r o t e i n c o r e . 3 3 2 The E a n d 2 - g l y c o s i d i c a l l y l i n k e d o l i g o s a c c h a r i d e s of t h e g l y c o p r o t e i n a p p e a r t o be r a n d o m l y The b r a i n p r o t e o g l y c a n appears t o d i s t r i b u t e d i n the proteoglycan. be c a p a b l e o f a t l e a s t a l i m i t e d d e g r e e o f i n t e r a c t i o n w i t h h y a l u r o n i c a c i d t o p r o d u c e l a r g e r a g g r e g a t e s . Some b o v i n e a o r t i c c h o n d r o i t i n s u l p h a t e- a n d d e rma t a n s u l p h a t e- co n t a i n i n g p r o t e o g l y c a n s have been i s o l a t e d and c h e m i c a l l y characterized.333 Disaccharides l i b e r a t e d from c h o n d r o i t i n s u l p h a t e s a n d d e r m a t a n s u l p h a t e by d i g e s t i o n w i t h c h o n d r o i t i n l y a s e A C o r c h o n d r o i t i n l y a s e ABC a r e c o m p l e t e l y s e p a r a t e d o n c e l l u l o s e a c e t a t e p l a t e s by e l e c t r o p h o r e s i s i n barium acetate or c a l c i u m acetate.334 G l y c o s a m i n o g l y c a n s have b e e n i s o l a t e d f r o m t h e f e m u r s o f o e s t r o g e n t r e a t e d male J a p a n e s e Chondroitin 4-sulphate and keratan s u l p h a t e were f o u n d i n t h e f e m u r s , b u t o n l y t h e k e r a t a n s u l p h a t e c o r r e l a t e s w i t h m e d u l l a r y bone p r o d u c t i o n . A proteoglycan isolated f r o m a s c i t e s f l u i d p r o d u c e d by a r a t y o l k - s a c t u m o u r h a s b e e n partially characterized as a chondroitin sulphate p r ~ t e o g l y c a n . ~ ~ ~ De rma t a n s u l p h a t e p r o t e o g l y c a n s h a v e b e e n i s o l a t e d a n d cha r a c t e r i z e d ’ proteoglycans, d i f f e r i n g with respect t o f r o m b o v i n e ~ c l e r a . ~ ~Two the nature o f t h e i r p r o t e i n cores and the co-polymeric s t r u c t u r e of Isopycnic centrifugation studies t h e i r s i d e c h a i n s , were i s o l a t e d . u s i n g caesium c h l o r i d e and caesium s u l p h a t e have been r e p o r t e d o n t h e s e p r o t e o g l y c a n s . 338 B o v i n e s c l e r a c o n t a i n s t w o p r o t e o d e rm a t a n sulphates, the l a r g e r exhibiting self-association i n both gel c h r o m a t o g r a p h y and l i g h t - s c a t t e r i n g e x p e r i m e n t s , and t h e smaller It is showing aggregation only i n l i g h t - s c a t t e r i n g experiments.339 p r o p o s e d t h a t t h e a g g r e g a t i o n i s s o l e l y o r p a r t l y m e d i a t e d by

208


interaction forms

of

between dermatan s u l p h a t e s i d e

self-association,

e d .

protein-protein interactions,

& v

chains,

although other

polysaccharide-protein

three or four

C a l f s k i n p r o t e o d e r m a t a n s u l p h a t e i s composed o f

wt.

chains o f dermatan s u l p h a t e (mol.

wt.

t o a core p r o t e i n (mol. A

proteodermatan sulphate

5.6 x

lo4)

from

rat

1.7 x l o 4 ) c o v a l e n t l y l i n k e d 2-glycosidic

wt.

3.6

via

46% p r o t e i n w h i c h i s bound t o d e r m a t a n s u l p h a t e Rat

t a i l tendon has

linkages.340

s k i n has been i s o l a t e d and

~ h a r a c t e r i z e d . ~ T h~e ~ p r o t e o g l y c a n ( m o l . linkage.

and

h a v e n o t been e x c l u d e d .

been s t a i n e d

x

lo4)

contains

an 2 - g l y c o s i d i c

with

a

cationic

p h t h a l o c y a n i n dye, C u p r o m e r o n i c B l u e , t o d e m o n s t r a t e t h e l o c a t i o n o f A dermatan s u l p h a t e - r i c h

p r o t e o g l y c a n by e l e c t r o n m i c r o s c o p y . 3 4 2 proteoglycan

i s

distributed

orthogonal array,

about

the

collagen

the transverse elements o f

fibrils

which are

i n

an

located

a l m o s t e x c l u s i v e l y a t t h e d b a n d i n t h e gap zone. Several different proteoglycan species, a Q-glucuronic acidr i c h and an L - i d u r o n i c with two different human

skin

acid-rich

fibroblast

cultures.343

dermatan p o l y s u l p h a t e p e p t i d e s notochord,

proteodermatan sulphate,

proteoheparan sulphates,

Three

I, 11, and

different

a l l contain I-serine,

x y l o s e , a n d Q - g a l a ~ t o s e . ~T~h e~ a - x y l o s y l - g - s e r y l

of

linkage

Rwas

I c o n t a i n s an 2 - g l y c o s y l

d e t e c t e d i n p o l y p e p t i d e 111. P o l y p e p t i d e

l i n k a g e between 2-acetamido-2-deoxy-P-galactose on SephadexR G-50

types

111, i s o l a t e d f r o m h a g f i s h

h a g f i s h skin, and s h a r k s k i n ,

Chromatography

together

have been i s o l a t e d f r o m

and L - s e r i n e .

results i n optimal isolation

o f a h e p a r i n component t h a t i s h i g h l y a c t i v e as m e a s u r e d by s e v e r a l assay

methods.345

highly

The b i n d i n g o f

co-operative,

factors.346

An

exothermic,

examination

of

Methylene Blue t o heparin i s and

stabilized

fundamental

by

entropic

conditions

for

h y d r o p h o b i c - i n t e r a c t i o n c h r o m a t o g r a p h y o f h e p a r i n on h y d r o p h o b i c g e l s has been r e p o r t e d . 3 4 7 applied,

flow

rate,

and t e m p e r a t u r e ,

Column d i m e n s i o n s , amount o f h e p a r i n

e l e c t r o l y t e a n d a c i d i t y o f t h e e l u t i o n medium,

i n f l u e n c e t h e d i s t r i b u t i o n o f h e p a r i n among t h e

f r a c t i o n s s e p a r a t e d on t h e g e l . A d i r e c t m e t h o d h a s been d e v e l o p e d f o r t h e d e t e r m i n a t i o n o f t h e dissociation constant

b e t w e e n h e p a r i n and b o v i n e s e r u m a l b u m i n ,

by

u s i n g f l u o r e s c e n t l y l a b e l l e d h e p a r i n and m o n i t o r i n g t h e f l u o r e s c e n c e i n t e n s i t y change i n d u c e d by t h e p r o t e i n . 3 4 8 Gel f o r m a t i o n o f thymus lymphocytes, c e l l s w i t h h e p a r i n has been observed.349 only,

source o f h e p a r i n i s t h e mast

spleen

or bone marrow

Since the major,

cell,

i f not

and s i n c e h e p a r i n i s

209


r e l e a s e d a l o n g w i t h h i s t a m i n e d u r i n g c e r t a i n t y p e s o f a l l e r g i c and inflammatory reactions, i t i s suggested t h a t heparin promotes the a c c u m u l a t i o n o f l e u c o c y t e s i n t h e ex t r a v a s c u l a r space. Heparin

and

chondroitin

glycosaminoglycans which

6-sulphate

a t p H 6.0

are

the

l i p o p r o t e i n f r o m aqueous s o l u t i o n s a n d i n t h e p r e s e n c e of However,

chondroitin 6-sulphate

immobilized low-density same pH.

The

only

p r e c i p i t a t e low-densi t y

i s the only

one t h a t

Ca2+.350 binds

to

l i p o p r o t e i n i n t h e p r e s e n c e o f Ca2+ a t t h e

binding o f

Ca2+ a n d M g 2 + i o n s t o h e p a r i n i n t h e

p r e s e n c e o f added u n i v a l e n t s a l t s has been s t u d i e d u s i n g a dye s p e c t r o s c o p i c method.351 The i n t e r a c t i o n o f M g 2 + w i t h h e p a r i n a p p e a r s t o be i n d e p e n d e n t o f t h e n a t u r e o f t h e c h a r g e d g r o u p s o n t h e polyanion,

b u t Ca2+ b i n d i n g i s c o n s i d e r a b l y s t r o n g e r t h a n Mg2+

b i n d i n g and i s i n excess o f t h e o r e t i c a l p r e d i c t i o n s , s u g g e s t i n g a l o c a l i z e d o r s p e c i f i c i n t e r a c t i o n with heparin.

A selective binding

o f Zn2+ i o n s t o h e p a r i n r a t h e r t h a n t o o t h e r g l y c o s a m i n o g l y c a n s has been observed.352

This observation suggests that,

between

the

negatively

charged

carboxy

g l y c o Sam ino g l y c a n s a n d Z n 2 + c a t i o n s c a n n o t b in d in g

.

Some

chemical,

disaccharides

enzymic,

derived from

d e g r a d a t i o n f o l l o w e d by reported.353 from

which a

Heparin,

and

beef

sodium yields

groups

physical

properties by

c l ea ve s t h e 2-&

conditions

of

of

the

heparin,

been p a r t i a l l y

before for

and a f t e r

sulpham i d a s e ,

to

characterized.354

acid

hydrolysis

that

s u l p h o - l - i d o py r a no s i d i c 1i n ka ge s. 355

substrates

prior

l a r g e r - molecular-w eight fragments,

p u r i f i e d r a d i o - l a b e l l e d d i - s a c c h a r i d e s (5-13) evaluated

o f

b o r o t r i t i d e r e d u c t i o n have been

t e t r a s a c c h a r i d e has

under

the

nitrous acid

a f t e r c a r b o x y l r e d u c t i o n w i t h sodium b o r o t r i t i d e ,

degraded

of

ex p l a i n t h e o b s e r ve d

lung heparin

P a r t i a l tj-desulphation

de g r a da t i ve deam i n a t i o n ,

contrary to a

s i m p l e e l e c t r o s t a t i c in t e r a c t i o n s

p r e v i o u s l y p r o p o s e d hypo t h e s i s,

tj-acetylation

h a s been

selectively The r e s u l t i n g

were c h a r a c t e r i z e d and

o r tj-sulphation,

as

ace t y l c o e n z y m e A:2-am i n o - 2 - d e o x y - a - p -

g l u c o s i de tj- a c e t y 1t r a n s f e r a se , 2 - ace t am i d o -2-deox y-a -Q-g1 uco s i da se,

-

o r 2- a c e t am ido 2 - de ox y f ib r o b l a s t s

.

Tritiated di-

-n-

gl uco s e 6- s u l pha t e s u l pha t a s e in h um a n s k i n

and t e t r a - s a c c h a r i d e s ,

v a r i o u s r e p e a t sequences o f heparin,

evaluated as substrates for the estimation o f present i n s k i n fibroblasts, t y p e A syndrome.356

representative o f the

have been p r e p a r e d and sulphamidase a c t i v i t y

and f o r the d e t e c t i o n o f S a n - f i l l i p 0


210

A s i m p l e m e t h o d f o r f r a c t i o n a t i n g h e p a r i n uses a n t i t h r o m b i n I 1 1 w h i c h i s n o n c o v a l e n t l y b o u n d t o i m m o b i l i z e d c o n c a n a v a l i n A.357

Active antithrombin i s e l u t e d from the immobilized l e c t i n using m e t h y l a-9-glucoside.

An e l e c t r o p h o r e t i c m e t h o d u s i n g a g a r o s e beads

has been d e v e l o p e d f o r t h e d e t e r m i n a t i o n o f h e p a r i n f r a c t i o n s h a v i n g a h i g h a f f i n i t y f o r a n t i t h r o m b i n III.358 A high-molecular-weight

heparin fraction that

includes

components w i t h a s much a s f i v e t o t e n t i m e s t h e a c t i v i t i e s o f t h e o r i g i n a l h e t e r o g e n e o u s p r e p a r a t i o n s has been i s o l a t e d f r o m lung.359

beef

The f r a c t i o n was c h a r a c t e r i z e d w i t h r e s p e c t t o i t s m o l e c -

u l a r w e i g h t (3.5

lo4),

x

c o m p o s i t i o n , and b i n d i n g t o a n t i t h r o m b i n

HOQoJQ HO NH2

*

'

(5)

R 1 = R2 = H

(6)

R1 = SO3-,

(7)

R1 = H,

OR2

NH2

R2 = H

R2 = SO3-

(8) R1 = R 2 = SO3-

R2

(9) (10)

R 1 = R2 =

(11)

R1

(12)

R 1 = R2 = S0-j-

R1 = SO3-,

= H,

H R2 = H

R2 = S O 3 -

CH20H

H NHAc

111.

OH

Hog rnucosal h e p a r i n , a f t e r p a r t i a l N - d e s u l p h a t i o n ,

co nj uga t e d t o f 1uo r e s c e i ny 1t h io ca r bam oy 1 g r o up s. 3 6 0 h e p a r i n was s e p a r a t e d i n t o l o w - a f f i n i t y w i t h respect

t o a n t i t h r o m b i n 111.

has been

The f 1uo r e s c e n t

and h i g h - a f f i n i t y

Substitution of

fractions

the heparin

m o l e c u l e s h a d no e f f e c t o n t h e b i o l o g i c a l i n t e r a c t i o n w i t h a n t i t h r o m b i n 111.

Evidence f o r a 3 - s - s u b s t i t u t e d

glucosyl residue i n the antithrombin-binding

T h i s s u l p h a t e d amino s u g a r i s a u n i q u e component

been r e p o r t e d . 3 6 1 o f high-affinity

2-amino-2-deoxy-Q-

sequence o f h e p a r i n has

heparin,

antithrom bin-binding

located a t a specific position i n the

sequence o f t h e m o l e c u l e .

Confirmation o f the

e x i s t e n c e o f t h i s component has been o b t a i n e d f r o m 1 3 C n.m.r. r o s c o p i c studies.362

spect-

A h i g h l y a c t i v e and a r e l a t i v e l y i n a c t i v e f o r m

o f w h a l e h e p a r i n have been s u b j e c t e d s e p a r a t e l y t o e n z y m i c d e g r a d a t ion.363

The

disaccharide

2-acetamido-2-deoxy-3-O-(4-deoxy-L-threo-


21 1

p y r a n o s y l u r o n i c acid)-!-glucose was f o r m e d e x c l u s i v e l y f r o m t h e h i g h l y a c t i v e f o r m a n d may a r i s e f r o m a n e s s e n t i a l b i n d i n g s e c t i o n f o r a n t i t h r o m b i n 111. Binding constants f o r t h e binding of high-affinity heparin t o a n t i t h r o m b i n 111 a t d i f f e r e n t i o n i c s t r e n g t h s h a v e b e e n d e t e r m i n e d by f l u o r e s c e n c e t i t r a t i o n s a n d e s t i m a t e d f r o m d i s s o c i a t i o n c u r v e s o f t h e h e p a r i n - a n t i t h r o m b i n I11 c o m p l e x . 3 6 4 A thermodynamic e v a l u a t i o n o f t h e d i s s o c i a t i o n o f t h e complex s u g g e s t s t h a t m a x i m a l l y f i v e t o six charged groups are involved i n t h e interaction. The b i n d i n g o f a n t i t h r o m b i n I11 t o h e p a r i n f r a c t i o n s w i t h d i f f e r e n t m o l e c u l a r Binding c o n s t a n t s and s t o i c h i o m e t r i e s w e i g h t s has b e e n s t u d i e d . 3 6 5 f o r t h e b i n d i n g o f t h e s e f r a c t i o n s t o a n t i t h r o m b i n I11 a r e p r e s e n t e d and c o r r e l a t e d with t h e a n t i c o a g u l a n t activities of the f r a c t i o n s . P o r c i n e i n t e s t i n a l h e p a r i n has been p a r t i a l l y degraded t o p r o d u c e a n o c t a s a c c h a r i d e w i t h h i g h a f f i n i t y f o r a n t i t h r o m b i n III!66 T h e o c t a s a c c h a r i de e x h i b i t e d m o r e p o t e n t i n a c t i v a t i o n a c t i v i t i e s f o r t h r o m b i n , f a c t o r Xa, a n d p l a s m a c o a g u l a t i o n t h a n t h e n a t i v e h e p a r i n . T h e h i g h - a f f i n i t y b i n d i n g o f h e p a r i n t o a n t i t h r o m b i n I11 r e q u i r e s t h e p r e s e n c e o f t w o c o n s e c u t i v e t j - s u l p h a t e d 2-am i n o - 2 - d e o x y - p glucosyl residues i n s p e c i f i c positions of the antithrombin-binding sequence.367 Loss o f e i t h e r o n e o f t h e s e N - s u l p h a t e g r o u p s , w i t h o r without tj-acetylation, r e s u l t s i n a d i s t i n c t and appreciable decrease i n binding a f f i n i t y and i n anticoagulant a c t i v i t y . P o r c i n e h e p a r i n h a s b e e n c l e a v e d r a n d o m l y by c h e m i c a l t e c h n i q u e s t o p r o d u c e h e x a saccha r i de s, o c t a s a ccha r i des, de ca sa ccha r i d e s, a n d f r a g m e n t s c o n t a i n i n g 1 4 a n d 16 r e s i d u e s t h a t a r e a b l e t o c o m p l e x with protease inhibitors.368 As a r e s u l t o f s t u d i e s o n t h e a v i d i t y of t h e s e f r a c t i o n s f o r p r o t e a s e i n h i b i t o r s , t h e i r b i n d i n g t o a n t i thrombin, a n d t h e c a t a l y s i s of t h e F a c t o r Xa-antithrom b i n i n t e r a c t ion, i t i s proposed that h e p a r i n p o s s e s s e s m u l t i p l e discrete s t r u c t u r a l d o m a i n s t h a t m o d u l a t e d i f f e r e n t f u n c t i o n s o f a n t i t h r o m b i n 111. T h e c h e m i c a l c o m p o s i t i o n a n d 1 3 C n.m.r. s p e c t r a o f h e p a r i n o c t a s a c c h a r i d e s h a v i n g h i g h a f f i n i t y f o r a n t i t h r o m b i n 111 a n d h i g h a n t i ( f a c t o r X a ) a c t i v i t y , a n d p r e p a r e d by t h r e e i n d e p e n d e n t a p p r o a c h e s , have been s t u d i e d and compared w i t h t h o s e of t h e corresponding i n a c t i v e species.369 Combined w i t h c h e m i c a l d a t a , t h e s p e c t r a of t h e active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The e f f e c t s o f h i g h - a c t i v i t y h e p a r i n f r a c t i o n s of d i f f e r i n g m o l e c u l a r w e i g h t o n a n t i t h r o m b i n I11 i n h i b i t i o n o f e a c h o f t h e s e r i n e p r o t e a s e s known t o o c c u r i n t h e i n t r i n s i c pathway of t h e

212


c o a g u l a t i o n cascade have been r e p o r t e d . 3 7 0 I n h i b i t i o n o f thrombin, F a c t o r IXa, a n d F a c t o r X I a showed s i m i l a r i t i e s i n t h e dependence o n molecular

weight

of

heparin,

and was

found

to

decreasing molecular weight o f the polysaccharide. F a c t o r Xa,

Factor XIIa,and

decrease

with

Inactivation o f

k a l l i k r e i n was l e s s dependent o n t h e s i z e

o f t h e p o l y sa ccha r i de. Low-molecular-weight

h e p a r i n o f l o w a n t i c o a g u l a n t a c t i v i t y and

h i g h - m o l e c u l a r - w e i g h t h e p a r i n o f c o r r e s p o n d i n g h i g h a c t i v i t y have been s u b j e c t e d t o N - d e s u l p h a t i o n

and t h e r e s u l t i n g amino groups r e -

a c y l a t e d w i t h d a n s y l c h l o r i d e o r r h o d a m i n e B i s ~ t h i o c y a n a t e . ~The ~ ~ results o f binding studies o f the modified heparins t o antithrombin I11 a r e c o n s i s t e n t w i t h t h e p r o p o s a l t h a t a s i n g l e h i g h - m o l e c u l a r weight high-activity

h e p a r i n o c c u p i e s t w o s i t e s when i t b i n d s t o

a n t i t h r o m b i n I11 w h e r e a s l o w - m o l e c u l a r - w e i g h t

low-activity

heparin

binds t o t h e two s i t e s s e p a r a t e l y . P r o t e o h e p a r a n s u l p h a t e p r e p a r e d from

r a t l i v e r p l a s m a membranes

i n h i b i t s t h e g r o w t h o f AH-130 a s c i t e s h e p a t o m a c e l l s . 3 7 2

Heparan

sulphate i s l e s s active. A has

non-collagen

been

occurring

protein,

identified rat

as

basement

a

i n a s s o c i a t i o n w i t h heparan sulphate, normal

constitutent

mem b r a n e . 3 7 3

The

a

naturally

material

of

resembles

p r o t e o g l y c a n s r e c e n t l y i s o l a t e d f r o m t h e EHS sarcoma a n d f r o m b o v i n e g l o m e r u l a r basement membrane. Heparan

sulphate

has

been

i d e n t i f i e d

as

the

major

g l y co s a m i n o g l y can i n b o v i n e r e t i n a l c a p i l l a r y b a s e m e n t m em brane.374 The b i n d i n g o f g l y c o s a m i n o g l y c a n s t o f i b r o n e c t i n a t a d h e s i o n s i t e s o f m u r i n e f i b r o b l a s t s has been r e p o r t e d . 3 7 5 s u l ph a t e p r o t eo g l y ca n s , co n t a in i n g h i gh 1y

l-s u l ph a t e d

Only heparan s e q ue nce s o n

t h e h e p a r a n s u l p h a t e , m e d i a t e s u b s t r a t u m a d h e s i o n t o t h e c e l l s by co-ordinate b i n d i n g t o

f i b r o n e c t i n o n t h e c e l l surface and serum

f i b r o ne c t i n. S e l f - a s s o c i a t i on

be t w een

various

hepa r a n s u l p h a t e s p e c i e s a n d

o l i g o s a c c h a r i d e f r a g m e n t s t h e r e o f h a v e b e e n s t u d i e d by a f f i n i t y

.

ch rom a t o g r a p h y 376 ,377

Se gm e n t s co m p r i s i n g bo t h

L - i d u r o na t e

and

g-

g l u c u r o n a t e may s e r v e a s c o n t a c t z o n e s f o r t h e s e l f - a s s o c i a t i o n , and t h e s t r e n g t h o f b i n d i n g i s dependent on c o o p e r a t i v e i n t e r a c t i o n s b e t w e e n a number o f s u c h zones.

However, v a r i o u s h e p a r a n s u l p h a t e

s u b f r a c t i o n s f r o m t r a n s f o r m e d c e l l s h a v e no a f f i n i t y f o r a g a r o s e g e l s s u b s t i t u t e d w i t h t h e c o r r e s p o n d i n g h e p a r a n s u l p h a t e species.378 T h e l a c k o f a s s o c i a t i o n o f t h e h e p a r a n s u l p h a t e may be c a u s e d by m i n o r changes i n t h e s e q u e n t i a l a r r a n g e m e n t s o f L - i d u r o n a t e -

and

0-

213

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides g l u c u r o n a t e - b e a r i n g r e p e a t i n g segments o f t h e p o l y s a c c h a r i d e . Heparan s u l p h a t e - c l e a v i n g o f a n a s c i t e s h e p a t o m a AH66, sulphate

at

the

cell

enzyme h a s been u s e d t o t r e a t c e l l s f o r which t h e occurrence o f heparan

surface

of

the

plasma

membrane has

been

e s t a b l i s h e d .379 Two p o o l s o f h e p a r a n s u l p h a t e p r o t e o g l y c a n s w i t h d i f f e r e n t b u o y a n t d e n s i t i e s h a v e been s e l e c t i v e l y s o l u b i l i z e d f r o m r a t l i v e r plasma

membranes

detergent.380

by

successive

incubations

with

heparin

and

The d e t e r g e n t - e x t r a c t e d h e p a r a n s u l p h a t e r e p r e s e n t s a

p r o t e o g l y c a n s p e c i e s t h a t has i t s c o r e p r o t e i n i n t h e l i p i d b i l a y e r o f t h e p l a s m a membrane. high

The p r e s e n c e o f h e p a r a n s u l p h a t e h a v i n g a

metabolic activity

has

been d e m o n s t r a t e d i n i s o l a t e d r a t

i n t e s t i n a l e p i t h e l i a l cells.381

A h i g h degree o f s u l p h a t i o n o f i t s

c h a i n s was d e t e c t e d . synthesis.382

H e p a r a n s u l p h a t e i s a p o t e n t i n h i b i t o r o f DNA

The i n h i b i t i o n i s n o t s i m p l y r e l a t e d t o g r o s s c h a r g e

density. B o v i n e v i t r e o u s body h y a l u r o n i c a c i d h a s been p u r i f i e d , t h e use o f

p r o t e o l y t i c enzymes,

by i o n - e x c h a n g e

without

~hromatography.~~~

1251 - L a b e l l e d

h y a l u r o n a t e- b i n d i n g

prepared.384

T h e i r a d s o r p t i o n on an i m m o b i l i z e d h y a l u r o n a t e g e l i n

p r o t e i n s f r o m c a r t i l a g e h a v e been

t h e p r e s e n c e o f f r e e c o m p e t i n g h y a l u r o n i c a c i d has been used as an assay f o r f r e e h y a l u r o n i c a c i d . o f 'H n.m.r. s p e c t r a i n {2H6}-dimethylsulphoxide dodecyltrimethylammonium s a l t s of c h o n d r o i t i n s u l p h a t e s and h y a l u r o n a t e o r s o d i u m s a l t s o f o l i g o m e r s f r o m h y a l u r o n a t e show u n a m b i g u o u s NH s i g n a l s . 3 8 5 some NH,

A s e c o n d a r y s t r u c t u r e i n h y a l u r o n a t e and

chondroitin sulphates, was o b s e r v e d .

i n v o l v i n g a hydrogen-bonded

homologous s e r i e s o f

oligosaccharides

t e s t i c u l a r h y a l ~ r o n i d a s e . ~C.d. ~~ showed t h a t t h e c.d.

acetamido

Sodium h y a l u r o n a t e has been c l e a v e d i n t o an by

the

a n a l y s i s of

action of

bovine

the oligosaccharides

s p e c t r u m o f h y a l u r o n a t e i n aqueous s o l u t i o n a t

n e u t r a l pH d o e s n o t r e f l e c t t o a n y s u b s t a n t i a l d e g r e e a p o l y m e r c o n f o r m a t i o n which r e q u i r e s c o o p e r a t i v e i n t e r a c t i o n between s e v e r a l repeating residues f o r stabilization. The s e q u e n t i a l h y d r o l y s i s o f r o o s t e r comb h y a l u r o n i c a c i d by e-glucuronidase

and f3-Q-2-acetarnido-2-deoxy-glycosidase

B-

has f a i l e d

t o d e m o n s t r a t e t h e p r e s e n c e o f any b r a n c h i n g i n t h e p o l y s a c c h a r i d e chain.387 sugars.

No e v i d e n c e

was

found for

the presence o f

neutral

The t e m p e r a t u r e dependence o f b i n d i n g h y a l u r o n a t e o l i g o m e r s

t o b o v i n e n a s a l p r o t e o g l y c a n has been reported.388 Hyalurononectin,

a human b r a i n g l y c o p r o t e i n c a p a b l e o f b i n d i n g


214 t o hyaluronic acid, by

hyaluronic

hyaluronidase

has been i s o l a t e d . 3 8 9

acid

and

by

the

but

not

by

either

The b i n d i n g i s i n h i b i t e d

products

hydrolysis

by

glycosaminoglycans

of

or

monosaccharides. The

interactions of

s u l p h a t e d p o l y s a c c h a r i d e s s u c h as

s u l p h a t e and c h i t i n s u l p h a t e

w i t h Solanum t u b e r o s u m

wheat germ a g g l u t i n i n have been r e p o r t e d . 3 9 0

keratan

a g g l u t i n i n and

The p r e s e n c e o f t h e

sulphate groups i n these polysaccharides d i d n o t i n t e r f e r e w i t h t h e i r s p e c i f i c i n t e r a c t i o n s with the l e c t i n s . The d e g r a d a t i o n o f k e r a t a n s u l p h a t e h a s b e e n a c h i e v e d by B - 2 -

For

a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s i d a s e s A a n d 8 (EC 3.2.1.52).391 e n z y m ic a c t i v i t y a s u b s t r a t e w i t h exposed n o n- s ulphat ed 2-deoxy-g-glucosyl

residues a t

t h e non-reducing

end o f

2-acetamidothe polymer

i s required. Keratan

-----Pseudo m o nas glucose

sulphate

=.

has

been

degraded

by

an

extract

of

a

t o g i v e 2 - a c e t a m ido -2-deox y-3-g-Q -ga l a c t o s y 1-B-0-

6 - s ~ l p h a t e . ~ ' ~T h i s

disaccharide

after

reduction with

s o d i u m b o r o t r i t i d e can be u s e d as a s u b s t r a t e f o r t h e measurement o f 2-acetamido-2-deoxy-q-glucose

6-sulphate

sulphatase,

w h i l s t the

d e s u l p h a t e d r e d u c e d d i s a c c h a r i d e c a n be u s e d as a s u b s t r a t e f o r t h e measurement

of

(1+3)-2-acetamido-2-deoxy-B-~-glucosidase.

keratan sulphate from hydrazinolysis,

bovine cornea,

deamination

and

d e g r a d e d by

sodium

releases two t e t r a s a c c h a r i d e f r a c t i o n s , which

(14)

has

been e s t a b l i s h e d . 3 9 3

borohydride

reduction,

t h e s t r u c t u r e o f one o f

80th

r e s i d u e s bear k e r a t a n s u l p h a t e ch a in s.

Peptido-

desulphation,

terminal

rj-mannosyl

A s t r u c t u r e i n w h i c h an

I-

a-Q-Manp-(l+6)-B-q-Mane-(l+?)-g-GlcENAc-L-Asn -

3

I 1 a-E-Manp (14) f u c o s y l r e s i d u e and an N " - d i a c e t y l c h i t o b i o s y l

2-acetamido-2-deoxy-~-glucosyl has

also

been

proposed.

sequence r e p l a c e t h e

residue attached t o the p r o t e i n chain Studies

on t h e

p o l y d i s p e r s i t y and

h e t e r o g e n e i t y o f p r o t e o k e r a t a n s u l p h a t e f r o m c a l f and p o r c i n e c o r n e a have

been

reported.394

The

linkage

region

of

bovine

corneal

p r o t e o k e r a t a n s u l p h a t e i s b r a n c e d a t a Q-mannose r e s i d u e (15).395 The s t r u c t u r a l c h a r a c t e r i s t i c s show t h a t t h e r e i s a g r e a t s i m i l a r i t y


m

I

n

n

I

hl 4 .-I

v

1

hl

u

9

E?

d

n

In 4

v

hl

n

v

hl I

0

n

I

a - m

v

u

n CI

v

I

u

I

c311

M

0

-

215


216

E-

b e t w e e n t h e l i n k a g e r e g i o n o f t h e p r o t e o k e r a t a n s u l p h a t e and t h e g l y c o s i d i c a l l y l i n k e d L - a s p a r a g i n e m o i e t y o f many g l y c o p r o t e i n s .

--

Biosynthesis. ectodermal effects

H y a l u r o n i c a c i d s y n t h e s i s by t h e

ridge of

of

chick

phase

of

l i m b bud has

growth,

c e l l

apical

been r e p o r t e d . 3 9 6

density

and

also

The serum

c o n c e n t r a t i o n a r e i m p o r t a n t c o n t r i b u t o r s f o r t h e i n c o r p o r a t i o n o f 2amino-2-deoxy-9-{

3H1 - g l u c o s e

t i c smooth m u s c l e c e l l s . 3 9 7

i n t o g l y c o s a m i n o g l y c a n s by r a b b i t a o r The s y n t h e s i s o f s u l p h a t e d g l y c o s a m i n o -

in

glycans i n r a t l i v e r explants prepared from regenerating tissue, which the f a c t o r s of

c e l l n e c r o s i s and c o n s e c u t i v e i n f l a m m a t i o n

a s s o c i a t e d w i t h most models o f e x p e r i m e n t a l h e p a t i c f i b r o s i s a r e shows a d e p r e s s i o n by a b o u t 50% o f t h e i n c o r p o r a t i o n o f 2-

excluded,

amino-Z-deoxy-Q-{ 14C) - g l u c o s e i n t o t o t a l

glyco~aminoglycan.~~~

R a t l i v e r p a r e n c h y m a l c e l l s have been e v a l u a t e d f o r

their

a b i l i t y t o s y n t h e s i z e and a c c u m u l a t e h e p a r a n s u l p h a t e as t h e m a j o r component and l o w - s u l p h a t e d c h o n d r o i t i n s u l p h a t e , chondroitin sulphate

dermatan sulphate,

and h y a l u r o n i c a c i d as t h e

minor

ones.399

G l y c o s a m i n o g l y c a n s a r e s y n t h e s i z e d by r a t g l o m e r u l i a n d t r a n s p o r t e d t o g l o m e r u l a r basement membranes. product synthesized, chondroitin

sulphates

are

also

produced.400

The

transfer

of

f a c t o r 4, a b a s i c t e t r a m e r i c p r o t e i n c o m p l e x e d w i t h a

platelet series of studied

Heparan s u l p h a t e i s t h e major

a l t h o u g h s m a l l e r amounts o f h y a l u r o n i c a c i d and

glycosaminoglycans,

by

derivative

measuring of

changes

platelet

factor

t o h e p a r i n has been d e t e c t e d and i n

the

anisotropy

4.401

The

f a c t o r 4 - p r o t e o g l y c a n complex f r o m t h e p l a t e l e t the t r a n s f e r o f the p r o t e i n t o heparin.

of

release

in

the of

dansyl

platelet

vivo results i n

voderate quantities o f

d e r m a t a n s u l p h a t e o r h e p a r a n s u l p h a t e do n o t p r e v e n t t h e t r a n s f e r . The e f f e c t o f c y c l o h e x i m i d e on t h e s y n t h e s i s o f p r o t e o g l y c a n s by c u l t u r e d c h o n d r o c y t e s f r o m t h e Swarm r a t c h o n d r o s a r c o m a h a s been reported.402

The c u l t u r e d c h o n d r o c y t e s c o n t a i n a l a r g e p o o l o f c o r e

protein available for

the addition o f

chondroitin sulphate

chains.

I n t h e absence o f e n t r y o f n e w l y s y n t h e s i z e d c o r e p r o t e i n i n t o t h i s pool following

cycloheximide treatment,

the remaining core p r o t e i n

molecules are processed t o completed proteoglycans w i t h n e a r l y f i r s t order

exponential

synthesis o f

kinetics.

The

biochemical pathways

for

the

c h o n d r o i t i n s u l p h a t e w e r e n o t d r a s t i c a l l y a f f e c t e d by

cycloheximide.

I n h i b i t i o n o f p r o t e i n s y n t h e s i s by c y c l o h e x i m i d e

reduces t h e i n c o r p o r a t i o n of { 35S)-sulphate i n t o heparan s u l p h a t e t o

217

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides about

5% o f

untreated hepatocytes.

4-Nitrophenyl f3-Q-xyloside

p a r t i a l l y reverses the i n h i b i t o r y effect.403 chains

not

bound t o

core p r o t e i n were

Free heparan sulphate

synthesized under

these

conditions. The p a t t e r n o f g l y c o s a m i n o g l y c a n s y n t h e s i s b y s m o o t h - m u s c l e c e l l s f r o m p i g a o r t i c media has been studied.404 s y n t h e s i z e d by media c e l l s e x h i b i t d i f f e r e n t

Proteoglycans

glycosaminoglycan

d i s t r i b u t i o n patterns according t o t h e i r l o c a l i z a t i o n . Cultured c e l l s from red dermal tissue,

containing a large

p r o p o r t i o n o f c e l l s from the h a i r f o l l i c l e s . , produce h y a l u r o n i c acid, dermatan sulphate, sulphate.405

The

c h o n d r o i t i n 4-sulphate,

patterns

of

synthesis

h e p a r i n and heparan

and s u l p h a t i o n

of

the

g l y c o s a m i n o g l y c a n s changed w i t h c o n t i n u e d t i m e i n c u l t u r e . Proteoglycan

synthesis

hydrocortisone-suppressed reported.406

i n

rabbit

normal

articular

and

chronically

cartilage

has

been

The r e p o r t c o n f i r m s t h a t h y d r o c o r t i s o n e c a n e f f e c t

p o o l s i z e s , and a l s o t h e l e v e l o f

UDP-2-acetamido-2-deoxy-p-hexose

a-galactosyltransferases, b u t n o t a - x y l o s y l t r a n s f e r a s e i n v o l v e d i n the synthesis of linkage region.

the chondroitin sulphate carbohydrate-protein

The m a j o r e f f e c t i s t h e d e p r e s s i o n o f p r o t e o g l y c a n

core-protein synthesis.

No n e t change i n t h e p r o t e o g l y c a n s t r u c t u r e

was o b s e r v e d . Insulin

and

r a t-derived,

multiplication-stimulating

somatomedin-like

activity

stimulates

s y n t h e s i s i n r a t c h o n d r o s a r c o m a c h o n d r o c y t e s .407

polypeptide proteoglycan

The s t r u c t u r e s o f

t h e p r o t e o g l y c a n s s y n t h e s i z e d i n response t o t h e hormones compared w i t h t h o s e s y n t h e s i z e d i n t h e medium a l o n e a r e s i m i l a r , chondroitin sulphate chains, hormones,

increase

i n

but the

synthesized i n the presence o f

molecular

weight

by

25-30%.408

The

d i s t r i b u t i o n o f g l y c o s a m i n o g l y c a n s y n t h e s i s by r a t g l o m e r u l i i n v i v o and

in

v i t r o has been reported.409

A d m i n i s t r a t i o n o f a s i n g l e dose o f

2-amino-2-deoxy-g-galactose

t o r a t s causes time-dependent b i p h a s i c changes g l y c o s a m i n o g l y c a n s y n t h e s i s i n l i v e r .410 A rapidly

o f t o t a l occurring

i n h i b i t i o n i s f o l l o w e d by a s i g n i f i c a n t l y enhanced p r o d u c t i o n o f { 35Sl-labelled

glycosaminoglycans i n l a t e r

stages.

Undersulphated

f3-e-xy 10s i d e g l y c o s a m i n o g l y c a n s a r e s e c r e t e d f r o m c h i c k - e m b r y o chondrocytes

i n the

presence o f

the

i o n o p h o r e monensin.411

S t i m u l a t e d p e r i p h e r a l b l o o d monocytes c o n t a i n a f a c t o r c s ) which affects synovial cells i n culture,

depressing markedly collagen

synthesis w h i l e enhancing glycosaminoglycan production.412

The

218


i n v o 1 vem e n t o f s u c h b i o l o g i c a 11y a c t i v e m o n o c y t e - d e r i ve d s u b s t a n c e (s ) i n c o n n e c t i ve-tiss u e me t a b o l i sm o f i n f 1am ma t o r y l e s i o n s i s discussed. The assembly of proteoglycan a g g r e g a t e s i n c u l t u r e s o f c h o n d r o c y t e s from b o v i n e t r a c h e a l c a r t i l a g e has been studied.413 P r o t e o g l y c a n monomers and h y a l u r o n i c acid a r e e x p o r t e d s e p a r a t e l y The s i z e o f t h e a g g r e g a t e s i n c r e a s e s and combined e x t r a c e l l u l a r l y . g r a d u a l l y w i t h time as t h e p r o p o r t i o n o f monomers bound t o Immunochemical a n a l y s i s of t h e hyaluronic acid increases. proteoglycans h a s i d e n t i f i e d c r o s s - r e a c t i v i t y of molecules i s o l a t e d from d i f f e r e n t species.414 The i n t r a c e l l u l a r p r e c u r s o r p o o l of c o r e p r o t e i n i n t h i s c h o n d r o c y t e system h a s been i d e n t i f i e d and p a r t i a l l y c h a r a c t e r i z e d .415 A s t u d y o f a c c e p t o r s a n d primers f o r c h o n d r o i t i n s u l p h a t e - c h a i n b i o s y n t h e s i s by m i c r o s o m a l p r e p a r a t i o n s f r o m c h i c k - e m b r y o e p i p h y s e a l Incorporation of sugars into c a r t i l a g e has been reported.416 g l y c o s a m i n o g l y c a n s has b e e n u s e d t o o b t a i n i n f o r m a t i o n o n t h e e n t i r e proteoglycan s t r u c t u r e a t t h e s i t e o f glycosaminoglycan synthesis. A s o l u b l e e n z y m e f r o m q u a i l o v i d u c t i n c o r p o r a t e s s u l p h a t e a t 06 o f t h e no n- r e d u c i n g 2 - a c e tam i d o - 2 - d e o x y - ~ - g a l a c t o s y 1 4 - s u l p h a t e r e a c t i o n c a t a l y s e d by t h i s r e s i d u e o f c h o n d r o i t i n ~ u l p h a t e . ~ ~ The ’ e n z y m e i s d i f f e r e n t f r o m t h e s u l p h a t i o n m e d i a t e d by o t h e r k n o w n s u l p h o t r a n s f e r a s e s. The f o r m a t i o n o f C-idopyranosyluronic acid during t h e s y n t h e s i s o f d e r m a t a n s u l p h a t e h a s been s t u d i e d i n human f i b r o b l a s t c u l t u r e s a n d m i c r o s o m e s o f t h e same c e l l s . 4 1 8 E p i m e r i z a t i o n o f ag l ucopy r a n 0 s y l u r o n i c a c i d t o L - i dopy r a n 0 s y l u r o n i c a c i d r e s i d u e s during the biosynthesis o f dermatan sulphate involves an abstraction o f t h e C-5 h y d r o g e n o f t h e u r o n o s y l r e s i d u e . S u b s t r a t e s f o r 2- ace tam i d o - 2 - d e o x y-Q-gl uco s y 1t ra n s f e r a se , w h i c h acts i n conjunction with Q-glucuronosyl transferase i n the b i o s y n t h e s i s o f h e p a r i n , h a v e b e e n p r e p a r e d f r o m a l o w s u l p h a t e d , aglucuronic acid-rich heparan sulphate fraction.419 U s i n g these o l i g o s a c c h a r i d e s , t h e products o f t h e r e a c t i o n have been c h a r a c t e r i z e d a n d some o f t h e k i n e t i c parameters o f t h e enzyme have been determined. S o m e o f t h e o l i g o s a c c h a r i d e s were s h o w n t o s u b s t i t u t e f o r t h e endogenous s u b s t r a t e s and s e r v e as p r i m e r s i n po 1y s a c c h a r i de c h a i n e l o n g a t i o n . T h e s e c r e t i o n o f h e p a r a n s u l p h a t e by c u l t u r e d r a t h e p a t o c y t e s i s i n c r e a s e d i n t h e p r e s e n c e of t h e f l a v e n o i d (+) ~ a t e c h i n . ~ ” T h i s i n c r e a s e i s d u e t o a new s p e c i e s o f h e p a r a n s u l p h a t e l a c k i n g t h e


219

c a r b o h y d r a t e - p r o t e i n l i n k a g e b e t w e e n I)-xylose and I - s e r i n e i n t h e no rm a1 h e p a r a n s u l p h a t e p r o t e o g l y c a n . E x t r a c e l l u l a r h y a l u r o n i c acid i s t h e major glycosaminoglycan s y n t h e s i z e d by t h e e p i d e r m i s w h e n i n t a c t s k i n s l i c e s a r e m a i n t a i n e d i n organ culture.421 I n t h e a b s e n c e o f t h e dermis t h i s s y n t h e s i s i s considerably reduced, and the synthesis of sulphated g l yco s a m i n o g l y c a n s , main1y hepa r a n s u l p h a t e , i s u n a f f e c t e d . T h e f o r m a t i o n o f b o t h N- a n d g - s u l p h a t e g r o u p s ( s u l p h a m i d o a n d ester s u l p h a t e groups, r e s p e c t i v e l y ) o f t h e 2-amino-2-deoxy-eg l u c o s y l r e s i d u e s i n p o r c i n e a o r t a h a s b e e n d e m o n s t r a t e d by t h e s t u d y o f s u l p h a t e i n c o r p o r a t i o n from l a b e l l e d 3’-phosphoadenyl sulphate t o heparan sulphate of microsomal f ractions.422 The p r e f e r e n t i a l N-sulphation is obtained f o r incorporation o f labelled precursor over a short period, with P-sulphation occurring on previously 3-sulphated heparan sulphate. The s t i m u l a t i o n o f h y a l u r o n i c a c i d s y n t h e s i s i n mouse s k i n i n r e s p o n s e t o e s t r o g e n t r e a t m e n t i s mediated through e s t r o g e n r e c e p t o r s and i n v o l v e s t h e i n d u c t i o n o f hy a 1 u r o n i c a c i d s y n t h e t a s e . 4 2 3 G l y c o Sam i no g l y c a n s e c r e t i o n from perfused monolayer c u l t u r e s o f r a b b i t ear c h o n d r o c y t e s has been r e p o r t e d . 4 2 4 C o l c h i c i n e has been shown t o a f f e c t t h e l e v e l of glycosaminoglycan synthesis and transport. A short-term incubation system, developed f o r the study of glycosaminoglycan synthesis during t h e early stages o f medullary bone f o r m a t i o n i n J a p a n e s e q u a i l i n t h e p r e s e n c e o f e s t r o g e n , h a s i d e n t i f i e d k e r a t a n s u l p h a t e and c h o n d r o i t i n ~ u l p h a t e . ~ ’ ~

-

The p o l y d i s p e r s i t y p r e s e n t i n a g g r e g a t i n g p r o t e o g l y c a n s n e w l y s y n t h e s i z e d by c h o n d r o c y t e s f r o m r a t chondrosarcoma has been a t t r i b u t e d t o t h e amount of c h o n d r o i t i n sulphate/core p r o t e i n and n o t t o any d e t e c t a b l e d i f f e r e n c e s i n t h e s i z e of t h e c o r e protein.426,427 Glycosaminoglycans i n u r i n e from p a t i e n t s representing t h e major d i f f e r e n t mucopolysaccharidoses have b e e n s e p a r a t e d a n d m e a s u r e d by u s e o f a p r o c e d u r e r e q u i r i n g s m a l l volumes of urine.428 Approximately h a l f t h e p a t i e n t s e x c r e t e d small amounts of heparin. T h e k e r a t a n s u l p h a t e i n samples f r o m M o r q u i o ’ s disease p a t i e n t s m i g r a t e d d i f f e r e n t l y from a u t h e n t i c k e r a t a n s u l p h a t e u n l e s s d i g e s t e d w i t h c h o n d r o i t i n s u l p h a t e l y a s e ABC. Total sulphated glycosaminoglycan l e v e l s i n commercially available preparations of hyaluronic acid and i n the urine of normal individuals and the synovial f l u i d o f individuals w i t h rheumatoid a r t h r i t i s and o s t e o a r t h r i t i s have been measured Pathology.

220


s p e c t r o p h o t o m e t r i c a l l y as t h e i r A l c i a n b l u e Lowe’s syndrome r e s u l t s i n an u n d e r s u l p h a t i o n o f c h o n d r o i t i n ~ u l p h a t e s . ~ ~ ’ T h i s i s now s h o w n t o be a c o n s e q u e n c e o f a l o w e r l e v e l o f a c t i v e sulphate

(adenosine 3’-phosphate

syndrome

fibroblasts,

caused

pyrophosphatase a c t i v i t y

5’-phosphosulphate) by

i n

an e l e v a t i o n o f

degrading

adenosine

Lowe’s

nucleotide

3’-phosphate

5’-

phosphosulphate. A

low

urinary

e x c r e t i o n o f heparan sulphate i n t h r e e p a t i e n t s

w i t h Lowe’s s y n d r o m e has b e e n o b s e r v e d , a l t h o u g h u r i n a r y e x c r e t i o n o f total

i s o m e r s shows no

g l y c o s a m i n o g l y c a n and c h o n d r o i t i n s u l p h a t e

significant

differences

b e t w e e n Lowe’s

syndrome s u b j e c t s

a n d age-

m a t c h e d c o n t r o l s . 431 A new D,

disease,

tentatively

has been r e c o r d e d . 4 3 2

designated Sanfilippo

It i s

f e a t u r e s o f t h e S a n f i l i p p o syndrome,

c h a r a c t e r i z e d by

disease type the

clinical

excessive e x c r e t i o n o f heparan

s u l p h a t e , a n d t h e i n a b i l i t y t o r e l e a s e i n o r g a n i c s u l p h a t e f r o m 2a c e t a m i d o - 2 - d e o x y - a - g 1 uco sy 1 6 - s u l p h a t e sulphate-derived oligosaccharides.

residues i n heparan

However, t h e r e q u i r e m e n t s f o r

e n z y m a t ic h y d r o 1y s i s o f 2 - a c e t am ido - 2-deox y - g - g 1 uco sy 1 6 - s u l p h a t e linkages are d i f f e r e n t f o r heparan sulphate and k e r a t a n sulphate, when c o m p a r e d w i t h t h o s e p o l y s a c c h a r i d e s f r o m

ordinary

Sanfilippo

syndrome p a t i e n t s . Glycosaminoglycan ne p h r o t i c

s y n drom e has

excretion

i n

urine

been i n ves t i g a t e d.433

from

patients

with

p r ot e i n-asso c i a t e d

A

l o w - c h a r g e f o r m o f c h o n d r o i t i n s u l p h a t e w h i c h i s n o t e x c r e t e d by n o r m a l s u b j e c t s was i d e n t i f i e d . C h o n d r o i t i n 6-sulphate w i t h low sulphate content i s e x c r e t e d i n t h e u r i n e o f p a t i e n t s w i t h an u n u s u a l f o r m o f s p o n d y l o e p i p h y s e a l d y ~ p l a s i a . ~ The ~ ~s e r a o f t h e s e p a t i e n t s show a l o w a c t i v i t y o f 3’phosphoadenos i ne 5’-phosphos u l phat e sulphotransferase, w h i l e the sulphate

i s

a much

urinary

better acceptor

chond r o it in

s u l p h a te

undersulphated chondroit i n of

sulphate

than

standard

c h o n d r o i t i n s u l p h a t e o n i n c u b a t i o n w it h 3 ’-p h o s p h o a d e no s i n e 5 ’pho sphos u l p h a t e a n d norm a1 s u l p h o t r a n s f e r a s e s . P r e c i p i t a t i o n w i t h c e t y l p y r i d i n i u m c h l o r i d e f o l l o w e d by u r o n i c

acid

assay

has

been

used

to

study

urinary

glycosaminoglycan

e x c r e t i o n i n normal subjects and calcium-stone

formers.435

No

s i g n i f i c a n t d i f f e r e n c e i n d a i l y l e v e l s o f g l y c o s a m i n o g l y c a n s was detected. P r o t e o g l y c a n s p r e s e n t i n norm a 1 and a t h e r o s c l e r o t i c human a o r t a s have been i s o l a t e d a n d p a r t i a l l y ~ h a r a c t e r i z e d . ~ ~ ~


22 1

The g l y c o s a m i n o g l y c a n c o m p o s i t i o n o f s a m p l e s o f l i v e r f r o m p a t i e n t s w i t h a l c o h o l i c c i r r h o s i s has been compared w i t h t h a t o f no rmal l i v e r . 4 3 7 I n c r e a s e d l e v e l s o f h y d r o x y - l - p r o 1 i n e , h y a l u r o n i c a c i d , h e p a r a n s u l p h a t e , c h o n d r o i t i n 4- a n d 6 - s u l p h a t e s , a n d d e r m a t a n s u l p h a t e were d e t e c t e d i n t h e c i r r h o t i c l i v e r s a m p l e s . Age-related changes i n hydrodynamic s i z e of the proteoglycan monomers o f human c a r t i l a g e a n d o f t h e s p e c i f i c { 3 5 S ) - l a b e l l i n g o f t h e i r chondroi t i n s u l p h a t e and k e r a t a n s u l p h a t e c h a i n s have been de s c r i be d. 4 3 I n o r d e r t o e x c l u d e de g e n e r a t i o n ( 0s t e o a r t h r o t i c ) processes that i n t e r f e r e w i t h age-specific changes, c a r t i l a g e of the p r o c e s s u s x y p h o i d was i n v e s t i g a t e d . A u t o l o g o u s h u m a n k n e e a n d s h o u l d e r c a r t i l a g e show a g e - r e l a t e d changes: t h e p r o t e o g l y c a n c o n t e n t decreases i n c o n t e n t a n d p r o t e o g l y c a n s u b u n i t s decrease i n s i z e . 4 3 9 In contrast, the p r o p o r t i o n s of keratan s u l p h a t e t o c h o n d r o i t i n s u l p h a t e , p r o t e i n t o glycosaminoglycan, and c h o n d r o i t i n 6 - s u l p h a t e t o c h o n d r o i t i n 4s u l p h a t e a l l i n c r e a s e w i t h age. The s t r u c t u r e s o f t h e p r o t e o g l y c a n s o f a r t i c u l a r c a r t i l a g e s o f b o v i n e f o e t u s e s a t v a r i o u s s t a g e s of development have been compared During f o e t a l l i f e , with those of the c a l f and a d u l t a r t i c u l a r c a r t i l a g e proteoglycans are rich i n c h o n d r o i t i n s u l p h a t e a n d c o n t a i n a t a l l a g e s some k e r a t a n s u l p h a t e a n d 2 - g l y c o s i d i c a l l y linked oligosaccharides i n both a keratan-rich region and i n the remainder o f the polysaccharide attachment region. An a n a l y s i s o f c h a n g e s o f s u l p h a t e d g l y c o s a m i n o g l y c a n s a n d related glycosidases during f o e t a l development shows t h a t c h o n d r o i t i n 6 - s u l p h a t e i n c r e a s e s i n c o n c e n t r a t i o n up t o f i f t y d a y s o f f o e t a l d e v e l o p m e n t , and t h e n decreases p r o g r e s s i v e l y u n t i l i t s complete disappearance i n a d u l t tissues.441 The l e v e l s o f h e p a r a t i n s u l p h a t e a n d d e r m a t a n s u l p h a t e do n o t c h a n g e d u r i n g t h i s p e r i o d . The m a j o r p r o t e o g l y c a n s i n t h e a r t i c u l a r c a r t i l a g e o f f o e t a l , calf, and a d u l t a n i m a l s d i f f e r i n t h e c o n t e n t , t y p e s o f s t r u c t u r e o f the chondroitin sulphate, keratan sulphate, and oligosaccharide constituents.442 These changes i n s t r u c t u r e r e f l e c t t h e g r o s s agerelated changes i n t h e chemical composition of t h e t i s s u e . Proteoglycans of t h e nucleus pulposus and annulus f i b r o s u s o f normal a n d d e g e n e r a t e i n v e r t e b r a l d i s c s have been examined.443 A l a r g e p r o p o r t i o n o f t h e proteoglycans found i n d i s c undergo a degree of d e g r a d a t i o n i n t h e h y a l u r o n a t e - b i n d i n g r e g i o n i n d e g e n e r a t e tissue. Glycosaminoglycans o f c u l t u r e d human-foetal melanocytes have

222 been

Carbohydrate Chemistry compared with

cells.444

those

p r o d u c e d by

c u l t u r e d human melanoma

By m a n i p u l a t i n g c e l l s u r f a c e s w i t h enzymes s p e c i f i c f o r

c l e a v i n g glycosaminoglycans from c e l l s , functional

i t i s p o s s i b l e t o change t h e impairment

r e l a t i o n s h i p s such a s adhesion and adhesion

among b o n e - m a r r o w

fibroblast-like

fi

cells cultured

vitro,

mature

l e u c o c y t e s and b l a s t s f r o m n o r m a l marrow and p e r i p h e r a l b l o o d o f leukaem i a p a t i e n t s . 4 4 5 The a b s e n c e o f p r o t e o g l y c a n c o r e p r o t e i n i n c a r t i l a g e f r o m omd/cmd

( c a r t i l a g e m a t r i x d e f i c i e n c y ) mouse446 a n d i n c a r t i l a g e

m u t a n t n a n ~ m - e l i ah ~a s~ ~been r e p o r t e d .

Cell and T i s s u e Glycoproteins

8

Glycoproteins from a d u l t - r a t

b r a i n s y n a p t i c v e s i c l e s h a v e been

f r a c t i o n a t e d by s e q u e n t i a l a f f i n i t y

chromatography i n t h e

presence

o f sodium d o d e c y l s u l p h a t e o n f o u r d i f f e r e n t i m m o b i l i z e d l e ~ t i n s . ~ ~ Nine f r a c t i o n s c o n t a i n i n g o n l y g l y c o p r o t e i n s and d i f f e r i n g i n t h e i r t e r m i n a l s u g a r s were com p o s i t i o n

and

s e p a r a t e d and a n a l y s e d f o r t h e i r c a r b o h y d r a t e

e l e c t rophor et i c

profiles.

considerable

A

h e t e r o g e n e i t y o f t h e g l y c o p r o t e i n p o p u l a t i o n was o b s e r v e d w h i c h c a n n o t be e x p l a i n e d s o l e l y by t h e m i c r o h e t e r o g e n e i t y o f t h e

glycans

o f the synaptic vesicle glycoproteins. A g r o u p of

s t r u c t u r a l l y r e l a t e d g l y c o p r o t e i n s f r o m human b r a i n

and f i b r o b l a s t s ,

which account f o r a l l t h e Thy-1

species cross-

r e a c t i v e antigenic a c t i v i t y present i n these tissues, purified.449

has

been

The i s o l a t e d p r o d u c t s a r e c l o s e l y

r e l a t e d t o each

o t h e r a n d c o u l d be t h e p r o d u c t o f a s i n g l e gene.

T h e y s h o w many

properties antigen.

i n common w i t h

p u r i f i e d forms o f

Human b r a i n g l y c o p r o t e i n s d e p l e t e d of Thy-1

been used f o r

monoclonal antibody

i n t e r a c t s with a determinant (cerebral,

the

production.450

rodent

Thy-1

a n t i g e n have The a n t i b o d y

p r e s e n t o n a l l human b r a i n s u b r e g i o n s

c o r t i c a l grey matter,

white matter,

caudate,

thalmus,

d e n t a t e n u c l e u s , putamen,and c e r e b e l l a r c o r t e x ) b u t i s a b s e n t f r o m a wide range o f o t h e r t i s s u e s (heart,

liver,

spleen).

The d e t e r m i n a n t

i s c o n s e r v e d i n mammalian e v o l u t i o n , a s t h e b r a i n s o f r a t a n d dog have amounts e q u a l t o t h a t f o u n d i n human b r a i n . Some b i o c h e m i c a l c h a r a c t e r i s t i c s , compositions,

examined and compared w i t h s i m i l a r

.

m o us e 45 1

i n c l u d i n g the carbohydrate

o f b r a i n T h y - 1 g l y c o p r o t e i n o f dog a n d man h a v e been c h a r a c t e r i s t i c s i n r a t and

223

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides A new

caprine n e u r o c e r v i c a l storage disease i n r e l a t e d Nubian

g o a t s h a s been c h a r a c t e r i z e d by t h e p r e s e n c e i n b r a i n t i s s u e o f t h e t r i s a c c h a r i d e O-B-g-mannopy r a n 0 sy 1-( 1+4) - 2 - a c e t a m i d o - 2 - d e o x y - Q--

(B-E-

g l u c o p y r a n o s y 1- ( 1 + 4 ) - 2 - a c e t a m id o - 2 - d e o x y - g - g l u c o s e

m a n n o s y l c h i t o b i o s e ) .452 The a c c u m u l a t i o n o f t h e t r i s a c c h a r i d e s u g g e s t s t h e p o s s i b i l i t y o f a g e n e t i c d e f e c t i n B-D=-mannosidase i n the

catabolic

pathway

for

d e f i c i e n c y o f B+-mannosidase

N-linked

complex

glycopeptides.

A

a c t i v i t y was d e m o n s t r a t e d i n a number

o f t i s s u e s f r o m t h e a f f e c t e d goats.453 The l e v e l s o f N - a c e t y l n e u r a m i n i c

a c i d i n w a t e r - s o l u b l e and

membrane f r a c t i o n s o f v a r i o u s r a t b r a i n c o r t e x t i s s u e s have been

.

m ea s u r e d 45 D e v e l o p m e n t a l changes i n t h e b i o s y n t h e s i s a n d c o n c e n t r a t i o n o f i n d i v i d u a l i-f u c o s y l - a n d !-ace t y l n e u r a m i n o s y l - c o n t a i n i n g glycoproteins

associated w i t h

s y n a p t i c membranes and s y n a p t i c

j u n c t i o n s have been r e p o r t e d . 4 5 5 The i n c o r p o r a t i o n o f L - f u c o s e

i n t o s y n a p t i c m i t o c h o n d r i a 1 and

p l a s m a-m em b r a n e g l y c o p r o t e i n s h a s b e e n r e c o r de d .456 mitochondria

s i t u have a g r e a t e r c a p a c i t y

Sy n a p t o som a 1

for the incorporation

o f &-fucose i n t o p r o t e i n t h a n f r e e mitochondria. S y n a p t i c membranes from

sheep b r a i n c o r t e x

incorporate

i n o r g a n i c s u l p h a t e i n t o a range o f g l y c o p r o t e i n s (mol.

lo4

-

1.6

wts.

1.6

x

1 0 ~ 1 . ~ 5 ~

E v i d e n c e h a s been p r e s e n t e d f o r

t h e i n c o r p o r a t i o n o f C-fucose

a n d t h e i d e n t i f i c a t i o n o f I - f u c o s y l g l y c o p r o t e i n s i n sheep c o r t e x sy na p t o som e s .458 The c a r b o h y d r a t e s t r u c t u r e o f g l y c o p r o t e i n s a s s o c i a t e d w i t h r a t c e n t r a l - n e r vo u s - s y s t em my e l i n i s de v e l a pme n t a l l y Although

r e g u l ated.459

t h e p r e c i s e a s s o c i a t i o n o f t h e s e membrane c o m p o n e n t s w i t h

t h e axolemma,

t h e o l i g o d e n d r o g l i a l p l a s m a membrane, o r m y e l i n has n o t

been d e t e r m i n e d ,

t h e o b s e r v e d changes t h a t o c c u r d u r i n g development

s u p p o r t a p r o b a b l e f u n c t i o n a l r o l e f o r many o f them i n t h e m e c h a n i s m o f m y e l i n o g e n e s i s. F l u o r e s c e n t - l a b e l l e d l e c t i n s have been u s e d t o examine t h e g l y c o p r o t e i n compo s i t i o n o f

sub fraction^.^^^

The

r a b b i t p e r i p h e r a l - n e r vo u s - s y s t em my e l i n

g l y c o p r o t e i n composition o f two major

f r a c t i o n s i s more complex t h a n p r e v i o u s l y r e p o r t e d . The s e q u e n c e o f e v e n t s i n t h e d e s t r u c t i o n o f m y e l i n p r o t e i n s and g l y c o p r o t e i n s i n experimental demyelination o f the r a b b i t o p t i c n e r v e h a s been r e p o r t e d . 461 The

presence

of

myelin-associated

glycoprotein

i n the

224


p e r i p h e r a l nervous system of rats has been demonstrated.462 The presence of t h i s g l y c o p r o t e i n i n t h e periaxonal region o f both peripheral and c e n t r a l m y e l i n s h e a t h s i s c o n s i s t e n t w i t h a similar i n v o l v e m e n t o f the g l y c o p r o t e i n i n axon-sheath c e l l i n t e r a c t i o n s i n t h e p e r i p h e r a l n e r v o u s system a n d t h e c e n t r a l n e r v o u s s y s t e m . Rat brain myelin contains several major glycoproteins i n addition t o t h o s e ( m o l . w t . 1 . 2 x l o 5 a n d 2.7 x l o 4 ) a l r e a d y ~ h a r a c t e r i z e d . ~ ~ ~ F o u r a d d i t i o n a l m a j o r g l y c o p r o t e i n s a n d many m i n o r g l y c o p r o t e i n s a r e r e v e a l e d a f t e r t r e a t m e n t o f i s o l a t e d m y e l i n membrane w i t h n e u r a m i n i dase b e f o r e ox i d a t i o n w i t h g a l a c t o s e ox i da se a n d subsequent r e d u c t i o n w i t h sodium b o r o t r i t i d e . A glycoprotein s o l u b l e i n o r g a n i c s o l v e n t s i s o l a t e d from r a t m y e l i n has b e e n p a r t i a l l y purified and characterized as a sulphated I-fucosyl g 1y c o p ro t e i n .464 The m a j o r g l y c o p r o t e i n (PO) o f c h i c k s c i a t i c - n e r v e m y e l i n h a s been p u r i f i e d and p a r t i a l l y characterized.465 The amino a c i d c o m p o s i t i o n i n d i c a t e s a d e f i n i t e s p e c i e s d i f f e r e n c e when compared w i t h mammalian PO g l y c o p r o t e i n s . Dopamine B-hydroxylase ( E C 1.14.2.1) i s a tetrameric g l y c o p r o t e i n w i t h s u b u n i t s o f m o l . w t . 7.5 x 104.466 I t i s p r e s e n t i n catecholamine s t o r a g e v e s i c l e s o f both a d r e n a l m e d u l l a and sympathetic nerve terminals. The p r e s e n c e o f n e u r a m i n i c a c i d residues on oligosaccharide chains g r e a t l y increases t h e resistance of t h e e n z y m e t o t r y p t i c p r o t e o l y s i s . An a g e - r e l a t e d i n c r e a s e o f n o n - e n z y m a t i c g l y c o s y l a t i o n i n It appears to normal bovine l e n s c r y s t a l l i n s has been observed.467 be a g e n e r a l c h a r a c t e r i s t i c o f t h e a g e i n g p r o c e s s . Bovine thyro g l o b u l i n has been s u b j e c t e d t o s e q u e n t i a l t r e a t m e n t w i t h glycohydrolases i n order t o define t h e components o f the oligosaccharide chain which are important i n binding the g l y c o p r o t e i n t o bovine t h y r o i d membranes.468 Preparations of a s i a l o g a l a c t o t h y r o g l o b u l i n e x h i b i t t h e best b i n d i n g , s u g g e s t i n g t h a t exposed 2-acetamido-2-deoxy-~-glucose r e s i d u e s o n t h e B c a r b o h y d r a t e chain of t h e hormone are i m p o r t a n t i n t h e i n t e r a c t i o n w i t h t h e t h y r o i d membranes. i - T y r o s i n e r e s i d u e s p l a y an i m p o r t a n t r o l e i n The t h y r o g l o b u l i n i n t e r a c t i o n s w i t h t h y r o i d membranes.469 attachment of carbohydrate t o thyroglobulin involves an intermediate (0-G 1 ce3 -p - M a n e g -p- G 1 ceNA c2- py r o pho s p h o r y 1d o 1 i c h o 1) a n d i s f o l 1ow e d r a p i d l y by t h e i n i t i a t i o n o f m o d i f y i n g r e a c t i o n s i n c l u d i n g t h e The r e m o v a l of Q - g l u c o s y l - a n d some Q-mannosyl r e s i d u e s . 4 7 0 i n h i b i t i o n o f i n c o r p o r a t i o n o f c a r b o h y d r a t e i n t o t h y r o g l o b u l i n by

n-

225

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides t u n i carny c i n has been r e p o r t e d .471 Parathyroid secretory protein c o n t a i n i n g 18% c a r b o y d r a t e )

(mol.

wt.

6.7

has been p u r i f i e d f r o m

x

lo4

and

extracts of

b o v i n e p a r a t h y r o i d glands.472 p r o t e i n I, a g l y c o p r o t e i n ( m o l .

Secretory unknown

biological activity,

c h a r a c t e r i z e d from ca r bo h y d r a t e

.

been

wt.

7.0

x 104) o f

isolated

and

partially

b o v i n e p a r a t h y r o i d glands.473

The ! - t e r m i n a l ACTH/LPH

has

fragment

precursor,

i s

a

of

porcine

major

I t c o n t a i n s 2.6%

pro-opiomelanocortin,

product

of

the

m a t u r a t i o n and

i s

g l y ~ o s y l a t e d . ~The ~ ~ a m i n o a c i d sequence i s h o m o l o g o u s t o s i m i l a r p e p t i d e s i n ox a n d man, L --Thr-45

w i t h two s i t e s o f g l y c o s y l a t i o n probably a t

and I - A s n - 6 5 .

Some

of

the

relationships

between

protein

synthesis,

g l y c o s y l a t i o n , cleavage, and s e c r e t i o n o f pro-ACTH-endorphin

i n mouse

p i t u i t a r y tumour c e l l s h a v e been d e s c r i b e d . 4 7 5 B-D-Glucuronidase gland.476

Two

has

been i s o l a t e d f r o m

oligosaccharide

r e d u c t i v e a 1ka 1i n e h y d r o 1y s i s,

alditols

rat preputial

(16-17),

released

by

have be e n c h a r a c t e r i zed.

P-Man3-a-{ ( a-Q-Mane-Q-GlcQNAc )-Q-Mang) -8 -Q-GlcgNA c-8 -Q-GlcNA c - 0 1 ( 16)

Q -M an^^ -4 -a {Q -M a ng2-a -Q -M an e} -B -Q - G 1CQ NA c - 8 -Q - G 1c NA c- 01 (17) Glycoproteins and pepsinogen a r e a s s o c i a t e d i n t h e secretory granules

of

fundic

epithelial

cells

isolated

from

guinea-pig

stomach.477 I n c o r p o r a t i o n o f monosaccharides i n t o r a t l i v e r f e r r i t i n can o c c u r n o n - e n z y m i c a l l y u n d e r p h y s i o l o g i c a l c o n d i t i o n s o f pH a n d i o n i c strength,

i n a process which i s dependent o n t h e c o n c e n t r a t i o n o f

monosaccharides, temperature.478

the

concentration

of

ferritin,

time,

and

Varying degrees o f g l y c o s y l a t i o n m i g h t account f o r

t h e occurrence o f i s o f e r r i t i n s . The s t r u c t u r e a n d d y n a m i c b e h a v i o u r o f t h e o l i g o s a c c h a r i d e s i d e c h a i n o f b o v i n e p a n c r e a t i c r i b o n u c l e a s e B have b e e n s t u d i e d b y 13C n.m .r.

spectroscopy.479

The c o n c l u s i o n s a b o u t t h e p r i m a r y s t r u c t u r e

of t h e c a r b o h y d r a t e s i d e c h a i n s a r e c o n s i s t e n t w i t h r e s u l t s b a s e d o n c h e m i c a l methods. Chem i c a l a n a l y s e s ,

t o g e t h e r w i t h h i s t o c h em i c a l a s s e s s m e n t s ,

226


have been o b t a i n e d f o r specimens of adenocarcinoma o f t h e colon and histologically normal c o l o n i c epithelium.480 In epithelial g l y c o p r o t e i n s o f normal t i s s u e , t h e m a j o r i t y of t h e n e u r a m i n i c a c i d r e s i d u e s c o n t a i n a s i d e - c h a i n g - a c y l s u b s t i t u e n t a t C - 8 , whereas s i d e - c h a i n s u b s t i t u t i o n o f t h e n e u r a m i n i c a c i d s of t u m o u r Differences i n the glycoproteins i s markedly reduced. s u s c e p t i b i l i t y t o n e u r a m i n i d a s e t r e a t m e n t were a l s o noted between t h e two s o u r c e s of g l y c o p r o t e i n s . Human c o l o n t u m o u r a n t i g e n w h i c h i s r e c o g n i z e d b y l e u c o c y t e adherence i n h i b i t i o n assay i s u n r e l a t e d t o c a r c i n o e m b r y o n i c a n t i g e n (CEA).481 The f o r m e r a n t i g e n , b u t n o t C E A , b i n d s s p e c i f i c a l l y t o immobilized human B2-microglobulin a n t i b o d i e s . B l o o d - g r o u p d e t e r m i n a n t s h a v e b e e n f o u n d on f i v e C E A p r e p a r a t i o n s . 4 8 2 One of t h e p r e p a r a t i o n s h a s an A 1 d e t e r m i n a n t , a n o t h e r h a s a B d e t e r m i n a n t , and a l l h a v e H , L e a , Leb,and MN b l o o d group d e t e r m i n a n t s . The f i n d i n g s a r e c o n s i s t e n t w i t h t h e i d e a t h a t incomplete o r unexpected glycosylation p a t t e r n s occur i n g l y c o p r o t e i n s produced by tumour c e l l s . Since antibodies d i r e c t e d a g a i n s t blood-group s u b s t a n c e s c r o s s - r e a c t w i t h c a r b o h y d r a t e d e t e r m i n a n t s on C E A , c l i n i c a l d e t e r m i n a t i o n s of C E A o r anti-CEA l e v e l s i n serum may be a d v e r s e l y a f f e c t e d . Concurrent measurements o f c a r c i n o e m b r y o n i c a n t i g e n , !-glucose phosphate isomerase, yg l u t a m y l t r a n s f e r a s e , and l a c t a t e dehydrogenase i n m a l i g n a n t , normal a d u l t , and f e t a l colon t i s s u e s g i v e r e s u l t s t h a t a r e c o n s i s t e n t w i t h e a r l i e r o b s e r v a t i o n s t h a t t h e a c t i v i t i e s of t h e s e m a r k e r s a r e i n c r e a s e d s i g n i f i c a n t l y i n t h e serum of p a t i e n t s w i t h m e t a s t a t i c colon cancer.483 Carcinoembryonic a n t i g e n - r e l a t e d a n t i g e n s have been p u r i f i e d and c h a r a c t e r i z e d i n normal a d u l t f a e c e s . 4 8 4 Glycoproteins metabolically l a b e l l e d w i t h 2-amino-Z-deoxy-g{ 3H}-glucose and i s o l a t e d from human r e n a l cancer and normal kidney e p i t h e l i a l c e l l c u l t u r e s h a v e been c o m p a r e d by t w o - d i m e n s i o n a l p o l y a c r y l a m i d e g e l e l e c t r o p h ~ r e s i s . ~Although ~~ o v e r a l l p r o f i l e s of g l y c o p r o t e i n s from tumours and normal c e l l s were e x t r e m e l y s i m i l a r , s e v e r a l s i g n i f i c a n t d i f f e r e n c e s were observed t h a t were c o n s i s t e n t even among a l l o g e n e i c comparisons. A g l y c o p r o t e i n i n h i b i t o r (mol. w t . 1 . 3 3 x l o 4 ) of c a l c i u m o x a l a t e m o n o h y d r a t e c r y s t a l g r o w t h h a s been i s o l a t e d f r o m human kidney t i s s u e c u l t u r e medium.486 T h i s g l y c o p r o t e i n c o n t a i n s 39.4% c a r b o h y d r a t e , c o n s i s t i n g of r e s i d u e s of I=-fucose, Q - g l u c o s e , Eg a l a c t o s e , and neuraminic a c i d . Studies a t the ultrastructural level indicate that the

227


g l y c o p r o t e i n GP-2 i s b o t h a c e l l m e m b r a n e a n d a b a s e m e n t m e m b r a n e p r o t e i n i n mouse k i d n e y c e l l s . 4 8 7 L - F u c o s e c a n be i n c o r p o r a t e d i n t o a s e r i e s o f l o w - m o l e c u l a r w e i g h t amino a c i d I - f u c o s i d e s i n normal r a t kidney cells.488 The m o n o s a c c h a r i d e sequence a n d p o s i t i o n o f t h e l i n k a g e o f one o f t h e fucosides

(18) and t h e anomeric c o n f i g u r a t i o n s o f

linkages o f three other L-fucosides

(19-21)

the

I-

carbohydrate

h a v e been i d e n t i f i e d . 4 8 9

T e n t a t i v e evidence i s given t o suggest t h a t these unusual glycosides a r e d e r i v e d f r o m an i - f u c o p r o t e i n .

a -L-Fucp-l+L-Thr (19) a --FucpI 1+I-Se r (20) B - Q - G l c e ( 1+)- a - & - F u c p - l + i - T h r (21) A p a r t i c u l a t e f o l a t e - b i n d i n g p r o t e i n f r o m human p l a c e n t a h a s

been is o l a t e d a n d c h a r a c t e r i z e d.490

I m m u n o f l uo r e s c e n t s t u d i e s u s i n g

antibody d i r e c t e d against t h i s g l y c o p r o t e i n demonstrate t h a t an i m m u n o l o g i c a l l y s i m i l a r p r o t e i n i s p r e s e n t o n t h e p l a s m a membrane o f human e r y t h r o c y t e s ,

s u g g e s t i n g t h a t i t may f u n c t i o n a s a f o l a t e

receptor. Some p h y s i c o c h e m i c a l

properties o f

an a c i d i c g l y c o p r o t e i n

s e c r e t e d by r a t e p i d i d y m a l c e l l s h a v e b e e n ~ h a r a c t e r i z e d . ~ ’ ~The g-galactose,

2-

unexpectedly,

Q-

From amino a c i d sequence s t u d i e s o f g l y c o s y l a t e d component

C3

c a r b o h y d r a t e m o i e t y c o n t a i n s r e s i d u e s o f Q-mannose, a c e t a m i do-2-deox y-1- g l ucose,

neuram in i c a c i d , and,

g l uco se. of

r a t p r o s t a t i c b i n d i n g p r o t e i n , a g l y c o p e p t i d e c o n t a i n i n g 77 amino

acids

has

been c h a r a c t e r i z e d . 4 9 2

An o l i g o s a c c h a r i d e c h a i n i s

a t t a c h e d t o t h e p e p t i d e by a n N - g l y c o s i d i c b o n d t o I - A s n - 1 7 . Bovine c a r t i l a g e m a t r i x g l y c o p r o t e i n (mol. co n t a i n s

three

s u b u n i t s.4 9 3

G l y c o p e p t i de s

wt.

1.48

isolated

x

lo5)

after

p r o t e o l y t i c d i g e s t i o n a r e heterogeneous o n g e l chromatography and c o n t a i n Q-manno s y l a n d 2 - a c e t a m i d o - 2 - d e o x y - a - g l uco s y l r e s i d u e s ,

228


p r o b a b l y d e r i v e d from o l i g o s a c c h a r i d e s o f the high-Q-mannose t y p e b o u n d t o p r o t e i n by 2 - g l y c o s i d i c l i n k a g e s . Tw o n o n - c o l l a g e n o us i n s o l u b l e s t r u c t u r a l g l y c o p r o t e i n f r a c t i o n s from u n c a l c i f i e d canine-puppy r i b c a r t i l a g e a r e both a s s o c i a t e d w i t h lipid.494 T h e c o m p l e x e s f o r m e d a r e s t a b l e e n o u g h t o be o f p o s s i b l e s i g n i f i c a n c e i n t h e metabolism of connective t i s s u e s . An i n t r i n s i c g l y c o p r o t e i n ( m o l . w t . 5.3 x l o 4 ) o f t h e s a r c o p l a s m i c r e t i c u l u m of r a b b i t s k e l e t a l m u s c l e has been separated f r o m t h e Ca2+ + Mg2+ a d e n o s i n e t r i p h ~ s p h a t a s e . ~ ' ~T h e g l y c o p r o t e i n c o n t a i n s 48% n o n p o l a r a m i n o a c i d s i n a d d i t i o n t o t w o g l y c a n c h a i n s , each c o n t a i n i n g n i n e Q-mannosyl a n d t w o 2 - a c e t a m i d o - Z - d e o x y - Qg l uco s y 1 r e s i d ue s A s u l p h a t e d g l y c o p r o t e i n , e n t a c t i n ( m o l . w t . 1.58 x l o 5 ) , h a s b e e n i s o l a t e d f r o m a n e x t r a c e l l u l a r b a s e m e n t mem b r a n e - l i k e m a t r i x e l a b o r a t e d i n c e l l c u l t u r e by a m o u s e e n d o d e r m a l c e l l l i n e . 4 9 6 I m m u n o e l e c t r o n m i c r o s c o p i c s t u d i e s show t h a t t h e m o u s e k i d n e y antigen is predominantly localized a t the surface o f e p i t h e l i a l c e l l s o f t u b u l e s a n d g l o m e r u l i a d j a c e n t t o t h e b a s e m e n t membrane. Laminin, p r e s e n t i n t h e l a m i n a l u c i d a o f t h e basement membrane, i s c l e a v e d by t h r o m b i n a n d p l a ~ m i n . ~ ' F~r a g m e n t s c l e a v e d by t h e enzymes have been assessed f o r t h e i r a b i l i t y t o m e d i a t e b i n d i n g o f e p i t h e l i a l c e l l s t o t y p e IV c o l l a g e n . P r o t e o l y s i s o f laminin from human p l a c e n t a l and r e n a l b a s e m e n t membranes has been a c h i e v e d w i t h The b i n d i n g o f l a m i n i n t o g l y c o s a m i n o g l y c a n s pepsin.498 (aggregating and non-aggregating subsets o f heparan sulphates and dermatan s u l p h a t e s a s well a s h e p a r i n , c h o n d r o i t i n s u l p h a t e s , and h y a l u r o n i c a c i d ) h a s b e e n s t u d i e d by a f f i n i t y c h r ~ m a t o g r a p h y . ~ ~ ' The b i n d i n g i s s p e c i f i c a n d o c c u r s o n a s i n g l e b i n d i n g s i t e o f laminin. S t u d i e s o n t h e b i o s y n t h e s i s o f l a m i n i n by m u r i n e p a r i e t a l endoderm cells have been r e p ~ r t e d . ~ " Evidence is given f o r the p r e s e n c e o f !-linked oligosaccharide side chains on a l l four p o l y p e p t i d e com po n e n t s o f t h i s g l y c o p r o t e i n . Two e x t r a c e l l u l a r m a t r i x g l y c o p r o t e i n s , s i m i l a r t o l a m i n i n , h a v e been i s o l a t e d from c u l t u r e s o f a mouse e n d o d e r m a l c e l l l i n e . 5 0 1 A s t r u c t u r a l g l y c o p r o t e i n from bovine ligamentum muchae exhibits a dual amine oxidase activity.502 If exposed t o copper i o n s t h e g l y c o p r o t e i n i s c a p a b l e o f f o r m i n g f i b r i l s a b o u t 11 nm i n diameter a n d o f t h e same s i z e a s t h e m i c r o f i b r i l s a s s o c i a t e d m a i n l y with e l a s t i c f i b r e s i n both embryonic and mature t i s s u e s . M o d i f i c a t i o n s i n the l e v e l s of glycosylamines and c r o s s l i n k s observed i n glomerular basement membranes i n s t r e p t o z o t o c i n d i a b e t i c

.

229

5: Glycoproteins, Glycopeptides, Proteoglycans, dnd Animal Polysaccharides

r a t s have been reported.503 Enhanced g - g l y c o s y l a t i o n o f in t e r m o l e c u l a r c r o s s l i n k s a n d i n c r e a s e d f o r m a t i o n o f g l y co s y l am ine c o u l d a f f e c t t h e p h y s i o c o c h e m i c a l p r o p e r t i e s o f t h e membrane. A

glycoprotein

endothelial

(mol.

wt.

1.9

c e l l s i n culture.504

x

lo5)

i s s e c r e t e d by

Immunological

aortic

and s t r u c t u r a l

studies i n d i c a t e t h a t the glycoprotein i s e i t h e r i d e n t i c a l t o o r c l o s e l y r e l a t e d t o thrombospondin,

which i s contained i n p l a t e l e t

granules and r e l e a s e d i n response t o thrombin-induced aggregation. A n t i b o d i e s t o human s e r u m a m y l o i d P b i n d i m m u n o s p e c i f i c a l l y t o the p e r i p h e r a l m i c r o f i b r i l l a r m a n t l e o f e l a s t i c f i b r e s i n s k i n and blood vessels o f normal adults.505 A s i a l o g l y c o p r o t e i n f r a c t i o n f r o m h e r r i n g eggs c o n t a i n s b o t h

a 1 k a l i - l a b i l e a n d a 1k a l i - s t a b l e c a r bohy d r a t e u n i t s .506

Each p e p t i de

c h a i n has an average o f f o u r o l i g o s a c c h a r i d e chains 2 - g l y c o s i d i c a l l y linked r e s i due s A

via

.

2-acetamido-2-deox

probable

structure

y-a- g a l a c t o s y l o f

the

glycan

and

L- t h r e o n i n e

portion

o f

a

s i a l o g l y c o p e p t i d e f r a c t i o n i s o l a t e d f r o m t h e s k i n o f t h e f i s h Labeo r o h i t a h a s been s u g g e s t e d f r o m m e t h y l a t i o n a n a l y s i s and e n z y m i c a n a 1y si s da t a. O7 The p r o t e i n c o m p o s i t i o n o f i s o l a t e d human p l a t e l e t a - g r a n u l e s has

been cha r a ~ t e r i z e d . ~ ' ~C r o s s e d a f fi n it y

i m m u n o e l e c tr o p h o r e s i s

u s i n g l e c t i n s i d e n t i f i e d a t l e a s t seven g l y c o p r o t e i n s , a p p e a r e d t o be s i a l o g l y c o p r o t e i n s . o r i g i n of

The a - g r a n u l e

s i x o f which

component i s t h e

t h e i r enhanced h a e m a g g l u t i n a t i o n a c t i v i t y . 5 0 9

insufficiency

of

the platelet-bound

An

l e c t i n may be t h e cause o f t h e

i n a b i l i t y o f gray p l a t e l e t s t o aggregate n o r m a l l y i n response t o thrombin. T h r o m b o s p o n d i n i s one o f t h e g l y c o p r o t e i n s r e l e a s e d f r o m human A hydrodynamic model o f

b l o o d p l a t e l e t s i n r e s p o n s e t o thrombin.510 thrombospondin i s proposed from molecular weight,

physical

s o l u t i o n measurements o f

p a r t i a l s p e c i f i c volume,

sedimentation coefficient,

and i n t r i n s i c v i s c o s i t y . P l a t e l e t g l y c o p r o t e i n s have been shown t o be u s e f u l p h e n o t y p i c markers

for

cells

o f m e g a k a r y o c y t e lineage.511

Immunofluorescent

s t a i n i n g w i t h an a n t i s e r u m t o h i g h l y p u r i f i e d p l a t e l e t g l y c o p r o t e i n s e x c l u s i v e l y l a b e l s human m e g a k a r y o c y t e s , p l a t e l e t s , a n d a p o p u l a t i o n o f s m a l l human bone-marrow m o n o n u c l e a r c e l l s . Differences

b e t w e e n human l e u c o c y t e a n d f i b r o b l a s t i n t e r f e r o n s

have been reviewed.512 dealing

w i t h

the

A book on i n t e r f e r o n i n c l u d e s a chapter

p u r i f i c a t i o n

and

characterization

of

230


.

in t e r f e r o n s 5 1 3 Human f i b r o b l a s t

i n t e r f e r o n h a s been p u r i f i e d

chromatography,514

as

well

as

by

S e p h a r ~ s e ~ .E ~ v i d~ e~n c e f o r t h e e x i s t e n c e o f dimer

(mol.wt.

reported. chelate

A

x

4.0

lo4>

and a f f i n i t y

on

Blue

t h i s i n t e r f e r o n as a

a s w e l l a s a monomer

com b i n a t i o n o f h y d r o p h o b i c

chromatography,

by z i n c - c h e l a t e

chromatography (2.0

lo4)

x

chromatography,

chromatography

i s

copper on

Blue

Sepharose* h a s been used i n t h e p u r i f i c a t i o n o f human l y m p h o b l a s t o i d i n t e r f e r o n .516 Size

characteristics

of

human

leucocyte

interferon

r e d u c i n g an d no n- r e d u c i n g co n d i t i o n s ha ve been de t e r m ine d

. l7

under

M e t a b o l i c c a r b o h y d r a t e l a b e l l i n g i n t h e mouse system shows t h a t n o t o n l y m i t o g e n - a c t i v a t e d T a n d B l y m p h o c y t e s , b u t some o f t h e i r subpopulations

can be

d i s t i n g u i s h e d by

display s p e c i f i c sets o f glycoproteins, metabolic

specific glycoprotein

Human l y m pho cy t e s u b p o p u l a t i o n s a 1 so

l a b e l l i n g p a t t e r n s .518 ,519 by

their

carbohydrate

which a r e e a s i l y d e t e c t a b l e

l a b e l l i n g

and

electrophoretic

techniques.520 The b i n d i n g p a t t e r n o f h o r s e r a d i s h p e r o x i d a s e - c o n j u g a t e d p e a n u t l e c t i n o n s e c t i o n s o f l y m p h o i d t i s s u e f r o m d i f f e r e n t s o u r c e s has been

investigated.521

dependent,

The

binding,

which

i s

highly

species-

i s g r e a t e s t t o lymphocytes i n t h y m i c c o r t e x and g e r m i n a l

c e n t r e s i n man, guinea pig,and

mouse, a n d s h e e p .

Lymphoid t i s s u e from hamster,

r a b b i t d i d n o t r e a c t , w h i l s t i n r a t o n l y weak b i n d i n g

t o c e l l s i n t h e t h y m i c c o r t e x and g e r m i n a l c e n t r e s was d e t e c t e d . The o x i d a t i o n o f human p e r i p h e r a l m o n o n u c l e a r c e l l s w i t h sodium periodate r e s u l t s i n lymphocyte activation.522 of

the

oxidant

i s

accompanied

by

the

The m i t o g e n i c e f f e c t

oxidation

of

membrane-

a s s o c i a t e d neuraminosy 1, ! - g a l a c t o s y 1, and I - f u c o s y l r e s i d u e s . The by

r e c e p t o r - m e d i a t e d p i no cy t o sis o f

Q-mannosyl

and

macrophages has been reviewed.523 L-fucosyl

o r P-mannosyl

by macrophages and

uptake o f

g l y copro t e i n s

2-acetam i d o - 2 - d e o x y - q - g l

ucosyl

terminated

r e s i dues

by

Glycoconj ugates b e a r i n g t e r m i n a l

residues are

r e c o g n i z e d and i n t e x n a l i z e d

t h e same r e c e p t o r t h a t m e d i a t e s p l a s m a c l e a r a n c e lysosomal

glycosidases.524

The macrophage

receptor

h a s a b r o a d s p e c i f i c i t y and i s a b l e t o b i n d v a r i o u s g l y c o c o n j u g a t e s . I n human f i b r o b l a s t s , c e l l - s u r face

receptors

i s

t h e r e c o g n i t i o n o f l y s o s o m a l enzymes by mediated

by

g-manno s y l

6-pho spha t e

residues l o c a t e d on oligosaccharide

c h a i n s o n t h e s e enzymes.525

B o t h c a t h e p s i n D and B-hexosaminidase

c o n t a i n t h e phosphate groups

w h i c h a r e d i e s t e r - l i n ke d t o 2-a ce t a m i do-2-de ox y

-a- g l uco s y l

residues.

I

23 1

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides Developmental

changes i n t h e

g l y c o p r o t e i n s s y n t h e s i z e d by

n o r m a l a n d d y s t r o p h i c s a t e l l i t e mouse c e l l s g r o w n i n c u l t u r e h a v e been

observed.526

glycoproteins

Similar,

appear

to

be

but

m u l t i n uc 1e a t e d m y o l u b e s , b u t

not

identical,

synthesized no t

by

I-fucosylated

exclusively

by

normal

dup 1ic a t i n g m ono n uc 1e a t e d

s a t e l l i t e c e l l s o r by o t h e r n o n - m y o g e n i c c e l l s f r o m b o t h s o u r c e s . Changes i n g l y c o p r o t e i n s (mol.

wts.

1.0

x

lo5

a n d 9.0

x lo4,

r e s p e c t i v e l y ) i n m a l i g n a n t t r a n s f o r m a t i o n have been shown t o

be

d i r e c t l y r e l a t e d t o t h e u p t a k e o f P - g l u c o s e by t h e s e c e l l s . 5 2 7 The

purification

and

partial

structures

of

the

major

g l y c o p e p t i d e s i s o l a t e d f r o m c u l t u r e d human melanoma c e l l s a n d f r o m t h e c u l t u r e m e d i a have been r e p o r t e d . 5 2 8 from

The g l y c o p e p t i d e s o b t a i n e d

t h e h i g h l y t u m o r i g e n i c human melanoma c e l l s a r e m a r k e d l y

d i f f e r e n t f r o m t h o s e o b t a i n e d f r o m n o n t u m o r i g e n i c human f e t a l u v e a l me1ano cy t e s . The p l a s m i n o g e n a c t i v a t o r s e c r e t e d by human melanoma c e l l s i n c u l t u r e i s very s i m i l a r to, activator

found

i n

normal

o r i d e n t i c a l with, tissue,

~ r o k i n a s e . ~ ~ The ’ g l y c o p r o t e i n (rnol. i m m o b i l i z e d c o n c a n a v a l i n A,

wt.

exists

but 7.2

as a

the

i s x

plasminogen

different

lo4),

from

p u r i f i e d using

dimer.

Receptors f o r

D o l i c h o s b i f l o r u s a g g l u t i n i n on e m b r y o n a l c a r c i n o m a c e l l s have been i s o l a t e d a n d s h o w n t o be g l y c o p r o t e i n s ( m o l .

wt.

The l e c t i n does n o t b i n d t o d i f f e r e n t i a t e d c e l l s . the content o f neuraminic acid of

x 1

>7.0

0 ~ 1 . ~ ~

Differences i n

low cancer c e l l s ,

high cancer

c e l l s , a n d n o r m a l mouse l u n g c o u n t e r p a r t s have been observed.531 Normal l u n g c e l l s c o n t a i n h i g h e r neuraminic a c i d l e v e l s per c e l l i n c o m p a r i s o n w i t h t h e t r a n s f o r m e d ones. Three

t y p e s o f mononucleosomes i s o l a t e d f r o m

Ehrlich ascites

n u c l e i have been c h a r a c t e r i z e d . 5 3 2 Specific glycoproteins are a s s o c i a t e d w i t h t w o of t h e m o n o n u c l e o s o m e t y p e s w i t h t h e p r i n c i p a l g l y c o p r o t e i n h a v i n g a n apparent mol. w t . A carbohydrate-rich,

lo4)

water-soluble

o f 1.3

x

lo5.

g l y c o p r o t e i n (mol.

wt.

4.5

x

has been i s o l a t e d f r o m d e l i p i d a t e d l u n g l a v a g e f l u i d f r o m a

patient

with pulmonary

alveolar proteinosis.533

The v e r y

high

c a r b o h y d r a t e c o n t e n t i s made up of r e s i d u e s o f n e u r a m i n i c a c i d ( 2 4 ) ,

-

2 ace t a m ido -2 -de o x y -p - g l uco se

ga 1a c t o se ( 23 1 , 2- ace tam ido - 2 -de o x y --!

( 6 1 , g - g a l a c t o s e ( 1 9 1 , p - m a n n o s e (41, I - f u c o s e (11, a n d ! - g l u c o s e (11, p e r m o l e c u l e o f g l y c o p r o t e i n . U n l i k e a number o f c o l l a g e n -

r e l a t e d g l y c o p r o t e i n s w h i c h have been i s o l a t e d f r o m i n s o l u b l e l u n g c o n t e n t s i n pulmonary

proteinosis,

this

g l y c o p r o t e i n does

co n t a i n hy d r oxy-&- p r o l i n e o r hy d r ox y - k - l y s i n e .

not


232 c v)

I

Q I

. + I nil

u

I nit I

I

m

m I

n

U

t

4 U

I

9 m

I

I L=yI

I

m I

n

M

t

4 v

A m

I I

v ) -

4

I

u

CJ t

I

II

I1

4

4

4

I

0

I

4 u

I

0 Q

z

2

4

U

I a 1 I

m I

n

U

t

4 v

AJ

4

m

U

I 0 1 1 I

m

I n v)

CJ

U

I

0

Q

-

1

b

C c m c z

b

cv t

l

w

oll

-

a

cv3

I K a I n Z

I

m n I

U

t

4 W

AI

4

m

U I

Ul I

m I

4

E


233

A g l y c o p r o t e i n (mol. w t . 6 . 2 x l o 4 ) , i s o l a t e d f r o m t h e l u n g l a v a g e m a t e r i a l of p a t i e n t s w i t h a l v e o l a r p r o t e i n o s i s , c o n t a i n s 72 r e s i d u e s of g l y c i n e and 5 r e s i d u e s o f hydroxy-I=-proline per m o l e c u l e , i n a d d i t i o n t o Q-mannose, a - g a l a c t o s e , I - f u c o s e , 2acetamido-2-deoxy-Q-glucose, and n e u r a m i n i c a c i d i n t h e m o l a r r a t i o o f 5:4:1:7:3.534 P a r t i a l amino a c i d sequence a n a l y s i s of p e p t i d e s d e r i v e d from t h e d i g e s t i o n o f t h e g l y c o p r o t e i n i n d i c a t e s t h e p r e s e n c e of a l t e r n a t i n g c o l l a g e n o u s and non-collagenous r e g i o n s i n t h e same p o l y p e p t i d e chain. A human a l v e o l a r g l y c o p r o t e i n r e l e a s e s two major g l y c o p e p t i d e f r a c t i o n s on p r o t e ~ l y s i s . ~From ~ ~ s t r u c t u r a l a n a l y t i c a l studies t h e t e n t a t i v e s t r u c t u r e s (22-23) have been proposed. Human a l - a n t i c h y m o t r y p s i n h a s been p u r i f i e d from human p l e u r a l f l u i d a n d from human serum.536 The g l y c o p r o t e i n (mol. w t . 5.8 x l o 3 ) i s o l a t e d from t h e two s o u r c e s h a s t h e same c h e m i c a l composition. A g l y c o p r o t e i n (mol. w t . 3.6 x l o 3 ) w i t h s i m i l a r p r o p e r t i e s t o c o l l a g e n has been i s o l a t e d from t h e l u n g l a v a g e of normal rabbit.537 B 1ood - gro up - spe c i f i c g l yco pro t e i n s o bt a i n e d from ova r i a n- cy st f l u i d s o f A1 a n d A 2 p e r s o n s h a v e b e e n s u b m i t t e d t o a l k a l i n e bo ro h y d r i de e l i m i n a t i on, de -! ace t y 1a t i on, pa r t i a 1 a c i d h y dro 1y s i s , and s u b s e q u e n t l y r e - i - a ~ e t y l a t i o n . ~The ~ ~ t r i s a c c h a r i d e s (24-26) w e r e c h a r a c t e r i z e d f r o m b o t h t h e A1 a n d A 2 m a t e r i a l s . O l i g o s a c c h a r i d e ( 2 6 ) h a s n o t p r e v i o u s l y been d e t e c t e d i n human g l yco pro t e i n s .

a-Q-GaleWc-( 1 + 3 ) - @ 3 - G a l p (1+3) -Q-GlcNAc (24)

-

a -Q Ga lgNA c- ( 1+3 ) -6 -Q - Ga le- ( 1+4) -Q - G 1 c NA c

(25) a-p-GaleNAc-( 1+3) -B-p-Galp( 1+3)-GalNAc-ol 26)

L u b r i c a t i n g g l y c o p r o t e i n 1 h a s been p r e p a r e d from b o v i n e synovial fluid.539 E l e c t r o n - m i c r o s c o p i c s t u d i e s show t h a t t h e g l y c o p r o t e i n i s an a s y m m e t r i c molecule. The m o l e c u l a r model t h a t best f i t s t h e m o l e c u l a r dimensions (222 nm l o n g and 1-2 nm d i a m e t e r ) i s t h a t o f a p a r t i a l l y extended f l e x i b l e rod. Human a m n i o t i c f l u i d c o n t a i n s an i r r e v e r s i b l e t i s s u e i n h i b i t o r


234 of

c ~ l l a g e n a s e . ~A~l t h~o u g h t h i s g l y c o p r o t e i n i s c l o s e l y a s s o c i a t e d

with

it

fibronectin,

does

not

cross-react

with

antibodies

to

f ib r o ne c t in.

The m i c r o h e t e r o g e n e i t y o f purified

rat

the d i f f e r e n t

a-fetoprotein

variants

carbohydrate chains o f

has

been s t u d i e d . 5 4 1

The

methodology used i n c l u d e s t h e r e l e a s e o f c a r b o h y d r a t e c h a i n s f r o m t h e g l y c o p r o t e i n by h y d r a z i n o l y s i s ,

-N - a c e t y l a t i o n labelled

with

{14C)-acetic

oligosaccharides

the l a b e l l i n g of

anhydride,

on

g l y c a n s by r e -

the f r a c t i o n a t i o n of

immobilized

concanavalin

c h a r a c t e r i z a t i o n o f t h e l a b e l l e d g l y c a n s by t.1.c. has been i s o l a t e d f r o m human a s c i t e s f l u i d . 5 4 2 (27-29)

were r e l e a s e d from

a-Fetoprotein

t h e p o l y p e p t i d e c h a i n by h y d r a z i n o l y s i s .

accumulated duodenal f l u i d , (mol.

wts.

x

5.4

lo4,

contains

1.02

the

and

Oligosaccharides

Human e n t e r o k i n a s e ( e n t e r o p e p t i d a s e EC 3.4.21.91,

subunit

the

A,

p u r i f i e d from

contains three glycosylated subunits x 105,and

1.4

active-site

x 10

1.5 4 3

i-serine

The s m a l l e s t

residue, and

the

o l i g o s a c c h a r i d e c h a i n s o f t h i s u n i t a p p e a r t o be b J - g l y c o s i d i c a l l y linked. The

NAO glycohydrolase

from

Bungarus

fasciatus

(banded k r a i t )

venom h a s b e e n p u r i f i e d t o e l e c t r o p h o r e t i c enzyme

i s a

glycoprotein

(mol.

wt.

1.2

The

lo5)

x

containing

two

s u b u n i t s.

-

Ce 11 s ur Pa ce G 1y Mp r o tei ns

9

with

Two r e c e n t l y p u b l i s h e d t r e a t i s e s h a v e i n c l u d e d c h a p t e r s d e a l i n g c a r b o h y d r a t e s and c e l l membranes,545 and w i t h t h e s t r u c t u r e s

a n d f u n c t i o n s o f t h e c a r b o h y d r a t e m o i e t i e s o f membrane g l y c o p r o t e i n s a n d g l y ~ o l i p i d s . ~R~e v~i e w s T-cell of

recognition,547

n o r m a l and cancerous

importance

of

membrane

and

dealing with glycosyltransferases

cells,548

glycoconjugates the

and

a n d w i t h g l y c o c o n j u g a t e s o f s u r f a c e membranes

multiple

i n

have

been p u b l i s h e d .

the organization

roles

played

co n s t i t ue n t s in m a 1ig na n t t r a n s f o r m a t i o n s

by

of

these

the

The cell

membrane

ha ve bee n r e v i ew e d .549

A

d e t a i l e d account o f h i s t o c h e m i c a l methods f o r c h a r a c t e r i z i n g complex cell-surface

glycoconjugates

by

l i g h t

microscopy

has

been

published. 550 Electron

microscopy

has

been

used

to

map

the

l o c i

of

i m m uno ch em ic a l a c t i ve s i t e s o n i n d i v i d u a l g l y cop r o t e i n m o l e c u l e s.551

This i n v o l v e s t h e complexing o f a g r o u p - s p e c i f i c macromolecule,

such


4

0 I

u

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z

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235

236


a s a l e c t i n o r an a n t i b o d y , w i t h an a s y m m e t r i c g l y c o p r o t e i n f o l l o w e d by t h e d i r e c t v i s u a l i z a t i o n o f t h e c o m p l e x i n t h e e l e c t r o n m i c r o s co pe A procedure f o r d e t e r m i n i n g membrane p u r i t y of i s o l a t e d c e l l s h a s been r e p ~ r t e d . ” ~ I n t a c t c e l l s a r e l a b e l l e d w i t h p e r o x i d a s e c o n j u g a t e d wheat-germ a g g l u t i n i n . A f t e r d i s r u p t i o n o f t h e i n t a c t l a b e l l e d c e l l s , p l a s m a membrane p u r i f i c a t i o n can be m o n i t o r e d by u l t r a s t r u c t u r a l h i s t o c h e m i c a l ex am i n a t i on o f t h e pe rox i d a s e conjugated l e c t i n . H i s t o c h e m i c a l f i x a t i o n o f c e l l g l y c o p r o t e i n s using p e r i o d a t e oxidation, followed by complexing w i t h L - l y s i n e and p a r a f o r m a l d e h y d e , h a s p r e v i o u s l y been a s s u m e d t o c r o s s l i n k s p e c i f i c a l l y p l a s m a r e s u l t s of an e v a l u a t i o n u s i n g membrane g l y c ~ p r o t e i n s . ’ ~ The ~ Novikoff r a t a s c i t e s h e p a t o c e l l u l a r carcinoma c e l l s suggest a com p l e x i n t e r a c t i o n b e t w e e n p e r i o d a t e - o x i d i z e d g l y c o p r o t e i n s a n d po 1ym e r i c com p l ex e s o f form a 1 de h y de and & - l y si ne C e l l - s u r f a c e g l y c o p r o t e i n s , b u t not g l y c o l i p i d s , from e r y t h r o c y t e s , c o l o n i c t u m o u r c e l l s , and s k i n f i b r o b l a s t s , b i n d t o i m m o b i l i z e d R i c i n u s communis a g g l u t i n i n . 5 5 4 The l a c k of b i n d i n g of t h e s o l u b i l i z e d g l y c o l i p i d s t o t h e i m m o b i l i z e d l e c t i n c o u l d be due t o t h e f a c t t h a t t h e i r m o n o v a l e n c y r e s u l t s i n t o o low a n a f f i n i t y f o r t h e l e c t i n t o c a u s e r e t e n t i o n on t h e column. A p p a r e n t m i n o r c h a n g e s i n c e l l - p r e p a r a t i o n m e t h o d s have been found t o r e s u l t i n major a l t e r a t i o n s i n c e l l b e h a v i o u r , r e f l e c t i n g d i f f e re nce s i n c e l l - s u r f a ce pro pe r t i e s of r a t h e pa t o cy t e s 555 P l a s m a membrane g l y c o p r o t e i n s from r a t h e p a t o c y t e s t h a t had been exposed t o s u b e n d o t h e l i a l s p a c e s of D i s s e have been s e l e c t i v e l y l a b e l l e d w i t h Q - g a l a c t o s e ox i d a s e - s o d i um b o r o t r i t i i d e . Approximately f o r t y gly c o p r o t e i n s were r e s o l v e d by two-dimensional e l e c t ro ph o r e si s. D i f f e r e n t c e l l - s u r f a c e g l y c o p r o t e i n s seem t o be i n v o l v e d i n t h e i n i t i a l r e a c t i o n s of c e l l - c e l l a n d c e l l - c o l l a g e n a d h e s i o n of r a t hepa tocy t e s . 5 5 7 G l y c o p e p t i d e s o b t a i n e d by pronase d i g e s t i o n o f r a t l i v e r plasma m e m b r a n e s have been f r a c t i o n a t e d by a f f i n i t y c h r o m a t o g r a p h y on i m m o b i l i z e d c o n c a n a v a l i n A.558 The primary s t r u c t u r e (29) of a b i a n t e n n a r y glycan from one of t h e f r a c t i o n s has been i d e n t i f i e d by h i g h - r e s o l u t i o n ‘H n.m . r . s p e c t r o s c o p y and m e t h y l a t i o n a n a l y s i s . T h e k i n e t i c behaviour of t r y p s i n i n non-covalent e l e c t r o s t a t i c i n t e r a c t i o n w i t h a s i a l o g l y c o p e p t i d e f r a c t i o n i s o l a t e d from major hepatoma c e l l s u r f a c e s has been reported.’”

.

.

.

5: Glycoproteins, Glycopeptides, Proteeglycans, and Animal Polysaccharides

237

Immunological evidence f o r t h e transmembrane nature o f t h e rat l i v e r r e c e p t o r f o r a s i a l o g l y c o p r o t e i n s has been presented.560 The r e c e p t o r i s exposed on t h e cytoplasmic, as well as t h e e x t e r n a l , s u r f a c e s o f t h e h e p a t o c y t e p l a s m a membrane, a n d d e t e r m i n a n t s o n e a c h s u r f a c e a r e a n t i g e n i c a l l y d i s t i n g u i s h a b l e . The b i o s y n t h e s i s a n d i n s e r t i o n of the r e c e p t o r i n t o endoplasmic reticulum membranes have been s t u d i e d u s i n g a n t i b o d y m o n o s p e c i f i c f o r t h e b i n d i n g protein.561 The b i n d i n g p r o t e i n i s e x c l u s i v e l y s y n t h e s i z e d o n t h e m e m b r a n e - b o u n d r i b o s o m e s and s p a n s t h e m i c r o s o m a l membrane, p r o b a b l y e x p o s i n g t h e C-terminal segment on the cytoplasmic surface and the N-terminal segment containing carbohydrate m o i e t i e s on t h e luminal surface. D e m o n s t r a t i n g a new r o u t e o f c l e a r a n c e o f g l y c o p r o t e i n s f r o m r a t p l a s m a , a b i n d i n g p r o t e i n s p e c i f i c f o r p - m a n n o s y l a n d / o r 2acetamido-2-deoxy-~-glucosyl r e s i d u e s has been i s o l a t e d from rat liver.562 This protein is analogous t o the binding protein s p e c i f i c f o r a s i a l o g l y c o p r o t e i n s , which m e d i a t e s t h e o r i g i n a l r o u t e of clearance, i n its predominant l o c a l i z a t i o n i n t h e l i v e r and i n its r eq u i r eme n t o f c a l c i um b i n d i n g. T h e a s i a l o g l y c o p r o t e i n r e c e p t o r , r e q u i r i n g Ca2+ f o r l i g a n d b i n d i n g , h a s been i d e n t i f i e d o n a c o n t i n u o u s human h e p a t o m a c e l l l i n e , Hep G2.563 There a r e a p p r o x i m a t e l y 150,000 l i g a n d m o l e c u l e s bound p e r c e l l a t 4OC. D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y h a s been used t o examine t h e t h e rm a 1 d e n a t u r a t i 0 n o f r a b b i t h e p a t i c 9 - g a l a c t o s y l - b i n d i n g Ca2+ a n d l i g a n d b i n d i n g i n f l u e n c e t h e d e n a t u r a t i o n protein.564 process. H u m a n a s i a l o t r a n s f e r r i n s , t y p e s 1 , 2, a n d 3 , a n d r a b b i t a s i a l o t r a n s f e r r i n exhibit unequal binding with p u r i f i e d rat l i v e r p l a s m a membranes.565 The a s i a l o g l y c o p r o t e i n r e c e p t o r o n c u l t u r e d rat h e p a t o c y t e s mediates t h e t o x i c e f f e c t s o f a c o n j u g a t e of a s i a l o f e t u i n and fragment A of d i p h t h e r i a toxin.566 A s i a l o g l y c o p r o te i n s b u t n o t t h e corresponding s i a l o gly coprot e i n s are able t o block t h e a c t i o n o f t h e conjugate. v i v o h e p a t i c u p t a k e by t h e p a r e n c h y m a l Evidence f o r the cells of a Q-galactosyl-terminated glycoprotein (asialofetuin) i n f i s h (Salmo a l p i n u s ) has been r e p o r t e d . 5 6 7 The l e c t i n - d e p e n d e n t r e c o g n i t i o n o f d e s i a l y l a t e d e r y t h r o c y t e s by K u p f e r c e l l s h a s b e e n d i s c u s s e d .568 The s p e c i f i c i t y and k i n e t i c s o f endocy t o s i s o f g l y c o p e p t i d e s a n d g l y c o p r o t e i n s by i s o l a t e d r a t r e t i c u l o e n d o t h e 1 i a l c e l l p r e p a r a t i o n s have been examined.569 The c e l l r e c e p t o r i s h i g h l y

238


selective

i n i t s

recognition of

subsequently internalized,

oligosaccharides

which

are

and t h e minimum s t r u c t u r e (30) r e q u i r e d

f o r r e c o g n i t i o n and e n d o c y t o s i s i s proposed. R - ( 1+6)-a-P-Mane-(

1+6)-B-B-Man~-( 1+4) -B-Q-GlceNAc-(

1+4)

-

8 -Q-GlceNAc-i-Asn

3

I 1 a -Q -Mane (30)

R = a-Q-Mane o r B-Q-GlCQNAC

The c e l l - s u r f a c e

glycoconjugates of

and o f

four

use o f

1251-substituted

and

by

the

ricin-resistant use o f

baby-hamster

kidney c e l l s

v a r i a n t s h a v e b e e n i n v e s t i g a t e d by t h e

r i c i n which binds t o 1-galactosyl

l e c t i n s which

bind t o

a c e tam ido - 2 -deox y -Q- g l uco sy 1 r e s id ue s .5 70 number o f l e c t i n a n d / o r r i c i n

of

the

2-

r i c i n r e c e p t o r s were found between t h e c e l l

resistance

mo d i f i c a t i o n s

and

M a j o r d i f f e r e n c e s in t h e

surface o f wild-type c e l l s and t h a t o f r i c i n - r e s i s t a n t The

residues,

neuraminosyl

o f

variant

concentration

cells of

i s

variants.

concomitant

certain

to

g l y c o c o nj u g a t e

structures which are accessible t o the sugar-binding proteins. Monkey k i d n e y c e l l s i n f e c t e d w i t h Yaba t u m o u r pox v i r u s e x h i b i t p l a s m a membrane a l t e r a t i o n s when t r e a t e d w i t h b o t h f l u o r e s c e i n l a b e l l e d a n d u n l a b e l l e d c o n c a n a v a l i n A.571 A c o m b i n a t i o n o f i m m u n o l o g i c a l and b i o c h e m i c a l methods have

been u s e d t o i d e n t i f y s u r f a c e membrane c o m p o n e n t s i n v o l v e d i n . c e l l substratum

adhesion.572

Highly p u r i f i e d adhesion-related

wt.

c o n s i s t i n g o f i n t e g r a l membrane g l y c o p r o t e i n s ( m o l .

materials 1.4

x

lo5)

t h a t a r e exposed t o the e x t r a c e l l u l a r environment were i s o l a t e d . Their r o l e i n the process o f cell-substratum adhesion i s n o t y e t un de r s t o o d. Mutant Chinese-hamster ovary c e l l s s e l e c t e d f o r r e s i s t a n c e t o a h a v e a l t e r e d Q-

c o n j u g a t e o f r i c i n and o-(6-phospho)-P-mannopentaose mannose 6 - p h o s p h a t e r e c e p t o r s . 5 7 3

These m u t a n t s e x h i b i t r e d u c e d

u p t a k e and a l t e r e d b i n d i n g o f exogenously added a c i d h y d r o l a s e . mutants

secrete

glucuronidase, and

two

L-f

to

six

times

more

ucosidase than the

a-Q-mannosidase, parent

e l e v a t e d s e c r e t i o n i s n o t due t o a l t e r a t i o n o f p h o s p h a t e r e c o g n i t i o n m a r k e r o n t h e enzymes,

cells.574

The

B-QThis

t h e Q-mannose 6 -

b u t appears t o r e s u l t

f r o m a l t e r a t i o n s i n t h e P-mannose 6 - p h o s p h a t e r e c e p t o r . A r e v i e w has been p r e s e n t e d o n t h e c e l l - s u r f a c e

properties o f

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides the

glycoconjugate

patterns o f

normal,

239

d i f f e r e n t i a t i n g , and

n e o p l a s t i c p a n c r e a t i c a c i n a r c e l l s .5 75 S o l u b i l i z a t i o n w i t h sodium d o d e c y l s u l p h a t e and subsequent e l e c t r o p h o r e t i c f r a c t i o n a t i o n has d e m o n s t r a t e d t h e h e t e r o g e n e i t y o f p r o t e i n s and g l y c o p r o t e i n s i s o l a t e d from

i n t h e i n t e s t i n a l b r u s h b o r d e r membrane

tadpole,

G l y c o p r o t e i n bands t h a t

y o u n g a n d a d u l t Rana c a t e s b e i a n a . 5 7 6 co-migrate

with

maltase,

glucoamylase, a n d

a l k a l i n e p h o s p h a t a s e a c t i v i t y have been i d e n t i f i e d . Sucrase-isomaltase,

an i n t e g r a l membrane g l y c o p r c t e i n t h a t

a p p e a r s i n i n t e s t i n a l m i c r o v i l l u s membranes d u r i n g d i f f e r e n t i a t i o n of intestinal epithelial cells,

has been p u r i f i e d o n i m m o b i l i z e d

m o n o c l o n a l a n t i b o d y - p r o t e i n A columns.577

The m a j o r p a r t o f

the

carbohydrate associated w i t h the g l y c o p r o t e i n i s i n t h e form o f complex o l i g o s a c c h a r i d e s l i n k e d t o A - a s p a r a g i n e . Lectin

binding t o

sealed and d i s r u p t e d d i s c

membranes o f

v e r t e b r a t e r e t i n a l r o d c e l l s supports t h e view t h a t r h o d o p s i n i s a transmem brane p r o t e i n w i t h i t s o l i g o s a c c h a r i d e

components o r i e n t e d

toward t h e i n t e r i o r o f the discs.578

I n d i r e c t evidence favouring

the

a

transmembrane

disposition o f

r o d outer-segment

disc

g l y c o p r o t e i n i s a l s o considered. A human s p e r m a t o z o a 1 a n t i g e n ,

acid,

amino sugars, a n d hexose,

containing residues o f neuraminic

has been p u r i f i e d . 5 7 9

The a n t i g e n

r e a c t s w i t h i m m u n o g l o b u l i n G and i m m u n o g l o b u l i n M a n t i b o d i e s a n d w i t h both types o f antisera. The m a j o r g l y c o p r o t e i n o n t h e p l a s m a membrane o f r a t t e s t i c u l a r

wt.

x

1.1

spermatozoa

(mol.

spermatozoa

pass through

lo5)

has

been i s o l a t e d . 5 8 0

the epididymis,

this

As

glycoprotein

d i s a p p e a r s a n d i s r e p l a c e d by a n o t h e r g l y c o p r o t e i n ( m o l . w t . 3.2 x lo4), which i s a m a j o r component o f e p i d i d y m a l s e c r e t i o n s . A g l y c o p r o t e i n (mol.

ga l a c t o s e

-

,

Q - m a nno s e

,

wt.

1.6

x

lo5)

containing residues o f

e-

2 - a c e t a m ido - 2 - d e o x y - E - g l uco se , a n d 2 -

a c e t am i do 2 -de ox y -9- g a l a c t o se has bee n ide n t i f i e d a s a c e l l - s u r f a ce r e c e p t o r o f b o a r spermatozoa f o r c o n c a n a v a l i n A.581 E n z y m i c r e m o v a l of c e l l - s u r f a c e unmasking and s t i m u l a t i o n o f

n e u r a m i n i c a c i d l e a d s t o an

gonado t r o p h i n - r e c e p t o r

p l a s m a membranes of b o v i n e c o r p u s l u t e u m .582 go na do t r o p h i n - r e c e p t o r

activity i n

A r e l a t i o n s h i p between

i n t e r a c t i o n and g l yco p r o t e i n-bo un d ne uram i n i c

a c i d has been d e m o n s t r a t e d . The sodium c h a n n e l s a x i t o x i n - b i n d i n g s a r c o l emma c o n t a i n s r e s i d u e s of 9-manno se, 2-deox y - Q - g a l a c t o s e ,

component o f r a t h i n d - l i m b

s- g a l a c t o se , 2-ace tam ido-

2-acetam i d o -2-deoxy-e-gl

ucose, and n e u r a m i n i c

Carbohydrate Chemistry acid.583

The t o x i n - b i n d i n g

s i t e i s s p a t i a l l y separated from b i n d i n g

s i t e s f o r l e c t i n s s u c h a s c o n c a n a v a l i n A,

wheat-germ

a g g l u t i n i n , and

R i c i n u s communis. The b i n d i n g s i t e s f o r agglutinin, have

been i d e n t i f i e d . 5 8 4

different 5.0

x

wheat-germ

agglutinin,

R i c i n u s communis

a n d c o n c a n a v a l i n A o n mouse n e u r o b l a s t o m a c e l l membranes glycopeptides,

lo4,

but four

major cell-surface

Concanavalin most

A

binds

w i t h molecular

to

over

weights

twenty

greater

than

of these g l y c o p e p t i d e s were i d e n t i f i e d a s t h e

receptors f o r the lectin.

D e t e r g e n t s have been u s e d t o s o l u b i l i z e t r a n s f e r r i n - t r a n s f e r r i n r e c e p t o r c o m p l e x e s from

r a b b i t r e t i c u l o c y t e membrane.585

Estimates

o f t h e m o l e c u l a r w e i g h t o f t h e t r a n s f e r r i n r e c e p t o r depend o n t h e conditions o f electrophoresis. p a r t i a l l y modified,

I t is s u g g e s t e d t h a t t h e r e c e p t o r i s

p e r h a p s by g l y c o s y l a t i o n .

A s o l u b l e h a e m a g g l u t i n a t i o n a c t i v i t y has b e e n o b t a i n e d f r o m

t e r a t o c a r c i n o m a stem c e l l s . 5 8 6

This haemagglutinin i s s e n s i t i v e t o

t r y p s i n a n d i s i n h i b i t e d by s p e c i f i c c a r b o h y d r a t e s s u c h a s [)-mannans and I - f u c a n s ,

indicating that i t i s a lectin.

The c a r b o h y d r a t e -

and

erythrocyte-binding

s p e c i f i c i t i e s o f t h e s o l u b i l i z e d l e c t i n a r e very

similar to

the cell-surface

that of

o n i n t a c t stem c e l l s , The

effect

of

carbohydrate-binding

component

s u g g e s t i n g i d e n t i t y o f t h e two components. dexamethazone

on

cell-surface

glycoprotein

a c c u m u l a t i o n i n HeLa c e l l s i s r e l a t e d t o i n c r e a s e d s u g a r - l i n k e d do 1i c h o l s y n t h e s i s . The

major

87

plasma

membrane

sialoglycoprotein

a s c i t e s mammary a d e n o c a r c i n o m a

of

13762 r a t

c e l l s i s released from

non-

x e n o t r a n s p l a n t a b l e a n d x e n o t r a n s p l a n t a b l e s u b l i n e s by p r o t e o l y t i c cleavage.588

The s i a l o g l y c o p r o t e i n p r o b a b l y

binds to

t h e membrane

v i a a hydrophobic p o r t i o n o f the p o l y p e p t i d e which remains w i t h t h e

membrane o n r e l e a s e . E v i d e n c e h a s been p r e s e n t e d f o r t h e i n v i v o a s s o c i a t i o n o f t w o c e l l g l y c o p r o t e i n s o f mammary a s c i t e s t u m o u r studies

on

the

redistribution

Redistribution of presence

of

concanavalin A

peanut

o f

cells,

fluorescent

based upon lectins.589

r e c e p t o r s was o b s e r v e d i n t h e

agglutinin, but

a g g l u t i n i n r e c e p t o r s was o b s e r v e d o n l y

redistribution of

peanut

upon t r e a t m e n t o f t h e c e l l s

w i t h c o n c a n a v a l i n A. A pea l e c t i n r e c e p t o r

f r o m 6C3HED m u r i n e a s c i t e s t u m o u r c e l l s

has been d e g r a d e d by t r y p s i n t o r e l e a s e a g l y c o p e p t i d e ( m o l . x

1 0 ~ ) .T h~ is ~ h i g~h - m o l e c u l a r - w e i g h t

wt.

g l y c o p e p t i d e i s absent

non-specific protease digests o f viable cells.

2.0 from

Sequential l e c t i n

24 1


a f f i n i t y chromatography o f detergent e x t r a c t s o f c e l l s has p e r m i t t e d the probable i d e n t i f i c a t i o n of the native glycoprotein. C e l l - f r e e m a t r i x m a t e r i a l f r o m human f i b r o b l a s t contains a cell-surface

glycoprotein

wt.

(mol.

lo5)

x

1.4

cultures which

seems t o be c l o s e l y a s s o c i a t e d w i t h t h e p e r i c e l l u l a r f i b r ~ n e c t i n . ~ ~ ~ Like fibronectin,

t h i s g l y c o p r o t e i n can f o r m a n e x t e n s i v e d i s u l p h i d e

crosslinked structure,

wt.

(mol.

2.5

covalent

x l

bonds

p o s s i b l y w i t h a s e c o n d g l y c o p r o t e i n component

~ The) p o. s s ~ i b i l i~ ty th ~a t c o v a l e n t and/or

~ bind

a l l

three

glycoproteins

i n

a

non-

matrix

was

discussed. Rat

Thy-1

antigen,

a glycoprotein o f

111 amino a c i d r e s i d u e s ,

i s a m a j o r membrane m o l e c u l e o f t h y m o c y t e s and b r a i n . 5 9 3 amino

acid

reported.

sequence o f

rat

An

sequence and

apparent

brain

i m m u n o g l o b u l i n was o b s e r v e d .

Thy-1

The f u l l

glycoprotein

structural

Evidence

for

has

been

homology

with

an evolutionary

r e l a t i o n s h i p b e t w e e n Thy-1 a n t i g e n and i m m u n o g l o b u l i n s has been p r e s e n t e d and a h y p o t h e s i s f o r t h e f u n c t i o n a l s i g n i f i c a n c e o f t h e Thy-1 a n t i g e n proposed.594 either

The a n t i g e n e x i s t s i n t w o a l l e l i c f o r m s ,

o r Thy-1.2.595

Thy-1.1

The T h y - 1 . 2

antigen has

been

i d e n t i f i e d by m o n o c l o n a l - a n t i b o d y t e c h n i q u e s a n d shown t o be l o c a t e d on t h e T h y - 1 g l y c o p r o t e i n . Metabolic

carbohydrate

labelling

for

the

analysis

of

g l y c o p r o t e i n s f r o m a c t i v a t e d human l y m p h o c y t e s has been u s e d i n a s t u d y of

the responding c e l l population.596

Numerous g l y c o p r o t e i n s ,

i s o l a t e d f r o m lymp h o cyte s u b s e t s a f t e r s t i m u l a t i o n w i t h m i t o g e n and alloantigen,

have been d e t e c t e d .

moieties of

h i gh-m o l e c u l a r - w e i g h t

lymphocytes

of

T

and

B origins

Differences i n the carbohydrate gly coproteins

have

been

monoclonal a n t i b o d i e s w i t h a n t i - I and a n t i - i

from

human

demonstrated

using

s p e c i f i ~ i t i e s . ~The ~ ~

c h i c k e n a n t i - ( bo v i ne a 2 - m a c r o g l o b u l i n ) a n t i bo d i e s t o

b in d i ng o f

N a m a l v a l y m p h o b l a s t o i d c e l l s has been d e m ~ n s t r a t e d . ~ ’ ~The p o s s i b l e r o l e o f t h e g l y c o p r o t e i n o n t h e c e l l s i s discussed. Treatment

of

c u l t u r e d human l y m p h o c y t e s w i t h

tunicamycin

r e s u l t s i n a decrease o f i n c o r p o r a t i o n o f c a r b o h y d r a t e i n t o p r o t e i n , together

with a

decrease i n the

hormone t o t h e c e l l s . 5 9 9 i n receptor-binding at

surface

functions

human

T

x

inhibit

Antibodies

cytolytic

lymphocytes.600

lo5

i n s u l i n and g r o w t h

t h e decreases a r e m o s t l y

c a p a c i t y and n o t a f f i n i t y .

glycoproteins

of

binding o f

F o r t h e hormones,

A

but

not

monoclonal

directed

suppressor antibody,

m o l e c u l a r w e i g h t f o r m o f T200 ( t h e m a j o r

specific for

t h e 2.2

cell-surface

glycoprotein on lymphoid cells),

has been u s e d t o show

242


t h a t t h e g l y c o p r o t e i n i s expressed o n l y on B c e l l s and a subset o f bone-marrow

c e l l s w h i c h i n c l u d e s B c e l l precursors.601

A comparison o f

b i n d i n g s i t e s f o r wheat-germ

agglutinin on R a j i

l y m p h o b l a s t o i d c e l l s a n d t h e i r i s o l a t e d n u c l e i a n d p l a s m a membranes i n d i c a t e s s i m i l a r i t i e s i n t h e l e c t i n r e c e p t o r s on t h e o u t e r surface o f lymphoblastoid c e l l s and the c e l l nuclei.602

D i f f e r e n c e s were

o b t a i n e d w i t h i s o l a t e d membranes, a n d t h e s e may be due t o i n v e r s i o n of

t h e membrane v e s i c l e s o r t o t h e i r d e c r e a s e d r i g i d i t y as c o m p a r e d

with the i n t a c t cell. bo r o h y d r ide r e d u c t i on, a n d a c i d h y d r o 1y s i s

P e r i oda t e ox ida t i on,

o f human p e r i p h e r a l lymphocytes, b u t n o t mouse s p l e n o c y t e s o r c a l f l y m p h node c e l l s ,

release

glycerol,

propan-1 ,Z-diol,

carbon analogue o f n e u r a m i n i c acid.603 a r i s e from residues.

Q-galactosyl,

and t h e s e v e n

These f r a g m e n t s p r o b a b l y

I-fucosyl,

and N - a c e t y l n e u r a m i n o s y l

A subpopulation o f chicken B lymphocytes t h a t reacts w i t h

t h e I - f u c o s e - s p e c i f i c l e c t i n f r o m L o t u s t e t r a g o n o l o b u s has been identified.604

The d i s t r i b u t i o n o f t h e l e c t i n - r e a c t i v e

w i t h t h e age o f t h e c h i c k e n .

c e l l s varies

D i f f e r e n t i a l glycosylation o f murine B

c e l l a n d s p l e e n a d h e r e n t c e l l t A a n t i g e n s h a s been r e p o r t e d . 6 0 5 Three p r e d o m i n a n t g l y c o p r o t e i n s have been i s o l a t e d f r o m r a t t h y m o c y t e p l a s m a membranes.606

Two o f t h e g l y c o p r o t e i n s h a v e a

ca r b o h y d r a t e com po s i t i on c h a r a c t e r i s t ic o f The o t h e r

glycoprotein,

carbohydrate similarities to erythrocytes.

units

1-g l yco sy l a t e d

containing about

per

100

glycophorin,

amino

acids,

the major

p r o t e i ns.

twenty 2-glycosylated shows

structural

sialoglycoprotein

o f human

The m o n o c l o n a l a n t i b o d i e s a n t i - T 1 a n d a n t i - T 3 b o t h

r e a c t w i t h a l l human p e r i p h e r a l t h y m u s - d e r i v e d l y m p h o c y t e s a n d w i t h 1 0 % of

t h y m o ~ y t e s . ~Each, ~ ~ however,

surface structures, g l y c o p r o t e i n (mol. glycoprotein

wt.

(mol.


recognizes

wt.

6.9

x

wt. 1.0

lo4>

1.9 x

x

lo5>

as

a

receptor

for

cell-

being a

and t h a t o f t h e a n t i - T 3 b e i n g a

lo4>.

A

human

cell-surface

t h a t i s s e l e c t i v e l y e x p r e s s e d by

p r o l i f e r a t i n g human l e u k a e m i c t h y m u s - d e r i v e d identified

different

with the target antigen o f anti-T1

serum

c e l l s has

transferrin.608

been This

g l y c o p r o t e i n c o n t a i n s b o t h complex and h i g h g-manno-oligo s a c c h a r i d e s linked

to

L-asparagine.60g

Glycosylation

i s

not

an

absolute

requirement f o r the receptor t o a c t as an acceptor f o r f a t t y acid, nor f o r transport t o the c e l l surface. Two Q a - 1 a n t i g e n s f r o m m i c e s p l e n o c y t e s have been i s o l a t e d f r o m both b i o s y n t h e t i c a l l y l a b e l l e d c e l l s and from surface i o d i n a t e d cells.610

These g l y c o p r o t e i n s have been shown t o

be d i s t i n c t f r o m


243

TL a n t i g e n s .

Primary s t r u c t u r e s of t h e t r a n s p l a n t a t i o n a n t i g e n o f t h e murine m a j o r h i s t o com p a t i b i l i t y c o m p l e x h a ve b e e n r e v i ewe d . 6 1 Partially purified, papain-solubilized mouse l i v e r H-2a h i s t o c o m p a t i b i l i t y a n t i g e n s h a v e b e e n p u r i f i e d f u r t h e r by r e v e r s e p h a s e h.p. 1.c. u n d e r a c i d c o n d i t i o n s , y i e l d i n g B 2 - m i c r o g l o b u l i n a n d H-2a h e a v y c h a i n s . 6 1 2 An a m i n o a c i d s e q u e n c e o f t h e f i r s t 98 a m i n o acid residues a t the N-terminus of the murine major h i s t o c o m p a t i b i l i t y a n t i g e n 6 1 3 a n d o f 98 r e s i d u e s o f t h e m u r i n e h i s t o c o m p a t i b i l i t y a n t i g e n H-2Kd h a s b e e n d e t e r m i n e d . 6 1 4 Comparison of t h e s e q u e n c e s w i t h H-2Kb a n d H-2Db a n t i g e n s r e v e a l s e x t e n s i v e localized differences. Comparison o f t h e primary s t r u c t u r e s o f m u r i n e H-2 a n t i g e n s a n d h u m a n HLA m o l e c u l e s s u g g e s t s a n e v o l u t i o n a r y o r i g i n o f m a j o r h i s t o c o m p a t i b i l i t y products.615 Data p r e s e n t e d o n t h e amino acid sequence o f a n intramembranous hydrophobic segment of t h e H-2Kb m u r i n e h i s t o c o m p a t i b i l i t y a n t i g e n b l 6 a n d o f t h e C t e r m i n a l h y d r o p h o b i c r e g i o n 617 h a v e p r o v i d e d t h e c o m p l e t e p r i m a r y s t r u c t u r e of the glycoprotein. Evidence f o r the existence o f three carbohydrate prosthetic g r o u p s o n m o u s e h i s t o c o m p a t i b i l i t y a n t i g e n s H-2kd a n d H-2Db h a s b e e n r e p o r t e d . l8 P u r i f i e d HLA-A2 a n t i g e n f r o m l y m p h o b l a s t o i d c e l l m e m b r a n e s which h a s been f l u o r e s c e n t l y l a b e l l e d s p e c i f i c a l l y i n i t s C - t e r m i n a l r e g i o n i n t e r a c t s w i t h l y m p h o b l a s t o i d c y t o s k e l e t a l p r o t e i n s when r e c o m b i n e d i n ~ i t r o T. h ~i s ~s y ~s t e m a l l o w s s t u d y o f t h e i n t e r a c t i o n s o f a m e m b r a n e p r o t e i n w i t h c y t o s k e l e t a l e l e m e n t s , a n d may be u s e d t o explore the structural basis and regulation of such interactions. A number o f unusual p r o p e r t i e s o f t h e i n v a r i a n t ( I i ) c h a i n o f t h e m u r i n e Ia a n t i g e n s has been described.620 While a l l of t h e Ia polypeptide chains p o s s e s s N-linked o l i g o s a c c h a r i d e u n i t s , they a l s o show t h a t t h e i n v a r i a n t c h a i n d i f f e r s f r o m t h e p o l y m o r p h i c c h a i n s i n C e l l - s u r f a c e HLAboth t h e number and t y p e o f c a r b o h y d r a t e u n i t s . DR, HLA-ABC a n t i g e n s , a n d g l y c o p h o r i n a r e a l l e x p r e s s e d a t d i f f e r e n t stages during erythroid di fferentiation.621 I n t h e b i o s y n t h e s i s o f HLA a n t i g e n s i n t w o l y m p h o b l a s t o i d c e l l l i n e s , Oaudi a n d Raji, t h e a n t i g e n heavy c h a i n s i n Daudi cells are s y n t h e s i z e d normally, but,although core g l y c o s y l a t i o n t a k e s place, t e r m i n a l g l y c o s y l a t i o n d o e s not.622 The b i o s y n t h e s i s o f t h e h e a v y c h a i n s o f mouse l y m p h o b l a s t o i d h i s t o c o m p a t i b i l i t y a n t i g e n s a n d o f i m m u n o g l o b u l i n M i n v o l v e s t h e same i n t r a c e l l u l a r p a t h w a y a s secretory proteins.623 These p o l y p e p t i d e s p a s s through t h e Golgi

H-2

244


subsi te,

d e f i n e d by monensin,

and a c q u i r e t e r m i n a l

2

residues d i s t a l to t h i s site.

neuraminic a c i d

v i t r o t r a n s l a t i o n studies on the

b i o s y n t h e s i s o f HLR-DR a n t i g e n s i n d i c a t e t h a t t h e a - a n d $ - c h a i n s a r e s y n t h e s i z e d a s p r e c u r s o r s w i t h s i g n a l sequences,

i n common w i t h

many o t h e r membranes and s e c r e t e d g l y c ~ p r o t e i n s . ~I n~ ~ t h e presence o f a h e t e r o l o g o u s m i c r o s o m a l system, t h e s i g n a l p e p t i d e i s c l e a v e d and ' c o r e '

o l i g o s a c c h a r i d e u n i t s a r e added.625

The e v e n t s i n v o l v e d

i n t h e b i o s y n t h e s i s a n d m a t u r a t i o n o f t h e a n t i g e n s i n v i v o have a l s o been d e s c r i b e d . The

histocompatibility

(H-Y)

Y

antigen

i s

a

minor

h i s t o c o m p a t i b i l i t y antigen detected on the c e l l surface from the h e t erogam e t i c sexes o f species.626

b i r d s , am p h i bian, and i n v e r t e b r a t e

mamm a1 s,

The s e r o l o g i c a l d e t e r m i n a n t o f

the antigen resides i n

t h e carbohydrate p o r t i o n o f the glycoprotein. Three

cell-surface

antigens,

two

g l y c o p r o t e i n s and a p r o t e i n ,

s t r u c t u r a l l y r e l a t e d t o t h e m a j o r human h i s t o c o m p a t i b i l i t y a n t i g e n s h a v e b e e n c h a r a c t e r i z e d f r o m t h e l e u k a e m i c T c e l l l i n e MOLT-4.627 One a n t i g e n i s a g l y c o p r o t e i n (mol.

w t . 4.9 x l o 4 ) and i s a s s o c i a t e d The o t h e r g l y c o p r o t e i n (mol. w t . 4.3 x l o 4 )

w i t h B2-microglobulin. i s also

associated w i t h

B2-microglobulin

d i s t i n c t from t h e h i g h e r - w e i g h t

but

glycoprotein.

i s

serologically

The p r e s e n t s t a t e o f

knowledge o f the primary s t r u c t u r e o f the murine H-2 a l l o a n t i g e n s has been r e v i e w e d . 6 2 8

A speculative model o f the a n t i g e n on the

membrane i s p r e s e n t e d , and i n t e r p r e t a t i o n s o f t h e r e s u l t s o f p r i m a r y s t r u c t u r a l comparisons a r e used t o probe p o s s i b l e answers t o t h e questions

of

the

generation

of

polymorphism

and

genetic

r e l at i o nships. Wax-bean

agglutinin

mimics

the

activities

of

insulin

by

m e d i a t i n g a n t i l i p o l y s i s and t h e o x i d a t i o n o f Q - g l u ~ o s e . ~I ~t ~does not

appear

receptor,

to

compete w i t h

but

exerts

i t s

the

binding of

insulin-like

the

hormone

action

iia

glycoproteins which are d i s t i n c t from insulin-binding

to

i t s

membrane sites.

The

p o s s i b i l i t y that a glycoprotein effector molecule i s an i n t e g r a l p a r t o f t h e h o r m o n a l m e d i a t e d s y s t e m and t h a t t h e l e c t i n - m e d i a t e d o x i d a t i o n of

Q-glucose and a n t i l i p o l y s i s

may

be t r i g g e r e d

via

d i f f e r e n t g l y c o p r o t e i n r e c e p t o r s i s discussed. The p r e v i o u s l y o b s e r v e d i n h i b i t i o n by c o n c a n a v a l i n A o f i n s u l i n b i n d i n g t o i n t a c t r a t f a t c e l l s may be due t o t h e i n t e r a c t i o n o f t h e l e c t i n w i t h a carbohydrate-containing moiety on the d i s t i n c t from,

but capable o f modifying,

p r e v i o u s l y postulated.630

c e l l membrane

the i n s u l i n r e c e p t o r as

I n s u l i n r e c e p t o r s f r o m human p l a c e n t a and

245

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides cultured IM-9

lymphocytes display

similar

specificites for

twelve

i m m o b i l i z e d l e ~ t i n s . Based ~ ~ ~ on b i n d i n g s t u d i e s w i t h l e c t i n s t h e ca rbohy d r a t e m o i e t y o f t h e r e c e p t o r c o n t a i n s 2-ace tam ido-2-deox y-pglucosyl,

a-Q-mannosyl,

and $ + - g a l a c t o s y l

residues.

The i n c r e a s e

i n a f f i n i t y o f the i n s u l i n receptor f o r i n s u l i n a f t e r i t s desorption from l e c t i n s may be due t o t h e r e m o v a l o f an a s s o c i a t e d i n h i b i t o r . Tunicamycin,

which i n h i b i t s N-linked oligosaccharide chain

a d d i t i o n t o nascent polypeptides, i n s u l i n receptor receptors.632

interrupts glycosylation o f the

adipocytes

giving rise to inactive

The i n a c t i v e a g l y c o i n s u l i n r e c e p t o r a c c u m u l a t e s p o s t -

translationally re-enters

i n 3T3-Ll

d u r i n g c h r o n i c t r e a t m e n t w i t h t u n i c a m y c i n and t h e n

t h e g l y c o s y l a t i o n pathway when t h e i n h i b i t o r i s removed,

g i v i n g r i s e t o a f u n c t i o n a l i n s u l i n receptor. A

wt.

1.6

guinea-pig x

lo5)

sensitivity

p e r i t o n e a l macrophage s u r f a c e g l y c o p r o t e i n (rnol.

i s u n i q u e among m a j o r s u r f a c e c o m p o n e n t s i n i t s

to

m i l d t r y p s i n treatment.633

Neuraminic a c i d

r e s i d u e ( s ) w e r e l o c a t e d s o l e l y i n one o f t h e f r a g m e n t s (rnol.

wt.

8.5

x 104).

Heterogeneity

i n s u r f a c e g l y c o p r o t e i n s o f mouse p e r i t o n e a l

macrophage p o p u l a t i o n s has been observed.634 m a c r o p h a g e s w i t h t h e c u l t u r e medium o f fluid,

a new

expressed.

surface

glycoprotein

Upon a c t i v a t i o n o f

the

c e l l - f r e e tumour a s c i t e s

(rnol.

wt.

x

1.35

10')

is

The p o s s i b i l i t y t h a t t h i s m o l e c u l e i s m e r e l y a v a r i a t i o n

o f a pre-existing protein d i f f e r i n g i n i t s extent o f glycosylation,

o r even o n l y a h i g h e r r a t e o f s y n t h e s i s o f a n o r m a l l y m i n o r s u r f a c e g l y c o p r o t e i n , was n o t excluded. Two p r e v i o u s l y unknown macrophage-speci f i c a n t i g e n s (rnol.

wts.

x l o 4 a n d 1.1 x l o 5 ) h a v e b e e n i s o l a t e d by u s e o f a c o m b i n a t i o n o f c e l l h y b r i d i z a t i o n and r e m o v a l o f p r e v i o u s l y r e c o g n i z e d a n t i g e n s

3.2

by immunoadsor be n t s .635 On t h e b a s i s o f a g g l u t i n a t i o n s t u d i e s w i t h a number o f l e c t i n s , guinea-pig

phagocytic

v e s i c l e s have

been shown

r e c e p t o r s o n t h e i r cy t o p 1 asm i c s i d e . 636

Q - m a nno s y 1,

2-deox y -Q- g a l a c t o s y l , deoxy-D - g l ucosy 1, and

N, " - d i a c e

to

Q - G a l a c t o s y 1,

D- g l uco sy 1,

have l e c t i n . 2-acetam i d o -

2 - a c e tam i do-2-

t y l c h i t o b i o s y 1 residues are probably

present on the receptors. L e c t in- l i k e

r e c e p t o rs c a p a b l e o f

b i n d ing Staphylococcus a l b us

h a v e been d e m o n s t r a t e d i n t h e m e m b r a n e s o f p h a g o c y t e s i n c l u d i n g macrophages,

n e u t r o p h i l s , and e o s i n o p h i l s f r o m

v a r i o u s s o u r c e s and

species.637 These r e c e p t o r s a r e l i k e l y t o c o n t r i b u t e t o adherence and p h a g o c y t o s i s i n t h e non-immune a n i m a l .

bacterial

246


Four independent monoclonal a n t i b o d i e s p r e c i p i t a t e a m u r i n e c e l l - s u r f a c e g l y c o p r o t e i n w h i c h has been i d e n t i f i e d a s a p o l y m o r p h i c d i f f e r e n t i a t i o n a n t i g e n o f m u r i n e mesenchymal c e l l s . 6 3 8 Treatment

of

melanoma

cells with

tunicamycin selectively

inh ib i t s t h e sh e d d i n g o f m e l ano m a- asso c i a t e d g l yco p r o t e i ns w it h o u t a f f e c t i n g t h e i r c e l l - s u r f a c e e x p r e s s i o n .639 K-562 c e l l s ,

o r i g i n a t i n g from the p l e u r a l e f f u s i o n o f a c h r o n i c

my e l o ge no u s l e u k a e m i a pa t i e n t , (erythroglycan, surface

mol.

wt.

e x p r e s s e s a n N - l i n ke d o l i go s a c c h a r i d e

lo3 -

x

7.0

g y ~ o p r o t e i n . ~T h~ e~ e a r l y

biosynthesis

are

the

by

specific cell-

of

erythroglycan

the transferrin-type

maturation o f the oligosaccharide

so

protein structure

that

expressed o n s p e c i f i c glycoproteins. foetal

lo4>,

stages

same a s t h o s e o f

o l i g o ~ a c c h a r i d e . ~H~o w ~ ever, influenced

1.1 x

i s

erythroglycan i s only

The K - 5 6 2 c e l l s e x p r e s s t h e

t y p e (L) a n t i g e n o n d i f f e r e n t g l y c o p r o t e i n s f r o m t h o s e o f

e r y t h r o c y t e s .642 Carbohydrate m o i e t i e s o f r e c e p t o r s f o r immunoglobulin E on r a t b a s o p h i l i c leukaemia c e l l s and r a t mast located i n the

binding s i t e o f

the

c e l l s are not

receptor.643

directly

However,

r e c e p t o r c a r b o h y d r a t e may p l a y a p a r t i n t r a n s p o r t o f

the

the

receptor

t o t h e p l a s m a membrane o r i n i t s o r i e n t a t i o n t h e r e a f t e r .

Cell-

i m m u n o g l o b u l i n E have been i s o l a t e d f r o m r a t

surface receptors f o r

basophilic leukaemia cells.644 The e f f e c t o f t u n i c a m y c i n o n t h e m o l e c u l a r p r o p e r t i e s o f t h e r e c e p t o r have been e v a l u a t e d . The u s u a l diffuse

r e c e p t o r b a n d (rnol.

so d i um

do de c y l s u l p h a t e

r e p l a c e d by one (rnol. i n d i ca t e

that

wt.

wt.

4.5

lo4

x

p o l y a c r y l a m ide

3.8

x lo4),

-

6.2

gel

lo4)

observed on

A number o f l i n e s o f e v i d e n c e

l o w e r - m o l e c u l a r- w e i g h t

this

x

electrophoresis i s protein

represents

ca r b o h y d r a t e-de f ic i e n t r e c e p t o r . Characteristic

g lycoprot e in

prof i l e s

and

carbohydrate

s t r u c t u r e s a r e expressed on d i f f e r e n t l e u k a e m i c c e l l l i n e s blocked at

different

s t a g e s of

stages of

m y e l o i d c e l l d i f f e r e n t i a t i ~ n . ~D ~i f ~f e r e n t

g r a n u l o c y t e d i f f e r e n t i a t i o n can be i d e n t i f i e d by s p e c i f i c

cell-surface

structures.

A murine cell-surface

g l y c o p r o t e i n (rnol.

wt.

8.0

x

lo4)

been i d e n t i f i e d a n d s t u d i e d by u s e o f m o n o c l o n a l a n t i b o d i e s . 6 4 6 d e t e r m i n a n t s o f t h i s m a j o r plasma-membrane by

the antibodies,

are allospecific,

constituent,

differentiation

cells.

The

recognized

and t h e expression o f

glycoprotein i s specific t o certain types o f

has

the

During

r e c e p t o r s f o r c o n c a n a v a l i n A have been shown t o

be

d i s t r i b u t e d i n patches over t h e e n t i r e cell-surface neuroblastoma

247

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides c e l l s . 647

The i m p o r t a n c e o f t h e p l a t e l e t - i s o l a t i o n t e c h n i q u e t o p l a t e l e t r e c o v e r y and p l a t e l e t - membrane i n t e g r i t y h a s b e e n s t r e s s e d . 6 4 8 The

loss o f p l a t e l e t - m e m b r a n e g l y c o p r o t e i n s d u r i n g w a s h i n g p r o c e d u r e s i s p r o b a b l y t h e r e s u l t o f membrane loss f r o m p l a t e l e t s a n d i s n o t due t o the i s o l a t i o n o f a selected p l a t e l e t subpopulation.

Membrane

g l y c o p r o t e i n s h a v e been i s o l a t e d f r o m human p l a t e l e t s a f t e r a f f i n i t y chromatography on i m m o b i l i z e d wheat-germ

a g g l u t i n i n ,649 a n d by h i g h -

voltage free-flow e l e c t r o p h o r e ~ i s . ~ ~ ~ Monoclonal antibodies

have

been

q u a n t i t a t i o n o f the platelet-membrane

used

i s o l a t i o n and

i n

glycoprotein deficient

i n

Glanzmann’s

t h r ~ m b a s t h e n i a . ~ The ~ ~ membrane g l y c o p r o t e i n i s a

complex o f

t w o p l a t e l e t g l y c o p r o t e i n s d e s i g n a t e d I I b and I I I a .

Membrane g l y c o p r o t e i n s o f p l a t e l e t s f r o m n o r m a l and Glanzmann’s thrombasthenic subjects are i d e n t i c a l i n terms o f i s o e l e c t r i c p o i n t s and m o l e c u l a r w e i g h t s ,

and d i f f e r o n l y i n t h e amount o f c e r t a i n

g l y c ~ p r o t e i n s . ~The ~ ~ molecular with

this

disease

i s

due

defect i n p l a t e l e t s from p a t i e n t s

t o

a

deficiency

of

two

membrane

g l y c ~ p r o t e i n s . ~ The ~ ~p o s s i b i l i t y o f a d d i t i o n a l membrane d e f e c t s i n thrombasthenic

platelets

g l y c o p r o t e i n (mol. membranes

from

may

be r e l a t e d t o a n o t h e r s u r f a c e

9.3 x lo4). The g l y c o p r o t e i n s o f p l a t e l e t n o r m a l p a t i e n t s and t h o s e w i t h Glanzmann’s wt.

or

t h r o m b a s t h e n i a have been compared u s i n g c a r b o h y d r a t e - s p e c i f i c protein-specific

l a b e l l i n g techniques

and h i g h - r e s o l u t i o n

d i m e n s i o n a l g e l e 1 e ~ t r o p h o r e s i . s . ~I ~n~ Glanzmann’s

two-

thrombasthenia

t h e absence o r r e d u c t i o n o f t w o m a j o r membrane g l y c o p r o t e i n s i s observed, w h i l e a number o f o t h e r g l y c o p r o t e i n s c o n t a i n i n c r e a s e d l e v e l s o f neuraminic acid. The e x i s t e n c e

i n human p l a t e l e t p l a s m a

m a c r o m o l e c u l a r component (mol.

wt.

>4.0

x

lo6)

membranes o f

c o m p o s i t i o n of a p h o s p h o g l y c o p r o t e i n has been confirmed.655 p h o s p h o g l y c o p r o t e i n i n h i b i t s t h e ADP, thrombin-induced plasma-membrane

a

with the chemical

epinephrine,

The

collagen, and

p l a t e l e t r e a c t i o n w i t h c o n c o m i t a n t changes of

the

structure.

A n a l y s i s of t h e g l y c o p r o t e i n s and p r o t e i n s o f p l a t e l e t s o f f o u r Bernard-Soulier possess

patients

a specific

lesion.656

has

established that

and c h a r a c t e r i s t i c

these

platelets

membrane-glycoprotein

A comparison w i t h t h e r e s u l t s obtained f o r

platelets after

treatment

control

w i t h n e u r a m i n i d a s e has shown t h a t

d e c r e a s e d n e u r a m i n i c a c i d c o n t e n t c a n n o t a l o n e a c c o u n t for observed Bernard-Soulier s u r f a c e a l t e r a t i o n s .

a

the

248

Carbohydrate Chemistry R e c e p t o r s f o r b o v i n e von W i l l e b r a n d f a c t o r have been i d e n t i f i e d

o n w h o l e human p l a t e l e t s , The k i n e t i c s o f

a s w e l l a s o n p l a t e l e t membranes.657

formation o f factor IXa-factor

V I I I complex o n

t h e s u r f a c e o f human p l a t e l e t s h a v e been r e p o r t e d . 6 5 8 T h r o m b i n - i n d u c e d p l a t e l e t a g g r e g a t i o n h a s been i n h i b i t e d by d e r i v a t i v e o f wheat-germ

agglutinin.659

the affinity-chromatographic

used f o r

membranes o f

a g l y c o p r o t e i n (mol.

a

The i m m o b i l i z e d l e c t i n was i s o l a t i o n from 7.4

w t .

i n h i b i t i n g p l a t e l e t a g g r e g a t i o n by t h r o m b i n .

lo4)

x

platelet

capable of

T h i s g l y c o p r o t e i n may

be a p h y s i o l o g i c r e c e p t o r o f t h r o m b i n i n human p l a t e l e t s a n d d i f f e r s the r i s t o c e t i n o r r i s t o c e t i n - v o n

from

Willebrand factor

Platelet

p l a s m a membranes r e t a i n f u n c t i o n a l

following

c e l l l y s i s a n d membrane i s o l a t i o n . 6 6 0

receptor.

aggregation

sites

Isolated platelet

plasma-mem b r a n e g l y c o p r o t e i n s h a v e a g r e a t e r a f f i n i t y f o r t h r o m b i n activated platelets

than control

An i m m u n o l o g i c a l

platelets.

p r o c e d u r e has been u s e d t o e x p l o r e t h e c o n c e p t t h a t a r e d i s t r i b u t i o n of

membrane

secretion.661 agglutinin

receptors

has

may

be

involved

non-agglutinating

A

been

prepared

i n

human

derivative o f

that

precipitates an antibody t o the lectin.

binds

to

platelet

wheat-germ

platelets

and

Pla'telets treated with this

i n a c t i v e d e r i v a t i v e r e l e a s e 5 - h y d r o x y t r y p t a m i n e when e x p o s e d t o bivalent antibody

.

(but

not

monovalent)

fragments

of

the

lectin

A s i a l o g l y c o p r o t e i n I b and a s i a l o g l y c o c a l i c i n have been i s o l a t e d from

the

membranes a n d

from

the

supernatant,

n e u r a m i n i d a s e - t r e a t e d human p l a t e l e t s . 6 6 2

respectively,

of

The t w o g l y c o p r o t e i n s

have been shown t o be c l o s e l y r e l a t e d ,

and evidence s u p p o r t i n g the

idea

from

that

glycocalicin

i s

derived

the

membrane-bound

glycoprotein i s reported. has

been

d e m o n s t r a t e d a t t h e e x t e r n a l s u r f a c e o f human p l a t e l e t s . 6 6 3

The

presence

of

ectosialyltransferase

activity

The

m a j o r endogenous a c c e p t o r i s t h e plasma-mem b r a n e g l y c o p r o t e i n G P I I b . Platelet-membrane

g l y c o p r o t e i n s I I b a n d I I I a h a v e been i s o l a t e d a n d

p u r i f i e d f r o m h u m a n p l a t e l e t mem b r a n e . 6 6 4

The g l y c o p r o t e i n s a r e

a n t i g e n i c a l l y d i f f e r e n t a n d s t r u c t u r a l l y d i s t i n c t g l y c o p r o t e i ns, w h i c h i n t h e n a t i v e s t a t e may f o r m a m a c r o m o l e c u l a r c o m p l e x w i t h a role

i n

mediating

platelet-platelet

c o n s t i t u e n t s o f human p l a t e l e t membranes

interactions.

The

major

G P I I b a n d G P I I I a have been

p u r i f i e d , and the

generation o f monospecific a n t i s e r a t o these a n t i ge n i ca 11y d i f f e r e n t a n d s t r u c t ur a 11y d i s t in c t g l y co p r o t e i n s h a s been d e ~ c r i b e d . ~ M ~ o~r p, h ~o l ~o g~ i c a l e v i d e n c e

demonstrating

249

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides macromolecular complex

formation o f these glycoproteins i n t h e

p l a t e l e t membrane a f t e r t h r o m b i n s t i m u l a t i o n i s a l s o r e p o r t e d . P l a t e l e t g l y c o p r o t e i n I11 and a - a c t i n i n have

been

d i f f e r e n t i a t e d on the basis o f the a b i l i t y o f the former to bind to immobi 1i t e d whea t-germ

a g g l u t i n i n. 6 6 7

F u r t h e r e v i d e n c e h a s been s h o w n t o s u p p o r t t h e h y p o t h e s i s t h a t y o u n g r a b b i t p l a t e l e t s a r e h e m o s t a t i c a l l y m o s t e f f e c t i v e when t h e y a r e r e l e a s e d f r o m bone marrow and t h a t t h e y undergo s i g n i f i c a n t changes i n

the

circulation as

decreased size,

they

age.668

This i s

shown

s y m m e t r i c a l l o s s o f membrane g l y c o p r o t e i n s ,

by and

decreased h e m o s t a t i c e f f e c t i v e n e s s o f the p l a t e l e t s .

wt.

1.1 x 10')

carrying binding

s i t e s f o r c o n c a n a v a l i n A and soybean a g g l u t i n i n

has been p u r i f i e d

A

m a j o r g l y c o p r o t e i n (mol.

f r o m m o s q u i t o p l a s m a membranes.669

The p r e s e n c e o f N - g l y c o s i d i c a l l y

l i n k e d o l i g o s a c c h a r i d e c h a i n s composed o f r e s i d u e s o f e-mannose a n d

2-acetamido-2-deoxy-Q-glucose

(in

the

molar

r a t i o 9:2)

g l y c o s i d i c a l l y l i n k e d 2-acetamido-2-deoxy-q-galacto

and

0-

s y l r e s i d u e s was

e s t a b l i s h ed. Concanavalin A binds t o a surface-membrane

receptor o f the

i n s e c t t r y p a n o s o m a t i d C r i t h i d i a f a s ~ i c u l a t a . ~ ~ The ' receptor

wt.

1.4

x

lo4>

(mol.

may be i d e n t i c a l t o a p r e v i o u s l y r e p o r t e d Q - m a n n a n

from t h a t source. The

in

v i v o synthesis,

membrane i n s e r t i o n , a n d g l y c o s y l a t i o n o f

a m u l t i s u b u n i t i n t e g r a l membrane p r o t e i n o f t h e T o r p e d o e l e c t r o p l a x acetylcholine

r e c e p t o r have

been studied.671

Each o f

s u b u n i t s i s synthesized as an i n d i v i d u a l polypeptide,

the

four

and a l l f o u r

p o l y p e p t i d e chains are independently i n t e g r a t e d i n t o dog pancreas m i c r o s o m e s as transmembrane p r o t e i n s . appears t o

The mechanism o f i n t e g r a t i o n

i n v o l v e a c o t r a n s l a t i o n a l process analogous t o t h a t

d e s c r i b e d f o r v i r a l membrane g l y c o p r o t e i n s .

10

G l y m p r o t e i n Hormones

The s t r u c t u r e a n d f u n c t i o n o f g l y c o p r o t e i n h o r m o n e s , 6 7 2 a n d t h e r e g u l a t i o n o f g l y c o p e p t i d e hormone s y n t h e s i s i n c e l l culture,673 A book d e a l i n g w i t h t h e e v o l u t i o n o f p r o t e i n have been reviewed. s t r u c t u r e and 74

ho rmo ne s

.

function

includes a

chapter

on

glycoprotein

Four g o n a d o t r o p h i n c o m p o n e n t s have been p u r i f i e d f r o m t h e b a s i c p r o t e i n f r a c t i o n o f b u l l f r o g p i t u i t a r y glands.675

The a m i n o a c i d

250


c o m p o s i t i o n o f t h e four components i s a p p r e c i a b l y d i f f e r e n t f r o m t h a t o f mammalian l u t e i n i z i n g hormone. The b i n d i n g s i t e s o f human g o n a d o t r o p h i n i n t h e i n t r a c e l l u l a r organelles of compared

bovine corpora

with

l u t e a have been c h a r a c t e r i z e d and

plasma-membrane

sites.676

The

synthesis o f

g l y c o p r o t e i n hormone a - s u b u n i t and p l a c e n t a l a l k a l i n e phosphatase has been f o l l o w e d i n t h e p r e s e n c e o f t u n i c a m y c i n , 2-deoxy-P-arabinohexose, and s o d i u m b ~ t y r a t e . ~ ” The g l y c o s y l a t i o n i n h i b i t o r s r e d u c e d t h e b u t y r a t e - s t i m u l a t e d s y n t h e s i s o f t h e s u b u n i t and a l k a l i n e phosphatase t o a greater e x t e n t than t h e i r b a s a l l e v e l s o f synthesis. P r e g n a n t - m a r e s e r u m g o n a d o t r o p h i n d i s s o c i a t e s a t a c i d pH w i t h a p a r a l l e l l o s s o f f o l l i c l e - s t i m u l a t i n g hormone and l u t e i n i z i n g hormone a c t i v i t i e s , s u p p o r t i n g t h e h y p o t h e s i s t h a t t h e same molecular e n t i t y bears the two binding s i t e s for the receptors o f

.

f o 11ic l e -s t im u l a t in g h o r m o n e a n d l u t e i n iz i n g h o r mone 678

Mo l e c u l a r

w e i g h t s t u d i e s o f t h e g o n a d o t r o p h i n i n d i c a t e a v a l u e o f 4.5 o p p o s e d t o p r e v i o u s l y r e p o r t e d v a l u e s o f (5.3-6.4)

x

lo4

x

as

lo4.

A book d e a l i n g w i t h t h e c h e m i s t r y o f a n d h o m o l o g y b e t w e e n human c h o r i o n i c g o n a d o t r o p h i n and p i t u i t a r y l u t e i n i z i n g h o r m o n e h a s been published.679

The

current

understanding

of

immunological

s p e c i f i c i t y o f t h e B - s u b u n i t o f HCG i s e l u c i d a t e d . The

human s e r u m h o r m o n e s

by

immobilized

lutrophin

wheat-germ

agglutinin,

f o l l i t r o p h i n and t h y r o t r o p h i n . 6 8 0 produced

i s

suitable

radioimmunoassay o f immunoassay

and t h e

B-subunit

of

a r e a d s o r b e d by i m m o b i l i z e d c o n c a n a v a l i n A and

choriogonadotrophin

f o r

as

a

matrix

t h e s e hormones. human

which

also

The h o r m o n e - f r e e for A

chorionic

adsorbs

serum thus

standards

i n

highly specific

the

enzyme

gonadotrophin has

been

e s t a b l i s h e d . 681 C u l t u r e d human c h o r i o c a r c i n o m a c e l l s s y n t h e s i z e h i g h 0 - m a n n o s y l

o l i g o s a c c h a r i d e - c o n t a i n i n g f o r m s o f t h e a - s u b u n i t ( m o l . w t . 1.5 x

l o 4 and lo4) o f and

1.8 x

lo4>

and B - s u b u n i t f o r m s (mol.

human c h o r i o n i c g o n a d o t r o p h i n . 6 8 2

B-subunits

of

the

hormone

wt.

1.8 x

involves

the

The f r e e a - s u b u n i t

a n d 2.4 x

formation

accumulation o f incompletely processed forms o f intracellularly.

lo4

The s e c r e t i o n o f t h e aand

these subunits

produced by t h e c e l l s i s a

l a r g e r p o l y p e p t i d e and has a d i f f e r e n t c a r b o h y d r a t e c o m p o s i t i o n t h a n the

corresponding

a-subunit

of

the

placental

human

chorionic

gonadotrophin. A one-step

procedure f o r

the

i s o l a t i o n o f human c h o r i o n i c

gonadotrophin i n m i l l i g r a m amounts, u s i n g s e l e c t i v e s t e a d y - s t a t e

25 1

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides s t a c k i n g o n p o l y a c r y l a m i d e gel,

has been r e p o r t e d . 6 8 3

The p r o d u c t i o n o f human c h o r i o n i c g o n a d o t r o p h i n s u b u n i t s by h o r m o n e - p r o d u c i n g human c a n c e r c e l l s g r o w i n g i n n u d e m i c e i s s i m i l a r but n o t i d e n t i c a l t o t h a t observed i n culture.684

There a p p e a r s t o

be a s h i f t t o w a r d more c o m p l e t e hormone p r o d u c t i o n a n d a d e c r e a s e i n free

s u b u n i t p r o d u c t i o n a s compared t o t h e p a t t e r n i n c u l t u r e d

cells.

A e r o b i c t u m o u r - a s s o c i a t e d b a c t e r i a have been i s o l a t e d a n d

i d e n t i f i e d from m a l i g n a n t t i s s u e s from patients.685 the

i s o l a t e s were

capable o f

A l l b u t one o f

producing t h e B-subunit

o f human

c h o r i o n i c gonadotrophin. D i f f e r e n c e s i n t h e m o l e c u l a r w e i g h t s o f t h e a - s u b u n i t o f human chorionic

gonadotrophin e x c r e t e d i n the

c e l l s have been r e p o r t e d . 6 8 6

u r i n e and s e c r e t e d

by HeLa

The s u b u n i t s e c r e t e d by t h e t u m o u r

c e l l s h a s a h i g h e r m o l e c u l a r w e i g h t a n d may be d u e t o a n i n c r e a s e i n t h e neuraminic a c i d content o f the protein. H um a n cho r i o n i c

to

i t s receptor

go na d o t ro p h i n has

i n r a t testes.687

bee n co v a l e n t l y

c r o s s l i n ke d

Analysis o f the crosslinked

c o m p l e x e s i n d i c a t e s t h a t t h e m e m b r a n e r e c e p t o r may c o n s i s t o f a dimer o f two binding subunits which binds t o the a-subunit o f the hormone. An

assessment

has

b e e n made

o f

the

effects o f

various

m o d i f i c a t i o n s o f t h e t e r m i n a l Q - g a l a c t o s y l r e s i d u e s o f human a s i a l o choriogonado trophin.688

The p r e p a r a t i o n o f t h e d e r i v a t i v e s and t h e

i n f l u e n c e o f d e r i v a t i r a t i o n on t h e i r r a t e s o f plasma clearance, u p t a k e by t h e l i v e r , described.

i m m u n o - r e a c t i v i t y , and t a r g e t t i s s u e b i n d i n g a r e

The r o l e o f c a r b o h y d r a t e i n t h e s u b u n i t i n t e r a c t i o n s a n d

i n t h e b i n d i n g t o t h e r e c e p t o r h a s been a s s e s s e d by d e g l y c o s y l a t i o n of

i n d i v i d u a l a- and B - s u b u n i t s o f human c h o r i o n i c gonado t r ~ p h i n . ~ ~

Removal o f a b o u t 9 0 % and 8 0 % o f t h e c a r b o h y d r a t e f r o m t h e a - and Bs u b u n i t s , r e s p e c t i v e l y , had no e f f e c t o n t h e i m m u n o l o g i c a l a c t i v i t i e s , o n t h e apparent m o l e c u l a r weights, o r on t h e a b i l i t y o f t h e s u b u n i t s

-

F 1uo r e s c e n t l a b e l l e d a-sub u n i t s o f hum an

t o unde r g o r e a s s o c i a t i on. chorionic

gonadotrophin recombine

n o r m a l l y w i t h n a t i v e B-subunits.

L a b e l l i n g p r o c e d u r e s appear t o compromise t h e a b i l i t y o f t h e Bs u b u n i t s t o recombine.690 fluorescent

Since the method o f i n t r o d u c i n g the

label involves p r i o r periodate oxidation o f

the i n t a c t

i t i s p o s s i b l e t h a t p r i m a r y amino g r o u p s o f t h e p r o t e i n compete w i t h added f l u o r e s c e n t amines f o r t h e g e n e r a t e d aldehyde groups t o produce c o v a l e n t c r o s s l i n k i n g between subunits.

hormone,

The p r e s e n c e o f t h e B-subuni t o f human c h o r i o n i c g o n a d o t r o p h i n on

various

tumours

i s

reported to

be

rare on

the

basis

of

252

Carbohydrate Chemistry v a r i o u s t u m o u r s w i t h a n t i - B -HCG

i m m u n o h i s t o chem i c a l s t a i n i n g o f antibodies.691 I-Methionine

residues

of

the

a- a n d B - s u b u n i t s

l u t r o p h i n and b o v i n e t h y r o t r o p h i n have w i t h i o d o a c e t i c acid.692

of

been s p e c i f i c a l l y

bovine

alkylated

The m o d i f i e d r e s i d u e s w e r e i d e n t i f i e d a n d

t h e effects of m o d i f i c a t i o n on recombination with the unmodified c o u n t e r p a r t s u b u n i t , r e c e p t o r - b i n d i n g a c t i v i t y , and c o n f o r m a t i o n w e r e assessed.

Terminal 2-sulphated

deoxy-Q-glucose

residues o f 2-acetamido-2-

h a v e b e e n i d e n t i f i e d i n b o v i n e l u t r ~ p h i n . Human ~ ~ ~

p i t u i t a r y l u t r o p h i n i s a t l e a s t p a r t i a l l y sulphated, i t s placental counterpart,

i n contrast to

LCG.

Evidence f o r t h e amino a c i d sequence o f t h e a - s u b u n i t s o f o v i n e f o l l i c l e - s t i m u l at i n g hormone

a n d 1u t e i n i z i n g h o r m o n e

has

been

presented.694 S i m i l a r s t u d i e s o f t h e amino a c i d sequence o f t h e B - s u b u n i t o f o v i n e f o l l i c l e - s t i m u l a t i n g h o r m o n e have shown a r e m a r k a b l e d e g r e e o f p r e s e r v a t i o n o f s t r u c t u r a l i n f o r m a t i o n i n t h e B - s u b u n i t o f t h i s and o t h e r hormones.695

Two f o r m s o f e q u i n e f o l l i c l e - s t i m u l a t i n g hormone

have b e e n i s o l a t e d . 6 9 6

A h i g h l y s p e c i f i c homologous r a d i o r e c e p t o r

assay f o r t h e hormone i s described. follicle-stimulating

covalently attached to hormone.697

A

An enzyme immunoassay f o r human

h o r m o n e h a s been d e v e l o p e d u s i n g t h e hormone 8-glucose

oxidase and antiserum

radioimmunoassay

to

the

p r o c e d u r e f o r human t h y r o i d -

s t i m u l a t i n g h o r m o n e h a s b e e n i m p r o v e d by r e d u c i n g t h e h e t e r o g e n e i t y o f t h e 1251-human

t h y r o i d - s t i m u l a t i n g hormone t r a c e r . 6 9 8

The c e l l u l a r p r o c e s s i n g , a s s e m b l y , a n d r e l e a s e o f s u b u n i t s i n

-

-

t h y r o id s t im ul a t in g h o r m one b i o sy n t h e s i s h a ve a- and B - s u b u n i t s

are

initially

bee n s t u d ie d.

99 The

s y n t h e s i z e d i n e q u i v a l e n t amounts.

A subsequent excess o f a-subunits

i s observed,

r e s u l t i n g from the

It i s proposed t h a t t h i s n e t production and s e c r e t i o n o f the subunits

i n t r a c e l l u l a r d e g r a d a t i o n o f B-subunits. imbalance i n the

originates a t the level of post-translational

degradation rather

than a t the t r a n s l a t i o n a l process level. The b i o s y n t h e s i s o f c a l c i t o n i n , w t. of

3.5 a

x

lo3),

newly

a 32-amino

a c i d hormone (mol.

i n v o l v e s the g l y c o s y l a t i o n and p r o t e o l y t i c cleavage

synthesized precursor.700

e x t e n s i v e co- a n d p o s t - t r a n s l a t i o n a l

The

precursor

undergoes

p r o c e s s i n g t o t h e s m a l l e r non-

gl y co sy 1a t e d h orm one. Sim i l a r

i - g l u c o sy l - c o n t a i n i n g

o l i g o s a c c h a r i de

lipids

are

s y n t h e s i z e d by t h y r o i d r o u g h m i c r o s o m e s a n d by t h y r o i d c e l l s . 7 0 1 The t r a n s f e r o f Q - g l u c o s e t o t h e s e o l i g o s a c c h a r i d e l i p i d s i s

253

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides d e p r e s s e d b y t h e a d d i t i o n o f GDP-a-mannose. k i n e t i c experiments,

From t h e r e s u l t s o f

i t seems u n l i k e l y t h a t t h e s o l e p a r t i c i p a t i o n

o f newly synthesized oligosaccharide l i p i d s r e s u l t s i n the extensive transfer

o f p-glucose

f r o m UDP-a-glucose

t o t h y r o i d endogenous

p r o t e i n s .702 Two l a r g e g l y c o p r o t e i n f r a g m e n t s r e l a t e d t o t h e N - t e r m i n a l p a r t o f t h e adrenocorticotrophin-8-lipoprotein

p r e c u r s o r a r e t h e end

products of t h e maturation process i n t h e r a t pars intermedia.703 A n o v e l human p i t u i t a r y g l y c o p e p t i d e composed o f 39 a m i n o a c i d

r e s i d u e s a n d one o l i g o s a c c h a r i d e c h a i n h a s been i s o l a t e d f r o m w h o l e pituitaries.704

T h i s g l y c o p e p t i d e e x h i b i t s masked s e q u e n c e h o m o l o g y

t o p i g posterior p i t u i t a r y glycopeptide, g l y c o p e p t i d e s i s o l a t e d f r o m ox,

and t o w h o l e p i t u i t a r y

sheep, a n d p i g .

The i s o l a t i o n and p u r i f i c a t i o n o f a human g l y c o p e p t i d e r e p r e s e n t i n g t h e m a j o r i m m u n o r e a c t i v e f o r m o f t h e p i t u i t a r y N-

It

t e r m i n a l segment o f p r o - o p i o m e l a n o c o r t i n h a v e been r e p o r t e d . 7 0 5 b e a r s an 2 - g l y c o s y l a t i o n

s i t e a t L-Thr-45

and an N - g l y c o s i d i c s i t e

a t L-Asn-65. Human a n d

rhesus-monkey

immunocytochemically

p i t u i t a r i e s have

with antibodies against

been s t a i n e d

the B-subunits

of

p i t u i t a r y g l y c o p r o t e i n h o r m o n e s .706

11

M i l k Glycoproteins

The s t r u c t u r e s ( 3 1 ) o f a h o m o l o g o u s s e r i e s o f o l i g o s a c c h a r i d e s f r o m t h e m i l k o f t h e tamar w a l l a b y (Macropus e u g e n i i ) have been e l u c i d a t e d m a i n l y by 13C n.m.r.

Interactions

of

spectroscopy.707

substrates

g a l a c t o s y l t r a n s f e r a s e have spectroscopy.708

The

and

been

conversion

of

a-lactalbumin

measured

by

with

p-

difference

native a-lactalbumin

to

a

c o n f o r m a t i o n I or D and a l s o t h e c o n v e r s i o n o f It o D have been s t u d i e d by

U.V.

d i f f e r e n c e s p e c t r o s c o p y a n d c.d.

spectroscopy.709

The d a t a h a v e been u s e d t o c a l c u l a t e s t a n d a r d f r e e - e n e r g y

changes

f o r each of t h e t r a n s i t i o n s .

Laser photo-chemically

n u c l e a r p o l a r i z a t i o n n.m.r.

studies f o r f i v e a-lactalbumins from

different

induced dynamic

a n i m a l species c o n f i r m a h i g h degree o f homology f r o m


254

species t o species with only minor differences i n chemical-shift e n v i r o n m e n t b e t w e e n them.710

A t i g h t l y bound i m p u r i t y f r o m m i l k h a s

a pronounced e f f e c t on t h e &-tryptophan fluorescence o f goat a1a c t a 1b um in. 71

'

The b i n d i n g o f o n e Ca2+ i o n t o b o v i n e a - l a c t a l b u m i n c a u s e s a c o n f o r m a t i o n a l change r e f l e c t e d i n a d e c r e a s e of t h e k - t r y p t o p h a n f l u o r e s c e n c e and a s p e c t r a l s h i f t towards s h o r t e r wavelengths.712 The

s i t e

of

crosslinking

on

a-lactalbumin

Q-

f o r

g a l a c t o s y l t r a n s f e r a s e i n t h e l a c t o s e s y n t h e t a s e complex has been s t u d i e d using t h e mutual e x c l u s i v i t y o f a c e t y l a t i o n and a m i d a t i o n w i t h b i s ( i m i d o e s t e r s ) .713 s i t u a t e d 6.1

4

t o 7.3

-

The r e s u l t s i n d i c a t e t h a t C - l y s i n e - 1 0 8

from

is

an a m i n o g r o u p o n g a l a c t o s y l t r a n s f e r a s e

i n t h e c r o s s l i n k e d complex. Electrophoretic examination (Bibos)

javanicus

has l e d t o

electrophoretically distinct

of

milk

B-lactoglobulins,

Bali cattle three

d e s i g n a t e d E,

Bas new

F, a n d

D e l e t i o n s o r s u b s t i t u t i o n s o f s p e c i f i c amino a c i d s i n the

G.714

p r o t e i n are responsible for the different of

from

the identification of

these

variants.

A

new

electrophoretic properties

a-lactalbumin

variant

was

also

iden t i f i e d. 715 G l y c o p e p t i d e s h a v e been p r e p a r e d f r o m b o v i n e c o l o s t r a l K - c a s e i n a f t e r t r e a t m e n t w i t h cyanogen b r o m i d e a n d pro tease^.^^^ A sequence o f t h i r t e e n amino a c i d r e s i d u e s o n one o f established.

t h e g l y c o p e p t i d e s was

Three o f f o u r L - t h r e o n i n e r e s i d u e s i n t h i s sequence

a r e t h o u g h t t o be t h e s i t e s o f a t t a c h m e n t o f t h r e e p o l y s a c c h a r i d e chains i n t h e casein.

Glycopeptides released from bovine colostrum

K - c a s e i n g l y c o p e p t i d e o b t a i n e d i m m e d i a t e l y a f t e r c a l v i n g c o n t a i n one a d d i t i o n a l p r o s t h e t i c sugar group l i n k e d t o L-threonine i n s t e a d o f o n l y one,

a n d up t o t e n i n t h e c a s e o f b o v i n e ( n o r m a l ) a n d h u m a n

.

ca s e i no g l y co pe p t i de s, r e s pe c t iv e l y 71 been

isolated

from

bovine

Th e t e t r a sa c c ha r i de ( 3 2 ) has

colostrum

K-casein

after

alkaline

One n e u t r a l bo r o h y d r i d e t r e a t m e n t o f c a s e i no g l y c o p e p t i de.718 o l i g o s a c c h a r i d e a l d i t o 1 (33) a n d t h r e e a c i d i c o l i g o s a c c h s a c c h a r i de a l d i t o l s (34-36) taken s i x

have b e e n i d e n t i f i e d f r o m b o v i n e c o l o s t r u m K - c a s e i n

hours a f t e r parturition.719

O l i g o s a c c h a r i d e s (33-34)

-

-

are

ch a r a c t e r is t ic o 1igo sa c ch a r ide s h a v i n g 2 ace t a m ido 2 -de ox y - 3 -2-0 Q galactosyl-g-glucosyl been r e p o r t e d t o

groups a t the non-reducing end occur

i n

normal

K-casein.

-

a n d have n o t

The o t h e r

two

o l i g o s a c c h a r i d e s (35-36) have a l r e a d y been i d e n t i f i e d i n K - c a s e i n from

normal

bovine

milk.

H c h a r a c t e r i z e d i n a 500 M H t '

Oligosaccharide n.m.r.

study.720

(34)

has also

been


255

The l o c a l i z a t i o n of g l y c o s y l a t e d K - c a s e i n i n i s o l a t e d b o v i n e c a s e i n m i c e l l e s has been s t u d i e d a t the u l t r a - s t r u c t u r a l l e v e l u s i n g g o l d g r a n u l e s l a b e l l e d w i t h R i c i n u s cgmmunis l e c t i n , w h i c h s p e c i f i c a l l y i n t e r a c t s w i t h 8 - Q - g a l a c t o s y l residues.721 No evidence was o b t a i n e d f o r t h e p r e s e n c e of g l y c o s y l a t e d K - c a s e i n o n t h e s u r f a c e o f g l u t a r a l d e h y d e - f i x e d m i c e l l e s w h e t h e r o r n o t t h e y had The g l y c o p r o t e i n was m a i n l y been t r e a t e d w i t h n e u r a m i n i d a s e . l o c a t e d i n t h e b r i d g i n g n e t w o r k i n t e r c o n n e c t i n g t h e m i c e l l e s , and appeared t o be l o o s e l y a s s o c i a t e d w i t h t h e m i c e l l e s . B-Q-GlceNAc-( 1+3) -g-Gale-( 1+3) -g-GalNAc-ol 6

I 2 a -Ne

ue5 Ac

(32)

B-Q-Galp-( 1+4) -B-Q-GlCeNAC-( 1+6) -Q-GalNAc-01 3

I 1

6 -Q-Galg 3

I 2

R

a -Ne ue5 Ac- ( 2+3 -8

(33)

R = H

(34)

R = a-Neue5Ac

-9 -Gale-

( 1+3)

-a -Gal NA c- 01 6

I 2 R

(35)

R = H

(36)

R = a-Neup5Ac

The i n t e r a c t i o n between bovine K-casein and 6 - l a c t o g l o b u l i n h a s been examined.722 The o l i g o s a c c h a r i d e p o r t i o n o f the g l y c o p r o t e i n i n f l u e n c e s complex formation, and Ca2+ i o n s have an i n h i b i t o r y e f f e c t o n the interaction.

256

Carbohydrate Chemistry S u b c e l l u l a r f r a c t iona t i o n o f o v i ne m am m a r y - g l a n d m em b r a ne s

i n d i c a t es

that

2 - a c e t am id o - 2 - d e o x y -Q- g a l a c t o s y 1 : p o l y p e p t i de

t r a n s f e r a se a n d Q - g a l a c t o s y 1 : 2 - ace t a m i d o - 2 - d e o x y-a-D=-galactosy 1 transferase,

which a r e i n v o l v e d i n t h e assembly o f disaccharide

u n i t s o f x-casein,

a r e l o c a l i z e d c h i e f l y i n G o l g i membranes.723

n e u r a m i n o s y l t r a n s f e r a s e a l s o o c c u r s i n t h e same f r a c t i o n ,

A

probably

s u g g e s t i n g a p o s t - t r a n s l a t i o n a l g l y c o sy l a t i o n p r o c e s s o f x - c a s e i n occurring simultaneously w i t h

lo4)

from

rat

the transport

A c a s e i n component

the Golgi region.

m i l k has

(C-2

been p u r i f i e d

.

by

of

these molecules

casein,

wt.

mol.

ion-exchange

to

3.4

and

x

gel

f i1t r a t i on ch rom a t o g r a phy 7 2 4

only

sugar

detected

i n

2- Ace tam i d o -2-deox y - p - g l uco se w as t h e this glycoprotein. Alkali-labile

o l i g o s a c c h a r i d e s l i b e r a t e d f r o m t h e g l y c o p e p t i d e s o f human m i l k f a t g l o b u l e membrane have been c h a r a c t e r i z e d and compared w i t h t h e corresponding o l i g o s a c c h a r i d e s i s o l a t e d from bovine m i l k f a t g l o b u l e The f o r m a t i o n o f l i p i d - l i n k e d s u g a r s i n t o e x p l a n t s o f

membranes.725

d e v e l o p i n g r a b b i t mammary g l a n d ,

stimulated t o differentiate i n

c u l t u r e w i t h hormones, a n d t h e a p p e a r a n c e o f n o v e l g l y c o p r o t e i n s , whose

synthesis

described.726

i s

dependent

on

this

pathway,

have

I n c u b a t i o n o f a membrane p r e p a r a t i o n from

been

lactating

bo v i ne mam m a r y t i s s u e w i t h UDP - 2 - ace t a m i d o -2-deox y-n-gl ucose and GDP-Q-mannose linked

r e s u l t s i n t h e s y n t h e s i s o f an e n t i r e range o f l i p i d -

saccharides

that

differ

from

one

another

by

one

m o n o s a c c h a r i d e u n i t.727 They a p p e a r t o be p r e c u r s o r - p r o d u c t t y p e o f i n t erm e d i a t e s

for

glycoproteins.

Two i s o m e r i c s t r u c t u r e s

-

the

g l y c o s y l a t i o n o f I - a s p a r a g i ne- l i n k e d for

t h e l i p i d - l i n k e d hexa-

a n d h ep t a sa ccha r i de s a r e p r o PO se d. 7 2 The e l u c i d a t i o n o f t h e s t r u c t u r e s o f t w o c a r b o h y d r a t e u n i t s (37-38),

1-glycosidically

lactotransferrin,

de s c r ibe d

.

l i n k e d t o an &-asparagine

u s i n g 5 0 0 MHz 'H

n.m.r.

residue o f

spectroscopy

has been

S t r u c t u r a l r e l a t e d n e s s b e t w e e n human l a c t o t r a n s f e r r i n a'nd

human

c e r u l o p l a s m i n has been e ~ t a b l i s h e d . ~ ~ The ' comparison o f amino a c i d sequences o f t w o c e r u l o p l a s m i n f r a g m e n t s c o r r e s p o n d i n g t o a t o t a l o f 5 6 4 a m i n o a c i d r e s i d u e s a n d 70% o f t h e l a c t o t r a n s f e r r i n m o l e c u l e (445 r e s i d u e s ) i n d i c a t e s t e n h o m o l o g o u s a r e a s i n c l u d i n g 1 3 3 a m i n o acids.


c v)

Q

I J I

t

4

I

4

u I

4'

cn I

.j

t

4

u I

0 Q

z al

4

u I

4' cn

II

U

t

n n

r.03

4

9

M

u

a

m w - 4 m w - - 4 m h l - 4 m I ZE I

4'

m I

4' ?

n

n

4

4

4l

4'

+

M

I

M

t

I

n

n

4 v

u

+

N

I

4' I

n

hl

-4 U

I

a

II

a a

I

n

+

hl

-4

$ m

5

4'

a

v

I

7'

M

u

257

258


Serum G l y c o p r o t e i n s

R a t a 2 - r n a c r o g l o b u l i n h a s been p u r i f i e d f r o m s e r a by s e q u e n t i a l use o f

dextran

sulphate,

ion-exchange

chromatography, and g e l -

p e r m e a t i o n ~ h r o m a t o g r a p h y . ~An ~ ~a n t i s e r u m p r e p a r e d a g a i n s t t h e g l y c o p r o t e i n was u s e d t o a s s a y t h e c o n c e n t r a t i o n o f a 2 - m a c r o g l o b u l i n i n t h e s e r a of n o r m a l r a t s and i n t h e s e r a o f r a t s undergoing an i n f l a m m a t o r y response. Evidence for which

i s

t h e e x i s t e n c e o f t w o t y p e s o f c o m p l e x e s , one o f

covalent

and

the

m a c r o g l o b u l i n and p r o t e a s e s ,

other

of

which

i s

not

has been reported.732

between

a2-

The c o n v e r s i o n

of t h e non-covalent t o t h e covalent form r e q u i r e s t h e presence o f u n b l o c k e d amino groups. Soybean t r y p s i n i n h i b i t o r h a s been u s e d t o a s s e s s t h e d e g r e e o f i n a c c e s s i b i l i t y of porcine t r y p s i n w i t h i n t h e a2-macroglobulint r y p s i n complex.733

Proteinase-binding

on t h e a 2 - m a c r o g l o b u l i n do

not

appear

arranged.

to

surface.

be i d e n t i c a l ,

Evidence

for

the

s i t e s h a v e been i d e n t i f i e d

The t w o s u b u n i t s o f t h i s p r o t e i n

or they

cleavage

are not

of

a

macroglobulin during proteinase-complex discussed.734

thiol

symmetrically

i n a2-

ester

f o r m a t i o n has been

The n a t i v e human g l y c o p r o t e i n c o n t a i n s one r e a c t i v e

l a b i l e t h i o l e s t e r i n each o f i t s four i d e n t i c a l subunits.735

The

c y s t i n e r e s i d u e c o n s t i t u t i n g one p a r t o f t h e t h i o l e s t e r s t r u c t u r e i s l o c a t e d i n an i d e n t i c a l sequence t o c o m p l e m e n t c o m p o n e n t C3. of

a2-macroglobulin

that

f o u n d i n human

Trypsin-induced a c t i v a t i o n o f t h i o l esters

generates

a short-lived

intermediate that

can

r e a c t r a p i d l y t o i n c o r p o r a t e n o t o n l y methylamine or p u t r e s c i n e b u t also

proteins

interaction of

lacking trypsin

protease with

activity.736

this

protease

A

model

inhibitor

for has

the been

p r o p o s e d i n w h i c h t h e i n h i b i t o r can b i n d t o t h e enzyme i n t h r e e d i s t i n c t modes. 737 Human

a2-macroglobulin,

after

derivatization

m o n o d a n s y l c a d a v e r i n e by t r a n s g l u t a m i n a s e - c a t a l y s e d

with

incorporation,

contains two y-glutamyl residues per conjugated molecule,

a n d shows

n o d i f f e r e n c e s i n t r y p s i n b i n d i n g o r m e t h y l a m i n e i n a c t i v a t i o n when compared w i t h t h e n a t i v e a2-macroglobulin.738 methylamine

treatment,

the

a2-macroglobulin

A f t e r t r y p s i n or conjugate

e f f e c t i v e l y r e c o g n i z e d by i t s c e l l u l a r r e c e p t o r . binding o f t r y p s i n t o a2-macroglobulin g r o u p on t h e p r o t e a s e ,

The

i s

not

covalent

o c c u r s by a p r i m a r y a m i n o

a n d i s i n h i b i t e d b y p r i m a r y a m i n e s when

p r e s e n t d u r i n g complex f o r m a t i o n . 7 3 9

Covalent i n c o r p o r a t i o n o f t h e

259


same p r i m a r y a m i n e s d u r i n g t r y p s i n b i n d i n g t o a 2 - m a c r o g l o b u l i n indicates transient activation o f the methylamine-reactive s i t e during complexation. The c o v a l e n t l i n k a g e o f n o n - p r o t e o l y t i c proteins t o a2-macroglobulin

d u r i n g i t s r e a c t i o n w i t h p r o t e a s e s has

been f u r t h e r c h a r a c t e r i z e d . 7 4 0 The a m i n o a c i d s e q u e n c e l o c a t i o n o f a p u t a t i v e t r a n s g l u t a m i n a s e c r o s s l i n k i n g s i t e i n human a 2 - m a c r o g l o b u l i n h a s b e e n i d e n t i f ied.741 The a m i n o a c i d s e q u e n c e o f a 3 5 - r e s i d u e i n t e r n a l s t r e t c h o f h u m a n a2-macroglobulin, one

site

cleaved

established.742

w h i c h c o n t a i n s t w o s i t e s c l e a v e d by e l a s t a s e and by t r y p s i n , plasmin, and t h r o m b i n , has been T r y p s i n cleaves a I - L y s - i - L e u bond i n a2-macro-

g l o b u l i n , w h i l e S t a p h y l o c o c c u s a u r e u s V8 p r o t e i n a s e c l e a v e s a n Glu-Gly

I-

bond .743

al-Acid

g l y c o p r o t e i n h a s been m e a s u r e d b y l a s e r n e p h e l o m e t r y i n

a q u i c k and p r e c i s e m e t h o d f o r

d e t e c t i o n and f o l l o w - u p

i n f e c t i o n s i n newborn infants.744 o f r a t al-acid

of

bacterial

The c o m p l e t e n u c l e o t i d e s e q u e n c e

g l y c o p r o t e i n m e s s e n g e r RNA h a s b e e n d e s c r i b e d . 7 4 5

The i n f e r r e d a m i n o a c i d sequence h a s b e e n c o m p a r e d t o t h e p r e v i o u s l y reported

sequence

focusing

has

of

human a l - a c i d

glycoprotein.

been used t o r e v e a l t h e t w o

p o p u l a t i o n s s e p a r a t e d by a f f i n i t y

Isoelectric

al-acid

glycoprotein

chromatography on i m m o b i l i z e d

c o n c a n a v a l i n A and d e r i v e d from w h o l e n o r m a l serum and s e r a f r o m patients

u n d e r g o i n g an

significant

difference

i s

acute

i n f lammatory

observed

response.746

A

between p a t t e r n s o b t a i n e d f r o m

n o r m a l and i n f l a m m a t o r y s e r a . al-Acid

glycoprotein

produces

immunoaffinoelectrophoresis dimension,

thus

with

implying a

three

peaks

concanavalin

degree

of

A

on i n

crossed

the

heterogeneity

first i n

the

These carbohydrate p o r t i o n o f t h i s g l y ~ o p r o t e i n . ~ ~ ~ v a r i a t i o n s have been s t u d i e d under c o n d i t i o n s a s s o c i a t e d w i t h a l t e r a t i o n s i n serum concentration

of

female

sex

hormones.

A

new

type

o f

m i c r o h e t e r o g e n e i t y w i t h r e g a r d t o t h e p o s i t i o n o f a t t a c h m e n t o f an I - f u c o s y l r e s i d u e o n al-acid

g l y c o p r o t e i n h a s been o b s e r v e d f r o m t h e

i m p r o v e d r e s o l v i n g p o w e r a n d e n h a n c e d s e n s i t i v i t y o f 5 0 0 MHz l H n.m.r.

spectroscopy.748 al-Acid

glycoprotein

and

i t s

deglycosylated

derivatives,

p r o d u c e d by e n z y m a t i c c l e a v a g e of n e u r a m i n o s y l , B - g a l a c t o s y l , mannosyl, t o

and 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s y l r e s i d u e s ,

regulate

immune responses

p a t h o l o g i c a l conditions.749

i n

various

Q-

may f u n c t i o n

physiological

and

The p r e c u r s o r f o r m s o f p l a s m a p r o t e i n s

s y n t h e s i z e d i n human l i v e r h a v e been i n v e s t i g a t e d . 7 5 0

By c o m p a r i n g

260


t h e chromatographic behaviour of t h i r t e e n d i f f e r e n t plasma p r o t e i n s i s o l a t e d from e i t h e r l i v e r o r blood, f i v e i n t r a c e l l u l a r precursor p r o t e i n s can be i d e n t i f i e d .

Two o f t h e m , t r a n s f e r r i n a n d a l - a c i d

g l y c o p r o t e i n , were p u r i f i e d and c h a r a c t e r i z e d by c o m p a r i n g t h e i r s t r u c t u r e s w i t h those of t h e corresponding plasma forms. forms o f b o t h g l y c o p r o t e i n s l a c k e d n e u r a m i n i c acid, two l i v e r forms

of

al-acid

The l i v e r

and i n a d d i t i o n

g l y c o p r o t e i n are described as h a v i n g

l o w e r n e u t r a l and a m i n o s u g a r c o n t e n t . Rat a - l - a n t i t r y p s i n

has been c h a r a c t e r i z e d w i t h r e s p e c t t o i t s

a m i n o a c i d and c a r b o h y d r a t e c o m p o s i t i o n . 7 5 1

Some o f

i t s chemical

p r o p e r t i e s h a v e b e e n c o m p a r e d t o t h o s e o f human a - l - a n t i t r y p s i n . A n t i b o d i e s p r e p a r e d t o t h e r a t g l y c o p r o t e i n h a v e b e e n s h o w n t o be monospecific.

a-l-Antitrypsin

h a s been f r a c t i o n a t e d on i m m o b i l i z e d

concanavalin A i n t o t w o components which d i f f e r i n t h e degree o f branching

of

their

oligosaccharide

side

chains.752

One

form

c o n t a i n s t h r e e b i a n t e n n a r y s i d e c h a i n s and t h e o t h e r c o n t a i n s t w o b i a n t e n n a r y and one t r i a n t e n n a r y s i d e c h a i n . s t r u c t u r e s have been t e r m e d i s o f o r m s ,

These a l t e r n a t i v e

a t e r m used t o d i f f e r e n t i a t e

them from v a r i a n t s o f g e n e t i c o r p o s t - s e c r e t o r y o r i g i n . A l a t e n t t r y p s i n i n h i b i t o r i s r e l e a s e d f r o m d e n a t u r e d human s e r u m p r o t e i n s b y d i g e s t i o n w i t h t h e r m ~ l y s i n . I~m~m~u n o l o g i c a l c r o s s - r e a t i o n i d e n t i f i e d t h i s i n h i b i t o r as a complex between t h e i n h i b i t o r y a c t i v e p a r t o f t h e g l y c o p r o t e i n and i m m u n o g l o b u l i n G. al-Protease

i n h i b i t o r f r o m r a t serum e x i s t s i n f i v e

These m u l t i p l e f o r m s laser

nephelometric

p r o c e d u r e 7 5 5 and

i m m u n o a ~ s a yh~a v~e~ b e e n d e v e l o p e d f o r The t h e r m a l d e n a t u r a t i o n o f h e p a r i n and a l s o e.d.t.a.757

by

a

sandwich

enzyme

m e a s u r i n g a n t i t h r o m b i n 111.

a n t i t h r o m b i n 111

anions

by

A m o d i f i e d end-

s e q u e n t i a l a d d i t i o n o f n e u r a m i n i c a c i d t o e a c h one. point

forms.754

are derived from three o r i g i n a l forms

such as

i s

s t a b i l i z e d by

phosphate,

sulphate, and

The e f f e c t s o f d i t h i o t h r e i t o l o n t h e m o d u l a t i o n o f

a n t i t h r o m b i n I11 a c t i v i t y by s u l p h a t e d p o l y s a c c h a r i d e s have been reported.758

K i n e t i c analysis o f the heparin-enhanced plasmin-

a n t i t h r o m b i n 111 r e a c t i o n s u p p o r t s t h e t h e o r y t h a t h e p a r i n m u s t b i n d t o

the

enzyme

reaction.759

t o

accelerate

the

plasmin-antithrombin

The b i n d i n g o f h i g h - a f f i n i t y

111

heparin t o antithrombin

I 1 1 h a s been i n v e s t i g a t e d f o l l o w i n g c h a r a c t e r i z a t i o n o f t h e p r o t e i n fluorescence

enhancement

of

I-tryptophan

residues

a n t i t h r o m b i n i n t h e presence and absence o f heparin.760 flow

i n

the

Stopped-

k i n e t i c s t u d i e s o f t h e b i n d i n g i n t e r a c t i o n have a l s o been

reported.761

26 1


I n a study o f t h e plasma o f p a t i e n t s c o n g e n i t a l l y d e f i c i e n t i n

a n t i t h r o m b i n 111, o n l y t w o o f f o u r e l e c t r o p h o r e t i c a l l y s e p a r a t e d f o r m s i d e n t i f i e d i n n o r m a l p a t i e n t s ’ p l a s m a c o u l d be detected.762 Isoelectric-focusing

profiles

of

human

antithrombin

demonstrated heterogeneity i n l y o p h i l i z e d s a l t - f r e e

have

111

p r o d u c t s and i n

aged p r o d u c t s .763 A s i n g l e p r o t e o l y t i c c l e a v a g e s i t e i n human a n t i t h r o m b i n h a s

been i d e n t i f i e d between L-Arg-385 w i t h human t h r o m b i n . 7 6 4 -

and I - S e r - 3 8 6

following digestion

T h i s i s a t a p o s i t i o n homologous t o t h a t

observed f o r t h e bovine glycoprotein. The s t r u c t u r e a n d f u n c t i o n o f f a c t o r V I I I c o m p l e x h a v e b e e n r e v i e w e d .765 Factor

VIII-related

f luoroimmunoassay.766

anti-factor with

antigen

The a s s a y ,

has

been

measured

using

a

which uses a f l u o r e s c e n t - l a b e l l e d

V I I I a n t i b o d y as t h e s i n g l e m a j o r r e a g e n t , c o r r e l a t e s

an e x i s t i n g method.

The

molecular

forms

of

factor

V I I I

c o a g u l a n t a n t i g e n i n c l i n i c a l c o n c e n t r a t e s o f f a c t o r I X have been measured u s i n g 1 2 5 1 - l a b e l l e d

factor VIII:C

antibodies.767

Factor

c l o t t i n g a n t i g e n has been measured w i t h t w o d i f f e r e n t

V I I I

i m m u n o r a d i o m e t r i c assays,

one u t i l i z i n g t w o a c q u i r e d a n t i b o d i e s and

one a h a e m o p h i l i c a n t i b o d y . 7 6 8

The r e s u l t s c o n f i r m t h e d i v i s i o n o f

b o t h m o d e r a t e and m i l d h a e m o p h i l i a A i n t o t h r e e sub-groups. h a e m o p h i l i a A,

I n

there i s a quantitative reduction rather than a

q u a l i t a t i v e defect i n the p r o t e i n responsible f o r the f a c t o r V I I I clotting activity. A r a d i o r e c e p t o r assay

factor

VIII/von

f o r q u a n t i t a t i n g p l a s m a l e v e l s o f human

Willebrand’s

factor

has

r e c e p t o r s i t e s o n human p l a t e l e t s . 7 6 9

been

developed

using

The p l a s m a g l y c o p r o t e i n

c o n c e n t r a t i o n s c o r r e l a t e w i t h l e v e l s m e a s u r e d by t h e r i s t o c e t i n i n d u c e d p l a t e l e t - a g g r e g a t i o n method. Aluminium hydroxide but not barium chloride absorption o f factor

VIII p r o c o a g u l a n t

antigen

i s

associated

with

variable

r e c o v e r i e s o f t h e antigen.770 Immobilized p u r i f i c a t i o n of

11, V I I I ,

phospholipid

a number o f

vesicles

have

been

coagulation factors,

used

in

the

including factors

I X , a n d X.771

F i b r i l s u s p e n s i o n s o f t y p e s I,

II,and

111 c o l l a g e n a r e a l l

c a p a b l e o f a d s o r b i n g r i s t o c e t i n c o f a c t o r a c t i v i t y and f a c t o r V I I I r e l a t e d a n t i g e n a c t i v i t y f r o m n o r m a l plasma.772

The b i n d i n g o f

factor VIII/von

W i l l e b r a n d f a c t o r h a s b e e n d e t e c t e d by f o l l o w i n g t h e

adsorption

the

of

labelled

antigen

to

collagen

films.773

In

262


c o n t r a s t t o r e p o r t s f r o m o t h e r workers, b i n d i n g t o bovine tendon f i b r e s was a l s o d e t e c t e d . The a p p l i c a t i o n o f a f f i n i t y - c h r o m a t o g r a p h i c t e c h n i q u e s f o r s e p a r a t i o n s and s t u d i e s o n molecular i n t e r a c t i o n s of t h e components of t h e blood-coagulation system has been reviewed.774 An e l e c t r o p h o r e t i c sys tern , employing l a r ge-pore polyacry lam i d e g e l s i n n e u t r a l b u f f e r i n t h e absence of s o d i u m d o d e c y l s u l p h a t e , has been used t o f r a c t i o n a t e s i n g l e s p e c i e s o f f a c t o r V I I I m u l t i m e r s under c o n d i t i o n s where b i o l o g i c a l p r o p e r t i e s of f a c t o r V I I I a r e p r e s e r v e d . 7 7 5 F a c t o r V I I I : C , f a c t o r VIII:RCof, and f a c t o r VII1:Ag a c t i v i t i e s a r e e x t r a c t a b l e from t h e g e l s a f t e r e l e c t r o p h o r e s i s and can be a s c r i b e d t o i n d i v i d u a l f a c t o r VIII m u l t i m e r s . Human a n t i - h a e m o p h i l i c f a c t o r (mol. w t . 1.16 x l o 5 ) , c o n t a i n i n g n o d e t e c t a b l e von Willebrand f a c t o r , has been prepared from normal human p l a s m a . 7 7 6 P a r t i a l r e d u c t i o n w i t h d i t h i o t h r e i t o l e x p o s e s c r i t i c a l s u l p h y d r y l g r o u p s , which when a l k y l a t e d m a i n t a i n t h e a n t i - h a e m o p h i l i c f a c t o r w i t h o u t i n a c t i v a t i n g procoagulant a c t i v i t y . F u r t h e r p u r i f i c a t i o n of t h e a n t i - h a e m o p h i l i c f a c t o r was a c h i e v e d a f t e r c o n v e r s i o n of p r o t e i n - 2 - p y r i d y l mixed d i s u l p h i d e c o n j u g a t e s f o l l o w e d b y a f f i n i t y chromatography o n t h i o p r ~ p y l - a g a r o s e . ~ ~ ~ F r a c t i o n a t i o n of p a r t i a l l y p u r i f i e d b o v i n e f a c t o r V I I I o n c a l c i u m c i t r a t e c o l u m n s p r o d u c e s a m a t e r i a l which c o n t a i n s h i g h l e v e l s of f a c t o r VIII procoagulant a c t i v i t y and f a c t o r V I I I - r e l a t e d a n t i g e n b u t d o e s n o t a g g r e g a t e human p l a t e l e t s . 7 7 8 The a p p a r e n t molecular weight of t h e procoagulant f a c t o r is s i g n i f i c a n t l y lower t h a n t h a t of t h e f o r m s of f a c t o r VIII which c o n t a i n p l a t e l e t a g g r e g a t i n g a c t i v i t y , and i t e x h i b i t s a h i g h e r m o b i l i t y o n electrophoresis. Bovine f a c t o r VIII h a s been p r e p a r e d f r e e from p l a t e l e t a g g r e g a t i n g a c t i v i t y . 7 7 9 The p r e p a r a t i o n migrated a s a t r i p l e t on sodium dodecylsulphate-polyacrylamide gel electrophoresis w i t h a p p a r e n t m o l . wts. 9 . 3 x L O 4 , 8.8 x l o 4 , and 8.5 x l o 4 . B o t h p l a s m i n and t h r o m b i n d e s t r o y a p r o t e i n ( m o l . w t . 8.5 x l o 4 ) , p r e s e n t i n h i g h l y p u r i f i e d f a c t o r VIII/von Willebrand f a c t o r , a t a r a t e t h a t p a r a l l e l s t h e loss of f a c t o r V I I I C . 7 8 0 T h i s protein may be n e c e s s a r y f o r f u l l e x p r e s s i o n o f f a c t o r V I I I C and may be d e f e c t i v e i n c a s e s o f haemophilia. The l e v e l s o f f a c t o r V I I I c o a g u l a n t a n t i g e n i n n o r m a l , t h r o m b i n - t r e a t e d , and haemophilic plasma have been a n a l y ~ e d . The ~~~ a n t i g e n d e t e c t e d i n normal plasma i s n o t c o v a l e n t l y l i n k e d t o f a c t o r V I I I / v o n Willebrand f a c t o r m u l t i m e r s and is a b s e n t i n plasma from a

263

5: Glycoproteins, Glycopeptides, ProteoRlycans, and Animal Polysaccharides

number o f h a e m o p h i l i a p a t i e n t s . Thrombin-induced p r o t e o l y s i s o f t h e a n t i g e n has been o b s e r v e d i n n o r m a l plasma. Thrombin i s more e f f e c t i v e

i n potentiating factor

V I I I

p r o c o a g u l a n t a c t i v i t y when t h e a m o u n t o f t h r o m b i n r e l a t i v e t o t h e concentration

of

stoichiometric

rather

activation,

the

factor than

VIII/von

Willebrand

catalytic.782

I n

factor

contrast

the i n a c t i v a t i o n o f thrombin-activated

factor

to

i s i t s

VIII/von

W i l l e b r a n d f a c t o r i s n o t a p r o t e o l y t i c p r o c e s s , and a c t i v a t i o n o f t h i s factor i s a prerequisite for i t s inactivation. F u r t h e r e v i d e n c e o f t h e f o r m a t i o n o f s t a b l e immune complexes b e t w e e n human f a c t o r V I I I a n d i t s h o m o l o g o u s a n t i b o d i e s h a s b e e n published.783

Some e v i d e n c e i s a l s o p r o v i d e d w h i c h s u p p o r t s t h e

view t h a t f a c t o r V I I I c o n s i s t s o f two d i s s i m i l a r subunits which a r e noncovalently linked. Heterogeneity o f molecular size o f factor

i n von Willebrand’s

factor

disease has

VIII/von

Willebrand

been d e m o n s t r a t e d by

a

c o m b i n a t i o n o f SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and c r o s s e d -

i m m u n o e l e c t r ~ p h o r e s i s . ~ T~h~e

factor

g l y c o p r o t e i n c o n s i s t s o f a s e r i e s of i n m o l e c u l a r w e i g h t f r o m 2.4

x

lo6

VIII/von

Willebrand

multimeric structures ranging t o g r e a t e r t h a n 1 0 x 106.785

Differences i n the s p e c i f i c a c t i v i t y o f the polymeric forms are a f u n c t i o n n o t o n l y of

t h e i r s i z e but also o f quaternary conformation

t h a t may b e d i c t a t e d by d i s u l p h i d e b o n d a r r a n g e m e n t s . A

technique

relationship

has

been

between f a c t o r

developed

i n

VIII-related

order

t o

study

the

a n t i g e n ( V I I I R A g ) and

The V I I I C p a r t o f t h e f a c t o r V I I I c l o t t i n g a n t i g e n (VIIICAg).786 complex i s pre-incubated w i t h 1251-anti-VIIICAg prior to immunoelectrophoresis. Autoradiography o f the immunoprecipitin l i n e produced w i t h a n t i - V I I I R A g i n d i c a t e d t h e presence o r absence o f VIIICAg ( V I I I C )

a s s o c i a t e d w i t h VIIIRAg.

Human f a c t o r

V I I I procoagulant a c t i v i t y i n t e r a c t s w i t h c e r t a i n

phospholipid^.^^^

F u r t h e r e v i d e n c e h a s shown t h a t t h i s a c t i v i t y and

the

factor

VIII/von

Willebrand

factor

V a r i a n t forms of p r o c o a g u l a n t - l i k e f a c t o r

are

separate

entities.

V I I I i n haemophiliacs have

b e e n r e p o r t e d .788 The a c t i v a t i o n o f blood-coagulation

factor

X

t h e i n t r i n s i c pathway i n t h e

s y s t e m i n v o l v e s p h o s p h o l i p i d and f a c t o r

VIII.789

The s t i m u l a t i n g e f f e c t o f p h o s p h o l i p i d i n f a c t o r X a c t i v a t i o n i s m a i n l y due

t o

an e f f e c t

stimulatory effect increase i n the

of

ymax

factor

on t h e V I I I

Km f o r

factor

X,

while

the

i s e x p l a i n e d by i t s 2 0 0 , 0 0 0 - f o l d

o f f a c t o r Xa f o r m a t i o n .

264


A metal-ion-catalysed conformational change a f f e c t i n g f a c t o r X i s a p r e r e q u i s i t e f o r g l y c o p r o t e i n - l e c t i n p r e c i p i t i n i n t e r a c t i o n . 79b

When Ca2+ o r Mn2+, b u t n o t Mg2+, i s p r e s e n t , s p e c i f i c s u g a r s o n t h e c a r b o h y d r a t e c h a i n s o f f a c t o r X1 b e c o m e a c c e s s i b l e t o w h e a t - g e r m agglutinin. Whether t h i s c a t i o n - g l y c o p r o t e i n i n t e r a c t i o n t a k e s p l a c e t h r o u g h a d i r e c t i n t e r a c t i o n between c a r b o h y d r a t e m o i e t i e s and c a t i o n or by a d i r e c t i n t e r a c t i o n b e t w e e n p r o t e i n m o i e t y a n d c a t i o n , o r b o t h , i s n o t known. A three-step method h a s been developed f o r t h e d e t e r m i n a t i o n o f f a c t o r VII u s i n g a c h r o m o g e n i c s u b s t r a t e . 7 9 1 A p r e p a r a t i v e scheme f o r t h e i s o l a t i o n o f m u l t i p l e c o m p o n e n t s o f complement from one l a r g e p o o l o f human p l a s m a i n which t h e p u r i f i e d p r o d u c t s are c h a r a c t e r i z e d f u n c t i o n a l l y , physicochemically, a n d i m m u n o c h e m i c a 1l y h a s b e e n d e s c r i b e d . 792 The p u r i f i e d s u b c o m p o n e n t C l q o f t h e f i r s t c o m p o n e n t o f m o u s e complement c o n t a i n s h y d r o x y - i - p r o l i n e , h y d r o x y - k - l y s i n e , g l y c i n e , and ~ a r b o h y d r a t e . ” ~ T h e i n t e r a c t i o n o f i s o l a t e d h u m a n c o m p l e m e n t c o m p o n e n t s Clr a n d Cls w i t h c e l l - b o u n d a n d f l u i d - p h a s e C l q h a s b e e n r e p o r t e d . 7 9 4 The b i n d i n g of Clq t o t h e F c p o r t i o n of an a n t i b o d y i n f l u e n c e s i t s a c t i v i t y t w o a r d s Clr a n d Cls a n d i t s a b i l i t y t o a c t i v a t e t h e s e C 1 components. B i n d i n g o f Clr a n d C l s t o C l q i n t h e f l u i d p h a s e a p p e a r s t o change or f i x t h e C l q c o n f o r m a t i o n so t h a t Clq is t h e n able t o bind only t o t h o s e binding sites which can t r i g g e r t h e internal activation. The i n i t i a l r e a c t i o n o f b i n d i n g o f h u m a n c o m p l e m e n t C 3 a n d C 4 t o s u r f a c e s i n v o l v e s similar mechanisms f o r both p r o t e i n s , w i t h t h e b i n d i n g b e i n g by m e a n s o f a c o v a l e n t b o n d ( e s t e r o r a m i d e ) o f w h i c h t h e c a r b o x y l g r o u p i s d o n a t e d by C 3 o r C4.795 Haemolytically active C 3 a n d C4 u n d e r g o p e p t i d e - b o n d c l e a v a g e i n t h e a - c h a i n when incubated under denaturing conditions. I n e a c h case o n l y a s i n g l e p e p t i d e bond i s s p l i t . T h e n o n c o v a l e n t l y a s s o c i a t e d a- a n d B - s u b u n i t s o f t h e e i g h t h component o f human complement ( C 8 ) r e c o m b i n e i n s o l u t i o n t o f o r m n a t i v e C8 w i t h c o n c o m i t a n t a p p e a r a n c e o f C 8 h a e m o l y t i c a c t i v i t y . 7 9 6 S t r u c t u r a l domains which mediate s p e c i f i c i n t e r a c t i o n o f C 8 with its n a t i v e c y t o l y t i c complex are l o c a t e d i n t h e B-subunit. Direct c h e m i c a l e v i d e n c e s h o w s t h a t n o n - e n z y m a t i c g l y c o s y l a t i o n of haemoglobin i n v o l v e s the i n i t i a l f o r m a t i o n o f a r e v e r s i b l e a l d i m i n e ( S c h i f f b a s e ) p r e c u r s o r which s l o w l y r e a r r a n g e s t o a stable k e t o a m i n e by t h e A m a d o r i r e a r r a n g e m e n t . 7 9 7 An e s t i m a t e o f t h e

265


d i s t r i b u t i o n o f l a b i l e a l d i m i n e and s t a b l e k e t o a m i n e i n e r y t h r o c y t e s f r o m n o r m a l and d i a b e t i c s o u r c e s i s o b t a i n e d f r o m t h e r a t e c o n s t a n t s f o r t h e p r o d u c t i o n o f t h e two forms. Unless s t r i c t l y standardized conditions are a p p l i e d t o the measurement o f n o n - e n z y m i c a l l y g l y c o s y l a t e d serum p r o t e i n s u s i n g t h e t h i o b a r b i t u r i c a c i d a s s a y , e r r o n e o u s r e s u l t s may b e o b t a i n e d . 7 9 8 The

accuracy

of

the

modifications t o

method has

assay

been

i m p r o v e d by

conditions.

a

number

The c o n d i t i o n s f o r

of the

t h i o b a r b i t u r i c a c i d a s s a y f o r n o n - e n z y m i c g l y c o s y l a t i o n o f human serum a l b u m i n have been examined, and t h o s e o f o p t i m a l s e n s i t i v i t y and r e p r o d u c i b i l i t y a r e r e p o r t e d . 7 9 9 A

colorimetric

haemoglobin, carbohydrate,

based

method on

for

the

the

acid

glycosylated reaction

of

i s c l a i m e d t o be more r e l i a b l e a n d more s e n s i t i v e t h a n

t h e t h i o b a r b i t u r i c a c i d method.800 carbohydrate

estimation of

phenol-sulphuric

content

of

I n a d i f f e r e n t approach,

the

g l y c o s y l a t e d h a e m o g l o b i n i s d e t e r m i n e d by

measuring t h e r e l e a s e o f formaldehyde f o l l o w i n g p e r i o d a t e o x i d a t i o n o f t h e carbohydrate moieties.801

The f o r m a l d e h y d e i s e s t i m a t e d a s

t h e f l u o r e s c e n t c o n d e n s a t i o n p r o d u c t f o r m e d w i t h a c e t y l a c e t o n e and ammonia.

An

improved colorimetric

assay

for

glycosylated

h a e m o g l o b i n depends o n t h e c o n v e r s i o n o f t h e p - g l u c o s e m o i e t y t o 5h y d r o x y m e t h y l f u r f u r a l d e h y d e by o x a l i c a c i d , estimation

of

t h i o b a r b i t u r i c acid.802 a f f e c t e d by

with

2-

The u t i l i t y o f g l y c o s y l a t e d - h a e m o g l o b i n

measurement as an i n d e x o f adversely

f o l l o w e d by c o l o r i m e t r i c

5-hydroxymethylfurfuraldehyde

the

chronic control i n diabetes

interference

from

a

labile

can be

glycosylated

f r a c t i o n t h a t changes r a p i d l y w i t h b l o o d Q - g l u c o s e c o n c e n t r a t i o n . 8 0 3 T h i s f r a c t i o n may b e m e a s u r e d b y h.p.1.c.

or by

electrophoresis.

I n t e r f e r e n c e by Q - g l u c o s e i n t h e c o l o r i m e t r i c e s t i m a t i o n o f g l y c o s y l a t e d p l a s m a p r o t e i n h a s b e e n e l i m i n a t e d by p r e c i p i t a t i o n o f t h e p r o t e i n s w i t h t r i c h l o r a c e t i c acid.804 G l y c o s y l a t e d m i n o r c o m p o n e n t s o f human f e t a l h a e m o g l o b i n h a v e been s e p a r a t e d by ion-exchange

chromatography,

functionally

Sources o f v a r i a t i o n i n t h e i o n -

characterized.805

exchange column

chromatographic

identified,

determination

of

and

glycosylated

h a e m o g l o b i n (HbA) h a v e b e e n d e s c r i b e d . 8 0 6 A

simple,

commercially

a v a i l a b l e method f o r separating

g l y c o s y l a t e d haemoglobin e l e c t r o p h o r e t i c a l l y been

assessed.807

The

method

correlated

on a g a r - g e l well

chromatographic technique. 8-Glucose a l s o b i n d s c o v a l e n t l y t o €-amino

with

f i l m s has a

column

groups o f I - l y s i n e


266 o f human a p o l i p o p r o t e i n s . 8 0 8

The l e v e l o f g l y c o s y l a t e d a p o p r o t e i n

B o f l o w - d e n s i t y l i p o p r o t e i n s i s i n c r e a s e d i n serum f r o m d i a b e t i c patients. Nonenzymatic g l y c o s y l a t i o n o f p r o t e i n s i n d i a b e t i c s extends beyond h a e m o g l o b i n t o t h e p r o t e i n s o f t h e e r y t h r o c y t e membrane, probably affects

and

o t h e r p r o t e i n s t h a t have s l o w t u r n o v e r a n d a r e

exposed t o h i g h c o n c e n t r a t i o n o f ~ - g l u c o ~ e . ~ ~ ~ Human p l a s m a c o n t a i n s a f a c t o r , factor,

terminal autorosette i n h i b i t i o n

w h i c h can i n h i b i t t h e a b i l i t y o f m u r i n e l y m p h o c y t e s t o b i n d

a u t o l o g o u s e r y t h r o c y tes.810

The p u r i f i e d f a c t o r h a s been i d e n t i f i e d

w t . 8.0 x l o 4 ) , b e i n g a l a r g e r - m o l e c u l a r f r o m w h i c h h i s t i d i n e - r i c h g l y c o p r o t e i n and,

a s a g l y c o p r o t e i n (mol. weight

precursor

possibly,

p l a s m i n o g e n - b i n d i n g p r o t e i n a r e d e r i v e d by p r o t e o l y s i s

during purification. H i s t i d i n e - r i c h g l y c o p r o t e i n f r o m r a b b i t serum b i n d s Cu2+, Zn2+, Hg2+, Cd2+, N i 2 + , consistent

a n d Co2+, w i t h h i g h a f f i n i t y ,

with

t h i s protein having a

vitro, which i s

role i n transport

or

h o m e o s t a s i s o f m e t a l s i n serum.811 Human B 2 - m i c r o g l o b u l i n glycoprotein.812 complex

(mol.

forms

After injection wt,

x

5.5

lo4

-

in

vivo

the

lo4),

x

6.7

with

complexes

serum

rat

with

but

serum

contains a

i;

vitro

i n c u b a t i o n t h e B 2 - m i c r o g l o b u l i n f o r m s a d d i t i o n a l c o m p l e x e s (rnol. 2.0

wt.

lo5). A

com pa r i so n

of

serum

a n a l y s i s by e n z y m e - l i n k e d

pregnancy- s p e c i f i c

B, - g l y c o p r o t e i n

i m m u n o s o r b e n t assay a n d r a d i o i m m u n o a s s a y

has g i v e n good c o r r e l a t i o n between t h e t w o methods.813 A g l y c o p r o t e i n (mol. w t . 5.0 x l o 4 > a s s o c i a t e d w i t h m a l i g n a n c y h a s been i s o l a t e d f r o m human s e r a from c a n c e r p a t i e n t s . 8 1 4 Reaction

of t h i s glycoprotein w i t h various antisera

directed against

normal

serum g l y c o p r o t e i n s h a s e s t a b l i s h e d t h a t i t i s n o t one o f t h e m a j o r ac u t e-phase r e a c t a n t g l y co p r o t e i n s a s s o c i a t e d w it h m a 1 ig n a n cy a n d can be d i s t i n g u i s h e d f r o m c a r c i n o e m b r y o n i c a n t i g e n and a - f e t o p r o t e i n by i t s m o l e c u l a r w e i g h t and c h r o m a t o g r a p h i c b e h a v i o u r . The i n f l u e n c e o f t e m p e r a t u r e ,

pH,

and v a r i o u s enzymes o n t h e

i n t e rme d i a t e s t r u c t u r e s f o r m e d i n t h e t r a n s f o r m a t i o n o f t o f i b r i n h a s been r e p o r t e d . 8 1 5 rod-like

intermediates are

a g g r e g a t i o n p r o c e s s when Babroxobin

(an enzyme

Light-scattering formed a t

fibrinogen

preparation

the

from

the

venom

of

the

beginning

i s treated with

for

the

B o t h r o p s a t r o x m a j o e n s i s l , and C o n t o r t r i x

f i b r i n o gen

s t u d i e s show t h a t

venom

of

of

the

thrombin, the

snake

(an enzyme p r e p a r a t i o n

copperhead snake Ancistrodon c o n t o r t r i x


.

261

contortrix) E x t e n s i v e i n a c t i v a t i o n o f t h e i r o n - b i n d i n g c a p a c i t y o f human t r a n s f e r r i n o c c u r s when a p p r o x i m a t e l y f o u r & - t y r o s i n e r e s i d u e s a r e o x i d i z e d w i t h sodium periodate.816 The c o r r e l a t i o n o f d e s t r u c t i o n of t h i s amino a c i d w i t h t h e l o s s o f i r o n - b i n d i n g a c t i v i t y s u g g e s t s t h a t t h e & - t y r o s i n e r e s i d u e s are t h o s e t h a t f u n c t i o n a s l i g a n d s t o metal i o n s bound t o the p r o t e i n . Human s e r u m t h y r o x i n e - b i n d i n g g l o b u l i n h a s b e e n a f f i n i t y l a b e l l e d w i t h N - b r om o a c e t y 1-I- t h y r o x i n e . &-Met h i o n i n e was i d e n t i f i e d as t h e major residue labelled. M i c r o h e t e r o g e n e i t y of a - f e t o p r o t e i n i n human s e r u m has been A practical and d e m o n s t r a t e d by l e c t i n a f f i n o e l e c t r o p h o r e s i s . 8 1 8 e c o n o m i c enzyme immunoassay f o r human a - f e t o p r o t e i n h a s been developed and its performance evaluated.819 Some s t u d i e s o n b i n d i n g a n d i n t e r n a l i z a t i o n o f a s i a l o g l y c o p r o t e i n s by i s o l a t e d r a t h e pa t o cy t e s h a v e b e e n r e p o r t e d .820 The c o nf orm a t i o n s o f t h e N-gl y c o s y l a t e d o l i g o sa c c h a r i d e c h a i n s o f a glycopeptide derived from f e t u i n h a v e b e e n e x a m i n e d by hydrogen-exchange techniques.821 Eight acetamido hydrogens are p r e s e n t , o ri gina t i n g from f i v e 2-acetamido-2-deoxy-eglucosyl r e s i d u e s a n d from three N - a c e t y l n e u r a m i n i c a c i d r e s i d u e s , t o g e t h e r w i t h one hydrogen from t h e oligosaccharide-peptide linkage. Seven of t h e hydrogens exchange a t a rate i n d i c a t i n g free c o n t a c t w i t h s o l v e n t , b u t t h e r e m a i n i n g t w o hydrogens exchange a t a s i g n i f i c a n t l y s l o w e r rate, i m p l y i n g t h e i r i n v o l v e m e n t i n i n t r a m o l e c u l a r hydrogen bonds. The t o t a l carbohydrate content and t h e monosaccharide c o m p o s i t i o n of p l a s m a a p o l i p o p r o t e i n s i s o l a t e d f r o m n o r m a l a n d 2am i no -2 - d e ox y -Q - ga 1a c t o se - t rea t e d r a t s h a ve be e n r e c o r de d. 8 2 2 Radioimmunoassay t e c h n i q u e s have been used t o e v a l u a t e t h e contribution of the carbohydrate moiety t o the immunological r e a c t i v i t y o f human serum l o w - d e n s i t y l i p o p r o t e i n . 8 2 3 Neuraminic a c i d a n d p-mannose, t h e t e r m i n a l r e s i d u e s o f low-density l i p o p r o t e i n g l y c o p e p t i d e s I a n d 11, a r e i m p l i c a t e d i n t h e a n t i g e n i c s i t e ( s ) . The i n t e r a c t i o n s o f f i b r i n o g e n and a s i a l o f i b r i n o g e n w i t h concanavalin A and the blood-clotting properties of the complexes have been described.824 Two o f t h e f o u r p o s s i b l e b i n d i n g s i t e s o n t h e fibrinogen molecule are not a c c e s s i b l e t o concanavalin A tetramers. Asialofibrinogen and its concanavalin A complexes c o a g u l a t e twice a s f a s t a s t h o s e o f f i b r i n o g e n . The t r a n s l a t e d p r o d u c t s o f r a t l i v e r m e s s e n g e r RNA, t o t a l p o l y s o m e s , a n d r o u g h


268

microsomes i n c l u d e g l y c o s y l a t e d s u b u n i t s o f fibrinogen.825 Both t h e BB- a n d t h e y - c h a i n s a r e g l y c o s y l a t e d I-asparagine moieties and t h e Aa-chains are n o t g l y c o s y l a t e d . The y - c h a i n i s g l y c o s y l a t e d a s a n e a r l y c o t r a n s l a t i o n a l e v e n t , w h i l e t h e BB-chain i s g l y c o s y l a t e d a t o r s l i g h t l y a f t e r t h e t e r m i n a t i o n of p o l y p e p t i d e s y n t h e s i s o f t h a t chain. The p r e s e n c e o f o l i g o s a c c h a r i d e c h a i n s o n I - a s p a r a g i n e - 2 8 8 o f human p l a s m i n o g e n d e c r e a s e d the b i n d i n g o f f r a g m e n t s c o n t a i n i n g t h e h i g h - a f f i n i t y L - l y s i n e - b i n d i n g s i t e ( r e s i d u e s 79-337 o r 7 9 - 3 5 3 ) t o both a2-antiplasmin and fibrin.826 A technique f o r the i d e n t i f i c a t i o n o f glycoprotein antigens i n immune c o m p l e x e s i s o l a t e d from sera of p a t i e n t s w i t h B u r k i t t ' s lymphoma and n a s o p h a r y n g e a l c a r c i n o m a h a s been reported.827 Two c l o s e l y r e l a t e d g l y c o p r o t e i n s were i d e n t i f i e d i n 80% o f t h e s e r a e x a m i n e d f r o m p a t i e n t s w i t h t h o s e t w o d i s e a s e s , b u t were n o t p r e s e n t i n p a t i e n t s w i t h a v a r i e t y of u n r e l a t e d tumours o r i n sera of heal t h y i n d i v i d u a l s . The c o m p a r a t i v e a n a l y s i s o f t h e l e c t i n - b i n d i n g p r o p e r t i e s o f p l a s m a g l y c o p r o t e i n s f r o m c o n t r o l a n d c y s t i c - f i b r o s i s p a t i e n t s has been reported.828 The r e s u l t s o f a comparison o f plasma g l y c o p r o t e i n s f r o m n o rm a 1 c o n t r o l s , c y s t i c - f i b r o s i s p a t i e n t s, a n d c y s t i c - f i b r o s i s h e t e r o z y g o t e s do n o t s u p p o r t t h e h y p o t h e s i s o f a g e n e r a l i z e d d e f e c t i n g l y c o p r o t e i n metabolism a s the b a s i c defect i n c y s t i c fibrosis.829 However, t h e p o s s i b i l i t y o f a m o r e l i m i t e d d e f e c t i n t h e metabolism of c e r t a i n o t h e r t y p e s o f glycoproteins, possibly those involved i n t h e processes o f exocrine excretion, is considered. T r a n s c o r t i n , t h e m a j o r human p l a s m a g l u c o c o r t i c o i d , h a s been i s o l a t e d a n d p a r t i a l l y c h a r a ~ t e r i z e d . ~ ~ 'S o m e o f i t s phy si c o chem i c a 1 pa ram e t e r s, pa r t i c u l a r l y i t s m i c r o h e t e r o g e n e i t y a n d i t s propensity f o r aggregation, are described. Isopiestic data indicate that fish antifreeze glycoproteins b i n d s i g n i f i c a n t l y m o r e water t h a n h a e m o g l o b i n o r c y t o c h r o m e c , o b s e r v a t i o n s w h i c h a r e c o n s i s t e n t w i t h n.m.r. spectroscopic s t u d i e s 831 An i m p r o v e d p r o c e d u r e f o r t h e d e t e r m i n a t i o n o f a n t i f r e e z e g l y c o p r o t e i n a c t i v i t y u s i n g a f r e e z i n g - p o i n t o s m o m e t e r has b e e n described.832 The s e n s i t i v i t y o f t h e m e t h o d h a s a l l o w e d t h e identification of a hitherto overlooked antifreeze a c t i v i t y i n the A t l a n t i c cod (Gadus morhua).

.

269

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides I m m urn g l o b u l i n s

13 A

r a p i d method f o r simultaneously

complexes o f numerous sera

for

the

a n t i g e n h a s been developed.833

s c r e e n i n g t h e immune

p r e s e n c e o f an

unsuspected

An a n t i b o d y - e n z y m e r e a g e n t c a n

i d e n t i f y f r e e x - c a s e i n when no a n t i b o d y i s p r e s e n t a n d a l s o x - c a s e i n i f i t i s bound i n a n immune complex a b s o r b e d o n t o R a j i c e l l s .

level of sensitivity

allowed the demonstration o f

The

the development

and subsequent d i s a p p e a r a n c e o f x - c a s e i n immune complexes i n t h e sera o f e i t h e r immunoglobulin A-deficient o r m i l k - a l l e r g i c p a t i e n t s who h a v e i n g e s t e d a s m a l l amount o f m i l k . I s o e l e c t r i c f o c u s i n g o f a n t i b o d i e s i n agarose g e l s has been r e p o r t e d u s i n g i n e x p e n s i ve l a b o r a t o r y - s y n t h e s i z e d am p h o l y t e s . 8 3 4 The r e s o l v i n g p o w e r o f

these and commercially a v a i l a b l e ampholytes

a r e compared, a n d t h e a d v a n t a g e s o f s u b s t i t u t i n g a g a r o s e g e l s b o n d e d t o p l a s t i c f i l m s f o r polyacrylamide g e l s a r e discussed.

Soluble

immune c o m p l e x e s h a v e been s t u d i e d u s i n g a p r o c e d u r e o f b i n d i n g t o i m m o b i l i z e d p r o t e i n A f o l l o w e d by i s o e l e c t r i c f o c u s i n g t o d e s o r b a n d separate

the

immune

complexes.835

By

this

approach

IgG-type

i m m u n o g l o b u l i n s a n d p u t a t i v e a n t i g e n s can be d e t e c t e d i n b i o l o g i c a l

f l u i d f r o m human specimens. Differences i n binding o f r a t immunoglobulin G sub-classes (IgG1,

IgG2,

IgGZb,

reported.836

a n d I g G 2 c ) t o s t a p h y l o c o c c a l p r o t e i n A have been

A p r o c e d u r e f o r t h e i s o l a t i o n o f p u r e I g G l and I g G 2 c

f r o m n o r m a l r a t s e r u m was d e v e l o p e d .

Immunoglobulins associated

w i t h human p l a t e l e t s b i n d t o i m m o b i l i z e d p r o t e i n A, o f the platelets.837 thrombocytopenic

with r e t e n t i o n

T h i s t e c h n i q u e may be o f v a l u e i n t h e s t u d y o f patients

where

greatly

increased

amounts

o f

a s s o c i a t e d I g G a r e found. The

covalent

attachment

of

and

immunoglobulin G

F(aby)2

f r a g m e n t s t o l i p o s o m e s i s a c h i e v e d by t h e p r i o r p e r i o d a t e o x i d a t i o n o f g l y c o s p h i n g o l i p i d s i n liposomes t o produce aldehyde groups o n t h e vesicle surface,

f o l l o w e d by

protein coupling

a m i n a t i ~ n . ~ U n~d ~ e r t h e c o n d i t i o n s used, external glycolipid, Encapsulated g l y c e r o l v e s i c l e s and r e t a ine d

.

but not internal glycolipid, l-phosphate

encapsulated

The F ( a b ’ I 2

by

fragment

i s

not

goat

i s oxidized.

oxidized

carboxyfluorescein i s o f

reductive

a large proportion o f the within

the

substantially

i m m u n o g l o b u l i n G h a s been

co v a l e n t l y c o u p l e d t o pho s p h a t i d y l e t h a n o l a m i n e w i t h s u b s e q u e n t

Carbohydrate Chemistry a s s o c i a t i o n of t h i s complex w i t h liposomes.839 B i n d i n g assays w i t h antigen-coated Staphylococcus aureus c e l l s a r e used t o e v a l u a t e t h e i m m u n o l o g i c a l l y s p e c i f i c r e c o g n i t i o n and t h e membrane s t a b i l i t y o f t h e liposomes.

Comparative k i n e t i c s t u d i e s o f

ligand dissociation

and D20-induced f l u o r e s c e n t enhancement have been p e r f o r m e d w i t h b o t h h e t e r o g e n e o u s and homogeneous a n t i f l u o r e s c e i n y l i m m u n o g l o b u l i n G antibodies.840

The a n t i - f l u o r e s c e i n

antibody active s i t e contains

b o t h s o l v e n t - a c c e s s i b l e and r e l a t i v e l y - i n a c c e s s i b l e components,

with

t h e b i n d i n g o f l i g a n d i n v o l v i n g b o t h exchangeable hydrogen atoms and other

as

yet

unresolved

interactions.

The

mechanism

of

020

fluorescence enhancement i s discussed i n t e r m s o f i t s c o m p l e x i t y i n v o l v i n g h e t e r o g e n e o u s r a t e mechanisms. Several b i o l o g i c a l effector F c r e g i o n of variant

f u n c t i o n s m e d i a t e d by s i t e s on t h e

human i m m u n o g l o b u l i n G 1 h a v e b e e n s t u d i e d i n t w o

i m m u n o g l o b u l i n G1K

monoclonal

proteins

which

contain

d e l e t i o n s corresponding t o t h e e n t i r e hinge r e g i o n o f t h e heavy chains.841

The f u n c t i o n a l l a c k o f p o t e n c y o f t h e s e i m m u n o g l o b u l i n s

i s r e l a t e d t o t h e c l o s e a s s o c i a t i o n between Fab and Fc r e g i o n s i n t h e s e m o l e c u l e s and t h e l i m i t e d degree o f s e g m e n t a l f l e x i b i l i t y p e r m i t t e d i n t h e absence o f t h e h i n g e r e g i o n .

A major r o l e f o r t h e

C-y2 d o m a i n i n m e d i a t i n g e f f e c t o r f u n c t i o n s i n n o r m a l i m m u n o g l o b u l i n G 1 i s proposed. A model system,

b a s e d on t h e d e s i a l y l a t i o n o f i m m u n o g l o b u l i n G

and g e n e r a t i n g one o f t h e s e r o l o g i c a l changes n o r m a l l y a s s o c i a t e d w i t h t h e p a t h o l o g i c a l c o n d i t i o n o f r h e u m a t o i d a r t h r i t i s , has been developed.842

Rabbit asialo-immunoglobulin

i s immunogenic i n autologous hosts.

G

t e n d s t o a g g r e g a t e and

The n e u r a m i n i c a c i d c o n t e n t o f

r h e u m a t o i d f a c t o r i s o l a t e d f r o m t h e serum o f a r h e u m a t o i d p a t i e n t and i d e n t i f i e d as i m m u n o g l o b u l i n G and i m m u n o g l o b u l i n M i s a l s o lower than t h a t o f the corresponding normal immunoglobulins. I m m o b i l i z e d p e p s i n has been used f o r t h e l i m i t e d c l e a v a g e o f human i m m u n o g l o b u l i n G.843

The F ( a b ’ ) 2 - r i c h

f r a c t i o n was

isolated

f o r i n t r a v e n o u s use. Proximity

relationships

within

the

Fc

segment

of

rabbit

i m m u n o g l o b u l i n G h a v e been a n a l y s e d b y r e s o n a n c e - e n e r g y t r a n s f e r . 8 4 4 The

principle

forces

maintaining

the

integrity

of

the

native

f u n c t i o n a l Fc fragment are t h e strong noncovalent i n t e r a c t i o n s of t h e CH3 d o m a i n s a n d t h e s i n g l e i n t e r - h e a v y - c h a i n

d i s u l p h i d e bond.

T h e c a r b o h y d r a t e s t r u c t u r e s ( 3 9 , 40) a n d t h e c o m p l e t e a m i n o a c i d s e q u e n c e o f a human X - t y p e Sm,

have been determined.845

immunoglobulin l i g h t chain,

One g - g l y c o s i d i c a l l y

protein

l i n k e d c h a i n (40)


o u

LL I JI I

Q : I 011

z

a

4

a I

1311

m I

h

U

+

4

u

AJ

m m - 4

r

I 1311

m

r

I LYI

m

a

n

n

I

UJ 4

u I

2

r

I 1311 I

I

0

ct

z al

rl

U I 1311

I

@l

4

4

u

I

o

Q:

z

3

4

U I

+

4

u I

al

4

(II

U I

011

m

m

CI

n

I

U 4

4

I

UJ

t

@l

u

u

4

q:

A m

U I

I

0

m

9

I

z

I

c4

a n v)

4

@l

u I

u

27 1

272


i s attached t o I-Ser-21.

T h i s a p p e a r s t o be t h e f i r s t r e p o r t o f a

tetrasaccharide containing two neuraminic a c i d residues i n the l i g h t c h a i n o f an i m m u n o g l o b u l i n . O l i g o s a c c h a r i d e s o f s i m i l a r s t r u c t u r e o c c u r i n human X - t y p e m o n o c l o n a l i m m u n o g l o b u l i n l i g h t c h a i n s .846

~-N~UQ~AC-(~+~)-B-Q-G~~Q-(~+~)-~-G~~QNAC-L-S~~ 6

I 2 o - Ne u ~ 5 cA

(40) A human m o n o c l o n a l i m m u n o g l o b u l i n G 1 p o s s e s s e s t w o s p e c i e s o f

l i g h t c h a i n ( m o l . w t s . 2.3 x l o 5 a n d 2.8 x lo5, r e ~ p e c t i v e l y ) . ~ ~ ’ The d i f f e r e n c e i n mass is due t o t h e p r e s e n c e o f a c a r b o h y d r a t e m o i e t y on t h e 2.8 K-chains.

x lo4 species, attached w i t h i n the J region o f the

Not a l l of

t h e K-chains

differences i n the primary

are glycosylated,

e v e n t h o u g h no

s t r u c t u r e between the

two

L-chains

s p e c i e s c o u l d be f o u n d . mouse myeloma c e l l l i n e p r o d u c e s an i m m u n o g l o b u l i n G2b w i t h

A

two carbohydrate attachment sites.848 also

occur

i n

two

variant

cell

Alterations i n glycosylation

lines.

The

complete

s t r u c t u r e o f the constant r e g i o n o f L-chains homogeneous

Balilea

rabbit

antibody

primary

derived from

specific

for

type

a I1

pneumococcal p o l y s a c c h a r i d e h a s been e ~ t a b l i s h e d , ~ a ~ s’ h a s t h e a m i n o a c i d s e q u e n c e o f an e u g l o b u l i n - l i k e

X Bence-Jones p r o t e i n NIG-

58. 850 Antibodies

have

been r a i s e d t o

glycolipids,and polysaccharides.

numerous

glycoproteins,

Those a n t i b o d i e s k n o w n t o i n t e r a c t

w i t h carbohydrate d e t e r m i n a n t s on t h e a n t i g e n molecule a r e l i s t e d i n T a b l e 11. Further studies of

the

inhibition of

the monoclonal a n t i -

Ma(group 1) a n t i b o d y i n q u a n t i t a t i v e p r e c i p i t i n a s s a y s h a v e been reported.852 o f

a

I t i s confirmed t h a t the antibody binds t o a portion

B-~-galactopyranosyl-(1+4)-~-B-~-2-acetamido-2-deoxy-

glucopyranosyl-(1+6)-~-~-~-galactopyranosylunit oligosaccharide

structure

i n

a

specific

conformational requirements indicate

that

of

conformation. the

an The

determinant

is

accepted i n t o the antibody-com bining s i t e along a hydrophobic portion of the structure

which includes t h e regions about

C6-Cl’-

;-Galactan

(Continued o v e r l e a f )

Glc~-(1+6)-a-~-Glc~-(1+3)-~-Glc B-(l+6)-!-galactopyranan

1+6)-g-Glc,

a-p-Glce-(

1+3)-a-!-Glce-(

m e t h y l a-g-ManQ

O e x t r a n 81355

Polysaccharides D e x t r a n 8512

!-Gal,

a-g-

p-GlcNAc,

g l ycopro te i n R e t r o v i r u s envelope g l y c o p r o t e i n

Monoclonal

Polyclonal

Monoclonal

Polyclonal

Po l y c l o n a l

I n t a c t oligosaccharide chain

Bovine leukaemia envelope v i r a l

856

855

854

21

12,

13

853

852

Mono c l o n a 1

Ma ( g r o u p 1)

Polyclonal

851

Ref.

Monoclonal

6-g - G a l e - ( 1+4)- 8-9-G lceNA c - ( 1+6 ) - 8-g - G a l e

;-Mane

a-g -Mane

Neue5Ac, a,B-Q-Galg, -

Antibody type

Glucoamy l a s e

a-g-Mane,

on a n t i g e n

g l y c o l i p i d or polysaccharide antigens

Determinant(s)

Antibodies i n t e r a c t i n g with glycoprotein,

Glycoproteins H i s t o c o m p a t i b i l i t y a n t i g e n (H-2K)

Antigen

Table I1

E

E.

3

w

N 4

8

s5

c,

5

h

k 2-

"2 g

0

s

2

Q.

-8

B=.

s

%-

Q

"

a

a-L -F u c~

1

I

3

1 a-L-F u c ~

I

a-~-Fuc~-(1+2)-B-~-Gale-(1+3)-B-~-Glc~NAc-~-~-Gal~-(l~4)-~-Glc 4

SSEA-1

B-g-Gale-(1+4)-g-Glc~NAc, a - L-- F u c e - ( l + Z ) B-q-Galp-(1+4)-q-GlcpNAc B-g-Galp-(1+4)-B-g-Glc~NAc

a - L-- F u c p - ( 1 + 4 ) - g - G l c e " c

Glycosphingolipids

See

Lewisb blood group

below

D e t e r r n i n a n t ( s ) on a n t i g e n

Lewis2 blood group

Glycolipids

Antigen

T a b l e I1 (Continued)

Monoclonal

Monoclonal

Polyclonal

Monoclonal

Antibody t y p e

860

859

858

857

Ref.

P

4

N


275

C3’-05”-C6” a s r e p r e s e n t e d i n S c h e m e 2. I t is a s s u m e d t h a t 03’ is i n t r a m o l e c u l a r l y h y d r o g e n b o n d e d t o 05’’ a n d t h a t t h e o t h e r h y d r o x y l groups o f t h e determinant remain engaged in bonding with solvent water. I

I I

combining site .hydrophobic

edge

D i s p l a y s h o w i n g t h a t t h e c o n f o r m a t i o n o f t h e I Ma a n t i genic determinant possesses a region along C6Cl’-C3’-05’’-C6’’ w h i c h c a n b e e x p e c t e d t o be compatible with a hydrophobic region f o r t h e combining site Scheme 2 R a b b i t a n t i - d e x t r a n €31355 h a s b e e n s e p a r a t e d i n t o t w o Gfractions.855 Antibodies of a non-binding f r a c t i o n t o Sephadex 75 are d i r e c t e d a g a i n s t ~-a-Q-glucopyranosyl-(l+6)-~-a-~glucopyranosyl-(1+3)-~-glucosyl r e s i d u e s . The a f f i n i t i e s of t w o m o n o c l o n a l anti-!-galactan immunoglobulin Fab’ fragments have been measured with a linear and a highly b r a n c h e d Q - g a l a ~ t a n . ~A~f f~i n i t y d a t a s h o w t h a t t h e i m m u n o g l o b u l i n s c a n bind i n t e r c a t e n a r i l y t o a (1+6)-B-Q-galactopyranan b u t n o t t o a ( 1+3)-B-~-galactopyranan. F o u r m o n o c l o n a l a n t i b o d i e s p r o d u c e d by hybridomas o b t a i n e d from a mouse immunized with a human a d e n o c a r c i n o m a c e l l l i n e a r e d i r e c t e d a g a i n s t t h e Leb a n t i g e n o f t h e T h e i r s p e c i f i c i t i e s w e re h u m a n Lewis b l o o d - g r o u p s y s t e m . 8 5 7 e s t a b l i s h e d by b i n d i n g s t u d i e s u s i n g p u r i f i e d Leb - a c t i v e g l y c o l i p i d s a n d by h a p t e n i n h i b i t i o n s t u d i e s w i t h o l i g o s a c c h a r i d e s o b t a i n e d f r o m h u m a n milk. T h e a n t i g e n s p e c i f i c i t i e s o f d i f f e r e n t a n t i - L e w i s z sera h a v e b e e n e x a m i n e d by i m m u n o a d s o r p t i o n s t u d i e s u s i n g a d s o r b e n t s w i t h well d e f i n e d c a r b o h y d r a t e u n i t s c o v a l e n t l y b o u n d t o a n i n o r g a n i c

276


matrix.858 I n c o n t r a s t t o t h o s e of normal a n t i - L e a and a n t i - L e i s e r a , t h e a n t i b o d y - b i n d i n g s i t e o f L e r a n t i b o d i e s was f o u n d t o b e c o n s i d e r a b l y smaller , c o m p r i s i n g t h e s t r u c t u r e a - & - F u c e - ( 1 + 4 ) - Q G lceNAc Two monoclonal a n t i c a r b o h y d r a t e a n t i b o d i e s d i r e c t e d t o w a r d t h e c a r b o h y d r a t e m o i e t y o f g l y c o s p h i n g o l i p i d s c o n t a i n i n g a lacto-!O n e is s p e c i f i c f o r g l y c o s y l T y p e I1 c h a i n h a v e b e e n p r e p a r e d . 8 5 9 t h e T y p e I1 c h a i n H d e t e r m i n a n t ( 4 1 ) a n d t h e o t h e r is d i r e c t e d t o t h e n o n - r e d u c i n g t e r m i n a l 2 - a c e t a m i d o - 2 - d e o x y -4-0-13- Q - g a l a c t o s y l - Q glucosyl disaccharide.

.

a-~-Fucp-(1+2)-B-Q-Gal~-(l+4)-~-Q-GlcpNAc-l+R (41) R = r e s t o f o l i g o s a c c h a r i d e chain A monoclonal antibody r e a c t i n g with e a r l y mouse embryos and m u r i n e e m b r y o n a l c a r c i n o m a cells d e f i n e s t h e s t a g e - s p e c i f i c e m b r y o n i c a n t i g e n ( S S E A - 1 ) a n d is s p e c i f i c f o r t y p e 2 b l o o d - g r o u p antigens.860 T h e c o m b i n i n g s i t e o f t h e a n t i b o d y is c o m p l e m e n t a r y t o t h e s e q u e n c e (42). The a n t i g e n has b e e n i d e n t i f i e d as a complex g l y c o l i p i d .861 ,862 A cold agglutinin resembling anti-I antibodies has been i s o l a t e d from a p a t i e n t s u f f e r i n g from i m m u n ~ b l a s t o m a . ~ ~ ~ H i g h - t i t r e a n t i s e r a s p e c i f i c f o r Lewisa, b, a n d d d e t e r m i n a n t s h a v e b e e n o b t a i n e d by t h e i m m u n i z a t i o n o f r a b b i t s w i t h a r t i f i c i a l a n t i g e n s p r e p a r e d by c o u p l i n g c h e m i c a l l y s y n t h e s i z e d h a p t e n s t o These r e a g e n t s h a v e b e e n u s e d i n t h e bovine serum albumin.864 immunohistochemical analysis of t h e s e antigens i n t h e mucosa of t h e human s t o m a c h a n d d u o d e n u m o f H g r o u p i n d i v i d u a l s .

B-Q-Gale-(1+4)-B-g-Glc~NAc-(l+6)R 3

I 1

-F u c p (42) R = remaining oligosaccharide chain

a-

Monoclonal antibodies, which bind t o an a n t i g e n p r e s e n t i n highly purified carcinoembryonic antigen (CEA) from various tumours, T h e y are c o n s i s t e n t with t h e c o n c e p t t h a t h a v e been isolated.865

277

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides different

immunological

distinguished

by

forms

appropriate

i m munlogical heterogeneity of

many

conventional

of

CEA

antisera. CEA

anti-CEA

exist

which

How e v e r ,

and t h e

sera,

the

in

can

be

of

the

view

polyspecific nature o f possibility

that

these

monoclonal sera are n o t recognizing a unique antigen n o t r e l a t e d t o CEA c a n n o t be excluded. Evaluation

of

a

baboon

antiserum

to

CEA

has

given

similar

r e s u l t s t o a c o m m e r c i a l l y a v a i l a b l e g o a t anti-CEA.866 S p e c i f i c antibody t o a - f e t o p r o t e i n has been prepared t o bind s p e c i f i c a l l y t o a-fetoprotein-producing Human antibodies t o f a c t o r

VIII h a v e b e e n i s o l a t e d f r o m n i n e

Subsequent i m munological c h a r a c t e r i z a t i o n s

h a e m o p h i l i c plasmas.868

revealing restricted heterogeneity i n both light-chain heavy-chain

sub-class

antigen

been

assays,

has

and f o u n d

tumour c e l l s .867

have been described.

measured

with

two

Factor

t y p e and

VIII c l o t t i n g

i m munoradiometric

different

one u t i l i z i n g t w o acquired antibodies and one a haemophilic

a n t i b o d y .768 Monoclonal antibodies i s o l a t e d and t h e i r

to

von

Willebrand’s

with

reactivity

factor

have

been

porcine and human antigens

reported.869 Monoclonal

antibodies

against

human a c i d

a-glucosidase

have

b e e n ~ r e p a r e d . ~ ” T h e a n t i b o d i e s w e r e u s e d t o show t h a t t w o m a j o r c o m p o n e n t s of

the

purified

enzyme

c o n t a i n a t l e a s t one a n t i g e n i c

component i n common. An e n z y m e i m munoassay f o r a u t o a n t i b o d i e s t o t h y r o g l o b u l i n i n h u m a n s e r u m h a s b e e n developed.871 comparison

with

an

indirect

E v a l u a t i o n o f t h e technique by

haemagglutination test

for

anti-

t h y r o g l o b u l i n a n t i b o d i e s gave a c o r r e l a t i o n c o e f f i c i e n t o f 0.85. Sera

from

patients

with

Graves’

disease

contain

antibodies

w h i c h i n h i b i t t h e b i n d i n g o f t h y r o t r o p h i n t o t h y r o i d membranes.872 Immunoglobulin

i s o l a t e d from

G,

t h e sera,

inhibits the binding o f

t h e hormone by b i n d i n g d i r e c t l y t o t h e hormone r e c e p t o r . Antibodies used i n an enzyme immunoassay f o r human c h o r i o n i c gonadotrophin ( h C G ) after

affinity

have been i s o l a t e d i n a h i g h l y

chromatography

on

colums

of

purified form i m mobilized

t r i c o s a p e p t i d e o f t h e hC G Rabbit

transferrin

methionine sulphone

has

been

modified

hydrazide either into

by

introduction

i t s neuraminic

of

acid

r e s i d u e s or i n t o b o t h n e u r a m i n i c a c i d and Q - g a l a c t o s y l residues.873 Rabbits i m munized with t h e modified t r a n s f e r r i n produced antibodies which r e a c t e d w i t h t h e n a t i v e antigen.

278

Carbohydrate Chemistry Mouse i m m u n o g l o b u l i n

G 3 antibodies t o t h e phosphocholine

d e t e r m i n a n t of p n e u m o c o c c a l C c a r b o h y d r a t e a n d t o t y p e 3 pneu m o c o c c a l c a r b o h y d r a t e a r e h i g h l y p r o t e c t i v e against experimental type 3 pneumococcal infection. 874 that

antibodies

of

the

against bacterial infections. ag a inst

A n t ib o d i e s

glucosyltransferase

from

ability to inhibit the The

cross-reactivity

This i s one o f t h e f i r s t r e p o r t s

immunoglobulin D=

G3

protect

- 1 y s y 1: D= -

embryos have been t e s t e d f o r t h e i r

e n r y me r e a c t i o n w i t h the

can

- g a l a ct 0 sy l - hy d r o x y -

chick

of

sub-class

v a r i o u s substrates.875

glucosyltransferase

between

different

species i s low. Monoclonal r a t immunoglobulin G2b

antibody,

a g a i n s t t h e Thy 1.2 a n t i g e n , h a s b e e n c o v a l e n t l y Addition

of

lactose

saturates

the

r i c i n and i n h i b i t s t h e t o x i n f r o m

P_-galactosyl-containing

which i s d i r e c t e d bound t o

Q-galactosyl- binding

sites

b i n d i n g and k i l l i n g c e l l s & a

receptors.

Ricin-monoclonal

h y b r i d s o f t h i s t y p e combine a h i g h degree o f c e l l - t y p e

on the

antibody selectivity

and may have p h a r m a c o l o g i c a l use as a n t i t u m o u r r e a g e n t s . H i gh l y

sp ec i fi c

antibodies

dire c ted

against

c e l l - s u r f ace

i m m u n o g l o b u l i n s on n o r m a l o r n e o p l a s t i c murine B l y m p h o c y t e s have been c o v a l e n t l y molecules

coupled t o

maintain

t o x i c properties.

both

the

their

A

chain

of

r i ~ i n . T~h e~ h~y b r i d

antigen-binding

capacity

and

their

Minute amounts o f t h e conjugates are e f f e c t i v e i n

k i l l i n g target cells i n vitro. The

8aB5-9

antibody

Lab,

deficient

in

cloned hybridoma c e l l l i n e produces a

d i r e c t e d against a major platelet-surface platelets

monoclonal antibody,

of

patients

with

monoclonal

glycoprotein

t h r o m b a ~ t h e n i a . ~ T~h i~s

which i s d i r e c t e d against a platelet-mem brane

complex consisting o f glycoproteins I I b and I I I a ,

has been used t o

enumerate t h e number o f t h e s e complexes o n n o r m a l p l a t e l e t s and t o indicate the

v i r t u a l absence

of the

complex on t h e s u r f a c e

of

thrombasthenic p l a t e l e t s . Four independent

monoclonal antibodies that

recognize

an

m urine cell-surface glycoprotein have been used t o c h a r a c t e r i z e t h e c o m p l e x i t y o f p r o t e i n polymorphism.879

alloantigenic s t r u c t u r e on a

E a c h o f t h e a n t i b o d i e s a p p e a r s t o r e a c t w i t h t h e same o r a c l o s e l y r e l a t e d e p i t o p e on t h e g l y c o p r o t e i n . The

localization

components i n t h e

i m munochemically a n t i s e r a. 880

of

chitosan

pea-Fusarium with

and

other

fungal

cell-wall

i n t e r a c t i o n have been studied

anti-chitosan

and a n t i - f u n g a l

cell-wall


279

Monoclonal antibodies specific f o r subunit 1 o f t h e l e c t i n from

--

Dolichos biflorus combine with t h e C -ter minal portion o f t h e s u b u n i t a n d may b e i n t e r a c t i n g w i t h t h e a c t i v e s i t e o r w i t h a d e t e r m i n a n t This antibody, c o n f o r m a t i o n a l l y i n d e p e n d e n t o f t h e a c t i v e site.155

w h i c h d o e s n o t r e a c t w i t h a n o t h e r l e c t i n - l i k e p r o t e i n f r o m t h e stems a n d l e a v e s o f t h e plant,156 c a n d i s t i n g u i s h s u b u n i t 1 f r o m s u b u n i t 2 o f t h e s e e d l e c t i n . 157 The binding o f human immunoglobulin t o t y p e C retroviruses has b e e n a n a l y s e d by r a d i o i m m u n o a s s a y . 1 2 B i n d i n g is d i r e c t e d t o t h e o l i g o s a c c h a r i d e c h a i n s o f t h e v i r a l g l y c o p r o t e i n s a n d is n o n specific in character. Cytotoxic antibodies present in bovine leukaemia virus-infected a n i m a l s are mostly directed a g a i n s t t h e c a r b o h y d r a t e p a r t O P t h e v i r a l e n v e l o p e g 1 y ~ o p r o t e i n . l ~I m m u n e s e r a p r e p a r e d f r o m p u r i f i e d e n v e l o p e g l y c o p r o t e i n s are d i r e c t e d a g a i n s t d e t e r m i n a n t s o f t h e polypeptide backbone of t h e antigen. The majority o f , i f n o t all, n o r ma1 h u m a n s e r a c o n t a i n n a t u r a l l y o c c u r r i n g h e t e r o p h i l a n t i b o d i e s t h a t react with t h e c a r b o h y d r a t e moieties of r e t r o v i r u s envelope glycoproteins.21 T h e s e a n t i v i r a l a n t i b o d i e s are n o t d e t e c t a b l e i n c o r d sera b u t show a n a g e - d e p e n d e n t p e a k i n e a r l y c h i l d h o o d a n d s u b s e q u e n t l y p e r s i s t l i f e -lo n g Monoclonal antibodies d i r e c t e d against herpes simplex virus g l y c o p r o t e i n s h a v e b e e n u s e d t o p r o t e c t mice a g a i n s t a c u t e v i r u s induced neurological disease.29 Virus glycoprotein g c expresses t y p e - s p e c i f i c a n t i g e n i c d e t e r m i n a n t s , w h e r e a s g l y c o p r o t e i n gD e x p r e s s e s t y p e - c o m mon d e t e r m i n a n t s . Monoclonal antibodies t o h e r p e s simplex virus t y p e 2 have been prepared and found t o precipitate t w o d i f f e r e n t v i r a l g l y c o p r o t e i n s . 33 The major glycoprotein (Gp 350/220) o f Epstein-Barr virus h a s b e e n d e t e c t e d b o t h o n t h e p l a s m a m e m b r a n e a n d i n t h e c y t o p l a s m by i m m u n o f l u o r e s c e n c e w i t h a n t i b o d i e s .’O T w o m o n o c l o n a l a n t i b o d i e s r e a c t i n g w i t h A K R l e u k a e m i c cells r e c o g n i z e c l o s e l y r e l a t e d , if n o t i d e n t i c a l , a n t i g e n i c d e t e r m i n a n t s o n t h e Glx g l y ~ o p r o t e i n . ’ ~ Monospecific antibodies t o e a c h of t w o envelope glycoproteins The of paramyxovirus h a v e been p r e p a r e d and characterized.74 e f f e c t s of t h e s e a n t i b o d i e s o n t h e i n f e c t i o u s p r o c e s s a n d t h e o t h e r biological a c t i v i t i e s of t h e virions and of t h e i s o l a t e d glycoproteins are discussed. Amino a c i d s e q u e n c e d i f f e r e n c e s i n t h e V r e g i o n o f murine antibodies t o a(l+3)-P-glucans (dextrans) have been correlated with

.


280 the

expression o f

cross-reactive

and i n d i v i d u a l i d i o t y p e s ,

an analysis o f t w e l v e d i f f e r e n t d e x t r a n antibodies.881 r e a g e n t s can r e c o g n i z e t w o amino a c i d d i f f e r e n c e s w i t h segments

of

variable

I n anti-dextran

regions.

antibodies,

r e a c t i v e i d i o t y p e s i n v o l v e V-region determinants, idotype determinants secretory The

correlate

immunoglobulin A

hinge

region

variation.

has been i s o l a t e d f r o m

containing

four

cross-

whereas i n d i v i d u a l

D -segment

with

through Idiotype V and D

human

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milk.882

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linked

o l i g o s a c c h a r i d e s i s r e l e a s e d en b l o c a f t e r p r o t e o l y t i c d i g e s t i o n . The s t r u c t u r e s ( 4 3 - 4 6 ) to

I=-serine

of t h e s e oligosaccharides,

which are l i n k e d

or I - t h r e o n i n e residues, have been i d e n t i f i e d .

oligosaccharides are oligosaccharides

more complex

linked

to

I-serine

These

and heterogeneous t h a n t h e residues

of

the

hinge

region

f r o m myeloma s e r a i m m u n o g l o b u l i n A. A

partially

characterized

immunoglobulin glycopeptides

has

A

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of

milk four

f o l l o w i n g h y d r a t i n o l y s i s and nitrous acid

d e a m i n a t i o n o f t h e g l y c o p e p t i d e . 883 The

membrane

receptor

for

immunoglobulin

A,

isolated

from

r a b b i t l i v e r a n d mammary g l a n d , i s s t r u c t u r a l l y r e l a t e d t o s e c r e t o r y component.884 groups

of

The

membrane s e c r e t o r y

amphiphilic

molecules

component which

is composed o f t w o

are

synthesized

transmem b r a n e p r e c u r s o r s and i n s e r t e d i n t h e c e l l - s u r f a c e

as

membrane

where t h e y a c t as r e c e p t o r s f o r p o l y m e r i c i m m u n o g l o b u l i n A .

R1-(2+3)-B-g-Galp-(1+3)-g-GalNAc 6

I 1

(44)

R 1 = H,

(45)

R 1 = H , R 2 = B-Q-Gale-(1+4)-B-a-Glce"c

(46)

R1 = a-Neup5Ac,

Spectroscopic properties MOPC-315

immunoglobulin

b e t w e e n c.d.

R2

R 1 = R2 = H

(43)

A

of

R2 = @ - g - G l C e N A C R2 = H

light-chain

derivatives o f

h a v e been reported.885

A

m urine

comparison

spectra o f t h e t h r e e f r e e and hapten-bound light-chain

derivatives demonstrates the existence o f conformational transitions i n t h e s e p r o t e i n s i n d u c e d by h a p t e n b i n d i n g . B i o s y n t h e t i c and

fi

v i t r o t r a n s l a t i o n studies have demonstrated


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t h a t membrane-bound and secreted i m m u n o g l o b u l i n A m o l e c u l e s c o n t a i n d i f f e r e n t a - p o l y p e p t i d e c h a i n s t h a t a r e e n c o d e d by d i f f e r e n t a-

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V a r i a n t s h a v e b e e n i s o l a t e d f r o m t h e 3558 B a l b / c m o u s e m y e l o m a (immunoglobulin A,X: anti-a-(1+3)-dextran) with altered g l y c o s y l a t i o n of its heavy c h a i n and decreased r e a c t i v i t y w i t h d e ~ t r a n . T~ h~e ~c a r b o h y d r a t e m u t a n t i m m u n o g l o b u l i n c o n t a i n s m o r e neuraminic acid than does t h e carbohydrate of t h e wild-type immunoglobulin. However, t h e mutant immunoglobulin a p p e a r s t o have t h e same r e a c t i v i t y p a t t e r n w i t h o l i g o s a c c h a r i d e s a s d o e s t h e w i l d t y p e immunoglobulin, and t h u s t h e s i z e o f t h e antibody-combining s i t e s may b e s i m i l a r . By m e a s u r i n g t h e a f f i n i t i e s a t v a r i o u s t e m p e r a t u r e s b e t w e e n a n t i - e - g a l a c t a n i m m u n o g l o b u l i n A 5539 a n d a n t i d e x t r a n i m m u n o g l o b u l i n A W3129, a n d t h e i r r e s p e c t i v e l i g a n d s , t h e e n t h a l p i e s a n d e n t r o p i e s The i n t e r p r e t a t i o n o f t h e of binding have been determined.888 r e s u l t s i s i n a g r e e m e n t w i t h p r e v i o u s w o r k s h o w i n g t h a t 5539 h a s a g r o o v e - t y p e c o m b i n i n g r e g i o n a n d W3129 h a s a c a v i t y - t y p e r e g i o n . The i n t e r a c t i o n of s a c c h a r i d e h a p t e n s w i t h three homogeneous i m m u n o g l o b u l i n A mouse myeloma p r o t e i n s which h a v e s p e c i f i c i t y f o r s a c c h a r i d e s h a s b e e n i n v e s t i g a t e d i n a 270 MHz n . m . r . study.889 D i f f e r e n c e s p e c t r a show t h a t c o m p a r a t i v e l y few p r o t e i n r e s o n a n c e s are p e r t u r b e d on b i n d i n g h a p t e n , s u g g e s t i n g t h a t a c c o m p a n y i n g c o n f o r m a t i o n a l changes are l i m i t e d t o t h e combining s i t e . The primary s t r u c t u r e of t h e I-asparagine-563-linked carbohydrate chain of an immunoglobulin M from a p a t i e n t w i t h W a l d e n s t r o m ’ s m a c r o g l o b u l i n e m i a h a s b e e n r e i n v e s t i g a t e d u s i n g 500 MHz n . m . r . s p e c t r o s c o p y , a n d a new s t r u c t u r e (51) h a s b e e n N o e v i d e n c e was o b t a i n e d t o s u p p o r t a n e a r l i e r proposed.890 claim891 t h a t o n l y o n e a m i n o s u g a r r e s i d u e i s p r e s e n t i n a n u n u s u a l core structure. I n t h e p r e s e n t w o r k t h e n.m.r. spectrum shows a l l t h e s p e c t r a l features c h a r a c t e r i s t i c of an N”-diacetylchitobiosyl u n i t w i t h h e t e r o g e n e i t y e x i s t i n g i n both t h e c a r b o h y d r a t e and peptide moieties. G l y c o s y l a t i o n is n o t r e q u i r e d f o r membrane l o c a l i z a t i o n or s e c r e t i o n o f i m m u n o g l o b u l i n M i n a mouse B c e l l lymphoma.892 However, g l y c o s y l a t i o n d o e s c o n f e r i n c r e a s e d i n t r a c e l l u l a r s t a b i l i t y and r e s i s t a n c e t o p r o t e o l y t i c d i g e s t i o n . The m o l e c u l a r w e i g h t o f t h e h i g h g-mannose c o r e o l i g o s a c c h a r i d e o f i m m u n o g l o b u l i n M h a s been m e a s u r e d by f i e l d - d e s o r p t i o n ~ I . s . ~ ’ ~ High-affinity monoclonal immunoglobulin M antibodies d i r e c t e d


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towards e p i t o p e s on h e p a t i t i s B s u r f a c e a n t i g e n have been used i n t h e i m m u n o d i a g n o s i s o f h e p a t i t i s B.894 A P-mannose-rich g l y c o p e p t i d e (52) from a human p a t h o l o g i c a l immunoglobulin M c o n t a i n s o n l y Q-mannosyl and 2-acetamido-2-deoxy-Pg l u c o s y l r e s i d u e s ( m o l a r r a t i o 10:2).895 The r e c o v e r y o f o n e m o l e c u l e o f t h i s g l y c o p e p t i d e per m o l e c u l e of heavy c h a i n a n d t h e d e t e r m i n a t i o n of t h e amino acid s e q u e n c e l e d t o the l o c a t i o n o f t h e g l y c o p e p t i d e o n L-Asn-402 o f t h e Fc p o r t i o n o f t h e h e a v y c h a i n p o f t h e immunoglobulin. The a m i n o a c i d s e q u e n c e a n d l o c a t i o n o f t h r e e g l y c o p e p t i d e s i n t h e Fc r e g i o n o f t h e y c h a i n o f h u m a n i m m u n o g l o b u l i n I g D h a v e b e e n These g l y c o p e p t i d e s h a v e o l i g o s a c c h a r i d e c h a i n s identified.896 l i n k e d t j - g l y c o s i d i c a l l y t o I - A s n - 6 8 , L-Asn-159, a n d I - A s n - 2 1 0 . A t e n t a t i v e a m i n o a c i d s e q u e n c e o f t h e JH r e g i o n , t h e C y l domain, and the hinge r e g i o n of t h e y heavy c h a i n of t h e immunoglobulin h a s been published.897 The h i n g e r e g i o n o f t h e y c h a i n has an u n u s u a l s t r u c t u r e , i n which f o u r or f i v e o l i g o s a c c h a r i d e s c o n t a i n i n g 2-amino-2-deoxy-!-galactose residues a r e a t t a c h e d a t t h e N - t e r m i n a l h a l f o f t h e c h a i n , w h e r e a s t h e Ct e r m i n a l h a l f lacks c a r b o h y d r a t e , is d i s s i m i l a r i n sequence,and has a high charge. A h y b r i d mouse c e l l l i n e (61-8) secretes i m m u n o g l o b u l i n D which i s h e a v i l y g l y c o s y l a t e d , a s i n d i c a t e d by t h e f a c t t h a t t h e u n g l y c o s y l a t e d a c h a i n h a s a m o l e c u l a r w e i g h t o f 4.4 x l o 4 a s c o m p a r e d t o t h e v a l u e o f 6.1 x l o 4 f o r t h e m a j o r s p e c i e s o f g l y c o s y l a t e d y chain.898 A l k a l i - e x t r a c t e d h u m a n e r y t h r o c y t e g h o s t m e m b r a n e s f r o m Rho(D)p o s i t i v e d o n o r s c o m p l e x w i t h human i m m u n e a n t i - R h o ( D ) i m m u n o g l o b u l i n D.899 T h e a n t i b o d y was s h o w n t o b i n d t o b a n d 3 g l y c o p r o t e i n s o f t h e e r y t h r o c y t e membrane.

14

Erythrocyte Glycoproteins

A review dealing with t h e red-cell membrane and its c y t o s k e l e t o n has been publishedgo0 A new a p p r o a c h f o r t h e i s o l a t i o n o f l e c t i n r e c e p t o r s w i t h o u t t h e use of detergents involves t h e mechanical shearing of cells bound t o l e c t i n - c o a t e d macroporous a g a r o s e beads, whereupon t h e r e c e p t o r s r e m a i n a t t a c h e d t o t h e b e a d s and are r e l e a s e d s p e c i f i c a l l y by i n h i b i t o r y s u g a r s . 9 0 1 I n t h i s way a g l y c o p r o t e i n r e l e a s e d f r o m


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n e u r a m i n i d a s e - t r e a t e d human e r y t h r o c y t e s a f t e r b i n d i n g t o immobilized peanut a g g l u t i n i n or t o immobilized soybean a g g l u t i n i n was c h a r a c t e r i z e d a s a s i a l o g l y c o p h o r i n . B l o o d - g r o u p H s i t e s o f h u m a n 0 c e l l s h a v e b e e n c o n v e r t e d in v i t r o i n t o g r o u p A s i t e s by t r a n s f e r o f 2 - a c e t a m i d o - 2 - d e o x y - g - { 14c}g a l a c t o s y l r e s i d u e s u s i n g t h e b l o o d - g r o u p A g e n e - d e p e n d e n t a-Q-2a c e t a m i d o - 2 - d e o x y - g a l a c t o s y l t r a n s f e r a s e f r o m h u m a n A1 p l a s m a . 9 0 2 The m a j o r i t y o f t h e r a d i o a c t i v i t y i s t i g h t l y bound t o t h e m e m b r a n e s a n d i s o n l y r e l e a s e d by p r o t e o l y s i s , i m p l y i n g t h a t t h e b u l k o f b l o o d g r o u p H d e t e r m i n a n t s a r e bound t o g l y c o p r o t e i n m a t e r i a l . T h e human e r y t h r o c y t e r e c e p t o r f o r t h e I - f u c o s e - s p e c i f i c l e c t i n p r o d u c e d by S t r e p t o m y c e s s p e c i e s b i n d s t o human e r y t h r o c y t e s b e a r i n g ABO d e t e r m i n a n t s. ’03 I n a h a e m a g g 1u t i n a t i o n - i n h i b i t i o n a s s a y p o l y ( g l y c o s y 1 ) - c e r a m i d e was t h e b e s t i n h i b i t o r w i t h a g l y c o p r o t e i n f r a c t i o n showing only s l i g h t i n h i b i t o r y a c t i v i t y . The d i s t r i b u t i o n o f A a n d B d e t e r m i n a n t s i n t h e p o l y g l y c o s y l p e p t i d e s o f human r e d c e l l s o f b l o o d g r o u p AB h a s b e e n s t u d i e d by i s o l a t i n g b l o o d - g r o u p AB ABH-active c o m p o n e n t s f r o m d e l i p i d a t e d human b l o o d - g r o u p e r y t h r o c y t e mernbranes.’O4 The A and B a n t i g e n i c sites a r e l o c a t e d i n d i f f e r e n t p o l y g l y c o s y l c h a i n s . A p p r o x i m a t e l y h a l f o f t h e bloodg r o u p ABH-active g l y c o p e p t i d e s c a r r y b l o o d - g r o u p A d e t e r m i n a n t s and h a l f c a r r y blood-group 8 d e t e r m i n a n t s . Human e r y t h r o c y t e s h a v e b e e n l a b e l l e d a t t h e c e l l - s u r f a c e a g a l a c t o s y 1 a n d 2 - a c e t a m i d o - 2 - d e o x y - g a l a ct o sy 1 r e si d u e s w i t h s o d i u m b ~ r o t r i t i d e . ~ ’ A~ s i g n i f i c a n t d e c r e a s e i n t h e n u m b e r o f r e s i d u e s d u r i n g a g e i n g o f t h e e r y t h r o c y t e s was d e t e c t e d . Two h e r e d i t a r y p l a t e l e t d i s o r d e r s , Glanzmann’s t h r o m b a s t h e n i a a n d B e r n a r d - S o u l i e r s y n d r o m e , a r e c h a r a c t e r i z e d by d e f e c t s i n v o l v i n g a d h e s i o n a n d a g g r e g a t i o n a n d a r e a s s o c i a t e d w i t h d i f f e r e n t membrane g l y c o p r o t e i n d e f i c i e n ~ i e s . ~ ’ N~ o g l y c o p r o t e i n d e f e c t s h a v e b e e n o b s e r v e d on a n a l y s i s o f t h e e r y t h r o c y t e membrane g l y c o p r o t e i n s of p a t i e n t s o f both d i s o r d e r s . Immunochemical evidence f o r t h e e x i s t e n c e of hybrid s i a l o g l y c o p r o t e i n s o f human e r y t h r o c y t e s h a s b e e n r e p o r t e d . ” ’ U s i n g human e r y t h r o c y t e m e m b r a n e s a s a m o d e l s y s t e m , t h r e e related procedures, based on t h e s p e c i f i c i n t e r a c t i o n of l e c t i n s w i t h membrane c a r b o h y d r a t e , h a v e b e e n d e v e l o p e d f o r t h e i s o l a t 5 o n o f i n s i d e - o u t v e s i c l e s from h e t e r o g e n e o u s p o p u l a t i o n s o f membrane fragments.908 The t e c h n i q u e s s h o u l d p r o v e a p p l i c a b l e t o o t h e r b i o l o g i c a l m e m b r a n e s where i t i s n o t p o s s i b l e t o i n d u c e c o n t r o l l e d s e a l i n g o f membrane f r a g m e n t s i n a d e f i n e d o r i e n t a t i o n a s i s t h e

-e


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case f o r e r y t h r o c y t e membranes. Glycophorin A h a s been r e c o n s t i t u t e d i n small l i p i d v e s i c l e s ( 2 5 0 - 300 I! i n d i a m e t e r ) by u s i n g c h o l a t e d e t e r g e n t s o l u b i l i z a t i o n f o l l o w e d by r a p i d r e m o v a l o f c h o l a t e o n a m o l e c u l a r - s i e v e c o l u m n . 9 0 9 Incorporation is i n a transbilayer fashion, with a high concentration of the molecules orientated with t h e carbohydrateInteraction of the c o n t a i n i n g N-terminus t o t h e v e s i c l e e x t e r i o r . p r o t e i n w i t h t h e h y d r o p h o b i c p o r t i o n o f t h e b i l a y e r c a n be s h o w n by 1~ n.m.r. spectroscopy. The t r a n s l a t i o n a l m o b i l i t y o f g l y c o p h o r i n i n b i l a y e r membranes o f dimyristoylphosphatidylcholine h a s b e e n reported.’1° Evidence f o r m u l t i p l e components of blood-group A and B a n t i g e n s i n human e r y t h r o c y t e m e m b r a n e s - s u g g e s t i v e o f d i f f e r e n t c a r b o h y d r a t e s t r u c t u r e s - h a s been reported.911 An L- - a s p a r a g i n e - l i n k e d o l i g o s a c c h a r i d e c h a i n ( 5 3 ) h a s b e e n i s o l a t e d f r o m a t r y p t i c f r a g m e n t o f h u m a n g l y c o p h o r i n A.912 S t r u c t u r a l similarities between t h i s o l i g o s a c c h a r i d e and t h e s u g a r c h a i n s of immunoglobulin A are e v i d e n t . The b l o o d - g r o u p M a n d N s p e c i f i c d e t e r m i n a n t s o f human e r y t h r o c y t e g l y c o p h o r i n A h a v e t h r e e i d e n t i c a l c a r b o h y d r a t e m o i e t i e s ( 5 4 ) p e r ~ e p t i d e . T~h e~ M~ o r N determinant is t h e N-terminal amino a c i d and a neuraminic a c i d r e s i d u e ( s ) . The a u t h o r s a r g u e t h a t t h e r e i s no c h e m i c a l b a s i s f o r a s s e r t i o n s i n t h e l i t e r a t u r e that M and N a n t i g e n s d i f f e r i n t h e i r oligosaccharide s t r u c t u r e or that t h e N antigen is b i o s y n t h e t i c a l l y t r a n s f o r m e d t o t h e M a n t i g e n by s i a l y l a t i o n . a-Neu~5Ac-(2+3)-B-~-Gal~-(l+3)-~-~-Gal~NAc-l-~-~-Ser/~-Thr 6

I

2 a - Ne u p 5 A c (54) The g - g l y c o s i d i c a l l y l i n k e d o l i g o s a c c h a r i d e c h a i n s o f g l y c o p h o r i n from bovine e r y t h r o c y t e membranes h a v e been i s o l a t e d as r e d u c e d o l i g o s a c c h a r i d e s by a l k a l i n e b o r o h y d r i d e t r e a t m e n t , a n d p u r i f i e d by i o n - e x c h a n g e c h r o m a t o g r a p h y f o l l o w e d by g e l The s t r u c t u r e s o f t h r e e o l i g o s a c c h a r i d e s (55-571, a filtration.914 ~-Gal~-(1+3)-g-Gal~-(l+4)-g-Glc~NAc-(1+3)Q-Gal~-(1+3)-g-GalNAc-o1 (551


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penta-,

a hepta-, a n d a d e c a - s a c c h a r i d e o b t a i n e d as m a j o r c o m p o n e n t s

o f a n e u t r a l f r a c t i o n , have been e s t a b l i s h e d . acid

of

sequence

established.’15

porcine

erythrocyte

The c o m p l e t e a m i n o

glycophorin

has

been

The t w e l v e o l i g o s a c c h a r i d e c h a i n s a r e l o c a t e d a t

t h e N - t e r m i n a l e n d o f t h e m o l e c u l e . Ten o f t h e s e o l i g o s a c c h a r i d e s are l i n k e d g-glycosidically

t o L-threonine

o r I - s e r i n e r e s i d u e s and

the remaining two are attached N-glycosidically residues

.

Horse

erythrocyte

membranes

contain

t o L-asparagine two

d i s t i n c t

glyc~phorins.’T ~ h~e m a j o r c o m p o n e n t h a s a b l o c k e d N - t e r m i n u s a n d consists

of

70% p r o t e i n and 30% c a r b o h y d r a t e ,

while

the

minor

component has I - t h r e o n i n e . The c h e m i s t r y a n d b i o c h e m i s t r y o f g l y c o p h o r i n A a s a m o d e l membrane g l y c o p r o t e i n h a v e b e e n reviewed.’17 Amino

acid

erythrocyte

and

carbohydrate

glycoproteins

structural

have

been

variants

of

major

reported.918

The

M M ( M i 1 t e n b e r g e r 111) g l y c o p r o t e i n a p p e a r s t o be a p r o d u c t o f t h e M gene and c o n t a i n s g - g l y c o s i d i c a l l y l i n k e d o l i g o s a c c h a r i d e c h a i n s partially

i d e n t i f i e d by t h e s t r u c t u r e (58). The

o f t h e M-N

locus,

i n t h e N gene.

M 9 i s an a l l o m o r p h

p r o b a b l y e v o l v e d f r o m a s i n g l e base s u b s t i t u t i o n

The r e s u l t i n g s i n g l e a m i n o a c i d s u b s t i t u t i o n a f f e c t s

the post-translational

glycosylation o f neighbouring i-serine

o r I-

threonine resides. The a m i n o a c i d s e q u e n c e o f a N - t e r m i n a l t r y p t i c g l y c o p e p t i d e of the

blood-group

MQ-specific

major

human

s i a l o g l y c o p r o t e i n has been reported.919

erythrocyte

membrane

The a m i n o a c i d s e q u e n c e a n d

t h e s i t e s o f g l y c o s y l a t i o n o f t h e t w e l v e a m i n o a c i d s a t t h e Nterminus

of

blood-group

Ms-specific

major

s i a l o g l y c o p r o t e i n have been established.920 represents

an

evolutionary

link

erythrocyte

The

between blood-group

variant M-

Mc

a n d N-

s p e c i f i c glycoproteins.

Neu~5Ac-Q-Gal~-(1+3)-Q-Gal~NAc-l+~-Thr

I P - G lCQNAC

(58) T h e h u m a n l e u k a e m i a c e l l l i n e K562 s y n t h e s i z e s g l y c o p h o r i n A.921

An i s o l a t e d m e s s e n g e r RNA f r a c t i o n f r o m t h e c e l l s h a s b e e n

used i n a r a b b i t r e t i c u l o c y t e c e l l - f r e e system f o r t h e s y n t h e s i s o f the glycoprotein.

The p r e s e n c e o f m e m b r a n e s i s r e q u i r e d f o r

N-


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291

2 92


g l y c o s y l a t i o n and 2 - g l y c o s y l a t i o n o f t h e a p o p r o t e i n . Despite t h e i r heterogeneous appearance a f t e r sodium dodecyl sulphate

gel electrophoresis,

glycoprotein o f

band 3 ( t h e

human e r y t h r o c y t e s )

a r e homogeneous p o l y p e p t i d e s . 9 2 2

and i t s

major

transmembrane

p r o t e o l y t i c fragments

The a p p a r e n t h e t e r o g e n e i t y o f b a n d

3 and i t s C - t e r m i n a l r e g i o n may r e f l e c t v a r i a b i l i t y o f g l y c o s y l a t i o n on s o d i u m d o d e c y l s u l p h a t e b i n d i n g .

The c h a r a c t e r i s t i c d i f f u s i o n o f

band 3 g l y c o p r o t e i n as s e e n by S D S - p o l y a c r y l a m i d e g e l e l c t r o p h o r e s i s has

been a t t r i b u t e d t o

heterogeneity

o l i g o s a c c h a r i d e chains.923 further been

of

o l i g o s a c c h a r i d e s o r i g i n a t i n g from

established.924

glycoprotein

The

i s similar

the

molecular

The s t r u c t u r e s ( 5 3 , mechanism

to

that

of

59,

size of

60) o f t h r e e

band 3 g l y c o p r o t e i n have

of

biogenesis

of

cotranslationally

g l y c o p r o t e i n s l i k e v e s i c u l a r s t o m a t i t i s v i r u s G,

band

3

inserted

a n t i g e n , and

HLA-A

g l y c ~ p h o r i n . ~ T h~e ~m a j o r s i a l o g l y c o p r o t e i n s o f r a t e r y t h r o c y t e membrane h a v e been p u r i f i e d by h o t p h e n o l p a r t i t i o n i n g f o l l o w e d by cation-exchange

chromatography.926

Further

chromatographic

r e s o l u t i o n has y i e l d e d a s i n g l e p u r i f i e d g l y c o p r o t e i n (mol. x

lo4)

and a

accounting

mixture of

for

at

least

higher- molecular- weight 23% o f

the

wt.

1.9

glycoproteins

rat-erythrocyte-membrane

neuraminic acid.

15

S a l i v a r y and Mucous G l y c o p r o t e i n s

The f o l l o w i n g a s p e c t s o f t h i s a r e a h a v e been r e v i e w e d :

gastric

m u c o s a l p r o t e c t i o n and g a s t r i c g l y c o p r o t e i n s ,927 n e u r a m i n i c a c i d and mucous

rheology,928

and t h e

neutral oligosaccharides cystic-fibrosis A

i s o l a t i o n and

characterization

of

f r o m human b r o n c h i a l g l y c o p r o t e i n s o f

patients.929

gel-filtration

method has

been

developed

t o

isolate

s i m u l t a n e o u s l y and q u a n t i t a t e s p e c i f i c a l l y r a d i o l a b e l l e d mucous g l y c o p r o t e i n f r o m m u l t i p l e s a m p l e s o f c u l t u r e media.930 Mucin,

i s o l a t e d from a p a t i e n t w i t h c y s t i c f i b r o s i s ,

consists

o f an e x t e n s i v e l y g l y c o s y l a t e d c o r e p r o t e i n w h i c h i s l i n k e d t o a t l e a s t two other proteins.931

D i r e c t evidence f o r the r o l e o f the

m u c i n s i n t h e r h e o l o g i c a l b e h a v i o u r o f s p u t u m h a s been s u m m a r i z e d . Human p a r o t i d I - p r o l i n e - r i c h


wt.

3.6

x

lo4)

contains a biantennary oligosaccharide structure s i m i l a r t o that d e s c r i b e d f o r s e v e r a l serum c ~ l y c o p r o t e i n s . ~ ~ ~ M i l d a c i d h y d r o l y s i s has

been used t o

cleave

a

sulphated

293


m o n o s a c c h a r i d e f r o m s a l i v a r y m u c i n o f t h e s t u m p t a i l monkey (Macaca a r ~ t o i d e s ) . T~ h~e ~m o n o s a c c h a r i d e was p u r i f i e d b y i o n - e x c h a n g e chromatography

and

identified

sulphate. The i n c o r p o r a t i o n o f { 14C)-mannose

into

as

2-amino-2-deoxy-Q-glucose

L-{ 3 H ) - l e u c i n e

proteins

4-

and 2-acetamido-2-deoxy-P-

and g l y c o p r o t e i n s

submandibular, and s u b l i n g u a l g l a n d s o f

the

of

the

mouse

parotid, has

been

compared. 934 novel

A

neuraminic

acid,

4-g-acetyl-9-g-lactyl-N-

a c e t y l n e u r a m i n i c a c i d , h a s b e e n i d e n t i f i e d as a c o n s t i t u e n t o f h o r s e submandibular- gland

g l y c ~ p r o t e i n s . ~The ~ ~ structure

has

been

e s t a b l i s h e d b y g.1.c.-m.s. A

fluorimetric

porcine

method f o r

submaxillary

mucine

measuring a-h-fucose by

(A')

released from

a-I=-fucosidase

has

been

developed.936 A l k a l i n e borohydride r e d u c t i v e cleavage o f hog submaxillary glycoproteins from three immunologically results

i n

the

release

of

a

series

determined phenotypes

of

neutral

and

acidic

o l i g o s a ~ c h a r i d e - a l d i t o l s . ~A~ l~ l h a v e b e e n i d e n t i f i e d a s p a r t i a l structures

r e p r e s e n t i n g t h e p o s s i b l e c o m p l e t e and b i o s y n t h e t i c a l l y

i n c o m p l e t e s t a g e s o f t h e c h a i n o f t h e p e n t a s a c c h a r i d e (611,

present

in

The

the

glycoprotein

with

blood-group

glycolylneuraminic acid residues,

A activity.

when p r e s e n t ,

2-

are only linked t o

2 - a c e t a m i d o - 2 - d e o x y - Q - g a l a c t it o 1 r e s i d u e s a n d n o t t o P - g a l a c t o s y l r e s i d u e s , as h a d b e e n p r e v i o u s l y r e p o r t e d .

a-Q-Gal~NAc-(1+3)-B-Q-Galp-(1+3)-~-GalNAc-o1 2

6

1

2

I

I

a-i-Fucp

a-N e up5A c

(61) A 2-acetamido- 2- de oxy- 9- gluc osyltr a nsf e r a s e

which a c t s on mucin

s u b s t r a t e s has been d e t e c t e d i n c a n i n e s u b m a x i l l a r y glands.938 Using mucin

o r b e n z y 1 2 - a c e t a m i d o - 2 - d e o x y -3-g-B-P-galactosyl-a-Pt h e t r a n s f e r o f a 2-acetamido-2-deoxy-9-

g a l a c t o s i d e as acceptors,

g l u c o s y l r e s i d u e t o e i t h e r 0-4 o r 0 - 6 o f t h e 2 - a c e t a m i d o - 2 - d e o x y - P galactosyl residue o f the acceptor

occurs.

Detailed substrate

s p e c i f i c i t y of

t h e 2-acetamido-2-deoxy-~-glucosyltransferase

t e s t e d with a

variety

of

when

p o t e n t i a l g l y c o p r o t e i n and s y n t h e t i c


2 94 disaccharide

a c c e p t o r s has been reported.939

Porcine l i v e r

m i c r o s o m e s c a n i n t r o d u c e r e s i d u e s o f n e u r a m i n i c a c i d f r o m CMPneuraminic a c i d i n t o porcine submaxillary asialo-afuco-mucin, a gg a l a c t o s y l o v i n e s u b m a x i l l a r y a s i a l o m u c i n a n d g a n g l i o s i d e GH1.940

No e v i d e n c e was o b t a i n e d t o i n d i c a t e any t r a n s f e r o f n e u r a m i n i c a c i d t o 2-acetamido-2-deoxy-D-galactosyl residues, and t h e enzyme t r a n s f e r s n e u r a m i n i c a c i d s o l e l y b y f o r m a t i o n o f a n a(2+3)glycosidic linkage t o e-galactosyl

residues.

Rat submaxillary

mucous g l y c o p r o t e i n has been p u r i f i e d a f t e r aqueous e x t r a c t i o n o f t h e g l a n d s f o l l o w e d by r e c y c l i n g g e l - f i l t r a t i o n

~hromatography.~~~

The c h e m i c a l c o m p o s i t i o n o f t h i s m u c i n r e s e m b l e s t h a t o f t h e human c o u n t e r p a r t and i s d i s s i m i l a r t o t h e c o m p o s i t i o n o f r a t s u b l i n g u a l glycoprotein. Chemical

properties of

and a f f i n i t y

of

lectins

for

human

b r o n c h i a l mucous g l y c o p r o t e i n s h a v e been compared.942 The a c t i o n s o f

m e r c a p t o e t h a n o l on s o l u b l e mucous g l y c o p r o t e i n s

o b t a i n e d f r o m b o t h c y s t i c - f i b r o t i c and c h r o n i c - b r o n c h i t i c sputum h a v e been c ~ m p a r e d . ~ The ~ ~ ,e ~ f f e~ c ~t o f t h e r e d u c i n g a g e n t u n d e r n o n - r e d u c i n g c o n d i t i o n s o n t h e g l y c o p r o t e i n s c a n n o t be e x p l a i n e d solely

by

the

disulphide

bond-breaking

action,

but

appears

i n v o l v e t h e e x i s t e n c e o f a mercaptoethanol-inducible

to

mucolytic

s y s t e m w h i c h v a r i e s f r o m one s p u t u m t o a n o t h e r . O l i g o s a c c h a r i d e s o b t a i n e d b y a l k a l i n e d e g r a d a t i o n o f human b r o n c h i a l mucous exchange,

glycoproteins

have been f r a c t i o n a t e d

gel-permeation chromatography,

by

ion

and h . p . l . ~ . ’ ~ ~

S u l p h a t e d g l y c o p r o t e i n s f r o m human g a s t r i c j u i c e h a v e b e e n i s o l a t e d a f t e r a f f i n i t y c h r o m a t o g r a p h y on c o l u m n s o f i m m o b i l i z e d l y ~ i n e . Two ~ ~ m ~ ajor glycoprotein f r a c t i o n s d i f f e r i n g i n t h e i r

LO-

s u l p h a t e a n d n e u r a m i n i c a c i d c o n t e n t were i s o l a t e d . The

nature

of

the

non-covalent

interactions

g l y c o p r o t e i n m o l e c u l e s o f human and p i g g a s t r i c mucous s t u d i e d by mechanical spectroscopy,

between has

been

u s i n g s t o r a g e and l o s s m o d u l i t o

.

c h a r a c t e r ize, r e s p e c t i v e ly, so l i d - 1ik e a n d l i q u i d - 1ik e be h a v i o u r 947 Oligosaccharides having blood-group

Ii a c t i v i t y

have been

i s o l a t e d f r o m sheep g a s t r i c g l y c o p r o t e i n s w h i c h h a d been e n r i c h e d

f o r t h e s e b l o o d - g r o u p a c t i v i t i e s b y a f f i n i t y c h r o m a t o g r a p h y o n an a n t i - I a d s o r b e n t column.948

The f r a c t i o n s o f s m a l l e s t m o l e c u l a r

w e i g h t w i t h b o t h I and i a c t i v i t i e s a r e m i x t u r e s o f h e x a - a n d o c t a saccharides.

From s t u d i e s on t h e hexa- t o o c t a - s a c c h a r i d e

a s t r u c t u r a l model (62) i s proposed which c o n s i s t s o f region,

(b)

a b a c k b o n e r e g i o n h a v i n g (1+3)-

fractions

(2)

a cor,e

and ( 1 + 6 ) - l i n k e d

2-

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides al

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acetamido-2-deoxy-4-~-B-Q-galactosyl-~-glucosyl b r a n c h e s w i t h I a c t i v i t i e s and l i n e a r r e p e a t i n g (l+3)-linked 2-acetamido-2-deoxy-4-O - 8 - ~ - g a l a c t o s y l - ~ - g l u c o s e u n i t s w i t h i a c t i v i t i e s , a n d (c) a p e r i p h e r a l r e g i o n w i t h b l o o d - g r o u p i s o t y p e a c t i v i t i e s . 949 A p r o t e i n ( m o l . w t . 7.0 x l o 4 ) i s l i n k e d t o p i g g a s t r i c m u c o u s g l y c o p r o t e i n by d i s u l p h i d e b r i d g e s . 9 5 0 On d i s s o c i a t i o n o f t h e g l y c o p r o t e i n i n t o s u b u n i t s by r e d u c t i o n o r p r o t e o l y s i s , t h e p r o t e i n is e i t h e r r e l e a s e d i n t h e reduced form or l o s t on p r o t e o l y t i c digestion.951 P i g g a s t r i c a n d s m a l l - i n t e s t i n a l mucous g l y c o p r o t e i n s a r e c l e a v e d i n t o f o u r s u b u n i t s by p r o t e o l y t i c enzymes.952 A model f o r t h e g a s t r i c mucous g l y c o p r o t e i n (mol. w t . 2 x l o 6 ) , w h e r e on a v e r a g e f o u r s u b u n i t s a r e e a c h j o i n e d t o a p r o t e i n ( m o l . w t . 7.0 x l o 4 ) by d i s u l p h i d e b i n d i n g , i s p r o p o s e d . The i n t e s t i n a l g l y c o p r o t e i n probably h a s a similar o v e r a l l s t r u c t u r e . Rat g a s t r i c m u c o s a l c e l l s a r e c a p a b l e o f t h e s y n t h e s i s and s e c r e t i o n o f a t l e a s t t w o f a m i l i e s o f mucous g l y c o p r o t e i n s o f w i d e l y d i f f e r e n t m o l e c u l a r w e i g h t s a n d r h e o l o g i c a l p r o p e r t i e s .953 Oligosaccharide chains bearing t h e Forssman a n t i g e n i c d e t e r m i n a n t are a s s o c i a t e d w i t h g l y c o p r o t e i n s of t h e dog g a s t r i c mucous.954 Enzymic and immunological evidence i n d i c a t e s t h a t t h e s e g l y c o p r o t e i n s , l i k e F o r s s m a n g l y c o s p h i n g o l i p i d s , c o n t a i n t h e 2a c e t a mido-2-deoxy-3 -O-(2-acetamido-2-deoxy-a-~ - g a l a c t o sy l ) - Q g a l a c t o s e s t r u c t u r e , which d e t e r m i n e s t h e immunological s p e c i f i c i t y of the Forssman a n t i g e n . The v i s c o s i t y a n d g e l - f o r m i n g p o t e n t i a l o f p i g s m a l l - i n t e s t i n a l mucous i n c r e a s e w i t h p r o g r e s s i v e r e m o v a l o f n o n - c o v a l e n t l y bound protein during isolation.955 T h i s g l y c o p r o t e i n ( m o l . w t . 1.7 x l o 6 ) e x h i b i t s A a n d H b l o o d - g r o u p a c t i v i t y a n d c o n t a i n s r e s i d u e s o f If u c o s e , 2 - m a n n o s e , Q - g a l a c t o s e , 2 - a c e t a m i d o - 2 - d e o x y - Q - g l u c o s e , 2acetamido-2-deoxy-Q-galactose, a n d n e u r a m i n i c a c i d . The u n d e g r a d e d g l y c o p r o t e i n c o n t a i n s i n t e r c h a i n d i s u l p h i d e b r i d g e s and is d i s s o c i a t e d by p r o t e o l y s i s o r r e d u c t i o n i n t o s u b u n i t s t h a t a r e d i f f e r e n t i n s i z e and s t r u c t u r e from p i g g a s t r i c mucous gly~oprotein.9~6 Rat c o l o n i c mucous g l y c o p r o t e i n s h a v e b e e n s o l u b i l i z e d i n t h e The absence of p r o t e o l y t i c enzymes or d e n a t u r i n g solvents.957 presence of s e v e r a l high-molecular-weight components, varying i n t h e i r c a r b o h y d r a t e and s u l p h a t e c o n t e n t s , blood-group a n t i g e n i c i t y , a n d n e t c h a r g e , was o b s e r v e d . Evidence f o r the e x i s t e n c e o f two immunochemically d i s t i n c t m u c i n s i s o l a t e d a f t e r p r o t e o l y t i c d i g e s t i o n o f human c o l o n i c m u c i n


I

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n

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4

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4

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CJ I

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4

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aI

rill

297

298

Carbohydrate Chemistry I t i s n o t c l e a r whether these two mucin a s i n g l e mucin molecule or from t w o

h a s been reported.958 fragments o r i g i n a t e d i f f e r e n t mucins.

from

G l y c o p r o t e i n s f r o m t h e p e r i o v u l a t o r y phase mucins o f t h e b o n n e t monkey,

Macaca

enzymes.959

radiata,

have

been

The p a r t i a l s t r u c t u r e s

degraded

(63-64)

with

proteolytic

o f two oligosaccharide

c h a i n s were i d e n t i f i e d .

Urinary G l y c o p r o t e i n s , Oligosaccharides

16

Some

biochemical

sialidoses,

findings

Glycopeptides,and

concerning

four

different

types

of

on t h e b a s i s o f t h e s p e c i f i c a - n e u r a m i n i d a s e d e f i c i e n c y

and t h e n a t u r e o f t h e s t o r a g e m a t e r i a l , h a v e been r e v i e w e d . 9 6 0 From t h e knowledge o f t h e numerous s t r u c t u r e s o f g l y c o p r o t e i n g l y c a n s , i t i s p o s s i b l e t o d e m o n s t r a t e t h a t t h e o l i g o s a c c h a r i d e s and g l y c o a s p . a r a g i n e s a c c u m u l a t i n g i n t i s s u e s and u r i n e s o f p a t i e n t s w i t h diseases c h a r a c t e r i z e d by o r i g i n a t e from

a

lack

i n lysosomal glycosidases,

g l y c o p r o t e i n g l y c a n s i n c o m p l e t e l y c a t a b ~ l i z e d . ~A ~ ~

scheme f o r t h e n o r m a l a n d p a t h o l o g i c a l c a t a b o l i s m o f g l y c o p r o t e i n s h a s been p r o p o s e d . Three s i a l o g l y c o p r o t e i n s (mol. x

5.3

lo3,

urine.962

of

r e s p e c t i v e l y ) have

been

w t s . 7.79 X lo4, 3.7 x 104,and isolated

from

normal

human

Alkaline borohydride degradation o f the sialoglycoprotein

lowest molecular

weight yielded two s i a l y l a t e d oligosaccharides

w h i c h were p a r t i a l l y c h a r a c t e r i z e d . Patients with tubular or e x c r e t e an a l - m i c r o g l o b u l i n

mixed glomerular-tubular

proteinuria

w h i c h has been shown t o have t h r e e

g l y c o s i d i c a l l y l i n k e d carbohydrate chains o f

l-

structural similarity

t o t h o s e d e r i v e d f r o m many o t h e r s e r u m g l y c o p r ~ t e i n s . ~ ~ ~ A g l y c o p r o t e i n ( m o l . w t . 2.0 x lo4) w h i c h c o m p l e t e l y i n h i b i t s t r y p s i n a t a 1:l m o l a r r a t i o h a s b e e n i s o l a t e d f r m human u r i n e . 9 6 4

I t i s generated from a precursor molecule which i n t u r n i s d e r i v e d from

plasma

inter-a-trypsin

i n h i b i t o r (mol. w t . Kunitz-type

3.0

domains.965

x

inhibitor.

lo4) The

An a c i d - r e s i s t a n t

trypsin

f r o m human u r i n e i s c o m p o s e d o f t w o C-terminal

domain i s r e s p o n s i b l e f o r

a n t i t r y p t i c a c t i v i t y b u t no i n h i b i t o r y a c t i v i t y h a s b e e n d e s i g n a t e d t o t h e o t h e r domain.

B o t h t h e N - t e r m i n a l e x t e n s i o n p e p t i d e and t h e

l a t t e r domain are l i n k e d g - g l y c o s i d i c a l l y respectively.

The 0 - g l y c o s y l

and N - g l y c o s i d i c a l l y ,

residues are attached t o I-serine-10

299

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides w h i l e t h e N--glycosyl

residues are t o I-asparagine-24

on t h e f i r s t

K u n i t z - t y p e domain. 966 G l y c o p e p t i d e s have been i s o l a t e d f r o m p r o t e o l y t i c d i g e s t s o f human T a m m - H o r s f a l l g l y c o p r o t e i n a n d i t s a s i a l o d e r i v a t i v e . 9 6 7 glycoprotein

contains

at

least

five

I-asparagine

The

residues

s u b s t i t u t e d by complex c a r b o h y d r a t e m o i e t i e s ,

t h r e e b e i n g o f one

type,

and t w o c o n t a i n i n g

r e l a t i v e l y r i c h i n ;-galactosyl

residues,

l e s s g - g a l a c t o s e b u t more n e u r a m i n i c a c i d . The

structures

(65-67)

of

three oligo-Q-mannoside-type

h_-

a s p a r a g i n e s i s o l a t e d f r o m t h e u r i n e o f a p a t i e n t w i t h Gaucher's disease

have

been

elucidated

using

MHz

500

'H

n.m.r.

s p e c t r o s c o p y .968 A

reduction or

absence o f

degrading lysosomal amidase

activity of

the glycoprotein

l-aspartamido-$-2-acetamido-2-deoxy-~-

g l u c o s e a m i d o h y d r o l a s e (EC 3.5.1.26)

i s t h e b a s i c enzymic d e f e c t i n

a s p a r t y l g l y c o ~ a m i n u r i a . ~U~r ~i n a r y s i a l y g l y c o c o n j u g a t e s h a v e b e e n s t u d i e d i n a number o f p a t i e n t s w i t h t h i s i n h e r i t e d d e f i c i e n c y . levels

of

the

main

metabolite,

i n v a r i o u s n e u r a l and e x t r a n e u r a l t i s s u e s o f a s p a r t y l g l y c o s a m i n u r i a have been measured

asparaginyl-Q-glucose, a patient suffering by

The

2-acetamido-2-deoxy-l-$-~-

from

g . l . ~ . ~ ~ OC o r r e l a t i o n o f

the

results

with

the

clinical

m a n i f e s t a t i o n s o f t h e d i s e a s e d i d n o t r e v e a l any d i r e c t r e l a t i o n s h i p between t h e amount of

2-acetamido-2-deoxy-l-f3-l=-asparaginyl-~-

g l u c o s e s t o r e d and t h e degree o f o r g a n d y s f u n c t i o n . Six

mono-

and

simultaneously Improvements

in

components a l l o w

di-saccharides

by

h.p.1.c.

a

detector

have

been

determined

o f d e i o n i z e d human urine.971 based on o p t i c a l a c t i v i t y o f t h e

t h e d e t e c t i o n o f 100 n g o f s u g a r .

R1-(1+3)-$-Q-Man~-(1+4)-$-~-G1c~NAc-(1~4)-$-~-G1c~NAc-1+~-Asn 6

I 1 R2-(1+3)-a-g-Mang 6

I 1 R3 (65)

R 1 = R2 = R3 = H R2 = R 3 = H

(66)

R1 = a-Q-Mane,

(67)

R1 = R 2 = R 3 = a-Q-Mane

3 00

Carbohydrate Chemistry The s t r u c t u r e o f

an o l i g o s a c c h a r i d e ( 6 8 ) c o n t a i n i n g n i n e Q -

m a n n o s y l r e s i d u e s and i s o l a t e d f r o m p a t i e n t s w i t h m a n n o s i d o s i s has been s i m i l a r l y a s s i g n e d . 9 7 2

Excessive g i n g i v a l hyperplasia with

s t o r a g e o f Q - m a n n o s e - r i c h o l i g o s a c c h a r i d e s a p p e a r s t o be a u n i q u e feature

present i n a p a t i e n t e x h i b i t i n g mannosidosis.973

c h a r a c t e r i s t i c 0-manno-oligosaccharides

Eight

each t e r m i n a t e d a t

r e d u c i n g end w i t h a 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s y l r e s i d u e i s o l a t e d from the patients' which

a trisaccharide

urines.

the were

I n contrast t o the urine i n

i s predominant,

tetrasaccharides

and

p e n t a s a c c h a r i d e s a r e more abundant i n g i n g i v a . The u n i q u e s t r u c t u r e ( 6 9 ) o f a t r i s a c c h a r i d e f r o m a p a t i e n t w i t h m a n n o s i d o s i s h a s b e e n d e d u c e d b y 13C n.m.r. terms o f sugar composition,

r i n g forms,

spectroscopy i n

anomeric configurations,

sequence, and i n t e r - r e s i d u e l i n k a g e p o s i t i o n s . 9 7 4 I n a c a s e o f f e l i n e m a n n o s i d o s i s , a m a r k e d d e f i c i e n c y o f a-D-

mannosidase i n b r a i n ,

kidney,and

l i v e r t i s s u e i s accompanied by h i g h

A a_concentrations o f n e u t r a l oligosaccharides i n t h e urine.975 m a n n o s y l - r i c h h e x a s a c c h a r i d e was f o u n d t o be t h e p r e d o m i n a n t

oligosaccharide. A s e t o f procedures f o r s c r e e n i n g o f p a t i e n t s '

urine t o detect

o l i g o s a c c h a r i d e - s t o r a g e d i s e a s e s h a s been d e s c r i b e d . 9 7 6 patients

with

U r i n e s from

mucolipidosis

aspartylglycosaminuria,

I, m a n n o s i d o s i s , f u c o s i d o s i s , and t y p e I g l y c o g e n - s t o r a g e d i s e a s e can be

d i s t i n g u i s h e d b y t.1.c.

B-Q-Galactosidase d e f i c i e n c y i n p a t i e n t s

c a n be d e t e c t e d by e x a m i n a t i o n o f t h e u r i n e u s i n g a c o m b i n a t i o n o f

a-P-Mane-(1+2)-a-a-Mang-(l+2)-a-Q-Man~ 1

I 3 B-B-Manp(l+4)-g-GlcNAc

6

I 1 a-Q-Mane-(1+2)-a-g-Man~-(l+3)-a-p-Manp 6

I 1 a-P-Man~-(1+2)-a-Q-Mang

(68)

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides ion-exchange

c h r o m a t o g r a p h y a n d t.1.c.

301

Excess s i a l y l o l i g o s a c c h a r i d e

e x c r e t i o n i s d e t e c t e d by g e l f i l t r a t i o n f o l l o w e d by e s t i m a t i o n of neuraminic acid. O l i g o s a c c h a r i d e p a t t e r n s o b t a i n e d by g e l f i l t r a t i o n o f u r i n e o f GMl

gangliosidosis

gangliosidosis

(Type

(Type

1) p a t i e n t s d i f f e r

2)

p a t i e n t s . 977

from

The

those

amount

of

of

GM1

total

o l i g o s a c c h a r i d e excreted i n t h e u r i n e of

Type 2 p a t i e n t s i s 10-20%

o f t h a t i n t h e u r i n e o f Type 1 p a t i e n t s .

Oligosaccharides having

t h e s t r u c t u r e s (70-84) o c c u r i n t h e u r i n e o f Type 1 p a t i e n t s .

The

same o l i g o s a c c h a r i d e s w i t h t h e e x c e p t i o n o f ( 7 6 , 7 7 , 7 9 , 8 4 ) a r e e x c r e t e d i n t h e u r i n e s o f Type 2 p a t i e n t s .

a-g-ManQ-(1+3)-B-;-ManE-(l+4)-g-GlcNAc (69)

Four p o s i t i o n a l isomers o f neuraminosyl o l i g o s a c c h a r i d e s have been i s o l a t e d from

t h e u r i n e of

a patient

with sialidosis

with

p a r t i a l d e f i c i e n c y o f B - ~ - g a l a c t o ~ i d a s e . T~h ~e i~r s t r u c t u r e s a r e identical to

oligosaccharides

(70-71)

neuraminosyl or (2+6)-a-neuraminosyl

bearing either

(2+3)-a-

t e r m i n a l residues.

Two s i b l i n g s w i t h n e u r o n a l c e r o i d l i p o f u c o s i n o s i s e x c r e t e d t h e b l o o d - g r o u p A t r i s a c c h a r i d e , 3-g-(2-acetamido-2-deoxy-a-;-galactopy r a n o s y 1) -2-2- (a-C-f u c o p y r a n o s y 1)-;-ga

17

.

l a c t o s e , i n t h e u r i n e 979

Avian Glycoproteins

Absorption spectra,

c.d.

spectra, and sedimentation-equilibrium

v a l u e s have been r e p o r t e d f o r

complexes

of

l u t e i n with

egg

o v a l b u m i n .980 Ovalbumin has been s u b f r a c t i o n a t e d on i m m o b i l i z e d c o n c a n a v a l i n A.981

A

d i s t i n c t i v e carbohydrate

fractions

was n o t e d .

used

demonstrate

t o

H i g h - f i e l d 'H

composition n.m.r.

heterogeneity

i n each

of

four

spectroscopy has been o f

chick

ovalbumin

g l y c ~ p e p t i d e s . ~An~ ~u n a m b i g u o u s a s s i g n m e n t o f a l l C1-H a n d Q m a n n o s e C2-H r e s o n a n c e s was made, e n a b l i n g t h e s t r u c t u r e s o f t h e g l y c o p e p t i d e s t o be assigned.983 The c o m p l e t e a m i n o a c i d s e q u e n c e o f

385 r e s i d u e s ,

has been determined.984

postsynthetic modification: terminus,

hen o v a l b u m i n ,

comprising

Ovalbumin has f o u r s i t e s o f

in addition t o

the

a c e t y l a t e d N-

t h e c a r b o h y d r a t e m o i e t y i s l o c a t e d a t I-Asn-292,

and t h e

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5: Glycoproteins, Glycopeptides, Proteoglycaris, and Animal Polysaccharides t w o p h o s p h o r y l a t e d L - s e r i n e s a r e a t r e s i d u e s 6 8 a n d 344. Two

glycopeptides

have

been i s o l a t e d f r o m

chicken ~ v a l b u m i n . ~ ’ B ~ oth h i g h Q-mannose-type

pepsin-treated and h y b r i d - t y p e

o l i g o s a c c h a r i d e s w e r e r e l e a s e d by an a l m o n d e m u l s i n g l y c o p e p t i d a s e . N a t u r a l - a b u n d a n c e 1 3 C n.m.r.

s p e c t r o s c o p y h a s been u s e d t o show

t h a t some a - Q - m a n n o p y r a n o s y l - ( 1 + 3 ) - Q - m a n n o p y r a n o s y l ovalbumin

glycopeptides

are

cleaved

much

l i n k a g e s i n hen faster

than

the

c o r r e s p o n d i n g ( 1 + 6 ) - l i n k a g e s b y j a c k - b e a n a - P - m a n n o ~ i d a s e . ~ I~n~ addition the

technique

was

used t o

examine

the

structures o f

p a r t i a l l y cleaved species produced d u r i n g t h e a c t i o n o f glycosidases on o l i g o s a c c h a r i d e s ,

glycopeptides,and glycoproteins.

Ovotransferrin,

i n a d d i t i o n t o human s e r u m t r a n s f e r r i n ,

i s

o x i d i z e d by s o d i u m p e r i o d a t e w i t h a r e s u l t i n g d e s t r u c t i o n o f I=t y r o s i n e r e s i d u e s and a loss o f i r o n - b i n d i n g a c t i v i t y . The

structural

determination o f

one

of

the

glycopeptides

o b t a i n e d by p r o t e o l y s i s o f t u r t l e - d o v e ovomucoid has r e v e a l e d t h e p r e s e n c e o f a t e r m i n a l t r i s a c c h a r i d e (851,

a s p e c i f i c blood-group

P1

a n t i g e n i c d e t e r m i n a n t p r e v i o u s l y d e s c r i b e d f o r human e r y t h r o c y t e P1 antigen.9e7 The (20%),

secondary

structures of

B-structure

(46%), a n d

chicken ovomucoid c o n t a i n a - h e l i x

random

coil

s t r u c t u r e p r e d i c t i o n s f o r chicken egg-white p~blished.~”

and d i s c u s s i o n i n c l u d e s c o n f o r m a t i o n a l

characteristics o f the glycoprotein,

18

Secondary-

ovomucoid have been

Some o f t h e e a r l i e r p r e d i c t i o n s o n s t r u c t u r e a r e

i n c o r p o r a t e d i n t h i s model, reactive site,

(18%).988

t h e secondary s t r u c t u r e a t t h e

a n d t h e s t r u c t u r a l homology among t h r e e domains.

M i s c e l l a n e o u s G l y c o p r o t e i n s and C h i t i n

The s t r u c t u r e s o f f i f t e e n o l i g o s a c c h a r i d e s ( 8 6 - 1 0 0 ) i s o l a t e d f r o m

B-Q-Gale-(1+3)-Q-GlcNAc (86) B-Q-Gale-(1+3)-Q-GalNAc (87)

3 08

Carbohydrate Chemistry B-Q-GalQ-( 1+4) -g-GlcNAc 3

I

1 a-C-Fucg

(88) R - ( 1+2) -B -Q-Gale-( 1+4) -Q-Glc

3

I 1 a-L-Fucp

(89) R = H (90) R = a-L-Fuce

1+-4) -B-Q-GlcpNAc-( 1+3) + - G a l B-Q-Galp-( 92) R1-(

1+2) -a-Q-Mane 1

I 6 R 2 - ( 1+2) -a-Q-Manp-(

1+3) -B +-Mane-(

1+4) -B-Q-GlcNAc

4

I 1 B-Q-GlCQNAC A = a-Neup5Ac-(2+6)-B-Q-Gale-(

B = B-Q-GlCeNAC C = B-Q-Galg-( 1+4) -B-P-GlcpNAc

1-41

-B-Q-GlceNAc

309

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides newborn meconium have been i d e n t i f i e d . ’ ”

I t i s proposed t h a t

e n d o g l y c a n a s e s a r e i n v o l v e d i n t h e p r o d u c t i o n o f t h e s e compounds from glycoproteins. Q - G a l a c t a n s have been i s o l a t e d f r o m t h e albumen g l a n d s f r o m s n a i l s o f t h e g e n u s B i ~ m p h a l a r i a . ~T h~e~ r e s u l t s o f m e t h y l a t i o n a n a l y s i s and p e r i o d a t e o x i d a t i o n d a t a i n d i c a t e t h e p r e s e n c e o f a multibranched

structure

c o n t a i n i n g [1+3)-

Polysaccharides containing b o t h

9-

and (1+6)-linkages.

and l = - g a l a c t o s y l r e s i d u e s have

been i s o l a t e d f r o m t h e a l b u m e n g l a n d s o f f o u r s p e c i e s o f A n t i s e r a t o t h e f o u r g a l a c t a n s showed v a r i o u s degrees o f c r o s s reactivity,

indicating structural differences, ascribable i n part t o

d e t e r m i n a n t s i n v o l v i n g g a l a c t o s e p h o s p h a t e , and p r o b a b l y a l s o t o t h e A B-

l i n k a g e and p o s i t i o n o f I - g a l a c t o s y l r e s i d u e s i n t h e m o l e c u l e . Q-galactopyranan,

--Pomacea

i s o l a t e d from t h e albumen gland o f t h e s n a i l

l i n e a t a , c o n t a i n s 3,4-Q-(l-carboxyethylidene)

groups, as

s h o w n by t h e l i b e r a t i o n o f p y r u v i c a c i d o n a c i d h y d r o l y s i s a n d b y m e t h y l a t i o n data.993

The s t r u c t u r e o f t h e 5-membered a c e t a l g r o u p s spectroscopy.

o f p y r u v i c a c i d was s t u d i e d by 1 3 C n.m.r.

The r o l e o f g l y c o s y l a t i o n i n t h e s e c r e t i o n o f v a r i o u s a v i a n and m a m m a l i a n p r o t e i n s s y n t h e s i z e d i n Xenopus l a e v i s o o c y t e s u n d e r t h e d i r e c t i o n of

heterologous

messenger RNA h a s been r e p o r t e d . 9 9 4

The

presence o f 3 - g l y c o s y l chains on t h e p r o t e i n s d i d n o t appear t o facilitate

secretion.

O l i g o s a c c h a r i d e s w i t h a new t y p e o f c o r e

s t r u c t u r e ( 1 0 1 ) h a v e been i s o l a t e d f r o m t r o u t egg glycoprotein.’’5 The o c c u r r e n c e o f N - g l y c o l y l n e u r a m i n i c a c i d l i n k e d (2-3) i n t e r n a l 2-acetamido-2-deoxy-Q-galactosyl

r e s i d u e has

t o the not

been

d e s c r i b e d i n o t h e r g l y c o p r o t e i n s or g l y c o l i p i d s . Neug5G 2

I 3

8-~-Gal~NAc-(1+4)-B-~-Gal~NAc-(l+4)-8-p-Gal~NAc-(l+3)8-g-Galp-(1+3)-GalNAc-ol (101) B o v i n e h y p o t h a l m i c mRNA t r a n s l a t e d i n a r e t i c u l o c y t e l y s a t e s y s t e m y i e l d s a common p r e c u r s o r [ m o l .

wt.

2.1

x

lo4)

by m i c r o s o m a l

membranes f r o m dog p a n ~ r e a s . ~ ’ ~T h i s p r e c u r s o r b i n d s t o i m m o b i l i z e d c o n c a n a v a l i n A and i s s e n s i t i v e t o t r e a t m e n t w i t h a-p-mannosidase. Purified variant-specific

g l y c o p r o t e i n a n t i g e n s o f Trypanosoma

310


brucei exist

i n

solution

as

dimers

and o c c a s i o n a l l y as h i g h e r

T.

Two v a r i a n t s u r f a c e g l y c o p r o t e i n s o f b r u c e i have o 1 igo m e r s .99’ been shown t o h a v e a c o n s e r v e d C - t e r m i n a l a m i n o a c i d sequence.998 The g l y c o s y l a t i o n o f t h e m a j o r v a r i a b l e s u r f a c e c o a t g l y c o p r o t e i n o f

-T. b r u c e i

i s i n h i b i t e d by t u n i c a m y ~ i n . N ~ -~L i~n k e d g l y c o s y l a t i o n

occurs subsequent t o s y n t h e s i s o f t h e p r o t e i n . V a r i a n t a n t i g e n s o f Trypanosoma c o n g o l e n s e h a v e b e e n p u r i f i e d by l e c t i n a f f i n i t y c h r o m a t o g r a p h y . loo0 A

molecular- weight

glycopeptides

of

the

analysis of protozoan

the

major

p o l y p e p t i d e s and

Toxoplasma g o n d i i

has

been

r e p o r t e d . lool A s u l p h a t e d g l y c o p r o t e i n (mol.

wt.

1.85 x

lo5)

i s synthesized

i n t h e m u l t i c e l l u l a r organism Velvox c a r t e r i o n l y d u r i n g t h e l i m i t e d p e r i o d o f embryogenesis.1002 function o f t h i s cell-surface

E v i d e n c e f o r an e m b r y o n i c c o n t r o l glycoprotein i s provided.

S m a l l a m o u n t s o f c h i t i n i n a r t h r o p o d c u t i c l e s c a n be m e a s u r e d b y e s t i m a t i n g t h e d e r i v a t i z e d s o l u b l e c h i t o ~ a n . The ~ ~ ~m e~ t h o d i s n o t a f f e c t e d by t h e p r e s e n c e o f n o n - c h i t i n o u s c u t i c u l a r components. C o v a l e n t l y bound c h i t i n - p r o t e i n complexes o f s e v e r a l s p e c i e s o f marine

invertebrates

deproteinization,

a l l

the

have

been

samples

isolated.1004

contained

small

After

amounts

of

r e s i d u a l amino a c i d s w i t h marked s p e c i e s v a r i a t i o n i n t h e i r i d e n t i t y and c o n t e n t . A c o m p a r a b l e s e r i e s o f c h i t i n s d e r i v e d f r o m b l u e , r e d , stone, and h o r s e s h o e c r a b s by t r e a t m e n t w i t h m i l d a c i d ,

H 4 e.d.t.a.,

and

a l k a l i h a v e b e e n i s 0 1 a t e d . l ~ ~The ~ polysaccharides comprise a family

of

closely related

molecular weight,

products

with

variable

solubility,

o p t i c a l r o t a t i o n , and a c e t y l v a l u e s , w h i c h a r e a

f u n c t i o n o f b o t h t h e s p e c i e s f r o m w h i c h t h e y o r i g i n a t e d and t h e i r method o f p r e p a r a t i o n . I n c r e a s e d r a t e s o f h y d r o l y s i s by c h i t i n a s e and l y s o z y m e o f c h i t i n and c h i t o s a n a f t e r o x i d a t i o n w i t h s o d i u m m e t a p e r i o d a t e h a v e been o b s e r v e d . l o o 6 19

The

Analysis o f Glycoproteins

structure,

carbohydrate

elucidation of

structures,

and f u n c t i o n s

of

r e s i d u e s on g l y c o p r o t e i n s have been r e v i e w e d . l o o 7

M e t h o d s f o r a n a l y s i n g c o m p l e x m i x t u r e s o f a n t i g e n s and g l y c o p r o t e i n s have been r e v i e w e d . l o o 8 S i a l o g l y c o p r o t e i n s h a v e been f r a c t i o n a t e d o n an i m m o b i l i z e d


31 1

neuraminic acid-binding l e c t i n from the horseshoe crab Carcinoscorpius rotunda ~auda.~"' The r e s o l u t i o n o f t h e i s o e n z y m e s o f a l k a l i n e p h o s p h a t a s e was a c h i e v e d . Polyacrylamide g e l e l e c t r o p h o r e s i s has been used t o r e s o l v e f l u o r e s c e i n - d e r i v a t i z e d asialoglycopeptides.lolo Specific e x o g l y c o s i d a s e d i g e s t i o n of t h e g l y c o p e p t i d e s g e n e r a t e s a series o f d e g r a d a t i o n p r o d u c t s t h a t are r e s o l v e d . A method f o r m o n i t o r i n g t h e r e m o v a l o f poly-e-mannosyl c h a i n s f r o m g l y c o p r o t e i n s by e n d o - 2 - a c e t a m i d o - 2 - d e o x y - Q - g l y c a n a s e H has been described.l15 A f t e r p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f t h e g l y c o p r o t e i n s and t h e i r e n z y m i c a l l y degraded p r o d u c t s , t h e g e l s are overlaid with iodinated lectins. The d e g r e e t o which these l e c t i n s b i n d i s t h e n m e a s u r e d by a u t o r a d i o g r a p h y . A peptide:!-glycanase h a s been p u r i f i e d from almond e m u l s i n and its s p e c i f i c i t y examined w i t h glycopeptides prepared from ovalbumin Both high g-mannose and complex and immunoglobulin M.l0l1 g l y c o p e p t i d e s are h y d r o l y s e d a t t h e B-I=-aspartylglycosylamine linkage. A method f o r t h e f l u o r e s c e n t l a b e l l i n g o f s i a l o g l y c o p r o t e i n s i n v o l v e s o x i d a t i o n w i t h s o d i u m p e r i o d a t e f o l l o w e d by c o n d e n s a t i o n w i t h f l u o r e s c e i n a m i n e o r m o n o d a n s y l - 1 , 2 - d i a m i n o e t h a n e f o l l o w e d by s t a b i l i z a t i o n o f t h e S c h i f f ' s b a s e s by r e d u c t i v e a m i n a t i ~ n . ~ ~ ' L e c t i n s have been used f o r t h e d e t e c t i o n of g l y c o p r o t e i n s transferred t o nitrocellulose sheets, a f t e r t h e i r electrophoretic s e p a r a t i o n on p o l y a c r y l a m i d e g e l s . l0l2 O l i g o s a c c h a r i d e s d e r i v e d f r o m g l y c o p r o t e i n s by e n z y m i c r e l e a s e w i t h e n d o g l y c o s i d a s e s or chemical r e l e a s e by h y d r a z i n o l y s i s h a v e b e e n r e d u c e d by s o d i u m b o r o t r i t i d e a n d s e p a r a t e d by h . p . l . c . 1 ° 1 3 T r e a t m e n t of g l y c o p r o t e i n s w i t h trifluoromethanesulphonic acid a t room t e m p e r a t u r e r e s u l t s i n t h e r a p i d c l e a v a g e o f p e r i p h e r a l s u g a r s , a s l o w loss o f I - s e r i n e a n d I - t h r e o n i n e - l i n k e d 2 - a c e t a m i d o 2-deoxy-p-galactosyl r e s i d u e s , and r e t e n t i o n o f 2 - g l y c o s i d i c a l l y l i n k e d 2-acetamido-2-deoxy-~-glucosyl residues.1°14 The r e a g e n t is safer t o u s e t h a n e i t h e r hydrogen f l u o r i d e or its p y r i d i n e complex. Glycoproteins have been i o d i n a t e d using t h e solid-phase o x i d i z i n g a g e n t 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril ( i o d ~ g e n ) . ' ~ ' ~ The method c o m p a r e s f a v o u r a b l y w i t h t h e s o l i d - p h a s e l a c t o p e r o x i d a s e and c h l o r a m i n e T methods. A method f o r d e t e r m i n a t i o n of p 1 volumes o f g l y c o p r o t e i n u s i n g s p o t a n a l y s i s on c e l l u l o s e acetate l a y e r s i s described.1°16 W i t h pg l u c o s e o x i d a s e and f l u o r e s c e i n - l a b e l l e d c o n c a n a v a l i n A s e n s i t i v i t y


312 o f 10 n g i s r e a c h e d .

This l e v e l of d e t e c t i o n i s lowered t o 1 ng o f

Q - g l u c o s e o x i d a s e when t h e r e a g e n t s a r e u s e d i n c o m b i n a t i o n w i t h horse-radish

peroxidase.

. .r . s p e c t r o s c o p ic

3C n m

da t a f o r 3 -0- ( 2-ace t a m ido - 2 - de o x y -a-p

-

galactopyranosy1)-N-acetyl-L-serine, 3-0-(2-acetamid0-2-deoxy-a-D- g a l a c t o p y r a n o s y l l - N - a c e t y l - i - t h r e o n i n e , 3-g-a9B-Q-galactopyranosylL_- - s e r i n e, a n d 3 -g-a,B-g - g a 1a c t o p y r a n o s y 1-L- - t h r e o n in e h a v e , b e e n recorded.1°17

These m o d e l compounds r e p r e s e n t common c a r b o h y d r a t e -

p r o t e i n l i n k a g e r e g i o n s of

many g l y c o p r o t e i n s .

The s t r u c t u r e s o f t h e o l i g o s a c c h a r i d e c o m p o n e n t s o f a number o f g l y c o p e p t i d e s have been e s t a b l i s h e d f o l l o w i n g h y d r a z i n o l y s i s - n i t r o u s a c i d deamination,

w h i c h l e a d s t o t h e s p e c i f i c c l e a v a g e o f 2-amino-2-

deoxy-g-glucosyl

l i n k a g e s .883

T h e d e r i v e d 2,5-anhydro-Q-mannose-

c o n t a i n i n g o l i g o s a c c h a r i d e s a r e r e d u c e d w i t h s o d i u m b o r o h y d r i d e and a n a l y s e d by g.1.c.-m.s.

o f t h e i r methylated ethers.

The f l e x i b i l i t y o f b i acetyl-lactosamine

and t r i - a n t e n n a r y

N-

glycans o f the

t y p e has been s t u d i e d by s p i n - l a b e l l i n g

the

n e u r a m i n i c a c i d r e s i d u e s i n g l y c o p e p t i d e s o f known s t r u c t u r e . l o l 8 A

detailed

analysis

of

the

360

MHz 'H

n.m.r.

spectral

p a r a m e t e r s f o r t h e a n o m e r i c a n d C2-H r e s o n a n c e s o f a l a r g e number o f g l y c o p e p t i d e s and o l i g o s a c c h a r i d e s of

known s t r u c t u r e r e v e a l s a

g e n e r a l method f o r t h e d e t e r m i n a t i o n of t h e p r i m a r y s t r u c t u r e o f g l y c o p e p t i d e s f o r m o s t c u r r e n t l y known c l a s s e s o f s t r u c t u r e s . 1 0 1 9 two-dimensional

d i s p l a y formed by p l o t t i n g q-mannosyl

C1-H

A

against

C2-H c h e m i c a l s h i f t s d e m o n s t r a t e s t h a t t h e s e p a i r s o f v a l u e s a r e s e n s i t i v e t o long-range

p e r t u r b a t i o n by r e m o t e s u b s t i t u t i o n by

hexoses as w e l l as t o d i r e c t s u b s t i t u t i o n forty-one

o f these chemical-shift

characterize unique s t r u c t u r a l microenvironments. s e q u e n c e and b r a n c h i n g p a t t e r n f o r

A total of

effects.

c l u s t e r s have been d e f i n e d w h i c h On t h i s b a s i s t h e

most s t r u c t u r e s

can be d e r i v e d .

The h y d r o d y n a m i c b e h a v i o u r o f r e d u c e d g l y c o p o l y p e p t i d e s h a s been

studied

conjunction

by

gel

with

filtration

h.p.l.c.lo2'

in

guanidine

Although

hydrochloride

i n

carbohydrate-rich

g l y c o p o l y p e p t i d e s may o c c a s i o n a l l y y i e l d u n d e r e s t i m a t e d v a l u e s o f molecular weights,

t h e method i s s u i t a b l e f o r t h e r a p i d e s t i m a t i o n

o f m o l e c u l a r w e i g h t s o f s i m p l e p o l y p e p t i d e s and g l y c o p o l y p e p t i d e s . Neuraminic immunodiffusion glycoprotein.1021

acid but

residues not

are

involved i n

the

the r a d i a l immunodiffusion

electro-

of

al-acid

D i f f e r e n t i a l d e t e r m i n a t i o n o f t h e g l y c o p r o t e i n by

t h e t w o i m m u n o l o g i c a l methods o f f e r s a method f o r t h e e s t i m a t i o n o f t h e d e g r e e o f s i a l y l a t i o n o f t h i s and o t h e r s e r u m g l y c o p r o t e i n s .


313

T h e s t r u c t u r e s o f s i a l o - o l i g o s a c c h a r i d e s a s d e t e r m i n e d b y 'H n.m.r. s p e c t r o s c o p y t o g e t h e r w i t h m e t h y l a t i o n a n a l y s i s h a v e b e e n shown t o vary w i t h t h o s e d e r i v e d from p e r i o d a t e o x i d a t i o n d a t a alone.1022 E x a m i n a t i o n o f a c i d i c t e t r a s a c c h a r i d e s o b t a i n e d f r o m A', ,'H a n d A - H - h o g s u b m a x i l l a r y m u c i n i n d i c a t e s t h a t n e u r a m i n i c a c i d r e s i d u e s a r e n o t r e l e a s e d f r o m o l i g o s a c c h a r i d e s when p e r i o d a t e o x i d a t i o n i s c a r r i e d o u t a t pH 4.5. The a n i o n i c p r o p e r t i e s o f t h e n e u r a m i n i c a c i d r e s i d u e s were u s e d t o s e p a r a t e t h e p e r i o d a t e o x i d a t i o n p r o d u c t s , t h e r e b y g i v i n g i n f o r m a t i o n on t h e l o c a t i o n of n e u r a m i n i c a c i d on t h e o l i g o s a c c h a r i d e c h a i n . The monosaccharide components of g l y c o p r o t e i n s have been reacted w i t h d a n s y l h y d r a z i n e and the r e s u l t i n g d e r i v a t i v e s s e p a r a t e d b y h . p . l . ~ . l ' ~ ~F l u o r e s c e n t d e t e c t i o n p r o v i d e s a s e n s i t i v i t y o f 10 p m o l per i n j e c t i o n . P a r t i a l l y methylated 2-methyl 2-acetyl methyl glycosides o b t a i n e d by m e t h a n o l y s i s a n d a c e t y l a t i o n o f g l y c o p r o t e i n s h a v e b e e n i d e n t i f i e d by g.1.c.-m.s. analysis.1024 A scheme f o r t h e r a p i d d e t e r m i n a t i o n o f t h e p o s i t i o n o f t h e m e t h y l and a c e t y l r e s i d u e s is proposed. T h e m e t h o d was a p p l i e d t o s i a l o g l y c o p e p t i d e s i s o l a t e d from al-acid glycoprotein. Methylation techniques used i n t h e s t r u c t u r a l a n a l y s i s of g l y c o p r o t e i n s and g l y c o l i p i d s have been reviewed.1025 A m o d i f i c a t i o n o f a g.1.c. m e t h o d a l l o w i n g s i m u l t a n e o u s s e p a r a t i o n o f n e u t r a l and amino s u g a r a l d i t o l acetates has been described.1026 The m e t h o d h a s b e e n a p p l i e d t o t h e m o n i t o r i n g o f t h e f r a c t i o n a t i o n o f complex mixtures of g l y c o p r o t e i n s and glycosaminoglycans. A r a p i d i s o c r a t i c h.p.1.c. method f o r t h e e s t i m a t i o n o f n e u r a m i n i c acid i n serum h a s been described.lo2' S e p a r a t i o n is a c h i e v e d on a c a t i o n - e x c h a n g e r e s i n u s i n g a 0.006N s u l p h u r i c a c i d m o b i l e p h a s e . Reviews d e a l i n g w i t h g l y c o s y l t r a n s f e r a s e s and their u s e i n a s s e s s i n g o l i g o s a c c h a r i d e s t r u c t u r e and s t r u c t u r e - f u n c t i o n r e l a t io n s h i p s h a v e b e e n p u b 1i s h e d l o28 9 02' Lysosomal enzymes contain e-mannosyl 6-phosphate m o i e t i e s which mediate t h e i r t r a n s l o c a t i o n t o lysosomes.1030 A new a s s a y f o r a-Q2-acetamido-2-deoxy-glucosyl p h o s p h o t r a n s f e r a s e using a-methyl-emannoside as a c c e p t o r has been r e p o r t e d . A c o u p l e d e n z y m e a s s a y f o r 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e : UDPa - g a l a c t o s e a - g a l a c t o s y l t r a n s f e r a s e h a s been developed t h a t a l l o w s t h e enzyme t o be assayed s p e c t r o p h o t o m e t r i c a l l y and i n d e n a t u r i n g p o l y a c r y l a m i d e gels.1031 I n a sequence o f l i n k e d enzyme r e a c t i o n s t h e l i b e r a t e d UDP i s s u c c e s s i v e l y c o n v e r t e d t o UTP, U D P - a - g l u c o s e ,

.


314

and f i n a l l y UDP-a-glucuronic a c i d w i t h t h e s u b s e q u e n t p r o d u c t i o n o f NADH. N A D H may t h e n be e s t i m a t e d s p e c t r o p h o t o m e t r i c a l l y o r d e t e c t e d i n polyacrylamide g e l s fluorimetrically.

20

B i o s y n t h e s i s o f Glycoproteins

A r e v i e w of t h e transmembrane a s s e m b l y of membrane and s e c r e t o r y

g l y c o p r o t e i n s h a s b e e n p ~ b 1 i s h e d . l ~ ~ B’ l o o d - g r o u p a n t i g e n s a n d t h e enzymes i n v o l v e d i n t h e i r s y n t h e s i s h a v e been reviewed.1033 Exposure of rat hepatoma t i s s u e c u l t u r e cells t o dexamethasone r e s u l t s i n t h e a p p e a r a n c e o f a new g l y c o p r o t e i n ( g p 3 5 - 5 0 , m o l . w t . 3.5 x l o 4 - 5.0 x l o 4 ) a n d i n c r e a s e d s y n t h e s i s o f a n o t h e r g l y c o p r o t e i n ( m o l . w t . 5.0 x 1 0 ~ 1 . ~ T’h e~ f~o r m e r g l y c o p r o t e i n i s e x p r e s s e d i n n o r m a l l i v e r , whereas t h e l a t t e r i s e x p r e s s e d i n h e p a t o m a c e l l s a n d i s r e g u l a t e d d i f f e r e n t l y by s t e r o i d h o r m o n e s . T r e a t m e n t o f human k i d n e y t u m o u r c e l l s w i t h b u t y r a t e i n c r e a s e s markedly t h e synthesis and sulphation of tumour-cell u n t r e a t e d c u l t u r e d cells release most o f g 1 y ~ o p r o t e i n s . l ~Whereas ~~ t h e i r s u l p h a t e d g l y c o p r o t e i n s i n t o t h e medium, b u t y r a t e - t r e a t e d cells p r e f e r e n t i a l l y accumulate t h e s e products i n t o t h e cell layer. The s y n t h e s i s and a c c u m u l a t i o n of Q - m a n n o s e - c o n t a i n i n g g l y c o p e p t i d e s i n human f i b r o b l a s t c e l l s h a v e been studied.1036 R e s u l t s are c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t m u l t i p l e pathways f o r L - a s p a r a g i n e - l i n k e d g l y c o p r o t e i n b i o s y n t h e s i s a r e p o s s i b l e . The i n c o r p o r a t i o n of Q-mannose, Q-glucose, and 2-acetamido-2-deoxy-Qg l u c o s e i n t o endogenous membrane p r o t e i n s i n calf p i t u i t a r y has been r e p o r t e d . lo3’ Glycosylation of proteins i n the protozoan Crithidia ----------fasciculata does not involve glucosylated lipid-bound oligosaccharides a s intermediates.1038 An o l i g o s a c c h a r i d e c o n t a i n i n g s e v e n P-mannosyl and t w o 2-acetamido-2-deoxy-~-glucosyl residues is t r a n s f e r r e d t o protein before undergoing processing. Carbohydrate-depleted or c h e m i c a l l y d e n a t u r e d g l y c o p r o t e i n s or s y n t h e t i c C-Asn-X-&-Ser/L-Thr-containing p e p t i d e s have been g l y c o s y l a t e d u s i n g hen o v i d u c t membranes as t h e enzyme source.1039 T r i p e p t i d e s may b e g l y c o s y l a t e d b u t t h e l e v e l o f g l y c o s y l a t i o n The i n c r e a s e s as t h e l e n g t h o f t h e p e p t i d e c h a i n i n c r e a s e s . p e p t i d e s were e x a m i n e d b y c . d . i n a q u e o u s l i p i d m i x t u r e s , w h e n i t was o b s e r v e d t h a t t h e p r e s e n c e o f s e c o n d a r y s t r u c t u r e i n t h e l i p i d state promotes the level of glycosylation. The s y n t h e s i s and

315

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides processing

of

I=-asparagine-linked

reviewed.lo40

The

assembly

of

o l i g o s a c c h a r i d e s have been

the

oligosaccharide through i t s transfer

lipid-linked

precursor

t o p r o t e i n and i t s c o n v e r s i o n

t o t h e d i v e r s e a r r a y o f f i n a l products i s discussed.

Glycoprotein

b i o s y n t h e s i s i n mouse L c e l l s i n v o l v e s l i p i d - l i n k e d s a c c h a r i d e s i n the synthesis o f L-asparagine-linked

glycoproteins.1041

The

f o r m a t i o n o f h i g h Q - m a n n o s y l o l i g o s a c c h a r i d e bound t o p r o t e i n can occur

independently o f

higher

lipid-linked

oligosaccharide

synthesis. D o l i c h o l phosphate appears

t o c o n t r o l t h e r a t e of

g l y c o s y l a t i o n d u r i n g t h e development o f sea-urchin

protein

embryos.1042

C o m p a c t i n , an i n h i b i t o r o f p o l y i s o p r e n o i d b i o s y n t h e s i s ,

inhibits A

d o l i c h o l p h o s p h a t e s y n t h e s i s and t h u s g l y c o s y l a t i o n o f p r o t e i n .

second e f f e c t o f t h e i n h i b i t o r i s t h a t a s i g n i f i c a n t f r a c t i o n o f t h e o l i g o s a c c h a r i d e chains synthesized i n t h e presence o f t h e i n h i b i t o r a n d t r a n s f e r r e d t o p r o t e i n a p p e a r t o b e a l t e r e d so t h a t t h e y a r e more n e g a t i v e l y charged.

B o t h de novo s y n t h e s i s o f d o l i c h o l

w e l l as i t s phosphorylation

as

may p l a y a n i m p o r t a n t r o l e i n t h e

i n c r e a s e i n g l y c o p r o t e i n s y n t h e s i s d u r i n g embryonic development o f t h e sea urchin.1043

The p o s s i b l e r o l e o f d o l i c h o l k i n a s e i n t h e

a c t i v a t i o n o f s t o r e d d o l i c h o l , and p e r h a p s t h e pathway f o r i t s

---novo

biosynthesis

i n this

and o t h e r

b i o l o g i c a l systems,

& is

discussed. Turpentine-induced

inflammation

in

rats

causes

increased

s y n t h e s i s o f g l y c o s y l a t e d d o l i c h o l phosphate d e r i v a t i v e s , which a r e intermediates glycoproteins,

i n

the

biosynthesis

of

I-asparagine-linked

and a l s o i n c r e a s e d l e v e l s o f a CTP-dependent

dolichol

k i n a s e . 1044 Immature chick oviduct responsible for

membranes c o n t a i n a t r a n s f e r a s e

transfer o f oligosaccharide pyrophosphoryl d o l i c h o l

t o p r o t e i n acceptors.1045

The enzyme i s s t i m u l a t e d b y e s t r o g e n

treatment. The

metabolism of

lipid-linked

B-mannose

intermediates i n

g l y c o p r o t e i n s y n t h e s i s d u r i n g l i v e r r e g e n e r a t i o n h a s been s t u d i e d i n rat

liver

micro some^.^^^^

i n c o r p o r a t i o n of

p-{

F o l l o w i n g p a r t i a l hepatectomy,

the

1 4 ~ 1 - m a n n o s e f r o m GDP-Q-{ 1 4 ~ 1 - m a n n o s e i n t o

d o l i c h y l p h o s p h o r y l e-mannose a n d p r o t e i n d e c r e a s e s a s c o m p a r e d w i t h controls.

E v i d e n c e i s g i v e n s u g g e s t i n g t h a t t h e i n c o r p o r a t i o n o f Q-

mannose i n t o g l y c o p r o t e i n i n r e g e n e r a t i n g r a t l i v e r may b e r e g u l a t e d a t t h e s t e p of

oligosaccharide transfer.

U s i n g t h e Thy-1'

m u t a n t mouse l y m p h o m a c e l l s o f t h e c l a s s E

316


c o m p l e m e n t a t i o n g r o u p w h i c h l a c k GDP-Q-mannose : d o l i c h o l phosphoryl mannosyltransferase and which are t h e r e f o r e unable t o i n t e r c o n v e r t GDP-Q-mannose a n d g - m a n n o s y l p h o s p h o r y l d o l i c h o l , f u r t h e r e v i d e n c e h a s been p r o v i d e d t h a t f i v e o f t h e n i n e e-mannosyl r e s i d u e s which are added t o the growing l i p i d - l i n k e d o l i g o s a c c h a r i d e s a r e d o n a t e d d i r e c t l y by G D P - Q - m a n n o s e . l o 4 ’ The r e m a i n i n g f o u r r e s i d u e s a r i s e from Q-mannosyl p h o s p h o r y l d o l i c h o l . The r e a c t i o n s i n v o l v e d i n t h e a d d i t i o n o f g - g l u c o s y l r e s i d u e s t o t h e o l i g o s a c c h a r i d e - l i p i d i n t e r m e d i a t e s by t h y r o i d m i c r o s o m e s have been studied.1048 The p r o p e r t i e s o f t h e enzymes i n v o l v e d i n t h e t r a n s f e r o f !&glucose f r o m i t s p h o s p h o r y l d o l i c h o l d e r i v a t i v e t o endogenous a c c e p t o r s are d e s c r i b e d and t h e p r o d u c t s are c h a r a c t e r i z e d i n terms o f t h e i r s t r u c t u r e a n d c a p a c i t y t o s e r v e a s d o n o r s i n t h e glycosylation of proteins. The b i o s y n t h e s i s and p a r t i a l c h a r a c t e r i z a t i o n of a P-glucose-containing i n s e c t l i p i d - l i n k e d o l i g o s a c c h a r i d e have been reported.lo4’ Its p r o p e r t i e s are c l o s e l y r e l a t e d t o t h e g-glucosylated d o l i c h y l oligosaccharide obtained from mammalian s y s t e m s . A m p h o m y c i n i n h i b i t s t h e t r a n s f e r o f Q - m a n n o s e , g - g l u c o s e , a n d 2acetamido-2-deoxy-!-glucose l-phosphate from their r e s p e c t i v e n u c l e o t i d e d e r i v a t i v e s t o d o l i c h o l p h o s p h a t e by m e m b r a n e p r e p a r a t i o n s f r o m c a l f b r a i n membranes.1050 O t h e r g-mannosyl-, g l u c o s y l - , 2-acetamido-2-deoxy-Q-glucosyl-transferases associated w i t h t h e same p r e p a r a t i o n a r e n o t a f f e c t e d by t h e a n t i b i o t i c . UDP-2-Deoxy-Q-arabino-hexose (UDP-2-deoxy-~-glucose) i n h i b i t s t h e formation o f P-glucosylphosphoryl d o l i c h o l i n chick-embryo cell m e m b r a n e s , b u t h a s n o e f f e c t o n t h e f o r m a t i o n o f N”d i a c e t y l c h i t o b i o s y l p y r o p h o s p h o r y l d 0 1 i c h o l . l ~ ~G~D P - 2 - D e o x y - P ------arabino-hexose inhibits the formation of both the lipid i n t e r m e d i a t e s by c o m p e t i t i o n w i t h p h y s i o l o g i c a l n u c l e o t i d e s u g a r s f o r d o l i c h o l phosphate. The i n f l u e n c e o f v a r i o u s a r y l p h o s p h a t e s and p h o s p h o n a t e s on t h e s y n t h e s i s o f g l y c o s y l p h o s p h o r y l d o l i c h o l , o l i g o s a c c h a r i d e s , and g l y c o p r o t e i n s by r a t l i v e r m i c r o s o m a l f r a c t i o n s h a s b e e n investigated.1052 Phenyl phosphate d i r e c t l y e f f e c t s t h e s y n t h e s i s o f a-mannosyl p h o s p h o r y l d o l i c h o 1 , a n d t h e i n h i b i t i o n o f l i p i d - l i n k e d o l i g o s a c c h a r i d e s a n d g l y c o p r o t e i n s may b e a c o n s e q u e n c e o f t h e effect. The a n t i b i o t i c t s u s h i m y c i n i n h i b i t s t h e f o r m a t i o n o f Qg l u c o s y l - , a-mannosyl-,and 2-acetamido-2-deoxy-~-glucosyl p h o s p h o r y l dolichol, but allows the incorporation of sugars i n t o lipid-linked

a-

317

5: Glycoproteins, Glycopeptides, Proteoglycans, and Animal Polysaccharides oligosaccharides aorta.1053

i n a particulate

enzyme

The m a j o r o l i g o s a c c h a r i d e

preparation

from

pig

f o r m e d f r o m GDP-D-mannose

in

t h e p r e s e n c e o f t h e a n t i b i o t i c i s (Q-Man~),-(g-GlceNAc)~. T u n i c a m y c i n h a s l i t t l e e f f e c t on t h e p r o t e i n s i n t h e e p i d e r m i s when p i g s k i n s l i c e s a r e m a i n t a i n e d i n o r g a n c u l t u r e ,

although the

synthesis o f s p e c i f i c epidermal glycoconjugates i s affected.1054 The g l y c o s y l a t i o n o f p a r t i c u l a t e g l y c o p r o t e i n s i s d e c r e a s e d , that o f soluble glycoproteins i s hardly affected.

whereas

Inhibition of

s u l p h a t e d g l y c o s a m i n o g l y c a n s b u t n o t o f h y a l u r o n i c a c i d was a l s o noted. The p e p t i d e a n t i b i o t i c t r i d e c a p t i n s t i m u l a t e s t h e i n c o r p o r a t i o n o f g-mannose f r o m GDP-;-(

14C)-mannose

glucose i n t o l i p i d - l i n k e d p i g aorta.1055

and ;-glucose

f r o m UDP-!-{

3H}-

m o n o s a c c h a r i d e s by enzyme f r a c t i o n s f r o m

The a n t i b i o t i c a l s o s t i m u l a t e s t h e i n c o r p o r a t i o n o f

sugars i n t o l i p i d - l i n k e d oligosaccharides. E p i t h e l i a l c e l l s of

the r a t

mannosyl phosphoryl d o l i c h o l dolicho1,which tissues.1056

small intestine synthesize

9-

and o l i g o s a c c h a r y l p y r o p h o s p h o r y l

are s i m i l a r t o those found i n a v a r i e t y o f other The

importance o f

m o n i t o r i n g and c o n t r o l l i n g t h e

d e g r a d a t i o n o f n u c l e o t i d e s u g a r s when g l y c o s y l t r a n s f e r a s e a c t i v i t i e s a r e b e i n g compared i n d i f f e r e n t i a t i n g c e l l s i s d e m o n s t r a t e d . Incubation o f cell-free s a l i n a ) w i t h GDP-g-mannose

e x t r a c t s of

the brine shrimp (Artemia

o r UDP-!-glucose

results i n the formation

o f t h e c o r r e s p o n d i n g d o l i c h y l d e r i v a t i v e s o f t h e sugars.1057

The

enzymic a c t i v i t i e s were d e t e c t e d e a r l y i n t h e development o f t h e e n c y s t e d A r t e m i a embryos. S t a r v a t i o n o f !-glucose

alters lipid-linked oligosaccharide

b i o s y n t h e s i s i n Chinese h a m s t e r o v a r y cells.1058

The c e l l s q u i c k l y

c e a s e s y n t h e s iz i n g t h e (Q- G I c e ) 3(D, - M ane)9-(P-GlceNAc)2 i n s t e a d make p r e d o m i n a n t l y

the

(p-Ma~),(g-GlceNAc)~

s p e c i e s and

species, w h i c h

i s g l u c o s y l a t e d and t r a n s f e r r e d t o p r o t e i n w h e r e i t i s s u b s e q u e n t l y processed,

t h u s d e m o n s t r a t i n g t h e use of

an a l t e r n a t e g l y c o s y l a t i o n

pathway. An o l i g o s a c c h a r y l p y r o p h o s p h o r y l l i p i d f r o m p o r c i n e l i v e r i s composed o f a t e t r a s a c c h a r i d e w i t h e q u a l q u a n t i t i e s o f g-mannose and 2-acetamido-2-deoxy -B-glucose. Q - M a n n o s y l t r a n s f e r a s e I 1 has been p u r i f i e d f r o m r a b b i t l i v e r microsomes.1060

The enzyme c a t a l y s e s

direct transfer

mannose t o o l i g o s a c c h a r y l - p y r o p h o s p h o r y l f o r m a t i o n of

from

GDP-P-

l i p i d s with the resulting

ct(1+3)-~-mannosyl-~-mannose l i n k a g e s .

A number o f c e l l l i n e s o f r i c i n - r e s i s t a n t

baby- h a m s t e r k i d n e y

318


c e l l s h a v e been a s s a y e d f o r g r o s s c a r b o h y d r a t e c o m p o s i t i o n of c e l l u l a r g l y c o p r o t e i n s , f o r g l y c o s i d a s e and g l y c o s y l t r a n s f e r a s e a c t i v i t y . l o 6 1 None of t h e changes i n g l y c o s y l - t r a n s f e r r e a c t i o n s i n t h e s e l i n e s i s due t o e n h a n c e d g l y c o s i d a s e or s u g a r n u c l e o t i d a s e a c t i v i t i e s i n t h e mutant c e l l s . L a c t o s e s y n t h e t a s e A p r o t e i n from human serum h a s been p u r i f i e d and c h a r a c t e r i z e d 1062 The r e mar kab l e i m muno l o g i c a l d i s s i m i l a r i t y between bovine serum g a l a c t o s y l t r a n s f e r a s e and t h i s enzyme may be r e l a t e d t o major d i f f e r e n c e s i n t h e c a r b o h y d r a t e m o i e t i e s of t h e two enzymes. The b i o s y n t h e s i s of e r y t h r o g l y c a n - l i k e p r o d u c t s b y i n c u b a t i o n of a microsomal f r a c t i o n d e r i v e d from c h r o n i c myelogenous leukaemiad e r i v e d c e l Is w i t h UDP -2-ace t a m i d o -2-deoxy - 9 4 6-3H} - g l u c o s e and UDPQ-{ 6 - 3 H } - g a l a c t o s e h a s been d e s c r i b e d . 1 0 6 3 The h i g h - m o l e c u l a r w e i g h t p r o d u c t s o f t h e t r a n s f e r a s e - c a t a l y s e d r e a c t i o n s were p a r t i a l l y c h a r a c t e r i z e d a f t e r enzymic d e g r a d a t i o n a s a d i - , t r i - , and unidentified oligo-saccharide. The p r e s e n c e of g l y c o s y l t r a n s f e r a s e s on c h r o m a t i n a c c e p t o r s i n monkey l i v e r n u c l e a r membranes h a s b e e n d e m 0 n ~ t r a t e d . l ' ~ ~The e v e n t u a l r o l e of these enzymes i n t h e g l y c o s y l a t i o n of n o n - h i s t o n e proteins is discussed. An a - k - m a n n o s e : B 1 , 2 - ( 2 - a c e t a m i d o - 2 - d e o x y - Q - g l u c o s y l ) t r a n s f e r a s e h a s been i s o l a t e d and p u r i f i e d f r o m p o r c i n e t r a c h e a l mucosa.1065 The enzyme f o r m s 8 1 , 2 bonds b e t w e e n 2 - a c e t a m i d o - 2 d e o x y - Q - g l u c o s e and t e r m i n a l b r a n c h e d p - m a n n o s y l r e s i d u e s o f g l y c o p r o t e i n s and g l y c o p e p t i d e s . I o d i n e i s r e a d i l y i n c o r p o r a t e d f r o m i o d i n e c h l o r i d e i n t o 2g a l a c t o s y l t r a n s f e r a s e from bovine m i l k w i t h t o t a l loss of e n z y m a t i c activity.1066 S u b s t r a t e s and a - l a c t a l b u m i n p r o t e c t a g a i n s t i n a c t i v a t i o n and t h e only amino a c i d modified i s L - t y r o s i n e . An a-Q-galactosyltransferase a c t i v i t y i n E h r l i c h a s c i t e s tumour c e l l s h a s been c h a r a c t e r i z e d . l o 6 ' T h e enzyme h a s b e e n shown t o t r a n s f e r Q - g a l a c t o s y l r e s i d u e s f r o m U D P - Q - g a l a c t o s e t o yacetyl-lactosamine i n t h e s y n t h e s i s of t h e unique t r i s a c c h a r i d e ( 9 9 ) . The e n d o g e n o u s l o c a l i z a t i o n o f U D P - g a l a c t o s e : a s i a l o m u c i n galactosyltransferase a c t i v i t y i n r a t l i v e r endoplasmic reticulum and Golgi a p p a r a t u s has been reported.1068 P a r t i a l l y p u r i f i e d U D P - g a l a c t o s y l t r a n s f e r a s e from bovine m i l k h a s b e e n u s e d t o s y n t h e s i z e m i l l i m o l a r a m o u n t s o f 4-0-B-ggalactopyranosyl-!-glucose, 2-acetamido-2-deoxy-4-~-8-Q-galactosylp- - g l u c o s y l - B - h e x a n o l a m i n e , a n d ~ - 8 - Q - g a l a c t o s y l - ( l + 4 ) - ~ - 8 - 2 -

.

319


acetamido-2-deoxy-~-glucosyl-(l~4)-2-acetamido-2-deoxy-~-~glucose.1069 13C-Enriched Q - g a l a c t o p y r a n o s y l m o i e t i e s o f o l i g o s a c c h a r i d e were a l s o s y n t h e s i z e d . Peptides containing a t r i - & - p r o l y l

t h e same

sequence c a r b o x y l t o an

t h r e o n i n e r e s i d u e c a n be 2 - g l y c o s y l a t e d

&-

by crude e x t r a c t s o f p o r c i n e

s u b m a x i l l a r y g l a n d s c o n t a i n i n g UDP-2-acetamido-2-deoxy-Q-galactose p o l y p e p t i d e 2-acetamido-2-deoxy-~-galactose

t r a n s f erase. l o 7 0

:

Using

i t i s revealed t h a t I-threonine

eleven synthetic peptide substrates,

c a n n o t be g l y c o s y l a t e d w i t h o u t a c a r b o x y l t r i - l - p r o l y l

sequence

and

w i t h o u t b l o c k i n g o f t h e N - t e r m i n a l group o f t h e L-threonine. One f o r m o f 2 - a - C - f u c o s y l t r a n s f e r a s e a n d o n e f o r m o f 3 - a - & f u c o s y l t r a n s f e r a s e h a v e b e e n d e t e c t e d i n h u m a n serum.1071 m i l k c o n t a i n s t h r e e f o r m s o f 3-a-L-fucosyltransferase

Human

and one f o r m

o f 4-a-&-fucosyltransferase. Three a - L - f u c o s y l t r a n s f e r a s e single

enzyme.

a c t i v i t i e s have been p u r i f i e d f r o m

The t h r e e a c t i v i t i e s a p p e a r t o b e c o n t a i n e d i n a

human milk.1072

Evidence

for

two

distinct

3-a-L-fucosyltransferase

a c t i v i t i e s i n h u m a n s a l i v a h a s b e e n r e ~ 0 r t e d . l ' ~ B~ o t h e n z y m e s appear t o c a t a l y s e t h e t r a n s f e r o f I - f u c o s e t o 2 - 3 of 2-acetamido-2deoxy-6-Q-glucosyl

residues o f

blood-group

glycoproteins,

t h e t r a n s f e r a s e dependent on t h e e x p r e s s i o n o f t h e

but only

gene h a s t h e

c a p a c i t y t o t r a n s f e r C-fucose t o 0 - 3 o f g - g l u c o s y l r e s i d u e s . E v i d e n c e f o r t h e e x i s t e n c e o f t h e p o s t u l a t e d UDP-2-acetamido-2: g l y c o p r o t e i n 2-acetamido-2-deoxy-~-glucose

deoxy-e-glucose

phosphotransferase involved

the

i n

has

been

e ~ t a b 1 i s h e d . l ' ~ ~T h i s

phosphorylation

o f

the

high

enzyme

1-

i s

e-mannose

o l i g o s a c c h a r i d e u n i t s o f l y s o s o m a l enzymes. The D - m a n n o s y l 6 - p h o s p h a t e on

lysosomal

m o i e t i e s a c t as r e c o g n i t i o n m a r k e r s The

sequential action o f

marker

i s

synthesized

UDP-2-acetamido-2-deoxy-~-glucose

enzyme 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e

Q-2-acetamido-2-deoxyglucosyl

l-phosphotransferase

phosphodiesterase.

by

the

: lysosomal

and an a-

I t is p r o p o s e d

t h a t t h e t r a n s f e r a s e c a t a l y s e s t h e i n i t i a l d e t e r m i n i n g s t e p by which newly synthesized acid hydrolases are distinguished from other newly synthesized

glycoproteins

and

thus

are

eventually

targeted to

lysosomes. Glycosylated derivatives o f substrates

for

the

lysozyme have been used as

determination

o f

the


o f

s i a l y l t r a n s f e r a ~ e s . ~C~h~a ~ r a c t e r i s t i c d i f f e r e n c e s between v a r i o u s s i a l y l t r a n s f e r a s e s were d e m o n s t r a t e d . Fetal-calf

liver,

embryonic-chicken

brain,

human p l a c e n t a , a n d

320


s e v e r a l o t h e r t i s s u e s c o n t a i n C M P - t j - a c e t y l n e u r a m i n y l : 6-ggalactosyl-(l+ 4~-2-acetamido-2-deoxy-~-glucosyl-a-(2~3)-sialylt r a n ~ f e r a s e . " ~ ~T h i s s i a l y l t r a n s f e r a s e a c t i v i t y a p p e a r s t o be due t o an enzyme w h i c h i s d i s t i n c t from a l l o t h e r known s i a l y l transferases. E l e v a t e d s i a l y l t r a n s f e r a s e a c t i v i t i e s have been d e t e c t e d i n t h e i n t e s t i n a l l y m p h of c o l c h i c i n e - t r e a t e d r a t s . 1077 Enterectomy does not p r e v e n t t h e r i s e of serum s i a l y l t r a n s f e r a s e , s u g g e s t i n g t h a t t h e i n t e s t i n e i s not t h e m a j o r s o u r c e of t h e serum enzyme. C o l c h i c i n e has an e f f e c t o n g l y c o p r o t e i n s y n t h e s i s i n r a t l i v e r G o l g i membranes b y a f f e c t i n g g l y c o ~ y l t r a n s f e r a s e s . ~A~l t~h~o u g h b o t h s i a l y l - and g a l a c t o s y l - t r a n s f e r a s e s a r e i n h i b i t e d , t h e i r s e n s i t i v i t y t o t h e drug d i f f e r s . ( R e f e r e n c e s begin o p p o s i t e )


32 1

Ref erenes

1 B.P. Wasserman and H.P. H u l t i n , Arch. Biochem. Biophys., 1981, 212, 385. 2 A. Ross and I.E.P. Taylor, I n f e c t i o n and Imnunity, 1981, 31, 911. 3 K. Toda, K. On0 and H. O c h i a i , J. Biochem. (Tbkyo),1981, 90, 1429. 4 W. Breuer and C.H. S u i , Proc. Natl. Acad. S c i . USA, 1981, 78, 2115. 5

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m.

fatyo,

Enzymes BY J. F. KENNEDY AND D. P. ATKINS

1

Introduction

--

G e n e r a l A s p e c t s and N o m e n c l a t u r e .

V o l u m e 1 o f ‘Enzymes’ c o v e r s

t h e d e v e l o p m e n t o f enzymology f r o m i t s b e g i n n i n g s i n t h e e i g h t e e n t h century,

r e p r e s e n t e d by t h e s t u d i e s o n d i g e s t i o n by Reaumur a n d

Spallanzani,

t o t h e modern o r g a n i c s y n t h e s i s o f r i b o n u c l e a s e by

M e r r i f ie1d.l

The p a p e r s s e l e c t e d emphasize t h e c h e m i c a l n a t u r e and

mode o f a c t i o n o f e n z y m e s r a t h e r t h a n t h e f i e l d o f i n t e r m e d i a r y metabolism.

More

than

twenty

classic

papers

appear

translation for the f i r s t time,

i n c l u d i n g the work of

P e r s o z on enzyme p u r i f i c a t i o n ,

Schwann on p e p s i n ,

here

i n

Payen and

Berzelius

on

c a t a l y s i s , L i e b i g on t h e n a t u r e o f f e r m e n t a t i o n , Buchner o n c e l l free

fermentation,

F i s c h e r on s t e r e o s p e c i f i c enzyme r e a c t i o n s ,

and

S o r e n s e n on pH. The

publication

I n t e r n a t i o n a l Union o f

by

the

Nomenclature

Biochemistry

(NC-IUB)

Committee

‘Enzyme

Recommendations 1978’ has seen a f u r t h e r s u p p l e m e n t .

of

the

Nomenclature, ‘Supplement

2:

C o r r e c t i o n s and A d d i t i o n s ’ i s t h e second l i s t i n g o f amendments t o ‘Enzyme

N o m e n c l a t u r e 1978’ (Academic P r e s s ,

s u p p l e m e n t was p u b l i s h e d i n Eur.J.Biochem., M e t h o d s o f Assay.

-- ‘M e t h o d s

continues t h e coverage

of

p r e s e n t e d i n Volume 70,

New York, 1980,

1979); t h e f i r s t

104, 1.2

i n E n z y m o l o g y ’ , V o l u m e 73, P a r t B,

general P a r t A.3

immunochemical techniques The p a p e r s i l l u s t r a t e t h e

i n g e n u i t y c h a r a c t e r i s t i c o f w o r k e r s who h a v e a d a p t e d t h e a n t i g e n antibody

reaction to

develop

a variety

of

assays which

are

a p p l i c a b l e t o numerous b i o c h e m i c a l and c l i n i c a l p r o b l e m s . The P r o c e e d i n g s o f t h e 4 t h I n t e r n a t i o n a l Symposium, The N e t h e r l a n d s ,

22-26 June,

1981,

and

t h e s t a t u s o f a f f i n i t y chromatography, increasing importance of a f f i n i t y techniques

b i omedical/diagnos t i c a p p l i c a t i o n s .

Veldhoven,

g i v e a s u r v e y o f new d e v e l o p m e n t s i l l u s t r a t i n g the i n i n d u s t r i a l and


I n Volume 74 o f ‘Methods i n Enzymology’ immunochemical t e c h n i q u e s a r e d e s ~ r i b e d . ~O n e s e c t i o n o f t h i s v o l u m e i s p a r t i c u l a r l y concerned w i t h t h e u s e o f a n t i b o d i e s t o s t u d y enzymes. The l a t e s t v o l u m e i n t h e s e r i e s ‘ A d v a n c e s i n Enzymology a n d R e l a t e d Areas o f M o l e c u l a r B i o l o g y ’ c o n t a i n s a c h a p t e r w h i c h describes t h e use of g l y c o s y l t r a n s f e r a s e s i n t h e assessment o f oligosaccharide structure.6 Kinetics. -- A v e r s a t i l e BASIC p r o g r a m h a s b e e n d e s c r i b e d f o r Direct c u r v e - f i t t i n g i s u n c o n t e s t e d a s t h e ‘Analyzing K i n e t i c method o f c h o i c e f o r e v a l u a t i n g k i n e t i c e x p e r i m e n t s . I f carried o u t by c o m p u t e r , t h e n u m e r i c a l s o l u t i o n o f s u c h p r o b l e m s i s s u p e r i o r t o customary g r a p h i c a l p r o c e d u r e s i n any r e p s e c t . However, most p r o g r a m s a v a i l a b l e t o d a t e c a n n o t b e e x e c u t e d by s m a l l s y s t e m s . T h e a u t h o r s describe a BASIC program f o r d e s k - t o p m i c r o c o m p u t e r s t h a t p e r f o r m s l i n e a r and n o n - l i n e a r r e g r e s s i o n a n a l y s i s o f k i n e t i c data. I t may be d i r e c t l y a p p l i e d t o a n u m b e r o f p r o b l e m s common i n e n z y m e k i n e t i c s , and is e a s i l y modified t o handle f u r t h e r ones. The p e r f o r m a n c e o f t h e p r o g r a m i s i l l u s t r a t e d by s o m e t y p i c a l applications. I n a d d i t i o n , fundamental s t a t i s t i c a l methods are discussed t h a t allow a critical examination of the r e s u l t s obtained. Mechanisms o f A c t i o n . -- A b o o k h a s b e e n p u b l i s h e d o n e n z y m e i n h i b i t o r s w h i c h d e r i v e s f r o m t h e p a p e r s p r e s e n t e d a t t h e 1980 s p r i n g symposium of t h e S w i s s Chemical Society.8 The program c o n s i s t e d o f 21 f u l l p a p e r s a n d 3 a b s t r a c t s f r o m i n d u s t r i a l a n d u n i v e r s i t y l a b o r a t o r i e s r e p o r t i n g on theory, s t r u c t u r e s , syntheses and v i t r o a s well as 2 v i v o e f f e c t s o f mechanism-based enzyme i n h i b i t o r s . These i n c l u d e s u i c i d e s u b s t r a t e s , t r a n s i t i o n - s t a t e a n a l o g u e s , p a r a c a t a l y t i c s e l f i n a c t i v a t o r s , a n d a number o f compounds b e l o n g i n g t o t h e class o f s u b s t r a t e and coenzyme a n a l o g s . The f u l l y indexed publication o f f e r s a wealth of valuable information t o i n v e s t i g a t o r s s e e k i n g novel s t r u c t u r e s i n t h e d e s i g n o f enzyme inhibitors.

-- V o l u m e 5 o f t h e s e r i e s e n t i t l e d ‘ E c o n o m i c M i c r o b i o l o g y ’ c o v e r s m i c r o b i a l e n z y m e s a n d b i o c o n v e r ~ i o n s . ~T h i s volume d i f f e r s from t h e p r e v i o u s volumes i n t h a t i t is concerned o n l y w i t h t h e d e v e l o p m e n t f o r i n d u s t r y o f i n d i v i d u a l e n z y m e s or s h o r t s e q u e n c e s o f enzymes. P a r t i c u l a r l y r e l e v a n t s e c t i o n s are amylases, amyloglucosidases and related glucanases, Q-glucose

Applications.

6: Enzymes

349

o x i d a s e , Q - g l u c o s e d e h y d r o g e n a s e , Q - g l u c o s e i s o m e r a s e , B-Eg a l a c t o s i d a s e and i n v e r t a s e , p e c t i c enzymes, c e l l u l a s e s , immobilized enzymes. A book on c e l l u l a s e and o t h e r n a t u r a l polymer s y s t e m s c o n t a i n s , i n a d d i t i o n t o c h a p t e r s on t h e b i o s y n t h e s i s and s t r u c t u r e o f c e l l u l o s e , c h a p t e r s which d e a l w i t h B-q-glucanases i n h i g h e r p l a n t s and c e l l u l o s e d e g r a d a t i o n . lo The p r o c e e d i n g s of t h e 2 n d Symposium o n Enzyme T h e r a p y i n G e n e t i c D i s e a s e s c o v e r : human t r i a l s : c e l l and o r g a n t r a n s p l a n t a t i o n / o t h e r t h e r a p e u t i c a p p r o a c h e s ; human t r i a l s : d i r e c t enzyme r e p l a c e m e n t ; enzyme m a n i p u l a t i o n : mechanisms and t h e r a p e u t i c t r i a l s ; a n i m a l model s t u d i e s : enzyme e n t r a p m e n t , n e u r a l d e l i v e r y and t r a n s p l a n t a t i o n , enzyme r e c o g n i t i o n and m o d i f i c a t i o n ; enzyme a v a i l a b i l i t y : p u r i f i c a t i o n and c h a r a c t e r i z a t i o n . "

-- A book on t h e u s e s o f i m m o b i l i z e d enzymes f o r food p r o c e s s i n g c o v e r s t h e f o l l o w i n g a r e a s : i m m o b i l i z e d enzyme e n g i n e e r i n g ; r e q u i r e m e n t s u n i q u e t o t h e f o o d and b e v e r a g e i n d u s t r y ; manufacture of h i g h - f r u c t o s e corn s y r u p u s i n g i m m o b i l i z e d g l u c o s e i s o m e r a s e ; p o t e n t i a l and use of i m m o b i l i z e d c a r b o h y d r a s e s ; a p p l i c a t i o n o f l a c t o s e and i m m o b i l i z e d l a c t a s e ; i m m o b i l i z e d p r o t e a s e s - p o t e n t i a l a p p l i c a t i o n , a p p l i c a t i o n and p o t e n t i a l o f aminoacylase, a s p a r t a s e , o t h e r enzymes i n f o o d p r o c e s s i n g : fumarase, glucose o x i d a s e - c a t a l a s e , s u l p h y d r y l o x i d a s e , and c o n t r o l l e d - r e l e a s e enzymes. l 2 I n t h e p r o c e e d i n g s of t h e 1 8 t h Prague IUPAC Micro Symposium on Macromolecules, p u b l i s h e d i n a s e r i e s of j o u r n a l i s s u e s , t h e r e i s a s e c t i o n which d e s c r i b e s t h e i m m o b i l i z a t i o n of enzymes t o d i f f e r e n t p o l y s a c c h a r i d e s by g r a f t cop0 l y mer i za t i o n . l 3 The use of r e v e r s i b l e enzyme i m m o b i l i z a t i o n h a s been r e p o r t e d t o f a c i l i t a t e t h e r e c o v e r y o f p e p t i d e a n t i b i 0 t i ~ s . l ~P r a c t i c a l a s p e c t s o f t h e f a c i l e i m m o b i l i z a t i o n of e n z y m e s on h y d r o u s m e t a l o x i d e s , a w e l l e s t a b l i s h e d means of enzyme-movement r e s t r i c t i o n , a r e described. Various enzymes (e.g. g l u c o a m y l a s e , p e r o x i d a s e , d e x t r a n a s e ) h a v e been i m m o b i l i z e d b y c h e l a t i o n o f s e v e r a l h y d r o u s m e t a l o x i d e s , t h o s e of t i t a n i u m ( 1 V ) and zirconium(1V) p r o v i n g t o be t h e most s a t i s f a c t o r y f o r p r a c t i c a l purposes. M o d i f i c a t i o n of t h e g e l i n t o a g r a n u l a r f o r m c o u l d be a c h i e v e d s u c c e s s f u l l y w i t h good enzyme-immobilization c h a r a c t e r i s t i c s by u s i n g ion-exchange r e s i n a s an i n t e r n a l m a t r i x . The i m m o b i l i z a t i o n p r o c e s s was h i g h l y e f f i c i e n t f o r t h e r e l a t i v e p r o p o r t i o n s of hydrous o x i d e t o enzyme used, w i t h

Enzyme I m m o b i l i z a t i o n .

350


u s u a l l y > 90% of t h e a v a i l a b l e p r o t e i n b e i n g i n s o l u b i l i z e d . R e t e n t i o n o f e n z y m e a c t i v i t y was g e n e r a l l y v e r y g o o d a n d t h e e n z y m e was s t a b l e t o r e u s e a n d t o c o n v e n t i o n a l b u f f e r c o n d i t i o n s . A c t i v i t i e s o f t h e i m m o b i l i z e d e n z y m e s were p a r t i a l l y s t a b l e t o l y o p h i l i z a t i o n or d r y i n g of the hydrous o x i d e g e l s . M o d i f i c a t i o n o f t h e h y d r o u s m e t a l o x i d e s u r f a c e by d r y i n g o r t r e a t m e n t w i t h p h o s p h a t e o r c a r b o n a t e l e d t o a decrease i n c o m p l e x i n g a b i l i t y . The e f f e c t o f c a r b o n a t e c a n b e c i r c u m v e n t e d b y l o w e r i n g t h e pH o f t h e s o l u t i o n t o a r o u n d 5 a n d r e m o v i n g a n y c a r b o n d i o x i d e f o r m e d , by a e r a t i o n . Such t r e a t m e n t a l l o w e d compounds t o c h e l a t e t o hydrous zirconium oxide(1V) i n t h e presence of carbonate, and t h e r e f o r e t h e h y d r o u s o x i d e c o u l d be a p p l i e d s u c c e s s f u l l y t o t h e c o n c e n t r a t i o n o f p e p t i d e a n t i b i o t i c s f r o m t h e f e r m e n t a t i o n medium i n w h i c h t h e y are b e i n g p r o d u c e d , i n c l u d i n g p r o d u c t i o n a t low c o n c e n t r a t i o n s . A s i m p l e a n d n o v e l m e t h o d f o r t h e i m m o b i l i z a t i o n o f e n z y m e s by their c o i m m o b i l i z a t i o n i n hen egg-white u s i n g g l u t a r a l d e h y d e h a s been described.15 Films of highly polymerized collagen prepared under i n d u s t r i a l c o n d i t i o n s by t h e C e n t r e T e c h n i q u e d u C u i r , L y o n , F r a n c e , h a v e b e e n r o u t i n e l y used after a c y l azide a c t i v a t i o n f o r the c o v a l e n t i m m o b i l i z a t i o n o f n u m e r o u s e n z y m e s f r o m d i f f e r e n t classes.16 T h e s t a b i l i t y of t h e r e s u l t i n g membranes t o o p e r a t i o n a l and s t o r a g e conditions and their e x c e l l e n t mechahical s t r e n g t h and r e s i s t a n c e t o bacterial degradation allowed their use for several purposes. Fundamental a s p e c t s of t h e i r heterogeneous enzymology, including The d i f f u s i o n a l e f f e c t s a n d s u b u n i t i n t e r a t i o n s , were e x a m i n e d . e n z y m e - e n g i n e e r i n g a s p e c t was d e v e l o p e d w i t h p o l y m e m b r a n e b i o r e a c t o r s and enzyme e l e c t r o d e s . Besides t h e protein environment p r o v i d e d by t h e c o l l a g e n m a t r i x w h i c h p r e v e n t s e n z y m e i n a c t i v a t i o n , t h e f o r m o f these m e m b r a n e s i s a d v a n t a g e o u s f o r i n d u s t r i a l p u r p o s e s and f o r fundamental research.

2

B-Q-2-Acetamido-2-deoxygalactosidases, 2-deoxyglucosidases, and

B-g-2-Acetamido-

B-Q-2-Acetamido-P-

deoxyhexosidases

The bulk of rat b r a i n n e u t r a l B-Q-2-acetamido-2d e o x y h e x o s i d a s e s h a v e b e e n shown t o be p r e s e n t i n t h e c y t o s o l f r a ~ t i 0 n . l ~ T h e y were n o t b o u n d by c o n c a n a v a l i n A - S e p h a r o s e w h i l e t h e a c i d B-Q-2-acetamido-2-deoxyhexosidases were a l l b o u n d . The

351

6: Enzymes

h a d a pH o p t i m u m o f 5.2

n e u t r a l B-~-2-acetamido-2-deoxyglucosidase andKm

of

0.57

while

mM,

the

neutral

B-Q-2-acetamido-2-

d e o x y g a l a c t o s i d a s e h a d t h e h i g h e s t r e a c t i o n r a t e a t pH 6.0 o f 0.12

No d i v a l e n t

mM.

lost

B-Q-2-acetamido-2-deoxyglucosidase a c t i v i t y

i n

min

30

deoxygalactosidase activity

after

a t

at

The

5OoC.

more

The

5OoC.

was h e a t - s t a b l e h

3

with a

i o n s a c t i v a t e d e i t h e r o f t h e enzymes. than

90% o f

5, The the

€4-D-2-acetamido-2-

and l o s t o n l y 10-20% o f neutral

i t s

B-g-2-acetamido-2-

d e o x y g l u c o s i d a s e was i n h i b i t e d b y f r e e 2 - a c e t a m i d o - 2 - d e o x y - B - Q glucose

but

by 2-acetamido-2-deoxy-B-~-galactose.

not

The r e v e r s e

was f o u n d f o r t h e n e u t r a l ~ - ~ - 2 - a c e t a m i d o - 2 - d e o x y g a l a c t o s i d a s e . Two enzymes were s e p a r a t e d a l m o s t chromatography.

completely

by h y d r o x y a p a t i t e

Heat s t a b i l i t y o f t h e separated a c t i v i t y peaks

s u g g e s t e d t h a t t h e n e u t r a l B-g-2-acetamido-2-deoxygalactosidase, w h i c h was n o t b o u n d t o h y d r o x y a p a t i t e , may,

may b e s p e c i f i c t o t h e Q -

The n e u t r a l B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e

galacto substrate.

o n t h e o t h e r h a n d , h a v e some a c t i v i t y t o w a r d t h e Q - g a l a c t o

substrate.

B o t h o f t h e n e u t r a l enzyme a c t i v i t i e s w e r e shown t o be

h i g h e s t d u r i n g t h e f i r s t p o s t n a t a l week i n r a t b r a i n , t h e a c i d i c enzyme,

i n contrast t o

w h i c h showed peak a c t i v i t i e s d u r i n g t h e second

a n d t h i r d weeks. Activity

levels

i n v e s t i g a t e d

i n

Dugesia lugubris.18

and

cytochemical

i n t a c t

localization

regenerating

have

been

p l a n a r i a n

I n 24 h o u r r e g e n e r a t i n g p l a n a r i a n s t h e 6-B-2-

acetamido-2-deoxyglucosidase lysosomes o f

and was

dedifferentiating

found t o

cells.

be

localized i n the

T h i s B-E-2-acetamido-2-

d e o x y g l u c o s i d a s e l o c a l i z a t i o n c o r r e l a t e d w i t h t h e i n c r e a s e i n B-9-2-

acetamido-2-deoxyglucosidase

and

B-D-2-acetamido-2-

deoxygalactosidase a c t i v i t i e s during the regeneration,

f i r s t

24 h o u r s o f

confirming the involvement o f the d e d i f f e r e n t i a t i o n i n

t h e o r i g i n o f blastema.

I n non-regenerating


activity

demonstrated i n t h e lysosomes o f

has

a n i m a l s t h e B-Q-2-

been

cytochemically

g a s t r o d e r m a l c e l l s and i n t h e

mucous g r a n u l e s o f b a s o p h i l i c s e c r e t o r y c e l l s .

This l a t t e r finding

is i n t e r p r e t e d a s p r o o f o f a s i m i l a r i t y b e t w e e n p l a n a r i a n a n d v e r t e b r a t e mucous s e c r e t i o n . High lysozyme

and c h i t i n a s e a c t i v i t i e s have been f o u n d i n

L e y d i g ’ s o r g a n o f E t m o p t e r u s s p i n a x , Z m n i o s u s m i c r o c e p h a l u s , and Tonpedo n o b i l i a n a ,

i n Leydig’s

and e p i g o n a l o r g a n s and s p l e e n o f

R a j r a d i a t a , and i n t h e e p i g o n a l o r g a n o f R h i n o p t e r a bonasus.” S t r o n g c h i t i n a s e a c t i v i t y w i t h l i t t l e o r n o l y s o z y m e a c t i v i t y was


352 n o t e d i n Leydig's i n

the

and e p i g o n a l o r g a n s o f S c y l i o r h i n u s c a n i c u l a ,

epigonal

organ

GiX.slymostom c i r r a t u m

of

----__-____---------__ Heterodontus francisci.

Very

high

and and

B-Q-2-acetamido-2-

d e o x y g l u c o s i d a s e a c t i v i t y was f o u n d i n l y m p h o m y e l o i d o r g a n s o f a l l s p e c i e s i n v e s t i g a t e d and i n t h e pancreas o f Z m n i o s u s m i c r o c e p h a l u s . Thirteen other and

glycoside hydrolases

d i g e s t i v e

t i s s u e s

were

o f

assayed i n

lymphomyeloid

cLf;at21~,

G i n g L y ~ ~ o s t o m a

H e t e-r o d o~ n t u s -f r a ~ n c i s c-i , a n-d E t m o p t e r u s s p i n a x . A c t i v i t i e s o f a-Qmannosidase, B - g a l a c t o s i d a s e , B-~-2-acetamido-2-deoxygalactosidase, B-!-glucuronidase,

and a- a n d B - n - g l u c o s i d a s e

u s u a l l y were h i g h e r i n

lymphomyeloid t i s s u e s than i n d i g e s t i v e tissues. Nine

glycoside

hydrolases

Trypanosoma b r u c e i b r u c e i S42 h a v e a-Q - -Glucosidase to

those o f

i n

bloodstream

been p a r t i a l l y

forms

o f

characterized.20

had s i m i l a r p h y s i c o c h e m i c a l and e n z y m i c p r o p e r t i e s

a-Q-galactosidase,

- -2-acetamido-2B-Q

f3-g-glucosidase,

d e o x y g l u c o s i d a s e , and B-;-2-acetamido-2-deoxygalactosidase.

I t was

s u g g e s t e d t h a t t h e g l y c o s i d a s e s s t u d i e d may p l a y a r o l e i n t h e turnover o f trypanosomal glycoproteins,

i n particular the variant-

s p e c i f i c surface antigen. a-;-Galactosidase

deficiency

h a s been d e m o n s t r a t e d i n l y m p h o i d

c e l l l i n e s e s t a b l i s h e d by E p s t e i n - B a r r v i r u s t r a n s f o r m a t i o n o f Blymphocytes from a Fabry p a t i e n t , lymphocytes.21

as i n b l o o d w h o l e l e u k o c y t e s a n d

The r e s i d u a l a c t i v i t y

was h e a t s t a b l e .

a-p-2-

A c e t a m i d o - 2 - d e o x y g a l a c t o s i d a s e was p r e s e n t a t n o r m a l l e v e l i n leukocytes, lymphocytes, o r lymphoid c e l l s from n o r m a l and Fabry p a t i e n t s a n d was h e a t s t a b l e i n a l l c a s e s .

a-e-2-Acetamido-2-

d e o x y g a l a c t o s i d a s e e l e c t r o f o c u s i n g p r o f i l e s p r e s e n t e d one m a j o r p e a k (PI 4.5)

and

one

minor

peak

Blood

(PI 5.1).

lymphocytes

and

l y m p h o i d c e l l l i n e s f r o m F a b r y d i s e a s e showed s i m i l a r d e f e c t s o f a l l the forms o f a-g-galactosidase

g r o u p A and t h e r e s i d u a l a c t i v i t y

proceeded f r o m t h e a-!-galactosidase

acetamido-2-deoxygalactosidase.

f o r m 11, w h i c h i s an a-e-2-

I n the

absence

of

animal

models,

t h o s e e s t a b l i s h e d l i n e s seemed t o be an a c c u r a t e c e l l u l a r s y s t e m f o r

-i n vitro

e x p e r i m e n t a l s t u d i e s o f Fabry disease.

The p h y s i o l o g i c a l a c t i v a t o r p r o t e i n f o r t h e d e g r a d a t i o n o f g a n g l i o s i d e GM2 by B-Q-2-acetamido-2-deoxygalactosidase employed t o assess t h e c a p a b i l i t y

of

extracts t o catabolize t h i s ganglioside.22 more

reliable

diagnosis

of

the

T h i s method p e r m i t s a

different

g a n g l i o s i d e s t h a n t h e m e t h o d s h i t h e r t o used. a r t i f i c i a l substrates or,

has been

c u l t u r e d human f i b r o b l a s t variants

of

GM2

These e i t h e r r e l y on

when n a t u r a l s u b s t r a t e s a r e used,

on

6: Enzymes

353

d e t e r g e n t s . The p r e s e n t method avoids a number of p o s s i b l e s o u r c e s o f e r r o r i n t r o d u c e d by t h e u n p h y s i o l o g i c a l d e t e r g e n t s , such a s a l t e r a t i o n o f t h e isoenzymes’ s u b s t r a t e s p e c i f i c i t y o r i n a c t i v a t i o n o f t h e enzymes. The r a n g e o f a p p l i c a t i o n of t h e new method i s discussed. The p a t t e r n s o f B - g - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e excretion a f t e r r e n a l t r a n s p l a n t a t i o n have been i n ~ e s t i g a t e d . ~8-Q-2~ A c e t a m i d o - 2 - d e o x y g l u c o s i d a s e a c t i v i t y was a s s a y e d i n 750 e a r l y morning u r i n e samples from 25 r e n a l - t r a n s p l a n t p a t i e n t s d u r i n g t h e p o s t - o p e r a t i v e p e r i o d . Eighty-four per c e n t of a l l a c u t e r e j e c t i o n e p i s o d e s were p r e c e d e d o r a c c o m p a n i e d b y a g r e a t e r t h a n t w o - f o l d Similar r i s e i n B-~-2-acetamido-2-deoxyglucosidase a c t i v i t y . i n c r e a s e s were caused by d i a l y s i s , gentamicin therapy, and u r e t e r i c dehiscence. O n l y 9% of a l l s i g n i f i c a n t i n c r e a s e s i n B-g-acetamido2 - d e o x y g l u c o s i d a s e e x c r e t i o n c o u l d n o t be a c c o u n t e d f o r b y any o f t h e s e four p r o c e s s e s . Analysis of t h e day-to-day p a t t e r n o f B-g-2a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e a c t i v i t y a s opposed t o i n d i v i d u a l B-Q2-acetamido-2-deoxyglucosidase values provided a c l u e t o t h e o c c u r r e n c e of r e j e c t i o n d u r i n g i m m e d i a t e p o s t - t r a n s p l a n t a t i o n oligur ia. Urinary e x c r e t i o n of B-~-2-acetamido-2-deoxyglucosidase and La l a n i n e a m i n o p e p t i d a s e h a s been measured i n p a t i e n t s r e c e i v i n g a m i k a c i n o r c L ~ - p l a t i n u m . ~U~r i n a r y e x c r e t i o n of a l a n i n e aminopeptidase and B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e was determined f o r 25-70 days i n f i v e p a t i e n t s r e c e i v i n g *-platinum and f o r 8-53 days i n s i x p a t i e n t s r e c e i v i n g amikacin. T h i s s t u d y was performed t o i n v e s t i g a t e i f t h e e x c r e t i o n o f u r i n a r y enzymes r e p r e s e n t s a s e n s i t i v e parameter f o r t h e e a r l y d e t e c t i o n o f t o x i c kidney damage. I n b o t h p a t i e n t g r o u p s an i n c r e a s e i n t h e e x c r e t i o n of t h e t w o enzyme a c t i v i t i e s c o u l d be d e m o n s t r a t e d . I n patients receiving a m i k a c i n , t h e e x c r e t i o n of a l a n i n e aminopeptidase was always h i g h e r t h a n t h a t o f B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e , whereas i n t h r e e p a t i e n t s r e c e i v i n g c i s - p l a t i n u m i t was t h e o p p o s i t e . I n t w o cisp l a t i n u m p a t i e n t s t h e e x c r e t i o n o f b o t h enzymes was o f t h e same s i z e . The c h a n g e s d u r i n g a m i k a c i n t h e r a p y seem t o be r e v e r s i b l e , p a t i e n t s t h e s e c h a n g e s seemed t o be whereas i n f o u r =-platinum partly irreversible. Serum c r e a t i n i n e c o n c e n t r a t i o n was l e s s s e n s i t i v e than t h e u r i n a r y enzyme e x c r e t i o n f o r d e t e c t i o n of kidney damage. P r e d i c t i v e v a l u e s of u r i n a r y B-Q - -2-acetamido-2deoxyglucosidase, i - a l a n i n e aminopeptidase,and B-2-microglobulin

3 54


have been used i n e v a l u a t i n g t h e n e p h r o t o x i c i t y o f g e n t a m i c i n . 2 5 I-slanine

C o n c e n t r a t i o n s o f B-e-2-acetamido-2-deoxyglucosidase, a m i n o p e p t i d a s e , and 1 3 - 2 - m i c r o g l o b u l i n

were

determined

daily

i n the

u r i n e o f 28 p a t i e n t s t r e a t e d w i t h g e n t a m i c i n ( 2 - 3 mg k g - l day-') a mean o f activity

15 days.

A l l

had n o r m a l r e n a l f u n c t i o n .

i n B-Q-2-acetamido-2-deoxyglucosidase

2 o r 3 days o f t r e a t m e n t .

serum

creatinine

levels.

This

The r e s u l t s w e r e c o m p a r e d w i t h

concentrations

study

and I - a l a n i n e

e i t h e r immediately or

a m i n o p e p t i d a s e was o b s e r v e d f o r a l l p a t i e n t s , after

for

Increased

indicates

and a

urinary

B-2-microglobulin

relationship

between

the

n e p h r o t o x i c i t y o f g e n t a m i c i n and i n i t i a l u r i n a r y e n z y m i c a c t i v i t y o f

B-Q-2-acetamido-2-deoxyglucosidase p r i o r t o any t r e a t m e n t . The degree o f B-Q-2-acetamido-2-deoxyglucosidase response d u r i n g the first

t e n d a y s o f t r e a t m e n t a p p e a r e d as a s e c o n d p r o g n o s t i c f a c t o r .

Renal f a i l u r e

was o b s e r v e d f o r o n e o u t

o f the 12 p a t i e n t s w i t h

n o r m a 1 B - Q - 2 - a c e t a m i d o - 2 - d e o x y g 1u c o s id a s e deoxyglucosidasei

< 200 p m o l day").

(B-D,

- 2 - a c e t am i do - 2 -

Seven o f t h e m showed a m a r k e d

enzyme a c t i v i t y r e s p o n s e ( > 1 5 0 0 p m o l day-')

w i t h an i n c r e a s e i n B-

2-microglobulin

the

activity.

Eleven out

of

16 p a t i e n t s

with

elevated

6-Q-2-acetamido-2-de~xyglucosidase~ (6-Q-acetamido-2d e v e l o p e d r e n a l f a i l u r e and d e o x y g l u c o s i d a s e i > 2 0 0 p m o l day") showed an e l e v a t e d m a x i m a l r e s p o n s e .

aminopeptidase

appears

to

be o f

The c o n c e n t r a t i o n o f C - a l a n i n e l i t t l e prognostic value.

v a r i a t i o n i n i n d i v i d u a l maximal urinary

enzyme

The

responses observed

among t h e 28 p a t i e n t s d u r i n g t h e f i r s t t e n days o f t r e a t m e n t p o i n t s t o the existence o f individual s e n s i t i v i t i e s t o gentamicin,

the

e x a c t mechanism o f w h i c h r e m a i n s u n c l e a r . T h e c o n d i t i o n s f o r m a x i m a l a c t i v i t y (pH, substrate concentration, enzyme

activity

concentration)

i n

versus the

range o f

buffer,

saturating

l i n e a r r e l a t i o n s h i p s between

incubation

time

f l u o r i m e t r i c assay

and versus of

several

enzyme

glycoside

h y d r o l a s e s o f l y s o s o m a l o r i g i n i n human p l a s m a and serum h a v e been e ~ t a b l i s h e d . ~T~h e

following

enzymes

were

g a l a c t o s i d a s e , B-Q-2-acetamido-2-deoxyglucosidase, B-Q-glucuronidase,

a-Q-mannosidase,

studied:

a-Q-

8-~-glucosidase,

a-C-fucosidase.

A l l examined

e n z y m e s t u r n e d o u t t o b e m o r e o r l e s s u n s t a b l e u p o n s t o r a g e a t 37'C, 4OC,

a n d -2OOC i n b o t h s e r u m a n d p l a s m a .

Generally t h e degree o f

i n s t a b i l i t y was g r e a t e r i n s e r u m t h a n i n p l a s m a .

The l e v e l s o f some

enzymes, i n c l u d i n g B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e , h i g h e r i n serum t h a n i n plasma. enzymes i n p l a t e l e t - f r e e

were markedly

C o n v e r s e l y t h e l e v e l s o f t h e same

serum e q u a l l e d those i n plasma.

This

6: Enzymes

355

s t r e s s e s t h e n e c e s s i t y t o use f r e s h l y p r e p a r e d p l a s m a f o r l y s o s o m a l g l y c o h y d r o l a s e assay. for

the

assay

the

Under t h e p r o c e d u r a l c o n d i t i o n s recommended methods

for

the

determination of

g l y c o h y d r o l a s e s i n p l a s m a a p p e a r e d t o be s i m p l e ,

lysosomal

s e n s i t i v e , and

reproducible. A

spectrophotometric

assay

for

u r i n a r y B-Q-2-acetamido-2-

d e o x y g l u c o s i d a s e a c t i v i t y h a s been d e v e l o p e d . 2 7

I t i n v o l v e s (a) g e l

f i l t r a t i o n t o s e p a r a t e t h e enzyme f r o m i n h i b i t o r s i n u r i n e , enzy mi c

g l u c o p y r a n o s i d e a t pH 4.4,and 4-nitrophenate.

(c) spectrophotometry o f the l i b e r a t e d

M e a s u r e m e n t s o f a c t i v i t y o f t h e enzyme i n 58 u r i n e

specimens c o r r e l a t e d c l o s e l y e s t a b l i s h e d procedure. was 3.7%.

(b)

4 - n i t r o p h e n y l 2-acetamido-2-deoxy-B-Q-

hydrolysis of

(2

= 0.998)

The w i t h i n - r u n

The b e t w e e n - r u n

with results

by

coefficient of variation

a v e r a g e d 6.8%.

an

( 2 3

Reference values f o r

t h e a c t i v i t y w e r e e s t a b l i s h e d by a s s a y s o r u r i n e s p e c i m e n s f r o m 135 h e a l t h y persons, for

aged t w o weeks t o 5 2 y e a r s .

d e t e c t i o n o f n e p h r o t o x i c i t y was

experimental induction o f

E f f i c a c y o f t h e assay

demonstrated i n r a t s a f t e r

reversible

r e n a l i n s u f f i c i e n c y by

intraperitoneal i n j e c t i o n o f nickel chloride.

Clinical application

o f t h e a s s a y i n a p p r o x i m a t e l y 1000 p a t i e n t s c o r r o b o r a t e d i t s u t i l i t y

f o r d e t e c t i o n and m o n i t o r i n g o f r e n a l d i s o r d e r s . The d i g e s t i o n o f

I-asparagine-linked

B-Q-2-acetamido-2-deoxyglucosidase obtained

from

substrate

fucosidosis


patients

o f

o l i g o s a c c h a r i d e s by e n d o -

i n t h e human s k i n f i b r o b l a s t s has

been

reported.28

The

human m d g - B - & - Z - a c e t a m i d o - 2 -

d e o x y g l u c o s i d a s e was s t u d i e d b y u s i n g t h e h o m o g e n a t e o f c u l t u r e d s k i n f i b r o b l a s t s o f f u c o s i d o s i s p a t i e n t s as an enzyme s o u r c e . results indicate that

biantennary-complex-type

s u g a r c h a i n s as w e l l as h i g h - P - m a n n o s e - t y p e by t h e enzyme a c t i o n .

The

I-asparagine-linked

sugar chains a r e cleaved

None o f t h e s u g a r c h a i n s w i t h a f u c o s y l

r e s i d u e on t h e p r o x i m a l 2 - a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e d i a c e t y l c h i t o b i o s e m o i e t i e s was c l e a v e d .

o f t h e i r N,"-

These r e s u l t s p r o v e d

e n z y m i c a l l y t h e mechanism o f p r o d u c t i o n o f o l i g o s a c c h a r i d e s d e t e c t e d i n t h e u r i n e o f various %-glycosidase

Degradation o f

deficiencies.

mucin oligosaccharides

of

human c o l o n s y s t e m s

has been f o u n d t o be a s s o c i a t e d w i t h e x t r a c e l l u l a r , cell-bound, and

neuraminidase.29

Among

but not with

B-Q-2-acetamido-2-deoxyglucosidase,

B-P-galactosidase,

the

seven

subjects

studied,

the

e s t i m a t e d m o s t p r o b a b l e n u m b e r s (MPN) o f f a e c a l b a c t e r i a p r o d u c i n g e x t r a c e l l u l a r B-Q-galactosidase,

B-~-2-acetamido-2-deoxyglucosidase,

and n e u r a m i n i d a s e r a n g e d f r o m

106-1010g-1

dry

faecal

w t , were


356 comparable

to

significantly

the

MPN

of

mucin-degrading

bacteria,

and

s m a l l e r t h a n t h e MPN o f t o t a l f a e c a l b a c t e r i a .

were These

f i n d i n g s were i n t e r p r e t e d as e v i d e n c e f o r t h e e x i s t e n c e o f b a c t e r i a l sub-populations

i n

the

normal

faecal

flora

that

produce

e x t r a c e l l u l a r g l y c o s i d a s e s , and t h a t t h e s e s u b - p o p u l a t i o n s have a m a j o r r o l e i n d e g r a d i n g t h e complex o l i g o s a c c h a r i d e s o f m u c i n i n t h e g u t lumen. Adjuvant-induced

a r t h r i t i s i n r a t s has been s t u d i e d by t h e

changes i n serum and u r i n a r y p r o t e i n - b o u n d

carbohydrate metabolites,

changes i n serum and t i s s u e l y s o s o m a l g l y c o h y d r o l a s e s and l y s o s o m a l fragility.30

The

investigated, & v

free

activities of

B-Q-glucuronidase,

-

lysosomal glycohydrolases

B-Q-acetamido-2-deoxygluco-

s ida s e , B -Q - a c e t a m ido 2 - d e o x y g 1u c o s id a s e , B - Q - g a l a c t 0 s idase , a-9mannosidase,and

c a t h e p s i n D , a r e i n c r e a s e d i n l i v e r and s p l e e n i n

t h e a c u t e phase.

The f r e e a c t i v i t i e s o f B - Q - g l u c u r o n i d a s e ,

acetamido-2-deoxyglucosidase, change,

whereas

increased. of

and c a t h e p s i n

those o f B-a-galactosidase

D of

kidney

B-a-

showed

and a-p-mannosidase

no are

I n t h e c h r o n i c phase o f t h e d i s e a s e t h e f r e e a c t i v i t i e s

a l l glycohydrolases

are s i g n i f i c a n t l y

Serum g l y c o h y d r o l a s e s a r e s i g n i f i c a n t l y c h r o n i c phases.

increased i n a l l tissues.

i n c r e a s e d i n b o t h a c u t e and

S t u d i e s i n l y s o s o m a l p r e p a r a t i o n s showed i n c r e a s e d

f r a g i l i t y of l y s o s o m e s d e r i v e d f r o m l i v e r and k i d n e y o f a r t h r i t i c r a t s i n b o t h phases o f t h e d i s e a s e . The s u b u n i t a n d p o l y p e p t i d e s t r u c t u r e o f B - Q - 2 - a c e t a m i d o - 2 d e o x y g l u c o s i d a s e s f r o m human p l a c e n t a has been r e p o r t e d . 3 1

Previous

r e p o r t s have i n d i c a t e d t h a t t h e B-~-2-acetamido-2-deoxyglucosidases a r e t e t r a m e r i c enzymes composed o f e i t h e r t w o d i m e r s o f @ - c h a i n s (B-

Q - 2 - a c e t a m i d o - 2 - d e o x ~ y g l u c o s i d a s e B) o r a B - c h a i n d i m e r and an a c h a i n d i m e r ( B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e A). 6-a-2Acetamido-2-deoxyglucosidase S c o n t a i n s o n l y a-chains. The a p p a r e n t m o l e c u l a r w e i g h t o f b o t h a- and B - c h a i n s i s 25,000 d a l t o n . 8-e-2Acetamido-2-deoxyglucosidase A and 6 were i s o l a t e d and p u r i f i e d more A prepared t h a n 6 0 0 0 - f o l d f r o m B-Q-2-acetamido-2-deoxyglucosidase and a more a n o d i c a l l y S,

m i g r a t i n g B-~-2-acetamido-2-deoxyglucosidase

which contained o n l y a-chains.

After either extensive reduction

and a l k y l a t i o n o r p e r f o r m i c a c i d o x i d a t i o n ,

B-Q-2-acetamido-2-

d e o x y g l u c o s i d a s e B gave o n l y a s i n g l e p r o t e i n band i n p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n sodium dodecyl sulphate. deoxyglucosidase

A

consistently

gave

two

B-a-2-Acetamido-2-

bands,

c o n t a i n e d chains o f two d i f f e r e n t m o l e c u l a r weights.

indicating

it

Other r e p o r t s

have n o t e d t w o bands i n B-Q-2-acetamido-2-deoxyglucosidase

A but

357

6: Enzymes have d i s m i s s e d t h e h i g h - m o l e c u l a r - w e i g h t either

band as a d i m e r c a u s e d by

h y d r o p h o b i c i n t e r a c t i o n o r an u n b r o k e n d i s u l p h i d e bond.

r u l e out

hydrophobic interaction,

the

molecular

weight

d e t e r m i n e d by g e l f i l t r a t i o n i n 6 M g u a n i d i n i u m c h l o r i d e . complete disulphide-bond

breakage,

To was

To e n s u r e

a h a r s h e r r e d u c t i o n and

a l k y l a t i o n p r o c e d u r e t h a n p r e v i o u s l y e m p l o y e d b y o t h e r s was u s e d . The r e s u l t s w e r e t h e n c o n f i r m e d b y t h e u s e o f t w o p e r f o r m i c a c i d o x i d a t i o n techniques, t h e s t r o n g e r method r e s u l t i n g i n e x t e n s i v e Samples o f B-Q-2-acetamido-2-deoxygluco-

peptide-bond oxidation. sidase A

under

either

r e d u c t i o n and

a l k y l a t i o n or

the

weaker

p e r f o r m i c a c i d o x i d a t i o n p r o c e d u r e a g a i n c h r o m a t o g r a p h e d as t w o approximately

e q u a l peaks

(50,000

B-D-2-Acetamido-2-

and 25,000).

d e o x y g l u c o s i d a s e B and t h e B - Q - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e prepared from 25,000

$-~-2-acetamido-2-deoxyglucosidase

d a l t o n peak.

S chromato-

The a u t h o r s c o n c l u d e t h a t e i t h e r

i n B-~-2-acetamido-2-deoxyglucosidase w i t h one a - c h a i n

w e i g h t o f 50,000

B

r e s u l t e d i n one

B-~-2-Acetamido-2-deoxyglucosidase

g r a p h e d a s a s i n g l e 50,000 p e a k . the a-chain

A

per a-subunit

d a l t o n c h a i n s a r e u n i t e d by a n o n - d i s u l p h i d e

has a m o l e c u l a r

o r t h a t t w o 25,000 c r o s s l i n k (-

an

i s o p e p t i d e bond). The d i s a c c h a r id e 2 - a c e t a m i d o -2-deox y - B - Q - g l u c o p y r a n o s y l - ( 1 + 3 ) -

a-{ l - 3 H ) - g a l a c t i t o l ,

prepared from keratan sulphate,

h a s been f o u n d

t o be r a p i d l y h y d r o l y s e d by t h e A a n d B i s o e n z y m e s o f n o r m a l human l i v e r B-~-2-acetamido-2-deoxyglucosidase and by t h e B i s o e n z y m e p r e p a r e d f r o m t h e l i v e r o f a Tay-Sachs

disease patient.32

The

d i s a c c h a r i d e s u b s t r a t e was a l s o h y d r o l y s e d b y e x t r a c t s o f n o r m a l , cultured-skin fibroblasts, Sachs d i s e a s e ,

and f i b r o b l a s t s o f p a t i e n t s w i t h Tay-

w h e r e a s i t was n o t h y d r o l y s e d by f i b r o b l a s t e x t r a c t s

of p a t i e n t s w i t h Sandhoff disease.

Thus,

defective degradation o f

k e r a t a n sulphate, secondary t o a d e f e c t o f t h e subunits present i n the

A

and B i s o e n z y m e s o f B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e ,

contribute

to

the

appearance

of

skeletal

lesions

i n

may

patients

a f f e c t e d by Sandhoff disease. The p h o s p h o l i p i d a c y l - c h a i n

d e p e n d e n c e o f t h e membrane-bound

l y s o s o m a l B-Q-2-acetamido-2-deoxyglucosidase c o n t r o l membranes f r o m r a t

h a s been e x a m i n e d on

b r a i n p r i m a r y c e l l c u l t u r e s and on

membranes m o d i f i e d by c u l t u r i n g t h e c e l l s i n m e d i a s u p p l e m e n t e d w i t h polyunsaturated fatty

a c i d s .33

acetamido-2-deoxyglucosidase acyl-chain

The r e l a t i o n s h i p b e t w e e n $ - Q - 2 -

a c t i v i t y and t h e membrane p h o s p h o l i p i d

c o m p o s i t i o n has been e v a l u a t e d .

An i n c r e a s e i n t h e

u n s a t u r a t i o n l e v e l of p h o s p h a t i d y l e t h a n o l a m i n e s and p h o s p h a t i d y l -

Carbohydrate Chemistry cholines,

t h e m o s t a b u n d a n t p h o s p h o l i p i d s i n t h i s membrane f r a c t i o n ,

i s r e l a t e d t o the enzymic reaction.

The A r r h e n i u s p l o t o f t h e

enzyme a c t i v i t y i n m o d i f i e d membranes showed break-temperatures, s t a r t i n g f r o m a p p r o x i m a t e l y 1 5 'C. below

and above t h e

The a p p a r e n t a c t i v a t i o n e n e r g y

break-temperature

was

not correlated with

phospholipid acyl-chain unsaturation.

Q -Man n o s e , m e t h y 1- a - g

- man n o p y r a n o s i d e ,

mannose have

been f o u n d t o

inhibit

glucosidase B

p u r i f i e d from

both

and 2 - a m i n o -2-deox y -#-

B-Q-2-acetamido-2-deoxy-

porcine

placenta

and

bovine

e p i d i d y m i ~ . ~The ~ i n h i b i t i o n i s c o m p e t i t i v e and t h e i n h i b i t o r y c o n s t a n t s a r e a b o u t 20 m M f o r

g-mannose and m e t h y l - a - 1 - m a n n o p y r a n o -

s i d e and 2 m M f o r 2 - a m i n o - 2 - d e o x y - ~ - m a n n o s e . o f t h i s i n h i b i t o r y e f f e c t i s discussed. The c h a n g e s

of

sulphate-containing

The p h y s i o l o g i c a l r o l e glycosaminoglycans

and

g l y c o s a m i n o g l y c a n a s e s d u r i n g b o v i n e f o e t a l development have been

t was shown t h a t a n a l y ~ e d . ~I ~

chondroitin 6-sulphate

increases i n

c o n c e n t r a t i o n up t o t h e 5 0 t h day o f f o e t a l d e v e l o p m e n t a n d t h e n decreases p r o g r e s s i v e l y u n t i l i t s complete disappearance i n most adult tissues.

Likewise,

h y a l u r o n i d a s e a l s o r e a c h e s a p e a k on t h e

5 0 t h day and d e c r e a s e s i n a c t i v i t y u n t i l i t s d i s a p p e a r a n c e i n a d u l t tissues.

On t h e o t h e r hand,

as w e l l as B - P - g l u c u r o n i d a s e

heparan s u l p h a t e and d e r m a t a n s u l p h a t e and B-~-2-acetamido-2-deoxyglucosidase

r e m a i n w i t h o u t s i g n i f i c a n t changes d u r i n g t h e w h o l e p e r i o d . f o e t a l chondroitin 6-sulphate molecular

weights

properties of described.

depending on t h e t i s s u e o f

foetal

The

i s tissue specific with different

m u s c l e - and

origin.

brain-hyaluronidase

Some

are

The p o s s i b l e r o l e o f c h o n d r o i t i n 6 - s u l p h a t e

also and

h y a l u r o n i d a s e i n t h e p r o c e s s e s o f d i f f e r e n t i a t i o n and d i v i s i o n i s discussed i n view o f these f i n d i n g s . The a - k - f u c o s i d a s e , mannosidase,

a-l-arabinofuranosidase muscle o f

B-~-2-acetamido-2-deoxyglucosidase,

B-p-glucuronidase,

B-;-xylosidase,

activities

of

d e v e l o p i n g and a d u l t

B-Q-glucosidase,

a-p-

and

n o r m a l and a t r o p h i c s k e l e t a l

r a t s have been c o l l e c t i v e l y

i n v e s t i g a t e d . 36

B-;-2-Acetamido-2-deoxyglucosidase mouse t e s t e s .

h a s been c h a r a c t e r i z e d f r o m

Only one o f t h e t w o i s o z y m e s o f B-Q-2-acetamido-2-

d e o x y g l u c o s i d a s e i n t e s t e s was o b t a i n e d i n t h e f i n a l p r e p a r a t i o n , having a specific

activity

of

4.44

p u r i f i e d e n z y m e s h o w e d a I&, o f 0.24 mg-l

protein.

u n i t s mg-l mM and

The o p t i m u m t e m p e r a t u r e

t e m p e r a t u r e (L112)

protein.37

The

lmax o f 0.165 pM m i n - '

i s 6OoC a n d t r a n s i t i o n

f o r h e a t d e n a t u r a t i o n i s 63OC.

The o p t i m u m pH o f

359

6: Enzymes the

enzyme

i s

filtration, potent

4.5.

The

molecular

inhibitors

weight,

dalton.

c o r r e s p o n d s t o 178,000

by

Ag',

G-200

a n d PCMB a r e

o f B-~-2-acetamido-2-deoxyglucosidase,

e t h y l m a l e i m i d e and 2 - a c e t a m i d o - 2 - d e o x y - Q - g l u c o s e enzyme a c t i v i t y .

Sephadex

Hg2',

though

3-

also i n h i b i t the

P r o t e c t i v e a c t i o n by L - c y s t e i n e f o r t h e i n h i b i t i o n

by HgC12 s u g g e s t s t h e r o l e o f a t h i o l g r o u p a t t h e c a t a l y t i c s i t e o f t h e enzyme.

The p u r i f i e d e n z y m e d o e s n o t c r o s s - r e a c t i n i m m u n o -

d i f f u s i o n p l a t e s w i t h anti-mouse t e s t i c u l a r h y a l u r o n i d a s e serum, s u g g e s t i n g t h a t B-Q-2-acetamido-2-deoxyglucosidase

does n o t have

common a n t i g e n i c d e t e r m i n a n t s i n h y a l u r o n i d a s e o f h y a l u r o n i d a s e - B - g complex o f acrosome o r t e s t e s .

2- a c et am i d o - 2 - d eo x y g l u c o s i d a s e

The a c t i v i t i e s o f v a r i o u s g l y c o s i d a s e s i n h o m o g e n a t e s o f t h e small

i n t e s t i n a l mucosa o f

wallabies

eurgenii)

(M.

two

aged

adults from

6

a n d 1 8 s u c k l i n g tamrnar to

i n v e ~ t i g a t e d . ~B ~- g - G a l a c t o s i d a s e , deoxyglucosidase,

a-k-fucosidase,and

50

weeks

have

been

B-Q-2-acetamido-2-

neuraminadase a c t i v i t i e s were

h i g h d u r i n g t h e f i r s t 3 4 weeks p o s t p a r t u m and t h e n d e c l i n e d t o v e r y low levels.

a-p-Glucosidase,

aa-trehalase

a c t i v i t i e s were v e r y l o w or a b s e n t d u r i n g t h e f i r s t 34

weeks,

l i m i t dextrinase,

and t h e n i n c r e a s e d .

a-p-glucosidase,

The B - Q - g a l a c t o s i d a s e

activity

and was

unusual i n being greater i n t h e d i s t a l than t h e middle or p r o x i m a l thirds o f the intestine,

a n d i n i t s l o w pH o p t i m u m ( p H 4.6),

i n h i b i t i o n by 4-chloromercuribenzene t h e l a c k o f 6-0-glucosidase

i t s

s u l p h o n a t e b u t n o t by T r i s ,

activity.

and

These p r o p e r t i e s w e r e t h o s e

o f a lysosomal a c i d B-Q-galactosidase r a t h e r than o f a brush border n e u t r a l B-g-galactosidase. characteristics

of

a

The a - q - g l u c o s i d a s e

lysosomal

acid

a c t i v i t y had t h e

a-a-glucosidase

early

l a c t a t i o n and o f a b r u s h b o r d e r n e u t r a l a - Q - g a l a c t o s i d a s e animals.

The s i g n i f i c a n c e o f

these findings

was

in

i n adult

discussed

i n

r e l a t i o n t o changes i n d i e t a r y c a r b o h y d r a t e s d u r i n g w e a n i n g and t o t h e mode o f d i g e s t i o n o f m i l k c a r b o h y d r a t e s by t h e p o u c h young. The

bulk

of

r a t

brain

neutral

B-g-2-acetamido-2-

deoxyhexosidases

have been shown t o

be p r e s e n t

f r a ~ t i 0 n . l ~ They

w e r e n o t bound by c o n c a n a v a l i n A-Sepharose

t h e a c i d B-P-2-acetamido-2-deoxyhexosidases

i n the

cytosol while

were a l l bound.

The

n e u t r a l B-~-2-acetamido-2-deoxyglucosidase h a d a pH o p t i m u m o f 5.2 and

Em

of

0.57

mM,

while

the

neutral

B-Q-2-acetamido-2-

d e o x y g a l a t o s i d a s e h a d t h e h i g h e s t r e a c t i o n r a t e a t pH 6.0 of

0.12

mM.

B-Q-2-acetamido-2-deoxyglucosidase a c t i v i t y

with a

No d i v a l e n t i o n s a c t i v a t e d e i t h e r o f t h e enzymes.

i n

30

min

a t

5OoC.

lost The

more

than

90%

of

Em The the

B-D-2-acetamido-2-


360 deoxygalactosidase activity

after

3

was h e a t - s t a b l e h

at

5OoC.

and l o s t o n l y

The

neutral

10-20% o f

i t s

B-Q-2-acetamido-2-

d e o x y g l u c o s i d a s e was i n h i b i t e d b y f r e e 2 - a c e t a m i d o 2 - d e o x y - B - Q-glucose but not

by

The r e v e r s e

2-acetamido-2-deoxy-B-~-galactose.

was f o u n d f o r t h e n e u t r a l B-~-2-acetamido-2-deoxygalactosidase. enzymes were s e p a r a t e d a l m o s t chromatography.

Heat s t a b i l i t y

completely

by

Two

hydroxyapatite

o f t h e separated a c t i v i t y peaks

suggested t h a t t h e n e u t r a l B-g-2-acetamido-2-deoxygalactosidase, w h i c h was n o t b o u n d t o h y d r o x y a p a t i t e , may,

on t h e o t h e r hand,

substrate.

may b e s p e c i f i c t o t h e Q -

The n e u t r a l B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e

galacto substrate. Both of

h a v e some a c t i v i t y t o w a r d t h e Q - g a l a c t o

t h e n e u t r a l enzyme a c t i v i t i e s w e r e shown t o be

h i g h e s t d u r i n g t h e f i r s t p o s t n a t a l week i n r a t b r a i n , i n c o n t r a s t t o t h e a c i d enzyme,

w h i c h showed peak a c t i v i t i e s d u r i n g t h e s e c o n d and

t h i r d weeks. The

separation

and

properties

of

B-e-2-acetamido-2-

d e o x y g l u c o s i d a s e A , B, a n d I f r o m h o r s e b r a i n h a v e b e e n r e p ~ r t e d . ~ ’ Three forms

of

B-~-2-acetamido-2-deoxyglucosidase

been s e p a r a t e d f o r chromatography

(A,

B, and

I) h a v e

t h e f i r s t t i m e f r o m h o r s e b r a i n by i o n - e x c h a n g e on

DEAE-cellulose.

Form

I has

properties

i n t e r m e d i a t e i n t h e e l u t i o n p o s i t i o n f r o m D E A E - c e l l u l o s e PI and thermal s t a b i l i t y , f o r m s A a n d B.

under t h e assay c o n d i t i o n s ,

between those o f

A f t e r i s o e l e c t r i c f o c u s i n g , e s p e c i a l l y f o r m s A and I

a r e t r a n s f o r m e d i n t o more t h e r m o s t a b l e f o r m s . deoxy-a-mannose,

Q-Mannose, 2 - a m i n o - 2 methy1-a-Q-

2-acetamido-2-deoxy-Q-g1ucose,and

mannopyranoside have been f o u n d t o i n h i b i t t h e t h r e e above-mentioned forms.

Q-Glucose

inhibitors

for

and 2-amino-2-deoxy-!-galactose

B-!-2-acetamido-2-deoxyglucosidase

w e r e weak

B a n d I.

52-

G l u c u r o n i c a c i d was an i n h i b i t o r f o r f o r m s A and I. The m e t a b o l i c r o l e o f t h e s e i n h i b i t i o n s on s p e c i f i c i t i e s o f B-e-2-acetamido-2deoxyglucosidase towards n a t u r a l s u b s t r a t e s i s discussed. The i n d u c t i o n o f B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e

activity in

t h e s u b m a x i l l a r y g l a n d s o f s t r e p t o z o t o c i n d i a b e t i c m i c e has been reported.40

S t r e p t o z o t o c i n d i a b e t e s s t r o n g l y r e d u c e d t h e B-Q-2-

a c e t a mido-2-deoxyg lucosidase mice.

a c t i v i t y i n the submaxillary glands o f

A r e c i p r o c a l change was o b s e r v e d b e t w e e n t h e enzyme a c t i v i t y

and b l o o d s u g a r i n d i a b e t e s .

I n s u l i n t r e a t m e n t o f t h e d i a b e t i c mice

returned the reduced a c t i v i t y t o t h e normal l e v e l . f o c u s i n g a n a l y s i s showed t h a t t h e s u b m a x i l l a r y - g l a n d

Isoelectrice n z y m e was

composed o f t w o i s o z y m e s h a v i n g i s o e l e c t r i c p o i n t s (PI) o f 4.8 8.8.

O n l y t h e PI 8.8 i s o z y m e was a f f e c t e d by t h e d i a b e t e s .

and

361

6: Enzymes Two

major

isoenzymes,

and

A

of

B,

B-Q-2-acetamido-2-

d e o x y g l u c o s i d a s e have been p u r i f i e d f r o m a d u l t - r a t muscle.41

gastrocnemius

S t u d i e s w e r e a l s o done o n a d u l t - r a t

a t r o p h i c muscle a f t e r

u n i l a t e r a l neurectomy o f t h e s c i a t i c nerve.

I n b o t h n o r m a l and

a t r o p h i c t i s s u e s t h r e e a d d i t i o n a l m i n o r f o r m s o f t h e enzyme w e r e identified. Sucrose

density

lysosome-containing

-----calcitrans

L.42

galactosidase,

B-e - g l u c u r

gradients

have

been

used t o

fractions from 0 h white

The a c i d i c g l y c o s i d a s e s a-;-mannosidase,

o n i dase, a n d

characterize

prepupae o f

Stomoxys

(a-Q-glucosidase,

B-g-glucosidase,

a-Q-

B-P-galactosidase,

B-Q-2-acet amido-2-deoxyglucosidase)

e q u i l i b r a t e a t t h e same d e n s i t y a s d o e s a c i d p h o s p h a t a s e .

The

m i t o c h o n d r i a 1 m a r k e r enzyme c y t o c h r o m e o x i d a s e a l s o e q u i l i b r a t e s a t t h e same d e n s i t y .

Gomori s t a i n i n g showed t h e p r e s e n c e o f

acid

phosphatase i n t h e lysosomes. B-~-2-Acetamido-2-deoxyglucosidase from

the digestive gland of

Mercenaria

h a s been p a r t i a l l y p u r i f i e d

t h e t h r e e c o a s t a l New E n g l a n d b i v a l v e s

m e r c e n a r i a , S p i s u l a s o l i d i s s i m a , a n d blya a r e n a r i a a n d

t h e i r p r o p e r t i e s h a v e been compared.

H e a t - i n a c t i v a t i o n s t u d i e s on

t h e B-Q-2-acetamido-2-deoxyglucosidase

p r e i n c u b a t e d a t 45OC r e v e a l e d

t h a t t h e p r e p a r a t i o n f r o m S. w h i l e t h a t f r o m M.

s o l i d i s s i m a was s t a b l e u p t o 6 0 min,

a r e n a r i a and M.

m e r c e n a r i a l o s t 47 a n d 9 1 % o f

t h e i r o r i g i n a l a c t i v i t i e s under these c o n d i t i o n s ,

respectively.

I n h i b i t i o n s t u d i e s i n d i c a t e d t h a t ~ - g l u c u r o n o - 6 , 3 - l a c t o n e i s more i n h i b i t o r y t o w a r d s t h e M. a r e n a r i a enzyme,

w h i l e HgC12 a p p e a r s t o be

l e s s i n h i b i t o r y t o w a r d s t h e S. s o l i d i s s i m a enzyme. for

B-~-2-acetamido-2-deoxyglucosidase Other

deoxyglucosidase

M.

The

lma value

mercenaria

was

g r e a t e r t h a n t h a t f r o m S. s o l i d i s s i r n a a n d

approximately 2.5-fold

M. a r enaria.

from

characteristics

such

as

pH

of

the

Km,

optimum,

B-Q-2-acetamido-2mol.

wt.,

energy

o f

a c t i v a t i o n , and e f f e c t o f i o n i c s t r e n g t h o n enzyme a c t i v i t y w e r e f o u n d t o be s i m i l a r f o r a l l t h r e e s p e c i e s . B-;-2-Acetamido-2-deoxyglucosidase

and B-g-2-acetamido-2-deoxy-

g a l a c t o s i d a s e a c t i v i t y l e v e l s and c y t o c h e m i c a l l o c a l i z a t i o n have been

investigated i n

Dugesia Lugubris.18

i n t a c t

and

regenerating

planarian

I n 2 4 h r e g e n e r a t i n g p l a n a r i a n s t h e B-Q-2-


was

lysosomes of

cells.

dedifferentiating

found

to

be

localized i n the

The B-Q-2-acetamido-2-deoxy-

glucosidase l o c a l i z a t i o n i n d e d i f f e r e n t i a t i n g c e l l s , correlated w i t h a n d B-Q-2i n c r e a s e i n B-Q-2-acetamido-2-deoxyglucosidase a c e t a m i d o - 2 - d e o x y g a l a c t o s i d a s e a c t i v i t i e s d u r i n g t h e f i r s t 24 h o f

the

362


r e g e n e r a t i o n , c o n f i r m s t h e involvement o f t h e d e d i f f e r e n t i a t i o n i n t h e o r i g i n of b l a s t e m a . I n n o n - r e g e n e r a t i n g a n i m a l s t h e B-Q-2acetamido-2-deoxyglucosidase a c t i v i t y h a s been c y t o c h e m i c a l l y d e m o n s t r a t e d i n t h e l y s o s o m e s o f g a s t r o d e r m a l c e l l s and i n t h e mucous g r a n u l e s of b a s o p h i l i c s e c r e t o r y c e l l s . T h i s l a t t e r f i n d i n g i s i n t e r p r e t e d a s p r o o f of a s i m i l a r i t y b e t w e e n p l a n a r i a n and v e r t e b r a t e mucous s e c r e t i o n . Nine g l y c o s i d a s e s i n b l o o d s t r e a m f o r m s of Trypanosoma ------------b r u c e i b r u c e i S42 have been p a r t i a l l y c h a r a c t e r i z e d . a-gG l u c o s i d a s e had s i m i l a r p h y s i o c h e m i c a l and e n z y m i c p r o p e r t i e s t o t h o s e o f a - Q - g a l a c t o s i d a s e , B - Q - g l u c o s i d a s e , B-Q-Z-acetamido-2d e o x y g l u c o s i d a s e and B - ~ - 2 - a c e t a r n i d o - 2 - d e o x y g a l a c t 0 s i d a s e . ~ ~ a-QMannosidase was c l e a r l y d i s t i n c t from t h e o t h e r g l y c o s i d a s e s w i t h r e s p e c t t o pH optimum, t h e r m o s t a b i l i t y , s p e c i f i c a c t i v i t y d u r i n g t h e c o u r s e of p a r a s i t a e m i a , and s u b c e l l u l a r l o c a t i o n . I t was s u g g e s t e d t h a t t h e g l y c o s i d a s e s s t u d i e d may p l a y a r o l e i n t h e t u r n o v e r of trypanosomal glycoproteins, i n p a r t i c u l a r t h e v a r i a n t - s p e c i f i c s u r f ace a n t i g e n . A c o m p a r a t i v e s t u d y of B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e from M e r c e n a r i a m e r c e n a r i a , Mya a r e n a r i a , a n d S p i s u l a s o l i d i s s i m a h a s been made.43 A marked i n c r e a s e i n B - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e a c t i v i t y h a s b e e n o b s e r v e d i n t h e g e r m i n a t i n g c o t y l e d o n of c o t t o n s e e d s . 4 4 The enzyme was i s o l a t e d 3 5 , and 2 0 % was i n s o l u b l e m a t e r i a l t h a t r e s e m b l e d yeast-bud s c a r s m i c r o s c o p i c a l l y . C e l l - f r e e e x t r a c t s , membranous f r a c t i o n s , and c e l l - w a l l p r e p a r a t i o n s from Schizosaccharomyces p o m k were examined f o r t h e p r e s e n c e of (1 + 3 ) - a - and (1 + 6)-B-D-glucanase a c t i v i t i e s . 3 8 1 The v a r i o u s g l u c a n a s e s were assayed i n c e l l s a t d i f f e r e n t growth s t a g e s . O n l y ( 1 + 3)-B-;-glucanase a c t i v i t y was f o u n d , a n d t h i s w a s associated with the cell-wall fraction. Chromatographic f r a c t i o n a t i o n of t h e c r u d e enzyme r e v e a l e d t w o e n d o - ( 1 + 3>-8-Qg l u c a n a s e s , d e s i g n a t e d a s g l u c a n a s e I and g l u c a n a s e 11. Glucanase I c o n s i s t e d o f two s u b u n i t s o f m o l e c u l a r w e i g h t s 7 8 , 5 0 0 and 8 2 , 0 0 0 , and g l u c a n a s e I 1 was a s i n g l e p o l y p e p t i d e o f 7 5 , 0 0 0 . A l t h o u g h b o t h enzymes had s i m i l a r s u b s t r a t e s p e c i f i c i t i e s and s i m i l a r h y d r o l y t i c a c t i o n on l a m i n a r i n , g l u c a n a s e I 1 had much h i g h e r h y d r o l y t i c On t h e b a s i s of a c t i v i t y on i s o l a t e d c e l l w a l l s of S. pombe. d i f f e r e n t i a l l y t i c a c t i v i t y on c e l l w a l l s , g l u c a n a s e I1 was shown t o be p r e s e n t i n c o n j u g a t i n g c e l l s and h i g h e s t i n s p o r u l a t i n g cells. Glucanase I1 appeared t o be s p e c i f i c a l l y i n v o l v e d i n c o n j u g a t i o n and s p o r u l a t i o n s i n c e v e g e t a t i v e c e l l s and n o n - c o n j u g a t i n g and nons p o r u l a t i n g c e l l s d i d n o t c o n t a i n t h i s enzyme. The a p p e a r a n c e o f g l u c a n a s e I 1 i n c o n j u g a t i n g c e l l s may be due t o de novo enzyme s y n t h e s i s s i n c e no a c t i v a t i o n c o u l d b e d e m o n s t r a t e d b y c o m b i n i n g e x t r a c t s from v e g e t a t i v e and c o n j u g a t i n g c e l l s . I n c r e a s e d g l u c a n a s e a c t i v i t y o c c u r r e d when w a l l s from c o n j u g a t i n g c e l l s were combined w i t h w a l l s from s p o r u l a t i n g c e l l s . S t u d i e s w i t h t r y p s i n and p r o t e o l y t i c i n h i b i t o r s s u g g e s t t h a t g l u c a n a s e 11 e x i s t s a s a zymogen i n conjugating c e l l s . A t e m p e r a t u r e - s e n s i t i v e mutant of S. pombe was i s o l a t e d which l y s e d a t 37OC. Glucanase a c t i v i t y was h i g h e r i n v e g e t a t i v e c e l l s h e l d a t 3 7 O C t h a n c e l l s h e l d a t 25'C. Unlike t h e w i l d - t y p e s t r a i n , t h i s mutant c o n t a i n e d g l u c a n a s e I1 a c t i v i t y d u r i n g v e g e t a t i v e growth and may be a r e g u l a t o r y mutant.

502


=-(

1

+

3)-8-g-Glucanases

I t h a s been r e p o r t e d t h a t g l y c o s y l a t i o n i s n o t n e c e s s a r y f o r the secretion of gxg-(l + 3 ) - B - D = - g l u c a n a s e by ----Saccharomyces c e r e v i s i a e p r o t o p l a s t s . 3 8 2 I t was shown t h a t t h e normal s y n t h e s i s and s e c r e t i o n of t h e major z - ( l + 3)-B-D-glucana s e s e c r e t e d b y S. c e r e v i s i a e p r o t o p l a s t s w e r e a f f e c t e d b y t u n i c a m y c i n , and a new, presumably n o n - g l y c o s y l a t e d form i s e x p o r t e d t o t h e c e l l u l o s e medium. A s i m i l a r f o r m was a l s o s e c r e t e d i n t h e p r e s e n c e of 2-deoxy-q-glucose. P a r t i a l p u r i f i c a t i o n of e n d o - and a - ( l + 3 ) - B - Q - g l u c a n a s e enzymes from Zea mays s e e d l i n g s and t h e i r involvement i n c e l l - w a l l a u t o h y d r o l y s i s have been i n v e s t i g a t e d . 3 8 3 Molecular-sieve chromatography of t h e c e l l - w a l l p r o t e i n r e s o l v e d endo-B-Q-glucanase and g ~ - ( +l 3 ) - B - Q - g l u c a n a s e a c t i v i t i e s when Avena g l u c a n and l a m i n a r a n , r e s p e c t i v e l y , were employed a s s u b s t r a t e s . The exoenzyme ( m o l . w t . 6 0 , 0 0 0 ) was s t r o n g l y i n h i b i t e d b y i n o r g a n i c m e r c u r y a t a c o n c e n t r a t i o n which s u p p r e s s e d t h e r e l e a s e of monosaccharide from a u t o l y t i c a l l y a c t i v e c e l l w a l l . The e n d o - B - Q - g l u c a n a s e ( m o l . w t . 26,000) showed a marked p r e f e r e n c e f o r s u b s t r a t e s of mixed-linkage and e x h i b i t e d f e a t u r e s i n d i c a t i n g t h a t i t i n i t i a t e s t h e a u t o l y t i c s o l u b i l i z a t i o n of w a l l glucan. * 3)-B-EThe p u r i f i c a t i o n and some p r o p e r t i e s of an = - ( l g l u c a n a s e f r o m b a s i d i o m y c e t e s p e c i e s h a v e been d e s c r i b e d . 3 8 4 An e x o - ( 1 + 3)-B-!-glucanase was p u r i f i e d f r o m t h e c o m m e r c i a l enzyme K i t a l a s e , w h i c h i s a y e a s t c e l l - w a l l l y t i c enzyme p r e p a r a t i o n , b y ammonium s u l p h a t e f r a c t i o n a t i o n , ion-exchange chromatography, and gel filtration. The optimum pH v a l u e was 5 . 8 , and t h e optimum t e m p e r a t u r e was 5 5 ' C . The enzyme was s t a b l e i n t h e pH r a n g e of 5.1 - 9.8 and a t t e m p e r a t u r e s below 53'C. The i s o e l e c t r i c p o i n t and t h e m o l e c u l a r w e i g h t w e r e e s t i m a t e d t o b e pH 9 . 3 a n d 7 3 , 0 0 0 , respectively. The enzyme was shown t o b y p a s s ( 1 -* 6 ) - l i n k a g e d b r a n c h e s t o c l e a v e ( 1 + 3 ) - l i n k a g e s when s c l e r o g l u c a n was u s e d a s s u b s t r a t e . T h e Em v a l u e s f o r l a m i n a r i n , l a m i n a r i p e n t a o s e , l a m i n a r i t e t r a o s e , and l a m i n a r i t r i o s e w e r e 0 . 1 6 , 2 . 0 1 , 2.24, a n d 1.34 m M , respectively.

503

6: Enzymes 26

endo-( 1

*

4)-B-p-Glucanases

The s p e c i f i c p r o p e r t i e s h a v e been e x a m i n e d o f t h e ( 1

4)-8-n-

+

g l u c a n a s e component o f T r i c h o d e r m a k o n i n q i i t h a t p a r t i c i p a t e s i n an e a r l y a n d e f f e c t i v e s t a g e o f random b r e a k d o w n o f n a t i v e c e l l u l o s e t o s h o r t fibres.385 components that

of

The enzyme was p u r i f i e d and f r e e d f r o m a s s o c i a t e d t h e c e l l u l a s e complex

s u c h enzymes.

B-q-glucosidase)

P u r i f i c a t i o n increased the s p e c i f i c a c t i v i t y 25-fold

over c u l t u r e f i l t r a t e s .

The enzyme h y d r o l y s e d C M - c e l l u l o s e

t h a n t h e p u r i f i e d B-g-glucosidase any

(particularly

i n t e r f e r e w i t h and c o m p l i c a t e i n t e r p r e t a t i o n o f t h e a c t i o n o f

of

i t s

substrates

specificity

of

d e r i v a t i v e s of

the

(cellobiose

glucanase

cellulose.

faster

f r o m t h e same o r g a n i s m h y d r o l y s e d was

or

cellodextrins).

directed

towards

The

soluble

The g l u c a n a s e ( t e m p e r a t u r e o p t i m u m 6 O o C )

attacked larger c e l l o d e x t r i n s (cellohexaose t o cellotetraose, i n t h a t o r d e r ) much m o r e r e a d i l y t h a n s m a l l d e x t r i n s ( c e l l o b i o s e a n d c e l l o t r i o s e ) and r e l e a s e d a m i x t u r e o f p r o d u c t s , cellopentaose.

Q - g l u c o s e up t o

S i m i l a r examination o f hydrolysates o f t h e reduced

c e l l o d e x t r i n s showed c l e a r l y t h e h i g h s p e c i f i c i t y o f t h e enzyme f o r t h e c e n t r a l bond o f i t s n a t u r a l s u b s t r a t e s ( t h e c e l l o d e x t r i n s ) , whatever t h e i r chain length,

and i n d i c a t e d t h e n a t u r e o f t h e enzyme

as an e n d o - g l u c a n a s e . A new u l t r a s o n i c m e t h o d h a s been d e v e l o p e d f o r

c o m p o s i t i o n and p r o p e r t i e s of

determining the

i n d i v i d u a l components o f

s y s t e m s w i t h o u t t h e i r p r e l i m i n a r y separation.’’’

polyenzyme

The m e t h o d i s

b a s e d on d e t e r m i n a t i o n o f t h e pH dependence o f t h e r a t e c o n s t a n t s o f i n a c t i v a t i o n o f i n d i v i d u a l c o m p o n e n t s o f t h e enzyme s y s t e m u n d e r t h e a c t i o n of c a v i t a t i o n a l ultrasound.

T h e m e t h o d was u s e d t o s t u d y

c e l l u l a s e complex from t h e fungus G e o t r i c h u m candidum. contains at glucanase,

least

exo-(l

-+

four

c e l l u l o l y t i c enzymes,

4)-B-!-glucanase,

T h i s complex

endo-(1

B-Q-glucosidase,

+

4)-B-g-

and a r y l - B - g -

g l u c o s i d a s e , w h i c h d i f f e r i n v a l u e s o f pK o f t h e i o n o g e n i c g r o u p s , c o n t r o l l i n g t h e pH p r o f i l e s o f

ultrasonic inactivation,

as w e l l as

i n values o f t h e r a t e constants o f i n a c t i v a t i o n o f the form o f the a c t i v e s i t e most s t a b l e t o t h e a c t i o n o f u l t r a s o u n d .

endo-(1

-------mocellum

-+

by

4)-B-!-Glucanase

was p u r i f i e d f r o m C l o s t r i d i u m t h e r -

centrifugation,

p r e c i p i t a t i o n , ion-exchange,

u l t r a f i l t r a t i o n ,

selective

Sephadex c h r o m a t o g r a p h y , a n d p r e p a r a t i v e

g e l e l e c t r o p h o r e ~ i s . T~h~e ~2 2 - f o l d - p u r i f i e d

enzyme behaved as a

homogeneous p r o t e i n under n o n - d e n a t u r i n g c o n d i t i o n s . The e n z y m e r e p r e s e n t e d a s i g n i f i c a n t component ( > 25%) o f t o t a l e x t r a c e l l u l a r

5 04


e n d o - g l u c a n a s e a c t i v i t y , b u t was p u r i f i e d i n low y i e l d b y t h e p r o c e d u r e s employed. The n a t i v e m o l e c u l a r weight of t h e endo-(1 + 4 ) - B - e - g l u c a n a s e was d e t e r m i n e d b y u l t r a c e n t r i f u g a t i o n a l a n a l y s i s , amino a c i d c o m p o s i t i o n , and p o l y a c r y l a m i d e - g e l e l e c t r o p h o r e s i s and T h e enzyme c o n t a i n e d 11.2% v a r i e d b e t w e e n 8 3 , 0 0 0 and 9 4 , 0 0 0 . c a r b o h y d r a t e and was i s o e l e c t r i c a t pH 6.72. The pH and t e m p e r a t u r e o p t i m a of t h e endo-glucanase were 5.2 and 62'C, r e s p e c t i v e l y . The enzyme l a c k e d B - c y s t e i n e and was low i n s u l p h u r - c o n t a i n i n g a m i n o acids. The p u r i f i e d e n d o - ( 1 + 4 ) - B - Q - g l u c a n a s e d i s p l a y e d : h i g h a c t i v i t y towards carboxymethylcellulose, celloheptaose, cellohexaose, and c e l l o p e n t a o s e , low a c t i v i t y t o w a r d s A v i c e l m i c r o c r y s t a l l i n e c e l l u l o s e and c e l l o t e t r a o s e , no d e t e c t a b l e a c t i v i t y t o w a r d s increased a c t i v i t y towards c e l l o c e l l o t r i o s e or cellobiose, The o l i g o s a c c h a r i d e s w i t h i n c r e a s i n g d e g r e e of p o l y m e r i z a t i o n . i n t e r n a l g l y c o s i d i c bonds of c e l l o - o l i g o s a c c h a r i d e s were c l e a v e d b y t h e enzyme i n p r e f e r e n c e t o e x t e r n a l l i n k a g e s . K m and l m a f ox r c e l l o p e n t a o s e and c e l l o h e x a o s e h y d r o l y s i s were 2.30 m M and 39.3 pmol m i n - l p e r mg o f p r o t e i n and 0.56 m M and 58.7 umol m i n - ' p e r mg of protein, respectively. The k i n e t i c p r i n c i p l e s of t h e f o r m a t i o n of !-glucose and c e l l o b i o s e i n t h e h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n of c e l l u l a s e complexes from e i g h t d i f f e r e n t s o u r c e s have By s u c c e s s i v e a d d i t i o n of been s t u d i e d e x p e r i m e n t a l l y . 1 9 4 i n d i v i d u a l c o m p o n e n t s of t h e c e l l u l a s e c o m p l e x ( e n d o - ( 1 + 4 ) - B - q g l u c a n a s e and B - a - g l u c o s i d a s e ) t o t h e r e a c t i o n s y s t e m , t h e s t e p s l i m i t i n g t h e r a t e of e n z y m a t i c h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e were r e v e a l e d . I t was shown t h a t i n most o f t h e c a s e s s t u d i e d t h e s t e p d e t e r m i n i n g t h e r a t e of f o r m a t i o n o f Q - g l u c o s e w i t h t h e p a r t i c i p a t i o n of i n t e r m e d i a t e c e l l o b i o s e i s t h e a c t i o n of O n l y i n one c a s e (complex from A s p e r g i l l u s f o e t i B-g-glucosidase. &, e n r i c h e d w i t h B-g-glucosidase) i s t h e r a t e o f f o r m a t i o n of g l u c o s e from m i c r o c r y s t a l l i n e c e l l u l o s e l i m i t e d by t h e a c t i o n of t h e I n accordance endo-(1 * 4)-B-Q-glucanase of t h e c e l l u l a s e complex. w i t h t h e k i n e t i c p r i n c i p l e s d e v e l o p e d , i t was shown t h a t when an e x c e s s of B-2-glucosidase i s added t o t h e r e a c t i o n s y s t e m t h e s t e p l i m i t i n g t h e r a t e of h y d r o l y s i s of m i c r o c r y s t a l l i n e c e l l u l o s e under t h e a c t i o n of a l l t h e c e l l u l a s e complexes s t u d i e d becomes t h e a t t a c k on t h e i n i t i a l i n s o l u b l e s u b s t r a t e b y e n d o - ( 1 + 4 ) - B - Q - g l u c a n a s e . U n d e r t h e same c o n d i t i o n s , a l i n e a r c o r r e l a t i o n was f o u n d b e t w e e n t h e s t e a d y - s t a t e r a t e o f f o r m a t i o n of Lj-glucose f r o m m i c r o c r y s t a l l i n e c e l l u l o s e u n d e r t h e a c t i o n of a l l t h e c e l l u l a s e c o m p l e x e s

e-

6: Enzymes

505

s t u d i e d , on t h e one h a n d , and t h e a c t i v i t y of e n d o - ( 1 + 4 ) - 8 - Q g l u c a n a s e i n t h e s e c o m p l e x e s , on t h e o t h e r . I t was shown t h a t t h e a c t i o n of a l l t h e c e l l u l a s e c o m p l e x e s s t u d i e d ( s e l e c t e d r a t h e r a r b i t r a r i l y ) i s d e s c r i b e d by e s s e n t i a l l y t h e same k i n e t i c p r i n c i p l e s , which i s e v i d e n c e o f t h e same m e c h a n i s m s of t h e h y d r o l y s i s of i n s o l u b l e c e l l u l o s e b y c e l l u l a s e r e p a r a t i o n s of different origin. During growth of B a c t e r i o d e s s u c c i n o g e n e s i n a l i q u i d medium w i t h c e l l u l o s e a s t h e s o u r c e of c a r b o h y d r a t e , g r e a t e r t h a n 8 0 % of t h e endo-( 1 + 4)-B-Q-glucanase , x y l a n a s e , and a r y l - B - a - x y l o s i d a s e and 5 0 % o f t h e a r y l - 6 - Q - g l u c o s i d a s e was r e l e a s e d from c e l l s i n t o t h e c u l t u r e fluid.227 B y u s i n g QAE-Sephadex A50 chromatography i n t h e p r e s e n c e of 6 M u r e a , i t was p o s s i b l e t o s p l i t t h e g l u c a n a s e complex o f C l o s t r i d i u m thermocellum i n t o d i s t i n c t p r o t e i n f r a c t i o n s . 3 8 7 One of t h e s e f r a c t i o n s c o n t a i n e d an e n d o - ( 1 + 4 ) - B - e - g l u c a n a s e which was i s o l a t e d a t a h i g h d e g r e e of p u r i t y and was i d e n t i f i e d b y i t s a b i l i t y t o h y d r o l y s e t r i n i t r o p h e n y l a t e d c a r b o x y m e t h y l c e l l u l o s e . The enzyme i s o f monomeric n a t u r e , w i t h a m o l e c u l a r w e i g h t o f 5 6 , 0 0 0 . I t h a s an i s o e l e c t r i c pH of 6 . 2 and an optimum pH of 6.0. It h y d r o l y s e d c a r b o x y m e t h y l c e l l u l o s e and, a t a s l o w e r r a t e , c e l l u l o s e powder. The m a j o r e n d - p r o d u c t s o f c e l l u l o s e d e g r a d a t i o n w e r e 9 g l u c o s e , c e l l o b i o s e , and c e l l o t r i o s e . C e l l o t e t r a o s e i s formed a s an i n t e r m e d i a t e product. T h e f o r m a t i o n and l o c a t i o n of (1 + 4 ) - B - Q - g l u c a n a s e s and ( 1 + 4)-B-!-glucosidases h a v e been s t u d i e d i n c u l t u r e s of P e n i c i l l i u m j a n t h i n e l l u m grown on A v i c e l , sodium carboxymethyl c e l l u l o s e , c e l l o b i o s e , g l u c o s e , mannose, and m a l t o s e . 2 2 2 e n d o - ( 1 + 4 ) - B - k Glucanases were c e l l f r e e , and t h e i r f o r m a t i o n was induced by c e l l o biose. (1 + 4)-B-P-Glucosidases, on t h e o t h e r h a n d , w e r e f o r m e d c o n s t i t u t i v e l y and were p r i m a r i l y c e l l f r e e , b u t w i t h a s m a l l amount strongly associated w i t h the c e l l wall. A rotational viscosimetric method was developed t o measure t h e t o t a l endo-(1 + 4)-B-Q-glucanase a c t i v i t y o f t h e c u l t u r e ( b r o t h and s o l i d s ) . B y t h i s method, i t was p o s s i b l e t o d e t e r m i n e t h e endo-(1 + 4)-B-Q-glucanase a c t i v i t y not o n l y i n t h e s u p e r n a t a n t o f t h e c u l t u r e b u t a l s o on t h e s u r f a c e of t h e m y c e l i u m o r a b s o r b e d on r e s i d u a l A v i c e l . D u r i n g a 70 l i t r e b a t c h c u l t i v a t i o n of P. j a n t h i n e l l u m , t h e a d s o r p t i o n of e n d o - ( 1 + 4 ) - B - Q - g l u c a n a s e s b y r e s i d u a l and newly a d d e d 1 0 % A v i c e l was m e a s u r e d . The a d s o r p t i o n o f s o l u b l e p r o t e i n and e n d o - ( 1 + 4)-B-Qg l u c a n a s e b y A v i c e l was f o u n d t o be l a r g e l y i n d e p e n d e n t of t h e pH


506 v a l u e b u t d e p e n d e n t on t e m p e r a t u r e . The k i n e t i c s o f

g r o w t h and e x t r a c e l l u l a r

cellulase production

by Talaromyces e m e r s o n i L grown on c e l l u l o s i c measured.198

material

were

The enzyme s y s t e m was f o u n d t o b e c o m p r i s e d o f f o u r t o

f i v e f o r m s o f ~ g - (+l 4 ) - B - Q - g l u c a n a s e a n d a t l e a s t t w o f o r m s o f + 4)-B-P-glucanase and t h r e e 6 - Q - g l u c o s i d a s e s . One o f t h e

endo-(1

l a t t e r , t e r m e d B - Q - g l u c o s i d a s e 111, i s i n d u c e d c o n c u r r e n t l y w i t h t h e c e l l u l a s e s b u t d i s a p p e a r s f r o m t h e medium b e c a u s e o f t h e l o w pH t h a t develops d u r i n g growth. stable.

The c e l l u l a s e s b y c o n t r a s t a r e m o r e a c i d

The p o s s i b l e f u n c t i o n s o f t h e s e enzymes a r e d i s c u s s e d .

Production o f

-F-u-s a r i u m

sp.

extracellular

cellulase

by

an

has been s t u d i e d i n shake c u l t u r e s ,

isolate of

appearance o f c e l l u l a s e components (8-g-glucosidase day,

endo-(1

third

day

*

4)-B-;-glucanase

of

growth

on

and

on t h e f i r s t

s-(l + 4I-B-g-glucanase

insoluble

cellulose)

a

and s e q u e n t i a l

was

on t h e

observed.211

Maximum p r o d u c t i o n o f a l l t h e s e c o m p o n e n t s was a c h i e v e d on t h e f i f t h day.

The i n f l u e n c e o f d i f f e r e n t n i t r o g e n a n d c a r b o n s o u r c e s on

c e l l u l a s e p r o d u c t i o n was

investigated.

Crude

cellulolytic

enzyme

was u s e d f o r h y d r o l y s i s o f common a g r i c u l t u r a l w a s t e s b o t h w i t h and w i t h o u t sodium hydroxide pretreatment.

Analysis o f hydrolysates

i n d i c a t e d g l u c o s e as t h e m a j o r c o n s t i t u e n t ( a b o u t

83% o f

total

r e d u c i n g s u g a r 1. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f t h e e x t r a c e l l u l a r and intracellular

--Talaromyces

6-g-glucosidases

t h a t B-g-glucosidase glucosidases

of

the

thermophillic

e m e r s o n i i have been d e s c r i b e d . while

fungus

The a u t h o r s c o n c l u d e

I a n d B - g - g l u c o s i d a s e I V a r e t r u e B-a-

B-g-glucosidase

111

i s

an

=-[l + 4)-B-Q-

g l u c a n a s e .199

27

endo-( 1

An e n d o - ( 1 the

*

+

6)-B-P-Glucanases 6)-B-Q-glucanase

o f F l a v o b a c t e r i u m M64 h y d r o l y s e s

octasaccharide repeating u n i t

tetra saccharide^.^^^

An

endo-(1

+

of

succinoglycan

6)-B-Q-glucanase

t o

capable

two of

hydrolysing the octasaccharide repeating u n i t o f succinoglycan t o t w o t e t r a s a c c h a r i d e s h a s been i s o l a t e d f r o m c e l l s o f F l a v o b a c t e r i u m . One t e t r a s a c c h a r i d e was c o m p o s e d o f Q - g l u c o s e , p y r u v i c a c i d (4:1:1,

molar r a t i o ) ,

g l u c o s e and P - g a l a c t o s e (3:1, t h e (1 + 6 ) - B - Q - g l u c o s i d i c

s u c c i n i c acid, and

and t h e o t h e r was composed of

molar r a t i o ) .

l i n k a g e t o t h e (1

a-

T h i s enzyme h y d r o l y s e d

*

6 ) - l i n k e d B-Q-glucose

507

6: Enzymes

r e s i d u e i n t h e o c t a s a c c h a r i d e r e p e a t i n g u n i t o f s u c c i n o g l y c a n and also

hydrolysed

the

octasaccharide

p o l y s a c c h a r i d e s p r o d u c e d by

many

repeating units

strains

of

of

similar

Agrobacterium

and

Rhizobium species. The

structure

succinoglycan

of

the

extracellular

from Alcaligenes

faecal&

acidic

polysaccharide

has been e l u c i d a t e d by

s u c c e s s i v e f r a g m e n t a t i o n o f t h e p o l y s a c c h a r i d e w i t h e x t r a c e l l u l a r Be - g l y c a n a s e ( s u c c i n o g l y c a n d e p o l y m e r a s e ) and i n t r a c e l l u l a r e n d o - ( 1 + 6)-8-P-glucanase tetrasaccharides

from

12

Flavobacterium

sp.

M64

i n t o

two

i t s o c t a s a c c h a r i d e r e p e a t i n g u n i t and t h e n

m e t h y l a t i o n a n a l y s i s and e n z y m i c h y d r o l y s i s o f t h e p r o d u c t s . 3 8 9

P-Glucanases (Miscellaneous)

28 The

use

of

dye-polysaccharide

interactions

has

i n v e s t i g a t e d f o r a $-!-glucanase

assay.390

i n p a r t i c u l a r c e r e a l B-8-glucans

and s u b s t i t u t e d c e l l u l o s e s ,

been

Certain polysaccharides, induce

b a t h o c h r o m i c s h i f t s i n t h e a d s o r p t i o n s p e c t r a o f s u c h d i r e c t dyes as Congo

Red.

interaction,

As

cello-oligosaccharides

showed

it

followed

of

that

formation

complexes m i g h t be u s e f u l i n s t u d i e s of

l i t t l e

or

no

dye-polysaccharide

B-e-glucanase

action.

This

p o s s i b i l i t y h a s been i n v e s t i g a t e d by m o n i t o r i n g t h e d i g e s t i o n o f sample of o a t B-Q-glucan w i t h p u r i f i e d B-Q-glucan-endohydrolase S o l u t i o n s o f oat B-Q-glucan

Bacillus subtilis.

a

from

(pH 6.5)

(0.5% w/v)

w e r e t r e a t e d w i t h e n z y m e a n d t h e i r v i s c o s i t y was m o n i t o r e d ,

and

aliquots

dye

were removed a t

intervals,

i n t e r a c t i o n and r e d u c i n g s u g a r . r e l a t i o n s h i p between

The

heated,

and t e s t e d f o r

results

clearly

showed a

loss i n v i s c o s i t y and l o s s i n d y e i n t e r a c t i o n ,

whereas measurements of

r e d u c i n g sugar were n o t u s e f u l u n t i l l a t e r

s t ages o f d e g r a d a t i o n . P a r t i a l p u r i f i c a t i o n o f endo-

and E-B-Q-glucanase

enzymes

f r o m Zea mays s e e d l i n g s h a s b e e n d e s c r i b e d and t h e i r i n v o l v e m e n t i n c e 11- w a 1 1 a u t o h y d r o l y s is

i n v e s t i g a t e d . 391

chromatography o f t h e c e l l - w a l l and exo-B-g-glucanase

a c t i v i t i e s when A v e n a g l u c a n and l a m i n a r a n ,

r e s p e c t i v e l y , were e m p l o y e d as s u b s t r a t e s . w e i g h t 60,000)

M o l e c u l a r - s i eve

p r o t e i n r e s o l v e d endo-B-g-glucanase The exoenzyme ( m o l e c u l a r

was s t r o n g l y i n h i b i t e d b y H g 2 + a t a c o n c e n t r a t i o n

which suppressed t h e r e l e a s e o f active c e l l wall.

monosaccharide f r o m

The e n d o - B - Q - g l u c a n a s e

(mol.wt.

autolytically

26,000),

which


508 showed

a

marked

exhibited

for

preference

features

substrates

it

indicating that

of

mixed

initiates

the

linkage, autolytic

s o l u b i l i z a t i o n o f w a l l glucan. (1

+

3),(1

+

4 ) - B - ~ - G l u c a n a s e has been p u r i f i e d 1 4 2 - f o l d f r o m

B a c i l l u s s u b t i l i s by c h r o m a t o g r a p h y on c e l l u l o s e and SE-Sephadex

C-

50 f o l l o w e d b y g e l f i l t r a t i o n t o h o m o g e n e i t y t h r o u g h A c r y l e x P SOS

60.392

electrophoresis

w e i g h t s f o r t h e enzyme o f

and

30,000

gel

filtration

a n d 33,000,

4.5) does n o t c o n t a i n t r y p t o p h a n , enzyme (PI

gave

molecular

respectively.

The

f r e e s u l p h y d r y l groups,

or carbohydrates. I t h a s been shown t h a t c e l l o b i o s e , i n c o n t r a s t t o a - g l u c o s e ,

is

a r e g u l a t o r o f t h e a c t i v i t y o f endoglucanases o f c e l l u l a s e complexes f r o m v a r i o u s sources.393 molecular-weight

C e l l o b i o s e i s an a c t i v a t o r o f t h e l o w -

m-;-glucanase

f r o m T.

k o n i n g i i and an i n h i b i t o r

o f t h e h i g h - m o l e c u l a r - w e i g h t endo-p-glucanases Kinetic

of

analysis

c e l l o b i o s e or

the

reaction

showed

rnethylcellobiose t o the

f r o m t h e same s o u r c e . that

the

binding

low-molecular-weight

of

e - 9 -

g l u c a n a s e l e a d s t o a s i x f o l d i n c r e a s e i n t h e r a t e o f d e g r a d a t i o n of carboxymethylcellulose.

The a c t i v a t i n g a c t i o n o f c e l l o b i o s e i s due

t o i t s a b i l i t y t o play t h e r o l e o f a supplementary n u c l e o p h i l i c agent

i n

the

reaction

of

transglycosylation

of

intermediate

oligosaccharides i n the enzymatic hydrolysis o f cellulose. A new Q - g l u c a n a s e p r o d u c e d b y a m a r i n e B a c i l l u s s p e c i e s h a s been r e p o r t e d . 3 9 4

An i s o l a t e o f b a c t e r i a f r o m m a r i n e mud was f o u n d

t o p r o d u c e a new enzyme c a p a b l e o f h y d r o l y s i n g i n s o l u b l e g - g l u c a n s produced by o r a l s t r e p t o c o c c i .

The s u b s t r a t e s p e c i f i c i t y o f t h e

enzyme i n d i c a t e d t h a t t h e enzyme was a new a - Q - g l u c a n a s e . The

structure

of

the

extracellular

s u c c i n o g l y c a n f r o m A l c a l i g e n e s f a e c a l i s var. e l u c i d a t e d by

successive

ex t r a c e 1l u l a r intracellular

fragmentation

a-Q-gly canase

endo-B-(1

+

i n t o two tetrasaccharides

polysaccharide

myxoqenes 10C3 h a s been the

polysaccharide

with

( s u c c i n o g l y c a n d e p o l y m e r a s e ) and

6)-Q-glucanase

via

of

acidic

of

F l a v o b a c t e r i u m sp.

M64

i t s octasaccharide r e p e a t i n g unit.389

Polyacrylamide g e l electrophoresis o f

t h e c e l l u l o l y t i c system

f r o m t h e c u l t u r e s u p e r n a t e s o f A c e t i v i b r i o c e l l u l o l y t i c u s h a s shown t h e p r e s e n c e o f f o u r m a j o r enzymes:

a B-Q - -glucosidase,an

g l u c a n a s e , and t w o & - ~ - g l u ~ a n a s e s . ~ ~ ~

w-Q-

509

6: Enzymes

Glucoamylases

29

method

A

for

the

automatic

measurement

of

a-amylase

and

g l u c o a m y l a s e a c t i v i t i e s d u r i n g f e r m e n t a t i o n h a s b e e n d e v e l o p e d . 314 F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f

endo-

ref.314.

A k i n e t i c equation which represents a s y n e r g i s t i c action of

and exo-enzyme

tested

by

upon p o l y s a c c h a r i d e s ,

experiments

using

adequacy

immobilized

has been

a-amylase

and

g l u c o a m y l a s e .268 P h y s i c a l e n t r a p m e n t h a s b e e n u s e d a s an a p p r o a c h t o a c h i e v e t h e r m a l s t a b i l i z a t i o n o f Q - g l u c o s e o x i d a s e and g l u ~ o a m y l a s e . ~The ~~

-t

values

for

the t h e r m o i n a c t i v a t i o n of

!-glucose

oxidase

and

g l u c o a m y l a s e were i n c r e a s e d s e v e r a l - f o l d by t h e i r e n t r a p m e n t i n polyacrylamide

gels.

I n polyacrylate

behaved d i f f e r e n t l y ,

probably

gels

owing t o

the

i n d i v i d u a l enzymes

microenvironmental effects

a r i s i n g by t h e p o l y e l e c t r o l y t e n a t u r e o f t h e c a r r i e r . I n t r i n s i c k i n e t i c constants

for

pore-diffusion-limited

i m m o b i l i z e d enzyme r e a c t i o n s have been e s t i m a t e d . 3 9 6

A simple

method u s i n g t h e A r i s - B i s c h o f f

intrinsic

rate

parameters

when

slow

modulus

that

establishes

pore d i f f u s i o n

of

substrate

limits

i m m o b i l i z e d enzyme r e a c t i o n s t h a t obey M i c h a e l i s - M e n t e n k i n e t i c s has been p r e s e n t e d .

Data a t h i g h s u b s t r a t e c o n c e n t r a t i o n s , where t h e

enzyme w o u l d b e s a t u r a t e d i n t h e absence o f d i f f u s i o n l i m i t a t i o n , and a t l o w s u b s t r a t e c o n c e n t r a t i o n s ,

where e f f e c t i v e n e s s f a c t o r s a r e

i n v e r s e l y p r o p o r t i o n a l t o r e a c t i o n modulus, maximum

rate

and

Michaelis

constant,

were used t o d e t e r m i n e respectively.

Because

M i c h a e l i s - M e n t e n and L a n g m u i r - H i n s h e l w o o d k i n e t i c s a r e f o r m a l l y identical,

this

parameters of

m e t h o d may

be used t o e s t i m a t e

many h e t e r o g e n e o u s

catalysts.

demonstrated u s i n g experimental data

from

intrinsic rate

T h e t e c h n i q u e was of

maize

d e x t r i n w i t h d i f f u s i o n - l i m i t e d immobilized glucoamylase.

This

system y i e l d e d a M i c h a e l i s constant of

the hydrolysis

0.14%,

c o m p a r e d t o 0.11% f o r

s o l u b l e g l u c o a m y l a s e and 0.14% f o r i m m o b i l i z e d g l u c o a m y l a s e f r e e o f diffusional effects. The e n t r a p m e n t calcium

alginate

of

chemical derivatives o f

g e l s has

retained i n calcium alginate gels, enzyme was i n c r e a s e d , intermolecular

glucoamylase

been i n ~ e s t i g a t e d . ~ ~ G ’ lucoamylase

when t h e m o l e c u l a r w e i g h t o f t h e

9. by a t t a c h m e n t

cross-linking.

t o p o l y m e r i c s u p p o r t s o r by

Alternatively,

the

enzyme

r e t a i n e d by c o v a l e n t a t t a c h m e n t t o t h e a l g i n a t e m a t r i x . majority

of

the

i n was

c o u l d be I n the

g l u c o a m y l a s e d e r i v a t i v e s m o r e t h a n 50% o f

the

510

Carbo hy d ra re Chemistry

i n i t i a l a c t i v i t y d e t e r m i n e d w i t h m a l t o s e or p a r t i a l l y h y d r o l y s e d s t a r c h s u c h as s u b s t r a t e was r e c o v e r e d , w h i l e u p o n e n t r a p m e n t i n g e l p a r t i c l e s l e s s t h a n 40% a n d 6% o f t h e i n i t i a l a c t i v i t y t o w a r d s m a l t o s e a n d p a r t i a l l y h y d r o l y s e d s t a r c h , r e s p e c t i v e l y , was r e c o v e r e d . Thus, d i f f u s i o n a l and s t e r i c l i m i t a t i o n s on s u b s t r a t e access a p p e a r e d t o i n f l u e n c e t h e a p p a r e n t e n z y m i c a c t i v i t y , i n s p i t e of t h e high permeability of calcium a l g i n a t e g e l s . Dextran-coupled g l u c o a m y l a s e e n t r a p p e d i n c a l c i u m a l g i n a t e g e l s h a s b e e n u s e d as a packed-bed r e a c t o r f o r c o n t i n u o u s h y d r o l y s i s of o l i g o d e x t r i n s d u r i n g a p e r i o d of t w o months w i t h no change i n a c t i v i t y . The t h e r m a l s t a b i l i t y o f i m m o b i l i z e d g l u c o a m y l a s e from R h i z o p u s n i v e u s e n t r a p p e d i n p o l y a c r y l a m i d e g e l s a n d b o u n d t o SPS e p h a d e x C-50 h a s b e e n i n v e s t i g a t e d . 3 9 8 The t h e r m a l s t a b i l i t y o f i m m o b i l i z e d g l u c o a m y l a s e e n t r a p p e d i n p o l y a c r y l a m i d e g e l s was e n h a n c e d s l i g h t l y c o m p a r e d w i t h g l u c o a m y l a s e i n f r e e s o l u t i o n , and was i n d e p e n d e n t o f t h e a c r y l a m i d e m o n o m e r c o n c e n t r a t i o n a n d N,"methylene-bis(acrylamide1 c o n t e n t . To e x p l a i n t h i s p h e n o m e n o n , t h e c e l l u l a r s t r u c t u r e o f p o l y a c r y l a m i d e g e l was t a k e n i n t o c o n s i d e r a t i o n i n a d d i t i o n t o i n t e r a c t i o n s between g l u c o a m y l a s e and g e l a n d a d e c r e a s e i n d i e l e c t r i c c o n s t a n t i n t h e g e l . On t h e o t h e r h a n d , i m m o b i l i z e d g l u c o a m y l a s e b o u n d t o S P - S e p h a d e x by i o n i c i n t e r a c t i o n s h o w e d l o w e r s t a b i l i t y t h a n f r e e g l u c o a m y l a s e , a n d much greater stability than glucoamylase i n the presence of dextran s u l p h a t e , a c o n s t i t u e n t of SP-Sephadex. T h e rm al s t a b i l i t i e s f o r t h e . f r e e a n d i m m o b i l i z e d e n z y m e s were a l s o c o m p a r e d a t t h e pH n o t i n t h e b u l k s o l u t i o n b u t i n t h e SP-Sephadex. The thermal s t a b i l i t y of i m m o b i l i z e d g l u c o a m y l a s e i n t h e p r e s e n c e o f a s u b s t r a t e h a s b e e n i n v e s t i g a t e d u s i n g a mass b a l a n c e The a p p l i c a b i l i t y o f t h i s method t o a m a l t o s e - i m m o b i l i z e d g l u c o a m y l a s e s y s tem was e x a m i n e d . The p H - d e p e n d e n t u n f o l d i n g o f g l u c o a m y l a s e f r o m r a b b i t s m a l l i n t e s t i n e by m e t h a n o l h a s b e e n d e s c r i b e d . 4 0 0 Glucoamylase from the b r u s h b o r d e r s o f t h e r a b b i t s m a l l i n t e s t i n e w a s s o l u b i l i z e d by p a p a i n a n d T r i t o n X-100. T h e T r i t o n - s o l u b i l i z e d f r a c t i o n , when In t h e presence f r e e d of T r i t o n , assumed a m i c e l l e - l i k e s t r u c t u r e . o f m e t h a n o l , t h e enzyme micelle as well as t h e p a p a i n - s o l u b i l i z e d e n z y m e monomer u n f o l d e d , o b e y i n g t h e f i r s t - o r d e r r a t e law. U n f o l d i n g o f t h e m i c e l l e was d e s c r i b e d b y t w o t i m e c o n s t a n t s (k = s-l), whereas p a p a i n - s o l u b i l i z e d enzyme had s-l a n d k = s-'). T h e r a t e was s i n g l e - p h a s e k i n e t i c s o f u n f o l d i n g (k = d e p e n d e n t on pH a n d t h e v a l u e o f a c t i v a t i o n e n e r g y l i e s b e t w e e n 1-6

511

6: Enzymes k c a l mol"

a t v a r i o u s pHs.

M e t h a n o l was d e v o i d o f a n y e f f e c t o n t h e

a c t i v i t y o f p a p a i n - s o l u b i l i z e d g l u c o a m y l a s e a t v a r i o u s pHs, w h e r e a s b o t h T r i t o n - 1 0 0 - s o l u b i l i z e d enzyme a n d enzyme m i c e l l e w e r e i n h i b i t e d t o t a l l y b y 3 0 % m e t h a n o l b e t w e e n pHs 4 a n d 8 . An u n u s u a l t y p e o f

g l y c o p r o t e i n s t r u c t u r e h a s been i d e n t i f i e d

f o r a glucoamylase.401

Glucoamylase occurs i n two isoenzyme forms

( g l u c o a m y l a s e I and g l u c o a m y l a s e 11) i n e x t r a c t s f r o m c e r t a i n f u n g i . The i s o e n z y m e s f r o m A s p e r g i l l u s n i g e r a r e g l y c o e n z y m e s C o n t a i n i n g Qmannose,

Q-glucose, and

components.

Q-galactose

as

integral structural

8-

New d a t a f r o m e x p e r i m e n t s o n r e d u c t i v e a l k a l i n e

e l i m i n a t i o n and f r o m m e t h y l a t i o n a n a l y s i s show t h a t t h e c a r b o h y d r a t e chains o f glucoamylase I a r e l i n k e d Q - g l y c o s i d i c a l l y

f r o m c-mannose

- - s e r i n e or I - t h r e o n i n e r e s i d u e s o f t h e p r o t e i n m o i e t y . residues t o L t h e c a r b o h y d r a t e r e s i d u e s a r e p r e s e n t as 20 s i n g l e

I n t h i s enzyme,

p-mannose

residues,

11 d i s a c c h a r i d e c o m p o n e n t s h a v i n g t h e s t r u c t u r e

2-Q-~-mannopyranosyl-~-mannose, 8

trisaccharides,

5

and

0-

t e t r a s a c c h a r i d e s composed o f v a r i o u s c o m b i n a t i o n s o f Q-mannose, glucose,

and e - g a l a c t o s e

glycosidic linkages.

r e s i d u e s j o i n e d by ( 1 + 3 ) a n d ( 1

+

6)

Such an a r r a y o f c a r b o h y d r a t e c h a i n s i n a

g l y c o p r o t e i n i s unusual,

a n d may a c c o u n t f o r some o f t h e u n i q u e

p r o p e r t i e s e x h i b i t e d by g l u c o a m y l a s e . A m y l o l y t i c enzymes p r o d u c e d by a s t r a i n o f A s p e r g i l l u s n i g e r c u l t i v a t e d on c a s s a v a s t a r c h i n l i q u i d o r s o l i d c u l t u r e w e r e f o u n d t o b e m a i n l y g l u c o a m y l a ~ e s . ~F o ~ r~ t h e same i n i t i a l a m o u n t o f s u b s t r a t e , t h e g l u c o a m y l a s e a c t i v i t y i n c r e a s e d e v e n a f t e r 60 h o f c u l t u r e o n s o l i d medium w h e r e a s i t d e c r e a s e d i n l i q u i d c u l t u r e .

The

pH o p t i m a a n d t h e r m o s t a b i l i t i e s f o r e n z y m e s p r o d u c e d i n s o l i d a n d l i q u i d c u l t u r e s were

different.

s o l u b l e s t a r c h (100 m l ) "

The

Xm

values

e x p r e s s e d as

c u l t u r e and 0.057% f o r c r u d e enzyme f r o m l i q u i d c u l t u r e . active-site-directed

mg

w e r e 0.1% f o r c r u d e e n z y m e f r o m s o l i d Studies o f

i r r e v e r s i b l e i n h i b i t i o n o f g l y c o s i d a s e s by t h e

c o r r e s p o n d i n g glycosylmethyl-(4-nitrophenyl)triazines

show t h a t t h e y

h a v e no a c t i o n on g l u c o a m y l a s e f r o m A s p e r g i l l u s r ~ i g e r . ~ ~ A m a j o r g l u c o a m y l a s e o f A s p e r g i l l u s n i g e r h a s been p u r i f i e d 2 3 fold

(21% y i e l d )

by

ultrafiltration

c h r o m a t o g r a p h y on DEAE-Sephadex, The

purified

enzyme

polyacrylamide

gel

was

f o l l o w e d by

successive

U l t r o g e l AcA 44, a n d S P - S e p h a d e ~ . ~ ~ ~

proved

homogeneous

electrophoresis,

as

isoelectric

judged

by

focusing,

u l t r a c e n t r i f u g a t i o n , and a l s o f r o m t h e absence o f t h e g l y c o s i d a s e activities.

The enzyme was a g l y c o p r o t e i n c o n t a i n i n g n e u t r a l s u g a r

(18%) and 2-amino-2-deoxy-Q-glucose

(0.77%),

and i t s

molecular

512


weight

was

(90,000)

estimated

by

polyacrylamide

SDS

gel

e l e c t r o p h o r e s i s and a m i n o a c i d c o m p o s i t i o n . The N - t e r m i n a l a m i n o a c i d was I - a l a n i n e . The pH o p t i m u m o f t h e p u r i f i e d enzyme was 4.5 w i t h s o l u b l e s t a r c h as a s u b s t r a t e .

The enzyme was s t a b l e b e t w e e n

p H 2.5

a n d 7.5

5OoC.

The e n z y m e a c t i v i t y was i n h i b i t e d b y H g 2 + a n d ,

extent,

and r e t a i n e d f u l l a c t i v i t y

by Pb2+ and Mn2+.

The

vmax v a l u e

c.

value w i t h

xi

thus resulting i n the

The k i n e t i c p a r a m e t e r s f o r

o t h e r s u b s t r a t e s s u c h as s o l u b l e s t a r c h , w e l l as t h e

t o a lesser

s u b s t r a t e i n glucose u n i t

2,

increased with

lmax/Km

increase i n the

up t o

value f o r m a l t o - o l i g o m e r ,markedly

decreased w i t h i n c r e a s i n g c h a i n l e n g t h of

( 5 ) and t h e

a t temperatures

g l y c o g e n , a n d i s o m a l t o s e as

v a l u e s f o r some s a c c h a r i d e s w e r e a l s o d e t e r m i n e d .

A t h e r m o s t a b l e g l u c o a m y l a s e has been p u r i f i e d from

the culture

f i l t r a t e s o f Thermomyces l a n u g i n o s u s a n d h a s b e e n e s t a b l i s h e d t o be homogeneous

by

glycoprotein

(mol.

10.12%.

a

number wt.

of

criteria.404 with

57,000)

a

The

enzyme

carbohydrate

The enzyme h y d r o l y s e d s u c c e s s i v e

9-glucose

was

content

residues

a of

from

t h e n o n - r e d u c i n g ends o f t h e s t a r c h m o l e c u l e b u t d i d n o t e x h i b i t any glucosyltransferase activity. m a l t o t r i o s e by t h e m u l t i - c h a i n optimum linkages

7 O o C ) was of

unable

isomaltose

increase i n the

The e n z y m e a p p e a r e d t o h y d r o l y s e mechanism.

to h y d r o l y s e

and

dextran,

The enzyme ( t e m p e r a t u r e

(1

and

-*

the

enzyme

lmax a n d d e c r e a s e i n Em v a l u e s

chain l e n g t h o f t h e s u b s t r a t e molecule.

exhibited

with increasing

The enzyme was i n h i b i t e d by

t h e s u b s t r a t e analogue q-glucono-l,4-lactone manner.

6)-a-Q-glucosidic

i n a non-competitive

The enzyme e x h i b i t e d r e m a r k a b l e r e s i s t a n c e t o w a r d s c h e m i c a l

and t h e r m a l d e n a t u r a t i o n . The

-R h

thermal

stability

and

kinetics

of

glucoamylase

i z w ---s n i v e u s has been i n v e s t i g a t e d i n t h e presence of

DEAE-dextran,

dextran sulphate,

t e m p e r a t u r e r a n g e f r o m 52.0

p o l y e t h y l e n e glyco1,and

t o 60.0°C.405

from

dextran,

Ficoll i n a

Protective effects of

t h e s e p o l y m e r s a g a i n s t t h e r m a l i n a c t i v a t i o n o f t h e enzyme i n c r e a s e d i n t h e order polyethylene g l y c o l < F i c o l l < dextran.

The o r d e r was

t h e same a s t h a t o f a f f i n i t i e s b e t w e e n g l u c o a m y l a s e a n d e a c h o f polymers two-phase

d e t e r m i n e d by

partition of

glucoamylase i n l i q u i d - l i q u i d

systems c o n s i s t i n g o f these polymers.

glucoamylase

with

dextran

sulphate

On t h e o t h e r hand,

e x h i b i t e d an i n t e r e s t i n g

phenomenon w h i c h was a t y p i c a l e l e c t r o s t a t i c i n t e r a c t i o n e x p l i c a b l e i n t e r m s o f an e l e c t r i c a l d o u b l e l a y e r . A F l a v o b a c t e r i u m s p e c i e s which produces c y c l o d e x t r i n - d e g r a d i n g g l u c o a m y l a s e has been i s o l a t e d . 4 0 6

The i n d u c i b l e , c e l l - b o u n d

enzyme

513

6: Enzymes was p u r i f i e d a b o u t 1 0 - f o l d t o 7 5 % p u r i t y i n 5 7 % y i e l d . o f t h e enzyme w i t h c y c l o h e x a - a m y l o s e , octa-amylose

and t y p i c a l

The a c t i o n

cyclohepta-amylose,and c y c l o -

glucoamylase s u b s t r a t e s

always

gave

D-

g l u c o s e as t h e f i n a l d e g r a d a t i o n p r o d u c t .

S m a l l amounts o f m a l t o s e ,

w h i c h c o u l d be d e t e c t e d i n t h e c o u r s e o f

cycloamylose degradation,

were h y d r o l y s e d a t a l o w e r r a t e .

A p p a r e n t l y t h e enzyme p r e f e r r e d

s h o r t e r a-Q-glucopyranosyl chains, p r o v e d t o be

and a m y l o p e c t i n and g l y c o g e n

very poor substrates.

30 Glycanases ( M i s c e l l a n e o u s ) The

occurrence

of

high

and

Mr

low

forms

p h o s p h o r y l a s e i n e x t r a c t s o f human b r a i n h a s Accorinding t o gel-exclusion

of

glycogen

been r e p ~ r t e d . ~ ”

c h r o m a t o g r a p h y and s u c r o s e d e n s i t y

g r a d i e n t s e d i m e n t a t i o n b o t h p h o s p h o r y l a s e s a and b a p p e a r i n a high

Mr

f o r m o f 400,000 a n d a l o w

d i f f e r s w i t h t h e method o f

Mr

to exist

f o r m whose a p p a r e n t s i z e

determination.

The

l o w blr

form

i s

p r o b a b l y an e q u i l i b r i u m m i x t u r e o f d i m e r a n d monomer w h i c h g i v e s different

apparent

Yr

values

depending

upon

the

position

of

equilibrium. The

assignment

of

the

human

gene

for

liver-type

6-

p h o s p h o f r u c t o k i n a s e i s o e n z y m e t o chromosome 2 1 h a s been a c h i e v e d by u s i n g s o m a t i c c e l l h y b r i d s and m o n o c l o n a l a n t i - L a n t i b o d y . 4 0 8 Substrate specificty

o f t h e glycanase a c t i v i t y associated w i t h

p a r t i c l e s o f K l e b s i e l l a b a c t e r i o p h a g e N0.6 h a s b e e n i n v e s t i c ~ a t e d . ~ ’ ~ The g l y c a n a s e c a t a l y s e s c l e a v a g e o f g - B - Q - g l u c o p y r a n o s y l - ( l

+

3)-

4 , 6 - ~ - ( 1 - ~ a r b o ~ y e t h y l i d e n e ) - ~ - ~ - m a n n o p y r a n o sl ei n k a g e s i n K l e b s i e l l a serotype-6

capsular

polysaccharide.

O f

74 h e t e r o l o g o u s

Klebsiella

p o l y s a c c h a r i d e s and t w o d e r i v a t i v e s o f t h e t y p e - 6 g l y c a n o n l y t h e t y p e - 1 a n d t y p e - 5 7 p o l y m e r s w e r e a d d t i o n a l l y d e g r a d e d by t h e phage-6 enzyme. 3eq,

The r e p e a t i n g u n i t s i n t h e t h r e e s u b s t r a t e s h a v e a l a x -

leg-eq-linked

chain g-gluco-

or Q-galacto-pyranosyl

common ( w h i c h c o n s t i t u t e s t h e r e d u c i n g e n d a f t e r

residue i n

glycanase a c t i o n )

and a c a r b o x y l group on t h e n e x t h e x o p y r a n o s y l r e s i d u e .

O f t h e 72

p o l y s a c c h a r i d e s n o t a f f e c t e d by t h e v i r a l enzyme, a t l e a s t t h e t y p e 11 a n d t y p e - 2 1 g l y c a n s a l s o c o n t a i n t h e same h o m o l o g y o f p r i m a r y s t r u c t u r e . T h i s i n d i c a t e s t h a t t h e c o n f o r m a t i o n of recognition site substrates.

also

constitutes

an

important

the glycanase

feature

of

the

514

Carbohydrate Chemistry 31 H e p a r i n Hydrolases

Purification

of

heparinase

and

heparitinase

c h r o m a t o g r a p h y on g l y c o s a m i n o l y c a n - b o u n d A H - S e p h a r o s e reported.410 heparinase

The and

respectively,

recoveries

of

heparitinase

the

were

purified

estimated

to

by

affinity

48 has been

preparations be

and

39

of 50%,

f r o m t h e c r u d e enzyme f r a c t i o n s o b t a i n e d by t h e f i r s t

column chromatography on h y d r o x y a p a t i t e . An e n & - Q - g l y c o s i d a s e

( h e p a r i n l y a s e ) a c t i n g on h e p a r i n and

h e p a r a n s u l p h a t e was p a r t i a l l y p u r i f i e d ( - 3 0 0

t i m e s ) f r o m human

p l a t e l e t s by a f f i n i t y c h r o m a t o g r a p h y on h e p a r a n s u l p h a t e - s u b s t i t u t e d S e p h a r ~ s e . ~ O n~l y~ h e p a r i n - l i k e t h e enzyme. intermediates requirement

p o l y s a c c h a r i d e s were degraded by

The s u s c e p t i b i l i t y indicated

for

that

sulphamino

of

the

but

various

biosythetic heparin

platelet

not

ester

heparitanase sulphate

had

groups.

a

No

a c t i v i t y toward other uronic acid-containing glycosaminoglycans A l t e r n a t e l i n k a g e s i n h e p a r i n or h e p a r a n

c o u l d be d e m o n s t r a t e d .

s u l p h a t e w e r e a t t a c k e d by t h e e n z y m e a s s h o w n b y a n a l y s i s o f

the

reducing

The

sugar

moiety

i n

oligosaccharide

anticoagulant a c t i v i t y o f heparin, a c t i v a t i o n assay, heparin lyase.

products.

d e t e r m i n e d i n an a n t i t h r o m b i n I 1 1

was m a r k e d l y r e d u c e d a f t e r

treatment with the

T h e e n z y m e was r e l e a s e d f r o m i t s s t o r a g e s i t e i n

platelets a f t e r induction of the platelet-release reaction. physiological function of

p l a t e l e t heparin lyase i s not

may be t o m o d i f y e x t r a c e l l u l a r h e p a r i n - l i k e

The

known b u t

polysaccharides i n the

v a s c u l a r system. H e p a r i n h y d r o l a s e p r o d u c t i o n by F l a v o b a c t e r i u m h e p a r i n u m i n c o m p l e x p r o t e i n d i g e s t medium,

w i t h h e p a r i n e m p l o y e d as t h e i n d u c e r ,

h a s b e e n i m p r o v e d 1 5 6 - f 0 l d . ~ l ~R a p i d d e a c t i v a t i o n o f h e p a r i n a s e activity,

b o t h s p e c i f i c and t o t a l ,

s t a t i o n a r y phase.

p r o d u c t i o n showed an o b l i g a t e vitamin

requirement.

h i s t i d i n e requirement. ammonium s u l p h a t e ,

was o b s e r v e d a t t h e o n s e t o f

requirement for

L-Methionine

I-histidine

partially

t h i s m e d i u m was 0.21 h - l ,

relieved

and the

no

I-

A d e f i n e d medium c o n t a i n i n g P_-glucose,

basal salts,

L-methionine,

and L - h i s t i d i n e

d e v e l o p e d f o r g r o w t h and h e p a r i n a s e p r o d u c t i o n . medium.

the

N u t r i t i o n a l s t u d i e s on g r o w t h and h e p a r i n a s e

was

The g r o w t h r a t e i n

w h i c h i s 40% h i g h e r t h a n t h a t i n c o m p l e x

The maximum v o l u m e t r i c p r o d u c t i v i t y

d e f i n e d m e d i u m was i n c r e a s e d t o 1,475

o f heparinase i n the

U 1-1 h - l ,

p r o v i d i n g a 640-

f o l d i n c r e a s e o v e r t h a t a c h i e v e d by p r e v i o u s l y p u b l i s h e d methods.

313

6: Enzymes No r a p i d d e a c t i v a t i o n was o b s e r v e d .

An e x a m i n a t i o n o f a l t e r n a t e

i n d u c e r s f o r h e p a r i n a s e showed t h a t h e p a r i n d e g r a d a t i o n p r o d u c t s , h y a l u r o n i c acid, h e p a r i n monosulphate, B-g-2-acetamido-2-deoxyg l u c o p y r a n o s e , and m a l t o s e , i n d u c e h e p a r i n a s e i n c o m p l e x medium.

An

A z u r e A a s s a y was d e v e l o p e d t o m e a s u r e t h e h e p a r i n c o n c e n t r a t i o n d u r i n g f e r m e n t a t i o n and t h e h e p a r i n a s e s p e c i f i c a c t i v i t y of c r u d e extracts of

heparinum obtained from sonication,

thus negating the

need f o r f u r t h e r p u r i f i c a t i o n t o measure a c t i v i t y .

32

Hyaluronidases

P u r i f i e d bovine t e s t i c u l a r butane-2,3-dione

h y a l u r o n i d a s e i s i n a c t i v a t e d by

i n e i t h e r b o r a t e o r H e p e s b u f f e r , pH 8.3.413

p r e s e n c e o f b o r a t e enhanced t h e i n a c t i v a t i o n p r o c e s s , pseudo-first-order

k i n e t i c s w i t h a c a l c u l a t e d second-order

c o n s t a n t o f 13.54 M - l

min-l.

The

which f o l l o w e d rate

U s i n g k i n e t i c d a t a i t was e s t i m a t e d

t h a t t h e m o d i f i c a t i o n o f 1 m o l L - a r g i n i n e p e r m o l e n z y m e was t h a t s u f f i c i e n t f o r i n a c t i v a t i o n t o occur, whereas amino a c i d a n a l y s i s i n d i c a t e d t h a t 4 m o l & - a r g i n i n e h a d been m o d i f i e d . process

was

partially

prevented

by

using

i n h i b i t o r s o r s u b s t r a t e s o f t h e enzyme, essential

L-arginine

hyaluronidase.

residue

i s

The i n a c t i v a t i o n

either

competitive

thus indicating that the

close

to

the

active

site

of

A f u l l k i n e t i c a n a l - y s i s o f t h e enzyme w i t h e i t h e r

hyaluronic acid or chondroitin 6-sulphate

as s u b s t r a t e showed t h a t

t h e a c t i v i t y o f h y a l u r o n i d a s e was u n c o m p e t i t i v e l y a c t i v a t e d b y either

H+ or

NaC1.

The

product

obtained

by

r e d u c t i o n of

the

c a r b o x y l groups o f h y a l u r o n i c a c i d t o t h e corresponding a l c o h o l groups

was

a competitive

inhibitor.

The p o s s i b i l i t y t h a t

the

m i c r o e n v i r o n m e n t o f h y a l u r o n i c a c i d was r e s p o n s i b l e f o r t h e o b s e r v e d k i n e t i c e f f e c t s o f pH and i o n i c s t r e n g t h was d i s p e l l e d . conclude t h a t these i n v o l v e s an

data are compatible

with a

The a u t h o r s

mechanism t h a t

i o n i c i n t e r a c t i o n between a c a r b o x y l group on t h e

s u b s t r a t e and an L - a r g i n i n e r e s i d u e on t h e enzyme. A

procedure has been d e s c r i b e d f o r

testicular

hyaluronidase

a d m i n s t r a t i o n of

the

enzyme.414

the reported non-specific presence of

t h e assay

i n human b l o o d f o l l o w i n g I n h i b i t a t i o n of

of

bovine

intravenous

h y a l u r o n i d a s e by

serum i n h i b i t o r i s m i n i m a l .

However,

the

human s e r u m does a l t e r t h e pH p r o f i l e o f h y a l u r o n i d a s e

and enhances

the

activity

of

the

enzyme

at

low

pH

values.

P r e l i m i n a r y d a t a i n d i c a t e t h e e f f e c t s c a u s e d b y s e r u m o n t h e pH

516


o p t i m u m and t h e p r e s e n c e o f endogenous serum h y a l u r o n i d a s e .

The

a c t i v a t i o n e f f e c t i s n o t s p e c i f i c f o r any p a r t i c u l a r b l o o d t y p e and A i s i n d e p e n d e n t o f w h e t h e r serum o f c i t r a t e d p l a s m a i s used. similar

effect

produced

by

to

that

of

different It

concentration.

serum

on

buffer

i s

hyaluronidase

or

mixtures

recommended

h y a l u r o n i d a s e be m e a s u r e d a t pH 4.0

that

activity

increased

bovine

i s

NaCl

testicular

i n 0.1 M sodium c i t r a t e b u f f e r

M NaC1, as u n d e r t h e s e c o n d i t i o n s t h e a d d i t i o n o f

c o n t a i n i n g 0.15

human serum o r c i t r a t e d p l a s m a does n o t a l t e r t h e pH o p t i m u m o f t h e enzyme. C o m p a r a t i v e k i n e t i c s t u d i e s h a v e b e e n p e r f o r m e d o n mouse a n d b o v i n e t e s t i c u l a r h y a l ~ r o n i d a s e . ~The ~ ~ mouse enzyme has an o p t i m u m pH o f

4.3

testicular

with

a b r o a d pH s p e c t r u m

hyaluronidase.

h y a l u r o n i d a s e s show energy

of

While

maximum a c t i v i t y

activation

are

mg m l - l

that

mouse

different

Arrhenius

f r m

of

for the

values

b o v i n e enzyme. The m o s t

HgC12 a t

M concentration.

the

value

testicular

mouse t e s t i c u l a r

that

Xm

for

enzyme i s m o r e s t a b l e t h a n t h e b o v i n e e n z y m e a t 55OC. for

reveal

for

plots

mouse

potent i n h i b i t o r s

experiments

bovine

testicular

Mouse h y a l u r o n i d a s e has a

as c o m p a r e d t o 2.1 mg m l - '

Thermal-denaturation

to

and

a t 37OC,

obtained

degradation o f hyaluronic acid. o f 0.9

similar

bovine

h y a l u r o n i d a s e a r e CuS04 and

L-Cysteine

appeared t o p r o t e c t t h e

enzyme i n h i b i t i o n caused by HgC12. A

r a p i d p u r i f i c a t i o n o f b o v i n e t e s t i c u l a r h y a l u r o n i d a s e by

c h r o m a t o g r a p h y on d e r m a t a n s u l p h a t e - s u b s t i t u t e d

AH-Sepharose

46

f o l l o w e d by c h r o m a t o g r a p h y o n a c e t y l a t e d A H - S e p h a r o s e 48 h a s b e e n reported.416

The p r o c e d u r e y i e l d e d a p u r i f i e d h y a l u r o n i d a s e w i t h a

specific activity of SDS-polyacrylamide

19.1

u n i t s pmol m i n - l

g e l electrophoresis of

r e v e a l e d t h e p r e s e n c e o f t w o c l o s e bands w i t h w e i g h t s o f 61,000

mg-l

i n high yield.

the purified material approximate molecular

and 67,200.

C h a r a c t e r i z a t i o n o f B-c-2-acetamido-Z-deoxyglucosidase

from

mouse t e s t e s s h o w e d t h a t t h e e n z y m e d o e s n o t c r o s s - r e a c t i n immunodif f u s i o n p l a t e s with anti-mouse t e s t i c u l a r hyaluronidase serum,

suggesting t h a t

have common a n t i g e n i c

% - ~ - 2 - a c e t a m i d o - 2 - d e o x y g l u c o s i d a s e does n o t determinants i n hyaluronidase or

hyalurono-

g l u c o s a m i n i d a s e complex o f acrosome o r t e s t e s . 3 7 H y a l u r o n i d a s e h a s b e e n p u r i f i e d 9 4 - f o l d f r o m mouse t e s t e s b y ion-exchange

chromatography,

chromatography.417 10-20% o f

gel

filtration,

and

affinity

The enzyme was r e l a t i v e l y h e a t s t a b l e and l o s t

i t s a c t i v i t y a t 50-55OC

for

10 min.

La

f o r heat

517

6: Enzymes d e n a t u r a t i o n o f e n z y m e was 4 2 - 4 5

k c a l b e t w e e n 45 a n d 63OC.

The

M i c h a e l i s c o n s t a n t o f mouse t e s t i c u l a r h y a l u r o n i d a s e was 1.1 mg m l - I hyaluronic acid. A n t i b o d i e s t o t h e p u r i f i e d enzyme showed a s i n g l e precipitin

line

by

Ouchterlony

h y a l u r o n i d a s e i n h i b i t e d enzyme mouse

testicular

gel

diffusion.

activity

hyaluronidase

by

was

Antiserum

to

Immunologically,

25%.

species

specific.

Tissue

e x t r a c t s o f mouse v i t a l o r g a n s , e x c e p t t e s t e s a n d e p i d i d y m i s , d i d not react w i t h the antisera, occur

though n o n - s p e c i f i c

precipitation did

between i n t e s t i n a l e x t r a c t s and a n t i - h y a l u r o n i d a s e

H y a l u r o n i d a s e was

localized i n testis

immunof luorescence. localized

on

spermatids

cell

and

A

specific

boundaries

appeared

on

sections

dark-green

extending

the

sperm

by

serum.

indirect

f l u o r e s c e n c e was

from

spermatogonia

acrosome.

to

Cytoplasm o f

s p e r m a t o g o n i a and s p e r m a t o c y t e s showed l i g h t - g r e e n f l u o r e s c e n c e w h e r e a s i n t e r s t i t i a l t i s s u e was d e v o i d o f f l u o r e s c e n c e . I n a s i m p l e p l a t e assay

for

hyaluronidase a c t i v i t y hyaluronic

a c i d i s i n c o r p o r a t e d i n t o a g a r o s e g e l s a n d t h e enzyme i s a l l o w e d t o d i f f u s e f r o m punched wells.418

The u n d i g e s t e d h y a l u r o n i c a c i d i s

t h e n p r e c i p i t a t e d w i t h c e t y l p y r i d i n i u m c h l o r i d e and t h e d i a m e t e r s o f t h e c l e a r c i r c l e s a r e p r o p o r t i o n a l t o t h e l o g a r i t h m o f t h e enzyme concentration applied t o

the

well.

The

assay

e x a m i n e c o m m e r c i a l l y a v a i l a b l e h y m e n o p t e r a venoms, use

i n

allergy

diagnosis

and

h y a l u r o n i d a s e as a measure o f permits

the

analyses

reproducibility,

of

without

a

treatment, lot-to-lot

large

for

ut'ilized t o

manufactured f o r their

content

consistency.

number

t h e need f o r

was

of

of

The a s s a y

samples

with

good

any s p e c i a l i n s t r u m e n t a t i o n .

Based on t h e q u a n t i t y o f p u r i f i e d h y a l u r o n i d a s e r e p o r t e d i n h o n e y b e e venom

(T.P.

Lichenstein,

King,

1976, A r c h .

i s estimated t o

detect

A.K.

Sobotka,

Biochem.

L.

70 ng m l - l

of

Kochoumain,

172, 661-671),

Biophys.,

a n d L.M. t h e assay

p u r i f i e d honey-bee

venom

hyaluronidase. The d e g r a d a t i o n p r o c e s s o f h y a l u r o n i c a c i d b y S t r e p t o m y c e s h y a l u r o n i d a s e has been i n v e s t i g a t e d . 4 1 9

S a t u r a t e d and

unsaturated

h y a l u r o n a t e o l i g o s a c c h a r i d e s w e r e p r e p a r e d f r o m human u m b i l i c a l - c o r d h y a l u r o n i c a c i d by p a r t i a l d i g e s t i o n w i t h b o v i n e t e s t i c u l a r

-Streptomyces

hyaluronidase, respectively.

Streptomyces hyaluronidase,

the d i s t r i b u t i o n o f degradation products

f r o m t h e s e o l i g o s a c c h a r i d e s was d e t e r m i n e d . s a t u r a t e d or

unsaturated,

T e t r a - and h e x a - s a c c h a r i d e s as f i n a l p r o d u c t s .

and

After treatment with Octasaccharides, e i t h e r

were s u b s t r a t e s o f were n o t

t h e minimum size.

degraded f u r t h e r

and r e m a i n e d

This i n d i c a t e s t h a t a t l e a s t f o u r succeeding

3-

518


a c e t y l h y a l o b i u r o n o s y l r e s i d u e s were r e q u i r e d f o r t h i s e n z y m a t i c degradation. S i n c e u n s a t u r a t e d o l i g o s a c c h a r i d e s were more s u s c e p t i b l e t o t h i s enzyme t h a n s a t u r a t e d o n e s of t h e same p o l y m e r i z a t i o n d e g r e e , and i n n e r l i n k a g e s were c l e a v e d i n p r e f e r e n c e t o t h o s e a t t h e o u t e r m o s t s i t e s , some g r o u p s a d j a c e n t t o t h i s segment seemed t o i n f l u e n c e t h e s u s c e p t i b i l i t y of t h e o l i g o s a c c h a r i d e t o t h i s enzyme. Some p r o p e r t i e s of bovine f o e t a l b r a i n h y a l u r o n i d a s e have been described.35 The p o s s i b l e r o l e of c h o n d r o i t i n s u l p h a t e C and h y a l u r o n i d a s e i n t h e p r o c e s s e s o f d i f f e r e n t i a t i o n and d i v i s i o n i s discussed.

33

Inulinases

T h e s e p a r a t i o n and p u r i f i c a t i o n of B - Q - f r u c t o f u r a n o s i d a s e and an i n u l i n a s e f r o m g e r m i n a t i n g g a r l i c ( A l l i u m s a t i v u m ) b u l b s have been d e s c r i b e d . 8 0 Both t h e e n z y m e s ( m o l . wts. 7 6 , 0 0 0 b y g e l chromatography) were i n a c t i v a t e d b y 4 - c h l o r o m e r c u r i b e n z o a t e , 5,5'dithiobis(2-nitrobenzoic a c i d ) , and h e a t t r e a t m e n t a t 5 5 O C f o r 5 m i n a t pH 7.0. The i n u l i n a s e h y d r o l y s e d i n u l i n , s u c r o s e , and r a f f i n o s e . The K m v a l u e s f o r i n u l i n a n d s u c r o s e w e r e 1 0 m M a n d 25 m M , respectively. While i n t h e e a r l y s t a g e s of p l a n t growth ( u p t o 6 days) t h e i n u l i n a s e and B - Q - f r u c t o f u r a n o s i d a s e a c t i v i t i e s i n c r e a s e d i n a p a r a l l e l fashion i n t h e b u l b s ; a t l a t e r stages the increase i n a c t i v i t y was more t h a n t h a t of i n u l i n a s e a c t i v i t y . The p r o d u c t i o n o f a l c o h o l f r o m f e r m e n t a b l e e x t r a c t s o f J e r u s a l e m a r t i c h o k e h a s been i n v e s t i g a t e d u s i n g y e a s t s w i t h i n u l i n a s e a c t i v i t y .420

34

Isoamylases

Immunoreactive t r y p s i n and p a n c r e a t i c i s o a m y l a s e a c t i v i t y i n s e r u m of p a t i e n t s w i t h c h r o n i c r e n a l f a i l u r e o r h e p a t i c c i r r h o s i s h a s been i n v e s t i g a t e d . 4 2 1 I n 121 p a t i e n t s w i t h e i t h e r l i v e r c i r r h o s i s o r c h r o n i c r e n a l f a i l u r e p a n c r e a t i c enzymes i n serum were I n r e n a l i n s u f f i c i e n c y a d e c r e a s e d r a t e of a frequent finding. enzyme e l i m i n a t i o n i s t h e most l i k e l y c a u s e of t h e a b o v e - n o r m a l v a l u e s observed f o r serum and p a n c r e a t i c i s o a m y l a s e .

519

6: Enzymes 35

Laminaranases comparative

A

invertebrates

has

study been

of

carbohydrase a c t i v i t i e s

performed.299

invertebrates belonging t o 7 types, Arhropoda,

Mollusca,

103

species

Spongia, Coelenterata,

Echinodermata,

Chordata,

were

i n

marine

of

marine

Annelida,

tested

for

l a m i n a r i n a s e , c e l l u l a s e , a n d amylase a c t i v i t i e s .

36

Lysozymes

A s i m p l e method f o r t h e u l t r a s e n s i t i v e q u a n t i t a t i o n o f l y s o z y m e

has

been

developed.422

The

l y t i c

a c t i v i t y

against

M i c r o c o c c u s l y s o d e i k t i c u s was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y a f t e r an 18 h i n c u b a t i o n p e r i o d .

The m e t h o d i s c a p a b l e o f q u a n t i t a t i n g

l y s o z y m e a t c o n c e n t r a t i o n s as l o w as 5 pg m l - l

a n d is a p p l i c a b l e t o

d e t e r m i n a t i o n s o f t h e enzyme i n c o m p l e x b i o l o g i c a l m i x t u r e s . A radioimmunoassay d e v e l o p e d f o r serum and u r i n a r y l y s o z y m e i n v o l v e s use o f an a n t i b o d y , u r i n e from

a patient

d i l u t e d 4000-fold,

raised i n rabbits,

with monocytic

leukemia.423

t o lysozyme from This antiserum,

i s incubated w i t h r a d i o l a b e l l e d lysozyme f o r

2 h

and a n t i b o d y - b o u n d l y s o z y m e is s e p a r a t e d f r o m f r e e l y s o z y m e w i t h dextran-coated charcoal.

V a l i d a t i o n o f t h e assay i n c l u d e d p r e c i s i o n

and p a r a l l e l i s m s t u d i e s w i t h s e r u m a n d u r i n e s a m p l e s f r o m p a t i e n t s w i t h m o n o c y t i c l e u k e m i a and a n a l g e s i c n e p h r o p a t h y .

Results o f the

radioimmunoassay and t h o s e o b t a i n e d w i t h a M i c r o c o c c u s l y s o d e i k t i c u s l y t i c assay c o r r e l a t e d w e l l assay

are

i t s

(r =

0.94).

The m a i n a d v a n t a g e s o f t h e

good p r e c i s i o n and r e p r o d u c i b i l i t y and i t s h i g h

sensitivity. A new l y s o z y m e a s s a y b a s e d o n f l u o r e s c e n c e p o l a r i z a t i o n o r fluorescence

-M i c r o c o c c u s

intensity u t i l i z i n g a peptidoglycan substrate from l y s o d e i k t i c u s subsequently l a b e l l e d w i t h fluorescein

i s o t h i o c y a n a t e (FITC) a t t h e amino group o f t h e p e p t i d e has been reported.424

When t h e F I T C - l a b e l l e d

lysozyme digestion, decrease of

an i n c r e a s e o f

fluorescence

s u b s t r a t e was s u b j e c t e d t o fluorescence intensity or a

p o l a r i z a t i o n value

(p

value)

was a p p a r e n t

i n f i v e m i n u t e s a t a l y s o z y m e c o n c e n t r a t i o n a s l o w a s 0.1 o r 0 . 0 1 p g ml-', respectively. The e f f e c t o f o t h e r h y d r o l y t i c enzymes, i n c l u d i n g a-P-mannosidase, p r o t e a s e s , a n d r i b o n u c l e a s e , o n t h e p v a l u e was f o u n d t o be n e g l i g i b l e . The m e a s u r e d v a l u e s r e p r e s e n t e d t h e s p e c i f i c i t y


520 and dose o f l y s o z y m e added.

Apparent

lmax and K m v a l u e s f o r t w o

d i f f e r e n t lysozymes, chicken egg-white d e t e r m i n e d by t h i s method.

and human,

c o u l d be

A s t u d y h a s b e e n made o f t h e r a t e s o f s t r u c t u r a l f l u c t u a t i o n s

of lysozyme i n t h e range of t h e r m a l - u n f o l d i n g t r a n s i t i o n . 4 2 5 hydrogen-deuterium

exchange

reaction for

The

the &-tryptophan residues

i n l y s o z y m e h a s b e e n f o l l o w e d i n 4.5M L i B r a t p H 7 . 2 i n t h e t e m p e r a t u r e range of

the unfolding t r a n s i t i o n

a n c e c h a n g e a t 2 9 3 nm.

by m e a s u r i n g t h e t r a n s m i t t -

The e x c h a n g e r e a c t i o n p r o c e e d e d i n t h r e e

r e l a t i v e l y exposed t r y p t o p h a n r e s i d u e s on t h e m o l e c u l a r s u r f a c e . The t h i r d p h a s e c o r r e s p o n d e d t o t h e H - D three buried i-tryptophan

residues.

exchange r e a c t i o n o f t h e

The H-D

exchanges

o f three

I-

t r y p t o p h a n r e s i d u e s b u r i e d i n f o l d e d m o l e c u l e s w e r e c a u s e d by f l u c t u a t i o n between t h e folded

and u n f o l d e d s t r u c t u r e o f t h e p r o t e i n

The r a t e s o f s u c h a f l u c t u a t i o n w e r e d e t e r m i n e d f r o m t h e

molecule.

r a t e s of t h e exchange r e a c t i o n a t v a r i o u s t e m p e r a t u r e s .

These r a t e s

agreed very w e l l w i t h those determined from the temperature-jump method.

T h i s means t h a t a p r o t e i n m o l e c u l e i n s o l u t i o n f l u c t u a t e s

b e t w e e n t h e Nion region,

and D - s t a t e s

a t every temperature w i t h i n t h e t r a n s i t -

where t h e N-form

i s the t i g h t l y folded native structure

and t h e D - f o r m t h e r a n d o m l y c o i l e d c h a i n . thermal unfolding of

ester-108-lysozyme

From measurements o f

and t h e b i n d i n g c o n s t a n t o f

i t was f o u n d t h a t a l m o s t a l l

(Q-GlcNAc)3,

t o ester-108-lysozyme,

cross-linked

m o l e c u l e s a r e i n t h e f o l d e d s t a t e n e a r 5OoC and pH 7.2

i n 4.5M

LiBr,

where i n t a c t m o l e c u l e s a r e u n f o l d e d .

A s t u d y was a l s o

In the

made o f t h e H - D e x c h a n g e r e a c t i o n o f e s t e r - 1 0 8 - l y s o z y m e . temperature

region

of

43-50°C,

about

t r y p t o p h a n r e s i d u e s of ester-108-lysozyme immediately after

the

m i x i n g of

D20,

70% o f

the

exchangeable

were exchanged w i t h i n 1 s i n spite of the fact that

almost a l l molecules are i n the folded state.

T h i s was c o n s i d e r e d

the p r e m e l t i n g o f t h e surface o f a cross-linked molecule. The

interaction of

,

lysozyme

with

mixed 1,2-dipalmitoyl-Q-

-I-phosph a t idy l c h o 1i n e

p h o s p h a t id ic a c id /1 2 - d im y r is t oy 1 has been i n v e s t i g a t e d by

laser

Raman s p e c t r o s c o p y . 4 2 6

l i p o s o mes

Substantial

changes were observed i n t h e s p e c t r a o f b o t h t h e l i p i d and p r o t e i n i n t h e m i x e d l i p o s o m e s o v e r t h e r a n g e 10-62OC. b e l o w 27OC,

A t temperatures

i n t e r a c t i o n w i t h l i p i d appears t o i n c r e a s e s l i g h t l y the

amount o f h e l i c a l s t r u c t u r e i n l y s o z y m e a t t h e expense o f random conformation.

A t t e m p e r a t u r e s above 3OoC, c o n s i d e r a b l e B-sheet i s Onset o f B - f o r m a t i o n a p p e a r s t o c o i n c i d e w i t h

i r r e v e r s i b l y formed. the

formation

of

disordered

lipid

side

chains

in

the

acidic

52 1

6: Enzymes component o f t h e l i p i d .

A t a l l temperatures,

t h e 0-P-0

diester

s t r e t c h i n g mode a t 7 8 2 cm-' i s much m o r e i n t e n s e i n t h e l i p i d p r o t e i n m i x t u r e than i n l i p i d alone. I t i s observed t h a t the d i m y r i s t o y l phosphatidylcholine chain-disorder t r a n s i t i o n i s lowered by 3 O C ,

w h i l e t h a t o f t h e p h o s p h a t i d i c a c i d i s l o w e r e d by 12'C,

yet

the post-transition conformation contains a s i g n i f i c a n t l y higher p r o p o r t i o n o f t r a n s segments i n t h e presence of lysozyme.

These

(1) a p o l a r i n t e r a c t i o n between

r e s u l t s are interpreted i n terms o f

a c i d i c p h o s p h o l i p i d and l y s o z y m e a t t e m p e r a t u r e s b e l o w e i t h e r c h a i n disorder transition,

i n which lysozyme i s e s s e n t i a l l y excluded from

the hydrophobic p o r t i o n o f

the

higher

involves

temperatures

dipalmitoyl

which

phosphatidic

acid

l i p i d , and (2) i n

the

the

an i n t e r a c t i o n a t

lipid

side

disordered

chains

state

and

of i s

m a n i f e s t e d by a s u b s t a n t i a l c o n f o r m a t i o n a l change. The e f f e c t s o f d e t e r g e n t s o r f a t t y a c i d s o n p r o t e o l y s i s o f lysozymes

have

been

studied.427

Low

concentrations

of

ionic

d e t e r g e n t s w i t h 0 o r more c a r b o n s i n t h e a l k y l c h a i n w e r e p r e d i c t e d t o s h i f t t h e native-denatured t r a n s i t i o n i n the lysozyme t o the denatured state.

F a t t y a c i d s were f o u n d t o e x e r t a s i m i l a r e f f e c t ,

a n d t h e i r p a r t i c i p a t i o n was p r o p o s e d i n p r o t e o l y s i s o f a l i m e n t a r y c a n a l d i g e s t i o n and i n t r a c e l l u l a r c a t a b o l i s m . Lysinoalanine

formation

i n

lysozyme has

dependent on a l k a l i c o n c e n t r a t i o n , time.428

The u p p e r

limits of

pH,

been found

to

be

t e m p e r a t u r e , and e x p o s u r e

lysinoalanine

f o r m a t i o n i n lysozyme

and a - l a c t a l b u m i n were r e l a t e d t o t h e number o f I - l y s i n e r e s i d u e s w i t h a c y s t i n e d i s u l p h i d e bond i n t h e a d j a c e n t p o s i t i o n r a t h e r t h a n t o the i n d i v i d u a l contents o f these residues. P r e p a r a t i o n s and i m m u n o l o g i c a l c h a r a c t e r i z a t i o n s o f lysozyme d e r i v a t i v e s d i n i t r o p h e n y l a t e d a t I - l y s i n e - 3 3 96,

r e s p e c t i v e l y , have been r e p o r t e d . 4 2 9

egg-white

Two d e r i v a t i v e s o f h e n

lysozyme w i t h s i n g l e s u b s t i t u t i o n s of

(DNP) r e s i d u e w e r e p r e p a r e d . f o l d molar excess of mono-DNP-substituted by i o n - e x c h a n g e presence of derivative

2,4-dinitrobenzene

chromatographies.

cellulose.

a 7-fold This

After

s u l p h o n i c a c i d p r o v i d e d one lysozyme),

amino

material

dinitrobenzene sulphonic

was

lysozyme)

m a l e y l a t i o n o f lysozyme i n the maleic anhydride,

the

g r o u p was p u r i f i e d o n OE-52 dinitrophenylated with

a c i d and t h e

d e r i v a t i v e was p u r i f i e d o n DE-52.

w h i c h was p u r i f i e d

The o t h e r one (DNP1-96

molar excess of

w i t h one f r e e

a 2,4-dinitrophenyl

T h e r e a c t i o n o f l y s o z y m e w i t h a 10-

l y s o z y m e (DNP1-33

was p r e p a r e d a s f o l l o w s .

two

and I - l y s i n e -

DNP1-96

2,4-

mono-DNP-substituted l y s o z y m e was f i n a l l y


522 p u r i f i e d on SE-Sephadex

C-25,

a f t e r d e m a l e y l a t i o n a t pH 3.5,

a t 37OC

f o r 5 days. DNP1-33 l y s o z y m e and DNP1-96 l y s o z y m e b o t h m i g r a t e d as On a s i n g l e band w i t h s l o w e r m o b i l i t y t h a n t h a t o f n a t i v e l y s o z y m e . reduction,

c a r b o x y m e t h y l a t i o n , a n d c h y m o t r y p s i n d i g e s t i o n , b o t h mono-

DNP-substituted lysozymes y i e l d e d a s i n g l e yellow peptide. amino a c i d compositions o r indicated

that

p a r t i a l sequence

C-lysine-33

and

of

these

I-lysine-96

were

the

d i n i t r o p h e n y l a t e d r e s i d u e s i n DNP1-33 l y s o z y m e and DNP1-96 respectively. antigenic antisera

DNP1-33

lysozyme

reactivities to

lysozyme.

a c c e s s i b l e t o anti-DNP lysozyme t o anti-DNP lysozyme.

equal

This

The

to

and

DNP1-96

that

DNP

of

residues

antibodies,

on

the

only

lysozyme,

lysozyme

native

The

peptides

showed

lysozyme

with

protein

were

b u t t h e a f f i n i t i e s o f DNP1-33

a n t i b o d i e s w e r e l o w e r t h a n t h o s e o f DNP1-96

result

i s

discussed with respect t o

the

local

e n v i r o n m e n t s o f t h e DNP r e s i d u e s i n t h e s e p r o t e i n s . Lysozymes f r o m hen,

duck 11, and goose e g g - w h i t e

lysozyme were compared a t t h e l e v e l o f using the solvent

a n d human

t h e i r L-tryptophyl

perturbation technique

and f l u o r e s c e n c e

residues emission

i n a d d i t i o n t o t w o c h e m i c a l m o d f i c a t i ~ n s . ~The ~ ~three C-tryptophyl r e s i d u e s o f goose l y s o z y m e were found

completely exposed t o the

solvent.

The s t u d y o f t h e s i t u a t i o n o f t h e a r o m a t i c r e s i d u e s o f hen,

duck 11,

and human l y s o z y m e s p r o v i d e d a d d i t i o n a l e v i d e n c e t h a t t h e

three-dimensional although

an

s t r u c t u r e s o f t h e s e enzymes a r e v e r y s i m i l a r ,

unexpected r e a c t i v i t y

of

~-tryptophan-28 of

human

lysozyme t o w a r d s N - b r o m o s u c c i n i m i d e c o u l d be evidenced.

As p a r t o f a s t u d y o f t h e whey p r o t e i n s o f v a r i o u s m a m m a l s a c o m p a r i s o n h a s been made o f t h e a - l a c t a l b u m i n s and l y s o z y m e s o f t h e kangaroo and horse.431 rufa)

there i s only

lactation,

b u t no

I n t h e m i l k o f t h e r e d kangaroo (Megaleia and i t o c c u r s

one a - l a c t a l b u m i n

l y s o z y m e has been d e t e c t e d .

throughout

T h e r e a r e t w o a-

l a c t a l b u m i n s i n t h e m i l k o f t h e g r e y kangaroo (Macropus g i g a n t e u s ) : one,

designated

lactation,

a-lactalbumin

and t h e second,

Zone

B,

i s

present

designated a-lactalbumin

present only i n l a t e lactation.

throughout Z o n e A,

One l y s o z y m e i s a l s o p r e s e n t .

i s The

m i l k o f t h e h o r s e (Equus c a b a l l u s ) c o n t a i n s one a - l a c t a l b u m i n and a t l e a s t one l y s o z y m e .

P a r t i a l amino a c i d sequences a r e p r o p o s e d f r o m

sequence

determination

compared

w i t h t h e known sequences o f

and

from

analyses other

of

tryptic

peptides

a-lactalbumins

and

lysozymes. M u l t i p l e forms of

l y s o z y m e found i n t h e r a t l i v e r have been

i s o l a t e d and c h a r a c t e r i z e d f r o m

cellular

organelles.432

Isolation

523

6: Enzymes of

the

e n z y m e s was a c h i e v e d b y Sephadex

gel

f i l t r a t i o n and

chromatography on carboxymethylcellulose. The p u r i t y o f t h e p r e p a r a t i o n s was e x a m i n e d by e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e g e l . The n u c l e a r l y s o z y m e moved as a s i n g l e band, i n d i c a t i n g h o m o g e n e i t y , whereas

other

subcellular

lysozymes

t h e p r e s e n c e o f m o r e t h a n o n e band,

appeared heterogeneous

due

to

thus showing p a r t i a l p u r i t y .

Although t h e s u b c e l l u l a r lysozymes were s i m i l a r w i t h r e s p e c t t o e n z y m a t i c p r o p e r t i e s , pH, mobility,

b u f f e r m o l a r i t y optima,and

electrophoretic

d i f f e r e n c e s were o b s e r v e d i n e l u t i o n p a t t e r n s ,

t o n u c l e a r i n h i b i t o r , and h e a t s e n s i t i v i t y . distinctly

different

by

these

criteria

responses

N u c l e a r l y s o z y m e was as

compared

to

other

s u b c e l l u l a r lysozymes. E v o l u t i o n a r y i m p l i c a t i o n s h a v e b e e n made a b o u t t h e r e l a t i o n b e t w e e n h e n e g g - w h i t e l y s o z y m e and b a c t e r i o p h a g e T4 l y ~ o z y m e . ~ ~ Hen e g g - w h i t e

l y s o z y m e a n d T 4 b a c t e r i o p h a g e l y s o z y m e h a v e t h e same

c a t a l y t i c f u n c t i o n , b u t have non-homologous amino a c i d sequences. Notwithstanding the differences i n t h e i r primary structures,

the two

l y s o z y m e s have s i m i l a r i t i e s i n t h e i r o v e r a l l backbone c o n f o r m a t i o n s , in their

modes

of

b i n d i n g substrates, and p r o b a b l y

mechanisms o f a c t i o n . t h e f o l d i n g of

By d i f f e r e n t c r i t e r i a ,

in their

t h e s i m i l a r i t y between

t h e t w o enzymes can be shown t o be s t a t i s t i c a l l y

significant.

A l s o t h e t r a n s f o r m a t i o n w h i c h o p t i m i z e s t h e agreement

between t h e

backbones

the t w o

of

accurately t h e i r active-site clefts, i n the A,

8,

C,and

molecules

i s

D s u b s i t e s o f hen egg-white

w i t h i n 0.1

t o 0.2

lysozyme.

Furthermore,

shown t o

align

so t h a t s a c c h a r i d e u n i t s bound lysozyme coincide

nm w i t h a n a l o g o u s s a c c h a r i d e s

bound t o phage

a number o f t h e s p e c i f i c i n t e r a c t i o n s

b e t w e e n enzyme and s u b s t r a t e w h i c h w e r e o b s e r v e d f o r h e n e g g - w h i t e l y s o z y m e , and t h o u g h t t o be i m p o r t a n t f o r c a t a l y s i s , a r e f o u n d t o o c c u r i n a s t r u c t u r a l l y a n a l o g o u s way i n t h e phage enzyme. four

atoms from

equivalent,

i n c l u d i n g saccharides

superimpose w i t h a root-mean-square structural

and

Fifty-

t h e r e s p e c t i v e a c t i v e s i t e s w h i c h a p p e a r t o be

functional

bound i n t h e

and C

B

d i s c r e p a n c y o f 1.35

similarities

suggest

sites,

nm.

that

These

the

two

l y s o z y m e s h a v e a r i s e n b y d i v e r g e n t e v o l u t i o n f r o m a common precursor.

This

i s

completely different probability,

the

first

case

i n which

two

proteins

a m i n o a c i d s e q u e n c e h a v e b e e n shown,

of

with high

t o h a v e e v o l v e d by d i v e r g e n t r a t h e r t h a n c o n v e r g e n t

evolution. The b i n d i n g mode o f s o d i u m d o d e c y l s u l p h a t e t o l y s o z y m e a n d t h e accompanying s t r u c t u r a l change o f

l y s o z y m e by b i n d i n g h a v e been

524


i n v e s t i g a t e d b y means o f t h e b i n d i n g i s o t h e r m ,

the precipitation

and t h e CD s p e c t r a i n p u r e w a t e r ,

and b o r a t e b u f f e r

curve,

solutions.434

NaC1,

The p r e c i p i t a t i o n p h e n o m e n a c o u l d b e e x p l a i n e d i n

t e r m s o f t h e n e u t r a l i z a t i o n o f t h e n e t c h a r g e o f l y s o z y m e due t o dodecyl s u l p h a t e - i o n b i n d i n g .

The a n a l y s i s o f t h e b i n d i n g i s o t h e r m s

by t h e u s e o f t h e B E T e q u a t i o n g a v e t h e s i t e n u m b e r o f t h e f i r s t l a y e r c o r r e s p o n d i n g t o t h e p o s i t i v e l y c h a r g e d r e s i d u e s a t t h e pH studied. helix

The c o n f o r m a t i o n a l change f r o m t h e B - s t r u c t u r e

has

been

environmental

observed

change

of

i n

the

the

second-layer

side-chain

t o t h e a-

binding.

residues

has

also

The been

observed. F l u o r e s c e n c e p o l a r i z a t i o n s t u d i e s h a v e been made o f s a c c h a r i d e binding

to

wheat-germ

- -glucopyranose deoxy-Q i n

the

polarization

saturating

levels

agglutinin

and

l y s o ~ y m e . ~2 ~ - A~c e t a m i d o - 2 -

has been shown t o c a u s e o n l y s l i g h t i n c r e a s e s of

wheat-germ

whereas

the

agglutinin

disaccharide

fluorescence

and

p r o d u c e d i n c r e a s e s i n t h e p o l a r i z a t i o n v a l u e f r o m 0.116 t o 0.151

0.154, r e s p e c t i v e l y .

at

trisaccharide and

These i n c r e a s e s have s u g g e s t e d t h a t r o t a t i o n a l

m o t i o n s o f t h e & - t r y p t o p h a n r e s i d u e a t t h e b i n d i n g s i t e s were b e i n g r e s t r i c t e d by an i n t e r a c t i o n b e t w e e n t h e s e t r y p t o p h a n s and t h e bound sugars.

A m o d e l o f t h e n a t u r e and l o c a t i o n o f t h e s e i n t e r a c t i o n s

was d i s c u s s e d .

Comparable r e s u l t s were o b t a i n e d w i t h l y s o z y m e ,

w h i c h showed a l a r g e r e f f e c t deoxy-P-glucopyranose

but

a

upon t h e b i n d i n g o f 2 - a c e t a m i d o - 2 -

maximal

increase

in

p o l a r i z a t i o n upon

b i n d i n g the corresponding disaccharide or trisaccharide. Bindings o f

calcium t o

l y s o z y m e and i t s d e r i v a t i v e s h a v e been

The s t u d i e d by UV d i f f e r e n c e s p e c t r o s c o p y a t v a r i o u s b i n d i n g c o n s t a n t was 40 M ' l a t n e u t r a l pH. The b i n d i n g c a u s e d p r o t o n r e l e a s e f r o m l y s o z y m e and d i d n o t i n h i b i t t h e b i n d i n g o f t r i -

2 - a c e t a m i d o - 2 - d e o x y - B - ~ - g l ~ ~ 0 p y r a n o s et o l y s o z y m e .

I n t h e presence

o f 0.2M Ca2+, l y s o z y m e s h o w e d 2 6 % o f t h e a c t i v i t y o f t h e f r e e e n z y m e t o w a r d h e x a - 2 - a c e t am id o -2-deox y - B - P - g l u c o p y r anose b u t t h e c l e a v a g e I t was p r e d i c t e d

p a t t e r n was s i m i l a r t o t h a t o f t h e f r e e e n z y m e .

t h a t c a l c i u m bound n e a r t h e c a t a l y t i c c a r b o x y l s c a u s e d i n h i b i t i o n o f lysozyme a c t i v i t y . digestion

that

I t was

calcium

found from

the r e s u l t s o f

binding shifted

the

protease

native-denatured

t r a n s i t i o n i n lysozyme t o w a r d t h e n a t i v e s t a t e . Oxindolealanine-62

lysozyme e q u i l i b r i u m ,

calorimetric,

and

k i n e t i c s of i t s r e a c t i o n w i t h B-~-acetamido-2-deoxyglucopyranose oligosaccharides

have

been

studied.437

r e a c t i o n w i t h N-bromosuccinimide

The

enzyme,

formed

by

and p u r i f i e d by u s i n g a f f i n i t y

525

6: Enzymes chromatography,

was

examined

i n

i t s

binding of

homologous

o l i g o s a c c h a r i d e s o f B-a-acetamido-2-deoxyglucopyranose catalysis

o f

hydrolysis

and

of

and i n i t s

transglycosylation

h e x a s a c c h a r i d e o f B-Q-acetamido-2-deoxyglucopyranose. binding constants

were

determined

by

changes

fluorescence associated with ligand binding.

of

the

Equilibrium

i n absorbance

or

Enthalpies of binding

The p a t t e r n o f v a r i a t i o n o f AGO a n d

were measured c a l o r i m e t r i c a l l y .

A H o o f b i n d i n g w i t h l i g a n d c h a i n l e n g t h a n d pH was d i f f e r e n t f o r oxindolealanine-62

lysozyme compared w i t h

native

lysozyme.

These

r e s u l t s i n d i c a t e t h a t t h e i n t e r a c t i o n s o f t h e ABC r e g i o n o f active

site

with

substrates

tryptophan-62

oxidation,

tryptophan-62

interactions.

intermediate

between

are

substantially

more t h a n e x p e c t e d f o r

oxidation o f I-tryptophan-62. hexasaccharide,

by

the

I-

l o s s o n l y o f t h e L-

The p a r t i t i o n i n g o f t h e g l y c o s y l enzyme

reaction

with

( t r a n s g l y c o s y l a t i o n ) and r e a c t i o n the

altered

a

with

Similarly,

predominantly t o

saccharide

water

i s

the p a t t e r n of

t e t r a - and

acceptor

unaffected

by

cleavage o f

di-saccharide,

is

u n a f f e c t e d by o x i d a t i o n o f I - t r y p t o p h a n - 6 2 .

The 2 0 0 0 - f o l d s l o w r a t e

of

enzyme

catalytic reaction

(relative

to

free

apparently r e f l e c t s a s t e r i c a l l y hindered f i t of t h e a c t i v e s i t e of

and

substrate)

the substrate i n t o

t h e m o d i f i e d enzyme and n o t a s p e c i a l c a t a l y t i c

i m p o r t a n c e o f I=-tryptophan-62.

The g e o m e t r y o f t h e t r a n s i t i o n s t a t e

f o r o l i g o s a c c h a r i d e h y d r o l y s i s i s i n f e r r e d t o be t h e same f o r n a t i v e and o x i d i z e d enzymes. Oxygen-18 l e a v i n g - g r o u p been measured f o r a s e t o f

kinetic isotope effects glycosyl transfer

n i t r o p h e n y l B-Q-glycosides

as

substrates.206

h y d r o l y s i s and a l k a l i n e h y d r o l y s i s e x h i b i t

-+

0.0015

and

1.0386

g l u c o s i d a s e A show

-+

0.0015

and 1.0377

0.0032,

K I E s on

2

Acid-catalysed

K I E s o f k1&18

respectively.

= 1.0355

Lysozyme and B-a-

respectively.

lmax/srn(!/K)o f

0.0061,

( K I E s ) have

r e a c t i o n s w i t h 4-

(2/5)1,/(1/5)18

= 1.0467

The l a r g e m a g n i t u d e o f

t h e s e K I E s r e q u i r e s t h a t c a r b o n - o x y g e n bond s c i s s i o n be f a r a d v a n c e d i n the t r a n s i t i o n states for these reactions.

Therefore,

i n the

t r a n s i t i o n states for the f i r s t i r r e v e r s i b l e steps i n these reaction sequences,

s c i s s i o n of

t h e g l y c o s i d i c bond must

be e s s e n t i a l l y

c o m p l e t e f o r t h e r e a c t i o n s c a t a l y s e d by l y s o z y m e and B - P - g l u c o s i d a s e A,

which

are

respectively.

thought

to

proceed

Acid-catalysed

fia

gN1

a t r a n s i t i o n s t a t e i n v o l v i n g a t l e a s t 80% C - 0 p a r t i a l proton transfer

and SN2

mechanisms,

h y d r o l y s i s i s shown t o p r o c e e d t h r o u g h bond c l e a v a g e and o n l y

t o t h e l e a v i n g 4 - n i t r o p h e n y l o x y g e n atom.

Binding o f 4-methylumbelliferyl

c h i t o t e t r a o s i d e t o hen lysozyme

526


h a s been s t u d i e d by m e a s u r i n g changes i n f l u o r e s c e n c e a t 375 nm.438 4-methylumbelliferyl lj-acetyl-chitotetraoside

Hydrolysis of

c a t a l y s e d b y l y s o z y m e was s t u d i e d b y m e a s u r i n g t h e r e l e a s e o f 4 methylumbelliferone

from


chitotetraoside fluorimetrically,

N-acetyl-

and t h e k i n e t i c c o n s t a n t s were

d e t e r m i n e d i n t h e pH r a n g e o f 2 t o 8 a t 0.1

i o n i c s t r e n g t h and 42’C.

The b i n d i n g a n d k i n e t i c d a t a s h o w e d t h a t 4 - m e t h y l u m b e l l i f e r y l acetyl-chitotetraoside

y-

binds t o subsites A t o E (productive binding)

and s u b s i t e s A t o D w i t h t h e n o n - r e d u c i n g s u g a r r e s i d u e e x t e n d i n g beyond s u b s i t e A (non-productive k i n e t i c constants

binding).

a t p H 8.5.

p r o d u c t i v e c o m p l e x was 0.77

The f r a c t i o n o f t h e

T h e pH d e p e n d e n c e o f t h e

was a n a l y s e d a s s u m i n g t h a t t h e m o l e c u l a r s p e c i e s

w i t h i o n i z e d I - a s p a r t i c a c i d - 5 2 and p r o t o n a t e d I - g l u t a m i c a c t i v e and I - a s p a r t i c a c i d - 1 0 1

pK v a l u e s o f t h e s e g r o u p s were d e t e r m i n e d . aspartic-52, a n d 4.20,

!=-glutamic-35,

respectively,

respectively, respectively,

acid-35 i s

participates i n the binding,

and t h e

The pK v a l u e s o f

a n d L - a s p a r t i c - 1 0 1 a c i d s w e r e 3.60, for

free

lysozyme,

3.40,

f o r t h e p r o d u c t i v e c o m p l e x , a n d 3.95, f o r t h e n o n p r o d u c t i v e complex.

6.55,

I-

6.20,

a n d 3.40,

6.55,and

3.30,

The pK v a l u e s f o r f r e e

lysozyme were i n e x c e l l e n t agreement w i t h t h o s e o b t a i n e d by a n a l y s i s of

for 4-methylumbelliferyl N-acetyl-

the kinetic constants

chitotrioside. pH 5.2.

The f r e e e n e r g y o f a c t i v a t i o n was 24 k c a l m o l ”

Comparison w i t h

the

corresponding

value

obtained

at for

h y d r o l y s i s o f c h i t o h e x a o s i d e suggests t h a t t h e i n t e r a c t i o n s o f 2-

acetamido-2-deoxy-~-glucopyranose r e s i d u e s w i t h s u b s i t e s E and F i n t h e t r a n s i t i o n s t a t e are i m p o r t a n t i n lysozyme c a t a l y s i s . Binding

of


methylumbelliferyl studied

by

chitotetraoside to

fluorescence

methylumbelliferyl

chitotrioside human

measurement.439

chitotrioside

and

and

4-

l y s o z y m e has been Hydrolysis

of

4-


c h i t o t e t r a o s i d e c a t a l y s e d by human l y s o z y m e was s t u d i e d by m e a s u r i n g the

release of

4-methylumbelliferone

fluorimetrically,

and t h e

k i n e t i c c o n s t a n t s w e r e d e t e r m i n e d i n t h e pH r a n g e o f 2 t o 8 a t 0.1 i o n i c s t r e n g t h a n d 42’C.

On t h e b a s i s o f b i n d i n g and k i n e t i c d a t a ,

i t was shown t h a t 4 - m e t h y l u m b e l l i f e r y l c h i t o t r i o s i d e b i n d s m a i n l y t o s u b s i t e s A t o D w i t h t h e t e r m i n a l m e t h y l u m b e l l i f e r y l g r o u p bound t o subsite D (non-productive

b i n d i n g ) and t h a t 4 - m e t h y l u m b e l l i f e r y l

c h i t o t e t r a o s i d e b i n d s t o s u b s i t e s A t o E [ p r o d u c t i v e b i n d i n g ) and s u b s i t e s A t o D w i t h t h e n o n - r e d u c i n g s u g a r r e s i d u e e x t e n d i n g beyond subsite A kinetic

(non-productive constants

for

binding). hydrolysis

The o f

pH d e p e n d e n c e s o f t h e 4-methylumbelliferyl

527

6: Enzymes chitotrioside

and


c h i t o t e t r a o s i d e were

analysed, assuming t h a t non-productive b i n d i n g occurs c o m p e t i t i v e l y , that

an i o n i z a b l e g r o u p i n a d d i t i o n t o t h e c a t a l y t i c g r o u p s

a s p a r t i c - 5 2 and & - g l u t a m i c - 3 5 ) that

the

molecular species

(L-

p a r t i c i p a t e s i n t h e c a t a l y s i s , and

with ionized &-aspartic

acid-52

and

protonated I-glutamic acid-35 i s active.

Analyses of t h e k i n e t i c

constants

chitotrioside

for


and

4-

m e t h y l u m b e l l i f e r y l c h i t o t e t r a o s i d e b o t h gave t h e same p E v a l u e s o f the

c a t a l y t i c groups

s t r e n g t h a n d 42OC).

( p K 5 2 = 3.63

a n d p 5 3 5 = 6.68

a t 0.1

ionic

These p E v a l u e s were v e r y c l o s e t o t h e v a l u e s

d e t e r m i n e d p r e v i o u s l y by s p e c t r o s c o p i c methods. A lysozyme-catalysed r e a c t i o n o f c h i t o - o l i g o s a c c h a r i d e s has

been d e s c r i b e d . 4 4 0

The t i m e - c o u r s e s

of

substrate

c o n s u m p t i o n and

p r o d u c t f o r m a t i o n i n t h e l y s o z y m e - c a t a l y s e d r e a c t i o n were d e t e r m i n e d w i t h c h i t o t e t r a o s e and c h i t o p e n t a o s e as s u b s t r a t e t o a c c u m u l a t e d a t a s u i t a b l e f o r t h e e s t i m a t i o n o f r a t e c o n s t a n t s by n u m e r i c a l a n a l y s i s .

o r h.p.1.c.

The l y s o z y m e - c a t a l y s e d r e a c t i o n s w e r e f o l l o w e d by t.1.c.

C h i t o t e t r a o s e decomposed a p p a r e n t l y t o s m a l l o l i g o s a c c h a r i d e s w i t h i n 5 h,

and c h i t o p e n t a o s e decomposed w i t h i n 15 m i n a t pH 5.0

The t e m p e r a t u r e

dependence o f

the rate of

i n i t i a l s u b s t r a t e showed a d i f f e r e n t

and 5 O o C .

disappearance of

the

p r o f i l e from t h a t observed w i t h

g l y c o l c h i t i n as s u b s t r a t e by t h e r e d u c i n g p o w e r method.

The o r d e r

( o r d i s t r i b u t i o n ) o f t h e amount o f p r o d u c t f o r m e d f r o m c h i t o p e n t a o s e i n the r e a c t i o n time-course d e t e r m i n e d by h.p.1.c. t h a n t h a t i n t.l.c., thought

d e t e r m i n e d by t.1.c.

d i f f e r e d from that

The r e l a t i v e e r r o r i n h.p.1.c. and t h e t i m e - c o u r s e

was much l e s s

d e t e r m i n e d by h.p.1.c.

was

t o be o f s u f f i c i e n t a c c u r a c y f o r t h e e s t i m a t i o n o f r a t e

c o n s t a n t s by c o m p u t e r a n a l y s i s . High

l y s o z y m e and c h i t i n a s e a c t i v i t i e s have been f o u n d i n

Leydig's organ of Etmopterus spinax

-Tompedo

nobiliana,

i n Leydig's

, Somniosus

m i c r o c e p h a i u s , and

and e p i g o n a l o r g a n s and s p l e e n o f

R a j a r a d i a t a , and i n t h e e p i g o n a l o r g a n o f R h i n o p t e r a bonasus.19 An

enzyme

s t r e p t o c o c c u s of

actively

lysing the

c e l l walls

of

a haemolytic

g r o u p A was shown t o be a l y s o z y m e mucopeptide-l\l-

acetylmuranoylhydrolase.441

I t was i s o l a t e d f r o m t h e c u l t u r e l i q u i d

of

ammonium

A c t i n o m y c e s l e v o r i s by

further

purification

chromatography.

by

gel

sulphate

f i l t r a t i o n

The m o l e c u l a r w e i g h t (12,5001,

p r e c i p i t a t i o n and and

ion-exchange

isoelectric point

(10.61, and a m i n o a c i d c o m p o s i t i o n o f t h e enzyme w e r e d e t e r m i n e d .

A

peptidoglycan

A

obtained

from

the

c e l l

walls

o f

a

group

s t r e p t o c o c c u s was u s e d a s t h e s u b s t r a t e i n a d e t e r m i n a t i o n o f t h e


528

s p e c i f i c i t y o f t h e enzyme. Lysozyrne a c t i v i t y h a s b e e n o b s e r v e d i n b a c t e r i o p h a g e T4 ghosts.442 T h i s enzyme is p r o b a b l y r e s p o n s i b l e f o r the l y s i s from w i t h o u t , observed a t high m u l t i p l i c i t y of i n f e c t i o n , a p r o c e s s independent of the p r e s e n c e of the e gene product which is a l s o a lysozyme. The g h o s t l y s o z y m e a n d e l y s o z y m e d i f f e r e d w i t h r e s p e c t t o t h e i r r e q u i r e m e n t s f o r maximal c a t a l y t i c a c t i v i t y and t o some extent in substrate specificity. The g h o s t l y s o z y m e w a s r e l e a s e d f r o m p h a g e p a r t i c l e by t h e a c t i o n o f T r i t o n X-100. B a c i l l u s c e r e u s peptidoglycan w i t h !-unsubstituted glucosamine r e s i d u e s has b e e n f o u n d t o b e i n s e n s i t i v e t o t r e a t m e n t w i t h b a c t e r i o p h a g e T 4 l y s o ~ y m e . ~A f~t e~r N - a c e t y l a t i o n w i t h a c e t i c a n h y d r i d e , T 4 l y s o z y m e - c l e a r e d s o l u t i o n s of t h e p e p t i d o g l y c a n and T h e d i g e s t i o n p r o d u c t s were m a i n l y r e d u c i n g s u g a r s were l i b e r a t e d . o f h i g h m o l e c u l a r w e i g h t , s i n c e t h e p e p t i d o g l y c a n is p e p t i d e c r o s s linked t o a great extent. !-Propylation d i d not convert the p a r t i a l l y !-unsubstituted p e p t i d o g l y c a n t o a s e n s i t i v e form. I t is concluded t h a t the acetarnido g r o u p s are r e q u i r e d f o r b i n d i n g and/or c a t a l y s i s by T4 l y s o z y m e .

37

~~~gO-1,6-~-Glucosidases

Detergent-solubilized pig intestinal sucrose-a-P- - g l u c o s i d a s e h a s b e e n p u r i f i e d 40 t o glucohydrolase-oligo-(1 + 6)-Q 100 t i m e s w i t h a y i e l d o f 1 0 t o 20% by a r a p i d i m m u n o a d s o r b e n t technique.444 T h e p u r i f i e d e n z y m e was s h o w n t o b e h o m o g e n e o u s by i m m u n o e l e c t r o p h o r e s i s a n d was e s s e n t i a l l y f r e e f r o m o t h e r k n o w n brush-border peptidases and disaccharides. It c o n s i s t e d o f two p o l y p e p t i d e c h a i n s w i t h a p p a r e n t m o l e c u l a r w e i g h t s o f 140,000 and 150,000, r e s p e c t i v e l y . I n c o n t r a s t , t h e enzyme isolated from p i g s i n w h i c h t h e p a n c r e a s was c o m p l e t e l y d i s c o n n e c t e d f r o m t h e duodenum 3 days before k i l l i n g migrated in polyacrylamide gel e l e c t r o p h o r e s i s i n d o d e c y l s u l p h a t e as a s i n g l e p o l y p e p t i d e c h a i n w i t h a n a p p a r e n t m o l e c u l a r w e i g h t o f 260,000. Treatment with pancreatic proteases i n v i t r o converted t h e large polypeptide chain i n t o bands w i t h m o l e c u l a r w e i g h t s e q u a l t o or somewhat l a r g e r t h a n t h o s e o f sucrose-a-e-glucohydrolase-oligo-(1 + 6 ) - P - g l u c o s i d a s e from normal pigs. I t was s u g g e s t e d t h a t t h e s i n g l e c h a i n r e p r e s e n t s a precursor, which is converted t o the f i n a l sucrose-a-kg l u c o h y d r o l a s e - o l i g o - ( 1 + 6 ) - Q - g l u c o s i d a s e i n v i v o by p a n c r e a t i c p r o t e o l y t i c e n z y m e s . T h i s i s o n e o f t h e few e x a m p l e s i n v e r t e b r a t e s

6: Enzymes of

529

a single polypeptide chain carrying two enzymatically active

sites.

The s i g n i f i c a n c e o f t h e r e s u l t f o r of

b i o s y n t h e s is

t h e mechanism o f t h e

s u c r 0 s e - a - Q - g l u c o h y d r o l a s e - 0 1ig o - ( 1

-c

6)-Q-

glucosidase i s discussed.

Pectate,

38

Pectin,

and P o l y - Q - G a l a c t u r o n a t e Lyases

P e c t a t e l y a s e h a s been p u r i f i e d t o a n e a r l y homogeneous s t a t e from the c u l t u r e f i l t r a t e o f Streptomyces nitrosporeus.445 m o l e c u l a r w e i g h t was e s t i m a t e d t o b e a b o u t 4 1 , 0 0 0 . p o i n t was pH 4.6.

The

Isoelectric

The e n z y m e was m o s t a c t i v e a t pH 10.0 a n d 5OoC,

a n d was r e l a t i v e l y s t a b l e a t a pH r a n g e o f 4 - 1 1 ( a t 2OC f o r 48 h ) and b e l o w

4OoC ( a t

pH 7.0

maximum a c t i v i t y .

for

10

min).

T h e e n z y m e was a n

Ca2+ was

required for

e n d o p e c t a t e l y a s e w h i c h was

more a c t i v e o n l o w m e t h o x y l p e c t i n t h a n on p o l y - P - g a l a c t u r o n i c

acid

and h a d m a c e r a t i n g a c t i v i t y on p o t a t o t i s s u e and G a n p i b a r k . The d e g r a d a t i o n o f p o l y - Q - g a l a c t u r o n i c a c i d b y r u m e n c i l i a t e p r o t o z o a has been i n v e s t i g a t e d . 4 4 6

The d e p o l y m e r a s e a c t i v i t y o f

c e l l - f r e e e x t r a c t s o f n i n e s p e c i e s o f r u m e n c i l i a t e p r o t o z o a and t w o mixed protozoal preparations, poly-Q-galacturonic

grown i n v i v o

a c i d was e x a m i n e d .

and i n v i t r o ,

towards

The h i g h e s t a c t i v i t y was

found w i t h E r e m o p l a s t r o n b o v i s and O s t r a c o d i n i u m o b t u s u m b i l o b u m while

there

was

none

i n

the

spined

or

spineless

forms

E n t o d i n i u m c a u d a t u m and l i t t l e i n P o l y p l a s t r o n m u l t i v e s i c u l a t u m . the b a s i s of t h e r a p i d drop i n v i s c o s i t y , p r o d u c t i o n of

u.v.-absorbing

material,

i n h i b i t i o n by EDTA,and

t h e enzymes

from

o f On the

a l l active

s p e c i e s were d e s i g n a t e d as e n d o p e c t a t e l y a s e s a l t h o u g h some p o l y - P galacturonase

may

be

present.

Neither

pectin

nor

poly-Q-

g a l a c t u r o n i c a c i d s u p p o r t e d t h e s u r v i v a l o r g r o w t h o f any o f t h e protozoal species tested. High-performance investigate

pectic

liquid

chromatography

enzymes.447

preparations Pectinex

Technical

has

been

pectic

used

to

multienzyme

U l t r a and Rohament P w e r e c h r o m a t o g r a p h e d on

an a n a l y t i c a l s c a l e u s i n g m e d i u m - p r e s s u r e l i q u i d c h r o m a t o g r a p h y on a g l y c o l methacrylate r i g i d macroreticular gel, ion-exchange derivatives. gradient

elution

(with

e m p l o y e d and f r a c t i o n s

S p h e r o n 1000 and i t s

A c o m b i n a t i o n o f i s o c r a t i c and l i n e a r -

gradients

i n

ionic

strength or

w e r e m o n i t o r e d by m e a s u r e m e n t s o f

c o n d u c t i v i t y , pH, and enzyme a c t i v i t y . (A285 a n d A_254), f o r r a p i d s e p a r a t i o n s of p e c t i c enzymes a r e e l a b o r a t e d .

pH)

was

absorbance Conditions The r e s u l t s

530


i n d i c a t e t h e p o s s i b i l i t i e s of s e p a r a t i n g t h e t e c h n i c a l l y undesirable p e c t i n - e s t e r a s e a c t i v i t y f r o m t h e o t h e r enzyme a c t i v i t i e s , and o f a more d e t a i l e d b i o c h e m i c a l i n v e s t i g a t i o n o f t h e s e enzymes, i m p o r t a n t f o r the .food i n d u s t r y . The s i m u l t a n e o u s s y n t h e s i s o f p e c t i n l y a s e a n d c a r o t o v o r i c i n n a l i d i x i c acid, or u l t r a v i o l e t l i g h t

has been i n d u c e d by m i t o m y c i n C,

irradiation i n e i n i a c a r o t ~ v o r a . ~ When ~ ~ K i n i a carotovora Er, a bacteriocinogenic ultraviolet mitomycin

l i g h t or

or

nalidixic

C

(designated

strain,

(UV)

was

induced a f t e r

inhibitors

carotovoricin)

acid,

of

pectin

activity

i r r a d i a t i o n by

DNA s y n t h e s i s , lyase

and

such as

bacteriocin

appeared i n t h e c u l t u r e

fluid.

The o p t i m a l d o s e o f e a c h o f t h e s e a g e n t s f o r p r o d u c i n g t h e e n z y m e o r b a c t e r i o c i n was essentially

identical,

t h e same.

and t h e

time-courses

for

b o t h were

The s y n t h e s e s o f t h e enzyme and b a c t e r i o c i n

w e r e a s s u m e d t o b e r e g u l a t e d b y t h e same m e c h a n i s m ,

i n which a

r e p r e s s o r i n a c t i v a t e d by UV l i g h t ,

m i t o m y c i n C , o r n a l i d i x i c a c i d was

involved.

bacteriocinogenic

E.

The

other

three

carotovora a l s o formed p e c t i n lyase,

i n

the

presence o f

syntheses

of

mitomycin

pectic

lyase

indicating that

C,

and

strains

of

i n addition t o carotovoricin

carotovoricin

simultaneous

were

widespread

phenomena i n b a c t e r i o c i n o g e n i c s t r a i n s o f E. c a r o t o v o r a . The p e c t i c enzyme a c t i v i t i e s o f b a c t e r i a a s s o c i a t e d w i t h r o t t e d o n i o n s h a v e been i n v e s t i g a t e d . 4 4 9 with soft

as a V i b r i o sp., Acinetobacter

-----Bacillus

The

aerobic bacteria associated

r o t i n o n i o n s ( A l l i u m cepa) were Micrococcus epidermidis,

sp.,

a

megaterium.

Xanthomonas

sp.,

With the cup-plate

i s o l a t e d and i d e n t i f i e d Pseudomonas c e p a c i a ,

and

B a c i l l u s polymyxa,

and

assay method, no p e c t i n

h y d r o l a s e c o u l d b e d e t e c t e d f r o m any o f t h e s e i s o l a t e s w h e n t h e y w e r e c u l t u r e d i n p e c t i n medium, detectable. for

P.

b u t l y a s e a n d p e c t i n e s t e r a s e s were

O n i o n t i s s u e c u l t u r e s showed p e c t i n h y d r o l a s e a c t i v i t y

c e p a c i a a n d B.

polyfiyza

and l y a s e and p e c t i n e s t e r a s e

a c t i v i t i e s for a l l of the isolates,

u s u a l l y a t h i g h e r l e v e l s of

a c t i v i t y t h a n t h o s e o f t h e p e c t i n medium c u l t u r e f i l t r a t e s . culture

media,

Vibrio

sp.

pectinesterase activities.

showed

the

highest

In the viscometric test,

I n both

lyase

i s o l a t e s a c h i e v e d a t l e a s t a 50% d e c r e a s e i n v i s c o s i t y f o r enzyme,

w i t h L e p i d e r m i d i s a n d V i b r i o sp.

d e c r e a s e s a s h i g h as 83%.

lyase

recording viscosity

The a b i l i t y t o c a u s e s o f t r o t i n o n i o n

b u l b s was d e m o n s t r a t e d b y P. a c i d a t a c o n c e n t r a t i o n o f 0.8 enzyme p r o d u c t i o n ,

and

a l l o f the

c e p a g a and Xanthomonas sp. mg m l - l

Benzoic

caused t o t a l suppression of

whereas sodium benzoate a t t h i s c o n c e n t r a t i o n

53 1

6: Enzymes

r e d u c e d p e c t i n e s t e r a s e p r o d u c t i o n by 71% and l y a s e p r o d u t i o n b y 72%. The p o s s i b l e u s e o f t h e s e p r e s e r v a t i v e s i n t h e c o n t r o l o f s o f t r o t i n o n i o n s is n o t e d .

P o l y-Q-Galacturonases

39

The d e g r a d a t i o n o f p o l y - Q - g a l a c t u r o n i c p r o t o z o a has been i n v e s t i g a t e d . 4 4 6 cell-free

a c i d by rumen c i l i a t e

The d e p o l y m e r a s e a c t i v i t y o f

e x t r a c t s of n i n e s p e c i e s o f rumen c i l i a t e p r o t o z o a and t w o

mixed protozoal preparations, poly-Q-galacturonic

g r o w n i n v i v o and i n v i t r o ,

a c i d was e x a m i n e d .

towards

The h i g h e s t a c t i v i t y was

f o u n d w i t h E r e m o p l a s t r o n b o v i s and O s t r a c o d i n i u m o b t u s u m b i l o b u r n , while

there

was

none

i n

the

or

spined

spineless

forms

E n t o d i n i u m c a u d a t u m and l i t t l e i n P o l y p a s t r o n m u l t i c e s i c u l a t u m . the basis o f the r a p i d drop i n viscosity, t h e p r o d u c t i o n o f u.v.-adsorbing

i n h i b i t i o n by H4 e d t a ,

material,

may b e p r e s e n t .

On and

t h e enzymes f r o m a l l

a c t i v e s p e c i e s w e r e d e s i g n a t e d as e n d o p e c t a t e l y a s e s , poly-Q-galacturonase

of

a l t h o u g h some

Neither p e c t i n nor poly-p-

g a l a c t u r o n i c a c i d s u p p o r t e d t h e s u r v i v a l o r g r o w t h o f any o f t h e protozoal species tested. The a p p l i c a t i o n o f c a r b o h y d r a s e s i n c l u d i n g p o l y - B - g a l a c t u r o n a s e t o

the

extraction

investigated.349

of

For

proteins further

from

details

wheat see

bran

has

been

i n i t i a l citation of

r e f .349. The e f f e c t o f i m m o b i l i z a t i o n o f A s p e r g i l l u s n i q e r e x t r a c e l l u l a r poly-a-galacturonase i n v e s t i g a t e d .450

on

kinetics

and

action

pattern

The e x t r a c e l l u l a r p o l y - B - g a l a c t u r o n a s e

was c o v a l e n t l y bound t o 4 - a m i n o b u t a n o i c a c i d , a m i n o h e x a n o i c a c i d , r e s p e c t i v e l y , as s p a c e r s . enzyme i n v a r i a b l y

has

been

o f A.

glycyl-glycine,

niqer and 6 -

Immobilization o f the

l e d t o decreased a c t i v i t y ,

the extent of

the

decrease being i n v e r s e l y p r o p o r t i o n a l t o t h e chain l e n g t h o f t h e spacer.

This fact,

as w e l l a s a p p a r e n t k i n e t i c p a r a m e t e r s o f t h e

i m m o b i l i z e d enzyme, i n d i c a t e d t h a t diffusion

s t e r i c hindrance

and,

probably,

e f f e c t s a r e r e s p o n s i b l e f o r t h e decrease i n a c t i v i t y .

c o v a l e n t b i n d i n g a l s o c a u s e d an a l t e r a t i o n o f

The

the action pattern o f

t h e enzyme on p o l y m e r i c and o l i g o m e r i c s u b s t r a t e s .

I n t h e case o f

t h e p o l y m e r s t h e r a n d o m n e s s i n d e g r a d a t i o n was l o w e r e d b e c a u s e o f restriction

of

the

s u b s t r a t e molecule.

enzyme

action

to

peripheral

Among o l i g o m e r i c s u b s t r a t e s ,

c h a n g e was o b s e r v e d i n t e t r a - ( g - g a l a c t o s i d u r o n i c

areas

of

the

t h e most i m p o r t a n t acid),

i n which (2

532 +


2) d e g r a d a t i o n

occurred

as

well

as

the

(1 +

3)

degradation

c h a r a c t e r i s t i c o f t h e s o l u b l e enzyme. The changes i n P o l Y - q - g a l a c t u r o n a s e ripening

of

normal

investigated.451

and

mutant

is

There

a

a c t i v i t y which occur d u r i n g tomato

sequential

have

been

of

two

during ripening.

These

i s o e n z y m e s h a v e been p u r i f i e d and t h e i r p r o p e r t i e s compared.

Poly-

isoenzymes,

1 and 2,

f r u i t

appearance

poly-Q-galacturonases

g-galacturonase

1 h a s a btr

and has a d e n s i t y

of

galacturonase 2 has a has a d e n s i t y of

1.343

Mr

1.300

1,000,000,

of

g cm-3

m

Poly-Q-

i s 5 0 % i n a c t i v a t e d a t 57'C,and

i ' n ~c a e s i u m c h l o r i d e .

i s o g e n i c l i n e s homozygous f o r ripen normally

i n caesium c h l o r i d e .

o f 42,000, g ~

i s 50% i n a c t i v a t e d a t 78OC

and c o n t a i n r e d u c e d a m o u n t s o f

F r u i t s from

m u t a t i o n do n o t

the ever-ripe (Nr)

poly-n-galacturonase.

O n l y p o l y a - g a l a c t u r o n a s e 1 was p r o d u c e d i n N r f r u i t .

Tomatoes f r o m

i s o g e n i c l i n e s homozygous f o r t h e r i p e n i n g i n h i b i t o r ( r i n ) m u t a t i o n do n o t r i p e n n o r m a l l y a n d p r o d u c e v e r y l i t t l e d e t e c t a b l e p o l y - a galacturonase. properties,

Although poly-0-galacturonases they

b o t h gave

rise

t o

a

1 and 2 had d i f f e r e n t

single

p o l y p e p t i d e on

e l e c t r o p h o r e s i s i n polyacryamide g e l s i n t h e presence o f sodium dodecylsuphate

(Mr

- 46,000).

p r o d u c e d by l i m i t e d with

chymotrypsin

A comparison o f

the major

fragments

p r o t e o l y s i s of poly-e-galacturonases suggests t h a t

isoenzymes were s i m i l a r .

the

polypeptides

from

1 and 2 the

two

The same c o n c l u s i o n was r e a c h e d f r o m a

comparison o f poly-P-galacturonases

1 and 2 by r a d i o i m m u n o a s s a y ,

using antibody prepared against poly-E-galacturonase

2 a n d 1251-

l a b e l l e d p o l y -g-ga l a c t u r o n a s e 2. Some p r o p e r t i e s o f

t h e p o l y g-galacturonase-elicitor

from

the

f i l t r a t e s o f R h i z o p u s s t o l o n i f e r c u l t u r e s h a v e been e x a m i n e d i n an a t t e m p t t o u n d e r s t a n d i t s mode o f a c t i o n as an e l i c i t o r o f casbene synthetase a c t i v i t y

i n castor-bean

seedlings.452

Both t h e poly-Q-

g a l a c t u r o n a s e a c t i v i t y and t h e e l i c i t o r a c t i v i t y a r e h e a t l a b i l e with

similar

heat-sensitivity

profiles.

a c t i v i t y o f t h e enzyme i s l o s t on t r e a t m e n t as h a d b e e n s h o w n p r e v i o u s l y f o r

Also,

the

the e l i c i t o r

activity.

o p t i m u m o f t h e enzyme a c t i v i t y w i t h p o l y - g - g a l a c t u r o n i c s u b s t r a t e i s 4.9.

catalytic

w i t n sodium periodate, T h e pH

a c i d as t h e

Exposures o f g e r m i n a t i n g castor-bean s e e d l i n g s t o

the e l i c i t o r for short-term

p e r i o d s of

w a s h i n g and i n c u b a t i o n i n s t e r i l e ,

1 t o 1 0 m i n u t e s f o l l o w e d by

d i s t i l l e d water are p a r t i a l l y

e f f e c t i v e i n e l i c i t a t i o n i n comparison w i t h t h e continuous exposure

o f t h e s e e d l i n g s o v e r 11 h o u r s t o t h e same a m o u n t o f t h e e l i c i t o r . The i n i t i a l r a t e o f r e a c t i o n c a t a l y s e d b y t h e enzyme is a b o u t 3

6: Enzymes

533 a c i d as a s u b s t r a t e t h a n w i t h

times faster w i t h poly-g-galacturonic partially

(50%) m e t h y l a t e d p o l y - e - g a l a c t u r o n i c

Em v a l u e

acid

o f t h e enzyme f o r p o l y - q - g a l a c t u r o n i c

(pectin).

The

a c i d i s a b o u t 4.2

m i l l i m o l a r i n t e r m s o f m o n o m e r i c u n i t s and a b o u t 0.07

millimolar i n

terms

the

of

polymer

concentration.

Examination of

types

of

p r o d u c t s f o r m e d by t h e a c t i o n o f t h e enzyme s u g g e s t s t h a t i t i s an

-endo-hydrolase.

The

amino

acid composition of

this

enzyme i s

s i m i l a r t o those o f other e x t r a c e l l u l a r fungal proteins reported. The c a r b o h y d r a t e m o i e t y o f t h e g l y c o p r o t e i n p o l y - ! - g a l a c t u r o n a s e e l i c i t o r i s c o m p o s e d o f 9 2 % q - m a n n o s e a n d 8 % 0 - g l u c o s a m i n e by g a s chromatography-mass

spectrometry

analysis.

The

linkage-group

a n a l y s i s o f t h e c a r b o h y d r a t e m o i e t y showed t h a t q - m a n n o s y l r e s i d u e s which

are

1,2-linked

comprise

about

70% o f

the

total

r e s i d u e s a n d d e m o n s t r a t e d t h e p r e s e n c e o f some 1 , 3 , 6 -

glycosyl

a n d 1,2,6-

l i n k e d b r a n c h i n g D-mannosyl r e s i d u e s .

t ur o n a s e e l i c i t or p u r i f i e d

A p p a r e n t 1y h o m o g e n e o u s p o 1y -;-galac

f r o m t h e f i l t r a t e s o f R h i z o p u s s t o l o n i f e r c u l t u r e s has been f o u n d t o stimulate

germinating castor-bean

increased

levels

of

casbene

seedlings t o produce g r e a t l y

synthetase

activity.453

The

p u r i f i c a t i o n procedure i n v o l v e d g e l - f i l t r a t i o n chromatography on Sephadex

G-25

a n d G-75

columns

followed

c h r o m a t o g r a p h y o n a Sephadex CM C-50 column. purified preparation

was

i n d i c a t e d by

the

by

cation-exchange

Homogeneity o f t h e results of

cationic

p o l y a c r y l a m i d e d i s c g e l e l e c t r o p h o r e s i s and i s o e l e c t r i c f o c u s i n g (PI = 8.0).

The i d e n t i t i e s o f t h e casbene e l i c i t o r a c t i v i t y and p o l y - Q -

galacturonase

were

i n d i c a t e d by

the

coincidence o f

the

two

a c t i v i t i e s a t a l l stages of p u r i f i c a t i o n , the coincidence o f both activities

with the single protein-staining

band d e t e c t e d on a

c a t i o n i c p o l y a c r y l a m i d e d i s c g e l and an i s o e l e c t r i c - f o c u s i n g g e l , and t h e i d e n t i c a l b e h a v i o u r o f b o t h a c t i v i t i e s on an agarose g e l affinity

column.

The p u r i f i e d p o l y - P - g a l a c t u r o n a s e

elicitor

i s

a

g l y c o p r o t e i n w i t h a p p r o x i m a t e l y 20% c a r b o h y d r a t e c o n t e n t a n d a n estimated molecular weight of

32,000

by p o l y a c r y l a m i d e d i s c g e l

electrophoresis. The

variability

isoelectric-focusing ~

of

poly-g-galacturonase

patterns

has

been

and

protein

investigated

i n

B o t 9 t i s cinerea isolates.454

Acetone p r e c i p i t a t e s f r o m c u l t u r e

filtrates

of

I _ I _

of

three

isolates

B. c i n e r e a w e r e r e s o l v e d b y

i s o e l e c t r i c f o c u s i n g on p o l y a c r y l a m i d e g e l s t o d e t e c t d i f f e r e n c e s i n the poly-Q-galacturonase four

and p r o t e i n p a t t e r n s .

Only a few

bands

-

i n t h e p r o t e i n p a t t e r n s and t w o i n t h e p o l y - E - g a l a c t u r o n a s e

534


p a t t e r n s - were common t o a l l t h e i s o l a t e s . D i f f e r e n c e s were a l s o detected i n poly-;-galacturonase and p r o t e i n p a t t e r n s o f a l l t h e s a m e i s o l a t e a t d i f f e r e n t a g e s of c u l t u r e ( 7 , 1 4 , a n d 21 d ) . I d e n t i c a l p o l y - g - g a l a c t u r o n a s e p a t t e r n s w e r e o b t a i n e d when i s o e l e c t r i c f o c u s i n g was a p p l i e d t o an a c e t o n e p r e c i p i t a t e e i t h e r d i r e c t l y o r a f t e r f u r t h e r p u r i f i c a t i o n by ion-exchange chromatography.

40

exo-Poly-Q-Galacturonate

Lyases

An m g - p o l y - Q - g a l a c t u r o n a t e l y a s e h a s been p u r i f i e d t o a homogeneous s t a t e from t h e c u l t u r e f i l t r a t e of S t r e p t o m y c e s m a s s a s p ~ r e u s . ~The ~ ~m o l e c u l a r weight was e s t i m a t e d t o be a b o u t 5 4 , 0 0 0 and t h e i s o e l e c t r i c p o i n t was pH 5 . 5 . The enzyme was most a c t i v e a t pH 9.5 and 4 O o C , and was r e l a t i v e l y s t a b l e a t pH Ca2+ was r e q u i r e d f o r maximum a c t i v i t y . The enzyme 3.0 f o r 10 m i n . was most a c t i v e on t r i - q - g a l a c t u r o n i c a c i d and had h i g h e r a c t i v i t y on low m e t h o x y l p e c t i n s r a t h e r t h a n on p o l y - q - g a l a c t u r o n i c a c i d . The enzyme removed t e r m i n a l u n s a t u r a t e d d i - Q - g a l a c t u r o n a t e u n i t s from t h e Q - g a l a c t u r o n i d e c h a i n s .

41

Pullulanases

The p r o p e r t i e s of two a m y l a s e s which d i f f e r i n t h e i r s u b s t r a t e s p e c i f i c i t y and s u b c e l l u l a r l o c a t i o n a s w e l l a s a c h l o r o p l a s t a s s o c i a t e d R-enzyme ( d e b r a n c h i n g a c t i v i t y ) have been r e p o r t e d . 3 0 1 The c o n v e r s i o n o f a c t i v e and i n a c t i v e d e b r a n c h i n g e n z y m e s i n r i c e s e e d s h a s been r e p o r t e d . 4 5 6 D e b r a n c h i n g - e n z y m e a c t i v i t y i n r i c e s e e d s i n c r e a s e d d u r i n g t h e e a r l y s t a g e of r i p e n i n g and t h e n d e c r e a s e d , and i n c r e a s e d a g a i n d u r i n g g e r m i n a t i o n . The i n a c t i v e enzyme accumulated r a p i d l y i n r i p e n i n g from t h e t w e n t i e t h day a f t e r flowering. R a d i o a c t i v e amino a c i d s were r e a d i l y i n c o r p o r a t e d i n t o t h e a c t i v e d e b r a n c h i n g enzyme a f t e r t h e i r a b s o r p t i o n i n t o immature r i c e seeds. Subsequently, t h e r a d i o a c t i v i t y increased i n the It i n a c t i v e enzyme, accompanying a d e c r e a s e i n t h e a c t i v e enzymes. was c o n c l u d e d t h a t t h e d e b r a n c h i n g enzyme i n r i c e s e e d s i s s y n t h e s i z e d d u r i n g r i p e n i n g i n a c t i v e form and t h a t i t a c c u m u l a t e s i n i n a c t i v e form, which can be r e a c t i v a t e d d u r i n g g e r m i n a t i o n . The i d e n t i f i c a t i o n o f an i n a c t i v e d e b r a n c h i n g enzyme i n r i c e

6: Enzymes

535

seeds

and i t s a c t i v a t i o n have been r e p o r t e d . 4 5 7

flour

with

1,4-dithiothreitol

pullulanase

activity.

Maximum

i n c u b a t i o n w i t h 6mM t h i o l .

I n c u b a t i o n of r i c e

(dithiothreitol) activity

generated

occurred

after

high 24

h

A l l of t h e t h i o l s a t l O m M a c t i v a t e d t h e

d e b r a n c h i n g enzyme, s u g g e s t i n g t h a t t h e p r o c e s s i n v o l v e d c l e a v a g e o f t h e d i s u l p h i d e bonds. other reductants. effective,

T h i s v i e w was c o n f i r m e d by t h e e f f e c t

b u t s o d i u m b o r o h y d r i d e was m o d e r a t e l y e f f e c t i v e i n s p i t e

o f t h e pH (-10) w h i c h was u n f a v o u r a b l e f o r e n z y m e s t a b i l i t y . debranching proteases,

of

S o d i u m s u l p h i t e and s o d i u m d i t h i o n i t e w e r e v e r y

enzyme

was

9. papain,

also

activated

a-chymotrypsin,

to

similar

extents

The by

and t r y p s i n .

D e b r a n c h i n g enzyme h a s been p u r i f i e d f r o m m a t u r e r i c e seeds.458 The enzyme was e x t r a c t e d f r o m

a supernatant s o l u t i o n o f r i c e f l o u r

f o l l o w i n g i t s homogenization i n l O m M sodium d i t h i o n i t e s o l u t i o n . DEAE-cellulose enzyme

was u s e d a s t h e e x t r a c t i o n m e d i u m f r o m w h i c h t h e

protein

was

eluted

with

P u r i f i c a t i o n was a c h i e v e d by t h e u s e o f a C M - c e l l u l o s e column.

sodium

phosphate

buffer.

a Sephadex G-100 c o l u m n a n d

The p u r i f i e d enzyme h a d no a m y l a s e ,

maltase,

and Q-enzyme a c t i v i t i e s and was homogeneous i n g e l f i l t r a t i o n , d i s c electrophoresis,

(c. 70,000) were

and i s o e l e c t r i c

focusing.

The

molecular

weight

d e t e r m i n e d by g e l f i l t r a t i o n and t h e o p t i m u m pH o f 5 . 6

i d e n t i c a l t o those of

the milky-stage

enzyme.

Substrate

s p e c i f i c i t i e s o f t h e enzyme w e r e a l s o s i m i l a r t o t h o s e o f t h e m i l k y s t a g e enzyme.

The enzyme r a p i d l y h y d r o l y s e d p u l l u l a n b u t s c a r c e l y

hydrolysed phytoglycogen, enzymes.

s i m i l a r t o other higher-plant

debranching

K i n e t i c d a t a s i m i l a r t o t h o s e o f t h e m i l k y - s t a g e enzyme

were a l s o obtained. B a c i l l u s a m y l o l y t i c u s produces a-amylase, glucosidase.

By s e l e c t i o n o f

p u l l u l a n a s e , and a-g-

c a r b o n s o u r c e i n t h e g r o w t h medium a-

Q - g l u c o s i d a s e was p r o d u c e d p r e f e r e n t i a l l y a n d w i t h e x c l u s i o n o f t h e other two activities.la2

42

Sucrose-a-g-Glucohydrolase

The a c t i v i t i e s o f a-!-glucohydrolase

various glycosidases

e.i n c l u d i n g

i n homogenates o f t h e s m a l l - i n t e s t i n a l

t w o a d u l t and 18 s u c k l i n g tammar w a l l a b i e s ( M .

e u q e n i i ) aged f r o m 6

t o 5 0 w e e k s h a v e b e e n i n ~ e s t i g a t e d . ~F ~o r f u r t h e r i n i t i a l c i t a t i o n o f ref.38. The

amounts

of

sucrosemucosa o f

sucrose-a-Q-glucohydrolase

d e t a i l s see

i n tangentially


536

s e c t i o n e d b i o p s i e s from j e j u n u m have been s t u d i e d by q u a n t i t a t i v e i m m u n o e l e c t r o p h o r e s i s and e n z y m i c a s s a y s . 1 2 9

For further

details

see i n i t i a l c i t a t i o n o f r e f . 1 2 9 . S u c r o s e -cr-q-g l u c o h y d r o l a s e -0 l i g o - ( 1 been p u r i f i e d from i n j e c t i o n of

rat

+

6)-a-!-glucosidase

intestinal microvillus

;-(2-3H)mannose

and & - ( 6 - 3 H ) f u c o s e ,

has

membranes

after

u s i n g a column o f

m o n o c l o n a l a n t i b o d y p r o t e i n A - S e p h a r ~ s e . ~A~f t~e r p r o n a s e d i g e s t i o n and

gel

f i l t r a t i o n

precursors,

of

the

glycopeptides

a major p a r t of

l a b e l l e d from

the radioactivity

a s p a r a g i n e - l i n k e d complex o l i g o s a c c h a r i d e s ,

was

both

recovered i n

a n d a s m a l l e r amount i n

p a r t i a l l y a l k a l i - l a b i l e high-molecular-weight

glycopeptides.

Only a

s m a l l a m o u n t o f ( 3 H ) m a n n o s e was f o u n d i n e n d o - B - Q - 2 - a c e t a m i d o - 2 d e o x y g l u c o s i d a s e H - s e n s i t i v e high-a-mannose

oligosaccharides.

D e t e r g e n t - s o l u b i l i z e d p i g i n t e s t i n a l sucrose-a-g-glucohydrolase oligo-(1

6 ) - Q - g l u c o s i d a s e h a s been p u r i f i e d 40 t o 100 t i m e s w i t h a

+

y i e l d o f 1 0 t o 20% by a r a p i d i m m u n o a d s o r b e n t t e c h n i q u e . 4 4 4

For

f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 4 4 4 .

43

aa- and B B - T r e h a l a s e s

The a c t i v i t i e s o f v a r i o u s g l y c o s i d a s e s i n h o m o g e n a t e s o f t h e small-intestinal wallabies

mucosa

(M. e u g e n i i )

of

two

aged

adult

from

and

6

t o

18 50

suckling weeks

tammar

have

been

i n ~ e s t i g a t e d . ~a a~- T r e h a l a s e a c t i v i t y was v e r y l o w o r a b s e n t d u r i n g t h e f i r s t 34 weeks,

and t h e n i n c r e a s e d .

I n order t o establish the s p e c i f i c i t y o f aa-trehalases t h e i r a c t i o n on m o n o m o d i f i e d a s y m m e t r i c a l d e r i v a t i v e s o f the

purification of

may-bug

(Melolontha vulgaris coleopterae)

t r e h a l a s e h a s been p u r i f i e d 1 6 , 0 0 0 - f 0 l d . ~ ~ The ~ was 307 u n i t s mg-'

of proteins,

previously reported. studied:

and

trehalose,

specific

z-

activi;ty

a p p r o x i m a t i v e l y 78 t i m e s h i g h e r t h a n

Some o f t h e p r o p e r t i e s o f t h e enzyme h a v e been

molecular weight,

isoelectric point,

a c t i o n of

various

e f f e c t o r s and i n h i b i t o r s . Effects

o f

a c r y l o n i t r i l e

T r i b o l i u m castaneum

I _ -

studied.461

and

on

trehalase

Trogoderma g r a n a r i u m

a c t i v i t y

everts

has

i n been

A c r y l o n i t r i l e i n h i b i t e d t r e h a l a s e and p h o s p h o r y l a s e s i n

l a r v a e and a d u l t s o f T r i b o l i u m c a s t a n e u m . l a r v a e p h o s p h o r y l a s e s a l o n e were i n h i b i t e d .

I n Trogoderma q r a n a r i u m

537

6: Enzymes 44

endo-( 1

The

mode o f

4)-B-a-Xylanases

+

action

of

an e n d o - ( 1

4)-B-Q-xylanase

-+

from

a

B a s i d i o m y c e t e S p o r o t r i c h u m d i m o r p h o s p o r u m h a s been r e p o r t e d and i t s utility

for

pattern

the structural investigation including substitution

o f

the

branched

me t h y 1g 1u c u r o no ) -9 been s t u d i e d . 4 6 2 region

of

the

- x y 1a n

L-afabing-(4-g-

water-soluble

f r o m r e d w o o d ( S e q u o ia s e mper v i r e n s ) h a s

The a c t i o n o f

t h e P-xylanase

polysaccharide

backbone

appears t o i n v o l v e a

having

three

P_-xylosyl

A mode o f a c t i o n t h a t r e q u i r e s u n s u b s t i t u t e d h y d r o x y l

residues.

C-3,

g r o u p s a t C-2,

a n d C-2’

o f a x y l o b i o s y l r e s i d u e was p r o p o s e d .

The b i n d i n g s i t e s e e m s t o c o r r e s p o n d t o a s h a l l o w

cavity.

The

c o m p o s i t i o n and s t r u c t u r e o f t h e f i n a l r e s i d u e o f a t t a c k shows t h a t the

enzyme

through

h a s no are

0-2

a c t i o n when t h e 2 - x y l o s y l

separated

by

only

residue.

This pattern o f action,

products,

and

the

production of

one

residues

branched

unsubstituted

P_-xylose

the nature of a

final

the dialysable

residue

i n

which

the

s u b s t i t u e n t s a r e a c c u m u l a t e d s u g g e s t t h a t t h e C - a r a b i n o s y l and

9-

g l u c o s y l u r o n i c groups a r e i r r e g u l a r l y d i s t r i b u t e d on t h e main c h a i n o f t h e 9 - x y l a n f r o m r e d w o o d a n d t h a t i n some r e g i o n s t h e y a r e i n c l o s e v i c i n i t y when n o t a c t u a l l y o n a d j a c e n t g - x y l o s y l r e s i d u e s . The

extracellular

endo-(1

+

4 ) - B - Q- - x y l a n a s e

of

the

yeast

C r y p t o c o c c u s a l b i d u s has been f o u n d t o c a t a l y s e t h e d e g r a d a t i o n o f aryl

B-q-xylosides

cleavage.463

by

reactions

Liberation

of

corresponding B-P-xylosides

other

phenol

or

than

simple

hydrolytic

4-nitrophenol

from

the

was a c c o m p a n i e d by f o r m a t i o n o f g - x y l o s e

o l i g o s a c c h a r i d e s and o n l y s m a l l a m o u n t s o f 2 - x y l o s e . of p h e n y l B-g-{U-14C)-xyloside

With the a i d

it

s y n t h e s i z e d f r o m P-{U-14C)-xylose,

was e s t a b l i s h e d t h a t t h e r e a c t i o n f o l l o w e d a c o m p l e x p a t t e r n w i t h t h e r a t e of

phenyl B-g-xyloside

d i g e s t i o n and a p p e a r a n c e o f v a r i o u s

products v a r y i n g markedly w i t h time.

The r e a c t i o n i n v o l v e d m u l t i p l e

transglycosylic reactions leading f i r s t t o phenyl-g-glycosides xylo-oligosaccharides,

of

9-

which are subsequently hydrolysed m a i n l y t o

g - x y l o b i o s e and P - x y l o t r i o s e . The a c t i o n p a t t e r n and r e a c t i o n mechanism o f t h e e n d o - ( 1 B-e-xylanase

of

the

yeast

i n v e s t i g a t e d u s i n g reducing-end

(1

+

was

4)-B-Q-xylo-oligosaccharides found

to

catalyse

Cfyptococcus

I l-3H)-labelled

albidus

xylotriose,

-+

4)-

been

and { U - 1 4 C ) - l a b e l l e d

up t o x y l ~ p e n t a o s e . ~The ~ ~ enzyme

degradation o f

oligosaccharides

pathways other than a simple h y d r o l y t i c cleavage. frequency of

have

xylotetraose,and

also

by

Bond-cleavage

x y l o p e n t a o s e was f o u n d t o

538 be

Carbohydrate Chemistry concentration

dependent.

r e a c t i o n s s u c h as x y l o s y l ,

high

A t

substrate

concentration

x y l o b i o s y l , and x y l o t r i o s y l t r a n s f e r o c c u r

and r e s u l t i n t h e f o r m a t i o n o f p r o d u c t s l a r g e r t h a n t h e s t a r t i n g substrate. reaction

g - X y l o s e and x y l o b i o s e t o a s i g n i f i c a n t e x t e n t e n t e r t h e p a t h w a y s as

glycosyl

acceptors.

None

of

the

g l y c o s y l i c r e a c t i o n s observed w i t h reducing-end-labelled

trans-

substrates

o r a c c e p t o r s was a c c o m p a n i e d by a s i g n i f i c a n t l a b e l r e d i s t r i b u t i o n from t h e reducing-end u n i t , intermediates effective

suggesting t h a t the enzyme-glycosyl

i n t h e t r a n s f e r r e a c t i o n s can be f o r m e d f r o m

the non-reducing-end u n i t s o f oligosaccharides.

Evidence for the

f o r m a t i o n o f a t e r m o l e c u l a r s h i f t e d complex o f 6-Q-xylanase

with

x y l o t r i o s e has a l s o been o b t a i n e d .

A l l features o f the degradation

o f o l i g o s a c c h a r i d e s by 6 - P - x y l a n a s e

were f o u n d t o be c o n s i s t e n t w i t h

t h e l y s o z y m e - t y p e r e a c t i o n mechanism. The s u b s t r a t e - b i n d i n g

s i t e o f endo-(1 + 4)-B-Q-xylanase

y e a s t C r y p t o c o c c u s a l b i d u s h a s been i n v e s t i g a t e d u s i n g (1 xylo-oligosaccharides Evaluation of

{ l-3H)-labelled

at

the

of the

+

reducing

4)-6-Qend.465

the a f f i n i t i e s o f t e n imaginary subsites pointed out

t h a t t h e s u b s t r a t e - b i n d i n g s i t e o f t h e enzyme i s composed o f f o u r s u b s i t e s and t h a t t h e c a t a l y t i c groups a r e l o c a l i z e d i n t h e c e n t r e . The i m a g i n a r y s u b s i t e s o n t h e l e f t - h a n d s i d e o f t h e b i n d i n g s i t e (non-reducing-end x y l o s y l residues.

s i d e ) s h o w e d l i t t l e o r no a f f i n i t y t o b i n d

b i n d i n g s i t e ('reducing-end' obtained,

9-

F o r t h e s u b s i t e s on t h e r i g h t - h a n d s i d e of the

s i d e ) n e g a t i v e v a l u e s o f a f f i n i t y were

w h i c h means t h i s r e g i o n o f t h e enzyme i s u n f a v o u r a b l e f o r

complexing

with

Q-xylosyl residues.

As

a

consequence

of

the

asymmetric d i s t r i b u t i o n o f n e g a t i v e values o f a f f i n i t y around t h e binding site,

t h e enzyme d i s p l a y s a s t r o n g p r e f e r e n c e f o r a t t a c h i n g

n e a r t h e r e d u c i n g end o f t h e s u b s t r a t e . {

l-3H)-xylo-oligosaccharides,

Regardless of t h e l e n g t h of

{ l-3H)-xylobiose

was t h e p r e v a i l i n g

r e a c t i o n p r o d u c t a t an e a r l y s t a g e o f h y d r o l y s i s ,

and frequency

d i s t r i b u t i o n o f bond c l e a v a g e decreased f r o m t h e second g l y c o s i d i c b o n d t o w a r d s t h e n o n - r e d u c i n g end. A d d i t i o n a l i n f o r m a t i o n on t h e s u b s t r a t e - b i n d i n g s i t e o f C. a l b i d u s B - p - x y l a n a s e w a s o b t a i n e d b y evaluating the

efficiency

of

p-xylose,

xylobiose,

m e t h y l 8-e-

x y l o p y r a n o s i d e , and p h e n y l 6 - Q - x y l o p y r a n o s i d e t o s e r v e as g l y c o s y l acceptors

i n

the

transglycosylic

concentrations of xylotriose.

reaction

proceeding

at

high

539

6: Enzymes 45

Xylanases (Miscellaneous)

The a c c u r a c y improved.466

of

a Q-xylanase

assay

has been t e s t e d and

The assay was u s e d t o m o n i t o r Q - x y l a n a s e p r o d u c t i o n by

a C e l l u l o m o n a s i s o l a t e and t o d e m o n s t r a t e t h a t t h i s a c t i v i t y i s d i s t i n c t f r o m t h e f 3 --p - x y l o s i d a s e

a c t i v i t y o f t h e organism.

The a p p l i c a t i o n o f c a r b o h y d r a s e s t o t h e e x t r a c t i o n o f p r o t e i n s from

wheat

b r a n has i n c l u d e d t h e e f f i c a c i o u s p r e t r e a t m e n t w i t h

xy l a n a s e .349 An e x t r a c e l l u l a r

9-xylanase

f r o m a s o i l fungus

( F u s a r i u m sp.)

g r o w n o n a medium c o n t a i n i n g g r o u n d n u t h e m i c e l l u l o s e B was p u r i f i e d 76-fold

by

ammonium

sulphate

fractionation,

c h r o m a t o g r a p h y , and g e l f i l t r a t i o n . 4 6 7

ion-exchange

The enzyme was homogeneous by

d i s c g e l e l e c t r o p h o r e s i s a t pH 8 a n d showed o p t i m a l a c t i v i t y a t pH 5.6

and

It

37'C.

hemicellulose B respectively)

by

was

were

observed degraded

that

groundnut

considerably

t h e p u r i f i e d Q-xylanase,

and

sesame

and

(-80

58%,

w h e r e a s g-gluco-!-mannan

and g - x y l a n f r o m g r o u n d n u t were c o m p a r a t i v e l y p o o r l y h y d r o l y s e d (-30-40%) The p r o d u c t i o n and c h a r a c t e r i z a t i o n o f t h e r m o s t a b l e R - x y l a n a s e f r o m T a l a r o m y c e s b y s s o c h l a f i y d o i d e s YH-50 h a v e b e e n d e s c r i b e d . 4 6 8 T h i s s t r a i n i s o l a t e d f r o m c o m p o s t heaps p r o d u c e d t h e h i g h e s t amount of

t h e r m o s t a b l e e - x y l a n a s e among 1 8 0 i s o l a t e s t e s t e d .

cultivated i n solid

wheat-bran

a d d i t i v e carbon source, was p r o d u c e d a f t e r

When i t was

medium c o n t a i n i n g

xylan

as

an

t h e m a x i m a l amount o f t h e r m o s t a b l e x y l a n a s e

3 d a y s a t 5OoC.

90% o f q - x y l a n t o g - x y l o s e .

This culture f i l t r a t e hydrolysed

The enzyme (pH o p t i m u m 5.5,

o p t i m u m 7 O o C ) was q u i t e s t a b l e a f t e r h e a t i n g a t 65'

temperature

f o r 5 min a n d

r e t a i n e d 55% o f o r i g i n a l a c t i v i t y a f t e r h e a t i n g a t 95OC f o r 5 min. T r i c h o d e r m a r e e s e i R u t C-30 high cellulase activities,

B-Q - -glucosidase. concentration, r a t i o of

The

organism

produced,

together

c o n s i d e r a b l e amounts o f i - x y l a n a s e s effect

of

temperature,

pH,

with and

Tween-80

c a r b o n s o u r c e , and s u b s t r a t e c o n c e n t r a t i o n on t h e

m y c e l i a l g r o w t h and e x t r a c e l l u l o s e enzyme p r o d u c t i o n was

d e s c r i b e d .208 A s p e r g i l l u s n i g e r s t r a i n 110.42 h a s been s e l e c t e d as a p r o d u c e r

of h i g h 0-xylanolytic a c t i v i t i e s . 4 6 9 The t i m e - c o u r s e o f x y l a n a s e a n d B - Q - x y l o s i d a s e p r o d u c t i o n a s w e l l a s t h e e f f e c t o f pH a n d t e m p e r a t u r e o n t h e a c t i v i t y o f t h e s e enzymes w e r e s t u d i e d . a n a l y s i s of

the enzymatic degradation of

H.p.1.c.

a r a b i n o x y l a n showed a

n e a r l y complete conversion t o pentose sugars.

The a u t h o r s d i s c u s s

540


t h e use o f c r u d e p - x y l a n a s e p r e p a r a t i o n s f o r t h e s a c c h a r i f i c a t i o n o f Q - - x y l a ns. The enhanced c e l l u l o l y t i c a c t i v i t y o f a C e l l u l o m o n a s m u t a n t has been shown t o

apply

also to

t o hydrolyse g-xylan-

i t s ability

c o n t a i n i n g h e m i c e l l u l o ~ e s . ~The ~ ~ h y d r o l y t i c a c t i v i t y was d i r e c t l y proportional t o the g-xylose content i n the hemicellulose substrate. A Cellulomonas s t r a i n , sugar-cane

bagasse,

has

carbohydrase a c t i v i t i e s

with potential for saccharification o f been

found

t o

possess

a

w h i c h c o u l d be n e c e s s a r y f o r

range

of

effective

h y d r o l y s i s o f t h e h e m i c e l l u l o s e f r a c t i o n o f bagasse.471 Inducible c o n s t i t u e n t of

B-xyloside

permease

has

been

reported

as

a

the e-xylan-degrading

enzyme s y s t e m o f t h e y e a s t

C r y p t o c o c c u s a l b i d ~ s . The ~ ~ y~e a s t ,

depending on whether i t i s

g r o w n on Q - x y l a n o r !-glucose, t a k e up i n d u c e r s synthesis.

of

d i f f e r s remarkably i n the a b i l i t y t o

extracellular

I n washed,

endo-(1

2-glucose-grown

*

4)-B-g-xylanase

c e l l s the i n i t i a l l y low

a b i l i t y t o t a k e up x y l o b i o s e o r m e t h y l B - 0 - x y l o p y r a n o s i d e

increases

d u r i n g i n c u b a t i o n w i t h t h e s e compounds a f t e r a l a g phase s h o r t e r than t h e i n d u c t i o n t i m e o f t h e e x t r a c e l l u l a r B-Q-xylanase.

Using

m e t h y l B - ~ - { U - 1 4 C ~ - x y l o p y r a n o s i d e as a v e r y s l o w m e t a b o l i z a b l e i n d u c e r o f B - 0 - x y l a n a s e i t h a s been e s t a b l i s h e d t h a t t h e i n c r e a s e i n t h e r a t e o f x y l o b i o s e o r m e t h y l x y l o s i d e u p t a k e i s due t o i n d u c t i o n o f an a c t i v e t r a n s p o r t s y s t e m f o r m e t h y l B - g - x y l o s i d e

P-xylo-oligosaccharides. permease. i t s

syste'm

The p e r m e a s e a c t i v i t y o f

absence o f B - 0 - x y l a n a s e

as

The

inducers.

inactivation

cycloheximide;

and ( 1

* 41-8-

c a l l e d B-e-xylosidase

induced c e l l s decreases i n t h e

The i n d u c t i o n o f p e r m e a s e as w e l l

(degradation)

can

be

prevented

with

t h u s b o t h e v e n t s a p p e a r t o b e d e p e n d e n t o n de n o v o

p r o t e i n synthesis. f3-e-xyloside

i s

I n analogy w i t h o t h e r a c t i v e t r a n s p o r t systems,

permease

function

can

be

blocked

effectively

by

i n h i b i t o r s o f energy metabolism i n t h e c e l l s . The

importance

of

cellulase

------------------B a c t e r o i d e s s u c c i n o g ---enes investigated.223

and

p-xylanase

release

from

i n t h e rumen e n v i r o n m e n t has been

During growth o f

B.

succinogenes i n a l i q u i d

medium w i t h c e l l u l o s e as t h e s o u r c e o f c a r b o h y d r a t e , g r e a t e r t h a n 80% o f t h e x y l a n a s e was r e l e a s e d f r o m c e l l s i n t o t h e c u l t u r e f l u i d .

F o r f u r t h e r d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 2 2 3 . Cultures

of

Streptomyces f l a v o g r i s E have

p r o d u c e c o n s i d e r a b l e amounts o f c o n t a i n i n g media.260

p-xylanase

been

found

to

when g r o w n on Q - x y l a n -

C o m p a r a t i v e l y l o w e r y i e l d s o f t h i s enzyme w e r e

o b t a i n e d when h a y o r A v i c e l s e r v e d as m a i n c a r b o n s o u r c e .

54 1

6: Enzymes Q - X y l a n a s e i n d u c t i o n by B - P - x y l o s i d e

was i n v e s t i g a t e d i n non-

g r o w i n g c o n d i t i o n s u s i n g non-induced m y c e l i a o f Streptomyces s p e c i e s h a r v e s t e d f r o m i - g l u c o s e medium.473 Q-xylanase w i t h o u t of

P-xylanase synthesis

x y l o s i d e added t o constants

of

the

various

inducing culture

mM,

mM,

The

induction

calculated from

i s o p r o p y l , b u t y l , and

Some a - D - -xylosides

respectively.

The r a t e

w e r e 10.53 m M , 3.83 m M , 0.55

mM,

repressed g-xylanase

Q-xylanase synthesis decreased suddenly

a f t e r the a d d i t i o n o f a-p-xyloside. methyl,

were

and t h o s e o f m e t h y l ,

The r a t e o f

synthesis.

medium.

B-g-xylopyranosides

Lineweaver-Burk p l o t s ,

was added.

d e p e n d e n t on t h e c o n c e n t r a t i o n o f 6-E-

was

ethylenecyanohydrin B-g-xylosides a n d 0.25

The m y c e l i a s t a r t e d t o p r o d u c e

l a g t i m e when B-;-xyloside

The i n h i b i t i o n c o n s t a n t s o f

e t h y l , and i s o p r o p y l a - q - x y l o p y r a n o s i d e s

a n d 33.33 m M , r e s p e c t i v e l y .

w e r e 8.80

mM,

12.50

T h e x y l a n a s e i n d u c t i o n was a l s o

r e p r e s s e d by P - g l u c o s e u n t i l c o n s u m p t i o n o f t h e a d d i t i o n a l g - g l u c o s e was c o m p l e t e . The p r o d u c t i o n o f x y l a n a s e by a S t r e p t o m y c e s s p e c i e s u s i n g n o n -

I t was f o u n d t h a t a

m e t a b o l i z a b l e i n d u c e r has been described.474 variety

of

non-metabolizable

B-g-xylosides

possessed a

marked

i n d u c i n g a b i l i t y i n comparison t o Q-xylan or i t s r e l a t e d m a t e r i a l s . I n t h e p r o d u c t i o n of difficulty basis.

P-xylanase u s i n g such a s y n t h e t i c i n d u c e r ,

i n preparing the inducer

the

i s c o n s i d e r e d on an e c o n o m i c a l

A s u i t a b l e c u l t u r e c o n d i t i o n f o r 0-xylanase p r o d u c t i o n by

methyl 6-g-xyloside

i n S t r e p t o m y c e s s p e c i e s No. 3 1 3 7 a n d a m e t h o d

f o r r e - u s i n g t h e i n d u c e r were i n v e s t i g a t e d .

46

C a r b o h y d r a t e Isomerases

Q-Xylose I s o m e r a s e s @ - G l u c o s e o f some p o l y o l s ( g - m a n n i t o l , x y l i t o l )

on

A-ctinomyces

the

-

Isomerase).

a c t i v i t y

~

--

The i n h i b i t o r y e f f e c t

Q-arabitol, Q-glucitol,

-

o f

--xylose Q

o l i v o c i n e r e u s has been studied.475

f o r g-mannitol,

0.200,

0.140,

g-arabitol,

0.030,

0.024,

g-glucitol, a n d 0.020

ribitol, M,

from

A l l the polyols

studied are purely competitive reversible inhibitors.

Xi

r i b i t o l , and

isomerase

The v a l u e s o f and x y l i t o l a r e

respectively.

The

i n h i b i t i o n i s p a r t i a l l y e l i m i n a t e d b y a n i n c r e a s e i n t h e Mg2+ a n d Co2+ c o n c e n t r a t i o n t o 5 x 1 0 - 2 and l ~ l O -M,~ r e s p e c t i v e l y .

The k i n e t i c p r o p e r t i e s o f

i m m o b i l i z e d and n o n - i m m o b i l i z e d

Q-

x y l o s e i s o m e r a s e c o n t a i n i n g A r t h r o b a c t e r s p e c i e s c e l l s have been i n ~ e s t i g a t e d . ~ ’ ~I n b o t h c a s e s t h e k i n e t i c s c o u l d b e d e s c r i b e d by a


542 modified

Michaelis-Menten

expression.

that i t was shown t h a t t h e p e r m e a b i l i t y o f t h e c e l l membrane was i n c r e a s e d by h e a t and toluene treatments. g - X y l u l o s e , an i n t e r m e d i a t e o f k - x y l o s e c a t a b o l i s m , h a s been i m m o b i l i z a t i o n c a u s e d no d e a c t i v a t i o n .

It

appeared

Furthermore,

o b s e r v e d t o be f e r m e n t a b l e t o e t h a n o l and c a r b o n d i o x i d e i n a y i e l d o f g r e a t e r t h a n 80% by y e a s t s ( i n c l u d i n g i n d u s t r i a l b a k e r ’ s y e a s t )

condition^.^"

under f e r m e n t a t i v e

T h i s c o n v e r s i o n a p p e a r s t o be

c a r r i e d o u t by many y e a s t s k n o w n f o r Q - g l u c o s e f e r m e n t a t i o n . some y e a s t s , xylulose. g-xylose

xylitol,

i n addition t o ethanol,

I n

was p r o d u c e d f r o m

!-

Fermenting yeasts are a l s o able t o produce e t h a n o l from when ! - x y l o s e - i s o m e r i z i n g

enzyme i s p r e s e n t .

The r e s u l t s

i n d i c a t e d t h a t e t h a n o l c o u l d be p r o d u c e d f r o m P - x y l o s e i n a y i e l d o f greater

than

8 0 % by

a

converted t o g-xylulose

two-step

process.

by g - x y l o s e

First,

isomerase.

Q-xylose

g-Xylulose

i s

i s then

f e r m e n t e d t o e t h a n o l by y e a s t s . C u l t u r e s o f S t r e p t o m y c e s f l a v o g r i s e u s has been f o u n d t o p r o d u c e i n t e r a l i a 9 - x y l o s e i s o m e r a s e when i n d u c e d by g - x y l o s e . 2 6 0 The e f f e c t o f g a m m a - i r r a d i a t i o n on Q - x y l o s e i s o m e r a s e p u r i f i e d f r o m t h e c e l l s o f S t r e p t o m y c e s phoeochromogenus i n d i l u t e s o l u t i o n has been i n ~ e s t i g a t e d . ~ ’ ~ The a c t i v i t y o f t h e e n z y m e d e c r e a s e d e x p o n e n t i a l l y w i t h t h e dose u n d e r a l l c o n d i t i o n s i n v e s t i g a t e d . i n a c t i v a t i o n y i e l d s (Go v a l u e ) i n n e u t r a l s o l u t i o n w e r e 0.069 a n d 0.115

i n nitrogen.

The

in air

The r o l e o f t h e r a d i c a l s p r o d u c e d by w a t e r

r a d i o l y s i s i n t h e i n a c t i v a t i o n o f a - g l u c o s e i s o m e r a s e was e s t i m a t e d by u s i n g n i t r o u s o x i d e o r t e r t - b u t a n o l scavengers.

as s e l e c t i v e r a d i c a l

Under t h e s e c o n d i t i o n s , t h e h y d r o x y l r a d i c a l and t h e

h y d r o g e n a t o m w e r e f o u n d t o be i m p o r t a n t i n t h e enzyme i n a c t i v a t i o n , and t h e h y d r a t e d e l e c t r o n c o n t r i b u t e d v e r y l i t t l e . The

radiosensitivity

i n v e s t i g a t e d under

i n a c t i v a t i o n o f !-glucose was e x p o n e n t i a l , an

oxygenated

of

various

E-xylose

irradiation

isomerase

has

been

conditions.479

The

isomerase i r r a d i a t e d i n a cell-bound s t a t e

and an i n c r e a s e i n i n a c t i v a t i o n was r e c o g n i z e d i n condition.

The

cell-free

enzyme

was

highly

r a d i o s e n s i t i v e and h a d a s m a l l o x y g e n e f f e c t c o m p a r e d t o t h a t i n a cell-bound state.

The o x y g e n enhancement r a t i o (OER) d e c r e a s e d w i t h

a d e g r e e i n enzyme p u r i f i c a t i o n .

R e l e a s e d Q - g l u c o s e i s o m e r a s e was

p r o t e c t e d by t h e a d d i t i o n o f g l u t a t h i o n e , and t h e i n a c t i v a t i o n c u r v e i n n i t r o g e n almost agreed w i t h t h a t i n t h e c e l l .

The p r o t e c t i v e

e f f e c t o f g l u t a t h i o n e i n oxygen d e c r e a s e d a t h i g h e r doses because g l u t a t h i o n e i n o x y g e n was e a s i l y decomposed b y i r r a d i a t i o n .

6: Enzymes

543

The s u b u n i t s t r u c t u r e h a s been d e t e c t e d i n g - x y l o s e

isomerase

f r o m S t r e p t o m y c e s q r i s e o f u s c u s by u s i n g d e n a t u r a n t s . 4 8 0 The enzyme was q u i t e s t a b l e t o s o d i u m d o d e c y l s u l p h a t e u n d e r m i l d c o n d i t i o n s , a n d d i s s o c i a t i o n i n t o s m a l l e r s u b u n i t s was d e p e n d e n t A t a c i d i c pH and h i g h t e m p e r a t u r e ,

temperature.

rapid dissociation

and i n a c t i v a t i o n .

The

o n pH a n d

t h e enzyme showed

dissociation

into

c o n s t i t u e n t s u b u n i t s p a r a l l e l e d t h e loss o f e n z y m a t i c a c t i v i t y .

The

enzyme was a l s o d i s s o c i a t e d i n t o a s i n g u l a r t y p e o f s u b u n i t b y 6 M g u a n i d i n i u m c h l o r i d e w h i c h gave a s i n g l e s y m m e t r i c a l e l u t i o n p e a k o n Sepharose 68 column chromatography i n t h e p r e s e n c e o f 6 M g u a n i d i n e hydrochloride,

and t h e e l u a t e showed n o a c t i v i t y .

estimations of

the subunit,

Molecular-weight

u s i n g both sodium dodecyl sulphate

polyacry1ami.de g e l e l e c t r o p h o r e s i s and g e l f i l t r a t i o n , was

w e r e t h e same

The N - t e r m i n a l a m i n o a c i d was i d e n t i f i e d as L - s e r i n e

(43,000).

estimated t o

derivatives.

be 43 p e r

cent

from

analyses

of

The s e q u e n c e f r o m t h e N - t e r m i n a l was f o u n d t o b e

....

Ser-L-Asp-L-Gln

enzyme as 180,000

and

PTH a n d DNP

i-

Considering the molecular weight o f the n a t i v e

and t h e r e s u l t s o b t a i n e d i n t h e e x p e r i m e n t s h e r e ,

i t was c o n c l u d e d t h a t n a t i v e ! - g l u c o s e

i s o m e r a s e was c o m p o s e d o f

f o u r i d e n t i c a l or very s i m i l a r s u b u n i t s w i t h e q u a l m o l e c u l a r w e i g h t s and d i m e n s i o n s . Some p h y s i c o c h e m i c a l p r o p e r t i e s o f

p u r i f i e d g-xylose

f r o m S t r e p t o m y c e s g r i s e o f u s c u s h a v e been examined.481

(gi&,)

coefficient 11.4

and

4.0,

coefficient

and i s o e l e c t r i c p o i n t

respectively.

( 5 j O w ) ,p a r t i a l

The

(PI) were d e t e r m i n e d t o be

values

specific

for

sedimentation

(I), d i f f u s i o n

volume

c o e f f i c i e n t ( g 2 0 w ) , a n d i n t r i n s i c v i s c o s i t y ({tj))

lo7

isomerase

The e x t i n c t i o n

w e r e 8.50 S, 0.73

m l g-l, respectively. The m o l e c u l a r w e i g h t was e s t i m a t e d t o b e 1 8 0 , 0 0 0 b y t h e s e d i m e n t a t i o n e q u i l i b r i u m m e t h o d a n d 185,000 by a g e l e l e c t r o p h o r e t i c a l method.481 Cm3

g-l,

4.6

x

cm2 S-l,

a n d 3.7

T h e e n z y m e was f o u n d t o c o n t a i n f o u r

cobalt ions per molecule i n

atomic-absorption spectrophotometrical analysis. d i c h r o i s m spectrum i n t h e f a r - u l t r a v i o l e t 280 nm a n d a m a x i m u m a t 1 9 8 nm.

The c i r c u l a r -

r e g i o n showed a m i n i m u m a t

Mean r e s i d u e e l l i p t i c i t y ( ( 0 ) )

at

220 nm was e s t i m a t e d t o b e - 1 1 , 2 0 0 deg.cm2/d mol. The c o m p u t e d v a l u e s f o r t h e c o n t e n t s o f t h e s e c o n d a r y s t r u c t u r e w e r e as f o l l o w s : a-helix

40%,

8-form

36%,

a n d r a n d o m c o i l 24%.

The enzyme showed a

v i s i b l e c i r c u l a r - d i c h r o i s m s p e c t r u m h a v i n g n e g a t i v e p e a k s a t 530 nm a n d 4 3 0 nm.

This suggested t h e f o r m a t i o n of a co-ordinated m e t a l

compound, Co-amino a c i d complex.

The m o l e c u l a r d i m e n s i o n o f t h e

enzyme was e x a m i n e d by m e a s u r i n g t h e h y d r o d y n a m i c p a r a m e t e r s .

The

544


(a/b)

frictional ratio

was 5 . 0

f o r p r o l a t e e l l i p s o i d a n d 0.2

for

0

S t o k e s ' r a d i u s was c a l c u l a t e d t o b e 47 A f o r a

oblate ellipsoid.

h y d r a t e d h y p o t h e t i c a l sphere. The p u r i f i c a t i o n a n d e n z y m a t i c p r o p e r t i e s o f E - x y l o s e i s o m e r a s e f r o m S t r e p t o m y c e s g r i s e o f u s c u s h a v e been d e s c r i b e d . 4 8 2 was p u r i f i e d 4 . 3 - f o l d ammonium

sulphate,

electrophoresis a c t i v i t y was 8.5

was

and

homogeneous

on

ultracentrifugation.

i n a c t i v e i n t h e absence o f

respectively,

!-glucose,

while

Vmax

values

The enzyme was q u i t e The enzyme c a t a l y s e d

b y

The

17.6

umol min-'

c o n t e n t t o !-glucose

s u g a r s ,

Em

x 10-1 M and 5.4 x

a t reaction equilibrium.

1.0

i n h i b i t e d

pH

and ! - r i b o s e .

were

The r a t i o o f [-I - f r u c t o s e

was a p p r o x i m a t e l y was

for

optimum

m e t a l i o n s b u t was r e m a r k a b l y a c t i v a t e d

v a l u e s f o r Q - g l u c o s e a n d c - x y l o s e w e r e 2.2 M,

gel

The

magnesium o r c o b a l t i o n s .

t h e i s o m e r i z a t i o n o f !-xylose,

respectively.

polyacrylamide

and o p t i m u m t e m p e r a t u r e 85OC.

by t h e a d d i t i o n o f

The enzyme

a n d o b t a i n e d i n c r y s t a l l i n e f o r m , by a d d i n g

mg-l,

content

The enzyme a c t i v i t y

s u g a r

a l c o h o l s ,

a n d

The Ei Q - s o r b i t o l 1.1 x 10-2

tris(hydroxymethy1)aminomethane i n a c o m p e t i t i v e manner. v a l u e s w e r e as f o l l o w s : g - m a n n i t o l 8.3

M,

3.2

x 10-1 M ,

g - x y l i t o l 1.2 x

x 1 0 - 2 M,

I - a r a b i n o s e 2.3

Chloromercuribenzoate, cyanide, effect

M,

x 10-1 M,

x 1 0 - 1 M, a n d T r i s 6.2

n-galactose x

i o d o a c e t i c a c i d , sodium azide,

sodium fluoride,and

M.

4-

potassium

2 - m e r c a p t o e t h a n o l h a d no i n h i b i t o r

on t h e a c t i v i t y o f !-glucose

a s i g n i f i c a n t l o s s of

lo3

n-mannose 3.4

isomerase,

w h i l e H4 e d t a c a u s e d

activity.

The i n v e s t i g a t i o n b y

'H

n.m.r.

spectroscopy o f the s i t e o f

p r o t o n e x c h a n g e c a t a l y s e d by p o l y ( g - m a n n u r o n i c

a c i d ) C-5 e p i m e r a s e ,

which c a t a l y s e s t h e conversion o f g-mannuronic a c i d r e s i d u e s i n t o

L-

g u l u r o n i c a c i d r e s i d u e s p r o v i d e d t h e s u b s t r a t e i s a glycuronan of

at

least

10 u n i t s ,

has been r e p o r t e d . 4 8 3

I n f o r m a t i o n i s l a c k i n g as t o

t h e enzyme's s p e c i f i c i t y f o r a p a r t i c u l a r l o c a l a r r a n g e m e n t o f u n i t s i n t h e chain.

I-Ribose

Isomerases.

--

I n d u c t i o n o f I - r i b o s e i s o m e r a s e by I - r i b o s e

i n M y c o b a c t e r i u m smegmatis has been i n v e s t i g a t e d . 4 8 4 which u n l i k e a-ribose

a growth substrate for isomerase possibly

i-Ribose,

was n o t a s u b s t r a t e o f t h e enzyme a n d a l s o n o t the organism,

i n d u c e d t h e same Q - r i b o s e

due t o t h e c h a r a c t e r i s t i c

conformation o f t h e r i b o s e molecule.

c o n f i g u r a t i o n and

545

6: Enzymes

Carbohydrate O x i d a s e s

47

--

0-Glucose O x i d a s e s .

e-Glucose o x i d a s e and an o x i d i z e d v e r s i o n o f

t h e enzyme h a v e been i m m o b i l i z e d c o v a l e n t l y t o t w o b a s i c t y p e s o f

-

sorbents

g l y c i d y l m e t h a c r y l a t e c o p o l y m e r s and bead c e l l u l o s e . 4 8 5

The p r o p e r t i e s o f t h e s a m p l e s t h u s o b t a i n e d were compared w i t h t h o s e o f i m m o b i l i z e d a-glucose

o x i d a s e bound on t o some common c a r r i e r s .

Samples w h i c h p o s s e s s e d n o t o n l y a h i g h a b s o l u t e a c t i v i t y adequate

m e c h a n i c a l and

flow

properties

were

but also

characterized

in

g r e a t e r d e t a i l w i t h r e s p e c t t o t h e i m m o b i l i z a t i o n e f f i c i e n c y and k i n e t i c p r o p e r t i e s o f bound i - g l u c o s e o x i d a s e . New

findings

i n

the

oxidation

of

glucose

by

means

i m m o b i l i z e d Q - g l u c o s e o x i d a s e have been r e p o r t e d . 4 8 6 p r o c e e d i n g p u b l i c a t i o n s t h e paper shows r e c e n t a s p e c t s , to

be

of

B a s e d on w h i c h seem

f o r an e c o n o m i c a l and t e c h n i c a l u s e o f t h e

important

i m m o b i l i z e d !-glucose

oxidase-catalase

system.

t r i a l s i n c o n t i n u o u s p r o c e s s i n g were n e g a t i v e ,

The r e s u l t s

of

since the conversion

r a t e s w e r e s u f f i c i e n t and t e c h n i c a l d i f f i c u l t i e s a r o s e i n k e e p i n g back

the

enzyme

processing

with

particles

i n

the

r e p e a t e d enzyme

reaction

vessel.

application

Batchwise

showed

the

major

i m p o r t a n c e o f o p t i m a l oxygen s u p p l y and k e e p i n g l o w t e m p e r a t u r e s o f a b o u t 2OC.

D i f f e r e n t types o f s t i r r e d - t a n k

r e a c t o r s were t e s t e d and

c l a s s i f i e d concerning t h e i r s u i t a b i l i t y .

The c h a n c e s f o r l a r g e -

scale a p p l i c a t i o n o f the immobilized g-glucose oxidase-catalase s y s t e m a r e j u d g e d t o be good. A m o d i f i e d l e c t i n m a t r i x has been u s e d t o s t u d y t h e t h r e e -

dimensional structure of

a dimeric glycoprotein,

g-glucose

w h i c h was a l l o w e d t o r e a c t w i t h I - l y s i n e - s p e c i f i c both

when

immobilized

on

a

succinoylated

oxidase,

cross-linkers,

lectin

matrix

at

a

c r i t i c a l l y l o w d e n s i t y and a l s o a t a h i g h d e n s i t y i n s o l u t i o n . 4 8 7 Analysis of the cross-linked following inferences Of

complexes t h u s o b t a i n e d l e d t o t h e

with regard t o the s t r u c t u r e o f t h i s protein.

t h e 1 5 I - l y s i n e r e s i d u e s on each g - g l u c o s e

oxidase protomer,

i s a v a i l a b l e on t h e n o n - i n t e r f a c i a l s u r f a c e s . protein

possesses

subunits,

C2

symmetry

with

none

Assuming t h a t t h i s

isologous

bonding

between

i t may be i n f e r r e d t h a t o n e a c h p r o t o m e r t h e r e a r e a t

least two I - l y s i n e clusters along or close t o the interprotomeric interface.

These i n t e r f a c i a l I - l y s i n e r e s i d u e s on each p r o t o m e r a r e

so o r i e n t e d t h a t t h e € - a m i n o g r o u p s o f L - l y s i n e r e s i d u e s 2 and protomer 1 face, lysine residues

and a r e very c l o s e t o ,

b'

and

s',

respectively,

t h e &-amino groups o f o n p r o t o m e r 2.

on

I-

General

546


i n f e r e n c e s on t h e g e o m e t r y o f d i m e r i c p r o t e i n s d e r i v a b l e f r o m an a n a l y s i s o f t h e c r o s s - l i n k e d c o m p l e x e s o b t a i n e d ( a s w e l l as t h o s e n o t seen) by u s i n g t h i s low - d e n s i t y m a t r i x c r o s s - l i n k i n g approach were enumerated. prove

useful

glycoproteins,

I t was s u g g e s t e d t h a t m o d i f i e d l e c t i n m a t r i c e s may

i n

studying

the

three-dimensional

structure

of

p a r t i c u l a r l y non-crys t a l l i z a b l e oligomers.

The e n h a n c e m e n t

of

oxygen a b s o r p t i o n by m a g n e t i t e - c o n t a i n i n g

beads of i m m o b i l i z e d g - g l u c o s e o x i d a s e has been i n v e s t i g a t e d . 4 8 8 R a t e s o f oxygen a b s o r p t i o n o f Q - g l u c o s e s o l u t i o n s were measured u s i n g an i m m o b i l i z e d - e n z y m e

reactor,

i n which m a g n e t i t e - c o n t a i n i n g

b e a d s o f i m m o b i l i z e d g - g l u c o s e o x i d a s e w e r e moved b y a r e v o l v i n g magnetic f i e l d t o reduce t h e mass-transfer l i q u i d i n t e r f a c e and a r o u n d t h e bead.

resistances at

t h e gas-

D a t a were a l s o o b t a i n e d f o r

solutions containing soluble

oxygen a b s o r p t i o n i n t o !-glucose

immobilized glucose oxidase (without magnetite),

or

as w e l l as f o r

p h y s i c a l a b s o r p t i o n f o r t h e r u n s w i t h t h e m a g n e t i t e - c o n t a i n i n g beads m e c h a n i c a l s t i r r i n g caused by s p i n n i n g o f

i n c r e a s e d because o f

beads a t t h e g a s - l i q u i d i n t e r f a c e . enhancement f a c t o r s

the

I n t h i s case t h e e x p e r i m e n t a l

were found t o be l a r g e r t h a n t h o s e p r e d i c t e d on

t h e b a s i s o f t h e f i l m t h e o r y f o r gas a b s o r p t i o n w i t h a p s e u d o - f i r s t order reaction. P h y s i c a l e n t r a p m e n t h a s b e e n u s e d as an a p p r o a c h t o a c h i e v e t h e r m a l s t a b i l i z a t i o n o f ;-glucose

oxidase.395

t h e t h e r m o i n a c t i v a t i o n o f e-glucose

for

I n polyacrylate gels the

f o l d by e n t r a p m e n t i n p o l y a c r y l a m i d e g e l s . enzyme behaved d i f f e r e n t l y ,

to.5 value

The

o x i d a s e was i n c r e a s e d s e v e r a l -

probably owing t o a microenvironmental

e f f e c t of t h e p o l y e l e c t r o l y t e n a t u r e of

the carrier.

I t has been r e p o r t e d f o u n d t h a t i n a d d i t i o n t o oxygen s i x r e d o x i n d i c a t o r s can s e r v e

as

Pencillium

The pH dependence o f t h e r a t e o f t h e r e a c t i o n

ita ale.^^'

c a t a l y s e d by g - g l u c o s e oxygen

and

substrates

for

Q-glucose

with

from

o x i d a s e was i n v e s t i g a t e d i n t h e p r e s e n c e o f

artificial

electron

I n contrast

acceptors.

r e a c t i o n w i t h o x y g e n , whose o p t i m u m pH i s 5.6, reactions

oxidase

phenazine

methosulphate,

to

the

t h e o p t i m u m pH o f

basic

dark

blue

2K,

p o l y v i o l o g e n , and t h e i o n - r a d i c a l s a l t o f b4,N,","-tetramethyl-4p h e n y l e n e d i a m i n e was o b s e r v e d a t pH 7.5. t h e r e d u c e d enzyme deprotonated)

can

exist

differing i n rate

i n of

The r e s u l t s i n d i c a t e d t h a t

two

forms

(protonated

o x i d a t i o n by

oxygen

and

and i n

a b i l i t y t o b i n d and r e d u c e a r t i f i c i a l e l e c t r o n a c c e p t o r s w i t h t h e f o r m a t i o n o f t h e semiquinone form of t h e c o f a c t o r . The u s e o f ! - g l u c o s e

oxidase-catalase

system

i n measuring

547

6: Enzymes aeration

capacity

of

fermenters

has

been

investigated

c o m p a r i s o n made o f t h e d y n a m i c a n d s t e a d y - s t a t e measurement.490

The

conditions

spontaneous h y d r o l y s i s o f

have

been

and

specified

where

l a c t o n e was s u f f i c i e n t l y r a p i d ,

thus I n Q-

e l i m i n a t i n g i n h i b i t o r y a c t i o n o f l a c t o n e on t h e o x i d a t i o n . glucose o x i d a s e - f r e e batches,

kla

the

a

kla

methods o f

v a l u e s were d e t e r m i n e d u s i n g

v a r i o u s m o d i f i c a t i o n s o f t h e dynamic method.

The d y n a m i c m e t h o d s i n

w h i c h gas i n t e r c h a n g e was e f f e c t e d w i t h o u t i n t e r r u p t i n g a e r a t i o n and

kla

agitation o f the batch yielded erroneously lower compared t o t h e r e s u l t s o f s t e a d y - s t a t e v a l u e was h i g h e r t h a n 0 . 0 3 s - ' .

v a l u e s as

methods i f t h e measured

kla

The v a l u e y i e l d e d b y t h e d y n a m i c

m e t h o d i n w h i c h t h e g a s i n t e r c h a n g e was e f f e c t e d a t t h e same t i m e w i t h t u r n i n g on a e r a t i o n and a g i t a t i o n of values r e s u l t i n g from the steady-state measured

kla

t h e b a t c h agreed w i t h

method,

provided that the

v a l u e s w e r e l o w e r t h a n 0.08 s - l a n d t h e s i m u l t a n e o u s

i n t e r f a c i a l t r a n s p o r t o f n i t r o g e n and oxygen had been t a k e n i n t o account i n the evaluation.

A t

kla

v a l u e s h i g h e r t h a n 0.08s"

kla

m o d i f i c a t i o n o f t h e dynamic method a l s o y i e l d e d l o w e r compared w i t h t h e outcome o f t h e s t e a d y - s t a t e experiments

performed

did

not,

however,

u n a m b i g u o u s l y on w h e t h e r t h e s e l o w e r

kla

allow

this

v a l u e s as

method. one

to

The

decide

v a l u e s w e r e due t o f a i l u r e

o f t h e adopted model t o descibe adequately t h e dynamic behaviour o f t h e system o r whether they were t r u e values d i f f e r i n g f r o m those y i e l d e d by t h e s t e a d y - s t a t e

m e t h o d on a c c o u n t o f d i f f e r e n t

physical

p r o p e r t i e s o f compared batches. The k i n e t i c s o f o x i d a t i o n o f ! - g l u c o s e o x y g e n i n t h e p r e s e n c e o f !-glucose been s t u d i e d b o t h t h e o r e t i c a l l y

s o l u t i o n by d i s s o l v e d

o x i d a s e and e x c e s s c a t a l a s e h a v e

and

experiment all^.^^^

The k i n e t i c

m o d e l u s e d was b a s e d o n t h e d e t a i l e d mechanism o f t h e r e a c t i o n .

The

d e s c r i p t i o n o f t h e m o d e l was g i v e n by t h e M i c h a e l i s - M e n t e n e q u a t i o n f o r t h e case o f t w o s u b s t r a t e s . a closed reactor commercial

at

enzymes.

The e x p e r i m e n t s w e r e c a r r i e d o u t i n

a t e m p e r a t u r e o f 25OC a n d p H 5 . 5 5 Kinetic

constants

of

the

using

reaction

were

e v a l u a t e d by f i t t i n g t h e oxygen c o n c e n t r a t i o n p r o f i l e s c a l c u l a t e d f r o m t h e model t o t h o s e f o u n d e x p e r i m e n t a l l y , by t h e method o f nonlinear

regression.

d i s t o r t i o n of

The r a t e o f

p r o b e were t a k e n i n t o account. the

reaction

were

i n

good

m u t a r o t a t i o n and

The d e t e r m i n e d k i n e t i c c o n s t a n t s o f agreement

l i t e r a t u r e f o r p u r i f i e d enzymes. data of

Q-glucose

d a t a caused by t h e dynamics o f t h e a p p l i e d oxygen with

those

given

i n

the

Adoption o f l i t e r a t u r e k i n e t i c

t h i s r e a c t i o n t o t h e d e t e r m i n a t i o n of

aeration capacity o f

548


f e r m e n t e r s by t h e Q - g l u c o s e o x i d a s e s y s t e m p r o v e d p o s s i b l e . A t pH 7 . 4 , i n 0 . 1 M p h o s p h a t e b u f f e r , Q - g l u c o s e o x i d a s e a n d B l u e D e x t r a n i n t e r a c t s t r o n g l y w i t h each o t h e r . 4 9 2 Under these c o n d i t i o n s a s o l u b l e c o m p l e x i s f o r m e d , as s h o w n by u l t r a f i l t r a t i o n e x p e r i m e n t s u s i n g a D i a f l o XM-300 m e m b r a n e ( n o m i n a l m o l e c u l a r w e i g h t c u t - o f f 300,000). I n t h i s c o m p l e x , t h e e n z y m e i s a b o u t 40% m o r e a c t i v e t h a n when f r e e i n s o l u t i o n , a n d i s a l s o m o r e s t a b l e t o w a r d s a c i d denaturation. Comparative s t u d i e s with a high-molecular-weight d e x t r a n d e v o i d o f dye r e s i d u e s ( D e x t r a n T - 2 0 0 0 ) s u g g e s t e d t h a t t h e dye moiety of Blue Dextran is d i r e c t l y i n v o l v e d i n t h e b i n d i n g of t h e p r o t e i n , and p o s s i b l y a l s o i n t h e enhancement o f i t s e n z y m a t i c activity. Q-Glucose o x i d a s e p u r i f i e d from A s p e r g i l l u s n i g e r on s u b j e c t i o n t o i s o e l e c t r i c f o c u s i n g a n d g e l e l e c t r o p h o r e s i s was f o u n d t o b e c o m p o s e d o f a t l e a s t s i x c o m p o n e n t e n z y m e s ( P I 3 . 9 t o 4.3).493 They a l l p o s s e s s an i d e n t i c a l p r o t e i n moiety, s i n c e the amino acid compositions, C-terminal sequences, catalytic parameters, q u a n t i t a t i v e and q u a l i t a t i v e immunological p r o p e r t i e s , and t h e e l e c t r o p h o r e t i c p a t t e r n s o f t h e p e p t i d e f r a g m e n t s o b t a i n e d by CNBr c l e a v a g e were p r a c t i c a l l y t h e s a m e . On t h e o t h e r h a n d , t h e c a r b o h y d r a t e c o n t e n t s o f t h e e n z y m e s were f o u n d t o b e d i f f e r e n t , a n d t h e s e d i f f e r e n c e s were a s s o c i a t e d i n t h e m a i n w i t h a p a r t i c u l a r peptide fragment. L-Galactonolactone Oxidases. -- L - G a l a c t o n o l a c t o n e o x i d a s e h a s b e e n f o u n d t o b e i n a c t i v a t e d by v a r i o u s s u l p h y d r y l r e a g e n t s : t h e o r d e r o f i n a c t i v a t i o n r a t e w a s HgC12, 4 - c h l o r o m e r c u r i b e n z o a t e , 4,4’dipyridyldisulphide, 2,2’-dipyridy l d i s u l p h i d e , 5 ,S9-dithio-bis-(2nitrobenzoate), and fj-ethylmaleimide iodoacetamide.494 The i n a c t i v a t i o n by 4,4’-dipyridyldisulphide w a s s t u d i e d i n d e t a i l , a n d i t was f o u n d t h a t t h e maximum d e g r e e o f i n a c t i v a t i o n a t t a i n e d w i t h i n c r e a s i n g r e a g e n t c o n c e n t r a t i o n was 93% a n d t h a t t h e k i n e t i c s o f i n a c t i v a t i o n were f i r s t o r d e r w i t h r e s p e c t t o r e a g e n t c o n c e n t r a t i o n . T h e pH d e p e n d e n c e o f t h e s e c o n d - o r d e r r a t e c o n s t a n t o f t h e i n a c t i v a t i o n r e v e a l e d t h a t a s u l p h y d r y l g r o u p w i t h a pKa o f 9.8 was involved i n the i n a c t i v a t i o n process. T h e v a l u e o f pKa i s h i g h compared w i t h t h a t of low-molecular-weight t h i o l s , i n d i c a t i n g t h a t t h e i o n i z a t i o n o f t h e s u l p h y d r y l g r o u p i s a f f e c t e d by t h e e l e c t r i c f i e l d of a n e g a t i v e l y charged group l o c a t e d i n its v i c i n i t y . The v a l u e s o f K m a n d lmax for the C-galactonolactone oxidase reaction d i d n o t c h a n g e g r e a t l y a r o u n d pH 9.8. This is consistent with the

549

6: Enzymes

v i e w t h a t t h e s u l p h y d r y l g r o u p does n o t p a r t i c i p a t e i n t h e c a t a l y t i c process.

The v e l o c i t y o f i n a c t i v a t i o n i n t h e p r e s e n c e o f s u b s t r a t e

was c o n s i d e r a b l y g r e a t e r

It

t h a n t h a t o b s e r v e d i n i t s absence.

a p p e a r s t h a t t h e s u l p h y d r y l g r o u p becomes more r e a c t i v e d u r i n g t h e catalytic

cycle of

the

enzyme.

I t was

also noted that

the

a b s o r p t i o n spectrum of t h e f l a v i n p r o s t h e t i c group ( t h e o x i d i z e d was m o d i f i e d b y a d d i n g 4 - c h l o r o m e r c u r i b e n z o a t e .

form)

I-Gulonolactone

--

Oxidases.

The

activity

of

I-gulonolactone

o x i d a s e i n l i v e r s o f 49 s p e c i e s o f e u t h e r i a n mammals h a s been f o u n d to

vary

intraspecifically

v a r i a t i o n w e r e 0.2

t o 0.4

--

Cellobiose Oxidases. produced

cellobiose

cellulose.496

among

individuals:

coefficients

of

i n many s p e c i e s . 4 9 5 A s p e c i e s o f t h e i m p e r f e c t fungus M o n i l i a

oxidase

extracellularly

when

grown

on

The i n d u c i b l e enzyme was b o t h bound t o t h e m y c e l i u m

and r e l e a s e d i n t o t h e g r o w t h medium and showed a h i g h d e g r e e o f specificity for

cellobiose,

but

a l s o o x i d i z e d l a c t o s e and 4-;-8-!-

glucopyranosyl-q-mannose.

The s p e c i f i c i t y

was

compounds

restricted V.

+0.22

t o those

4-Benzoquinone

n o t reduced.

of

the electron acceptor

having a redox

and s e v e r a l o t h e r q u i n o n e s ,

weight

48,000

of

t e c h n i q u e was d e v e l o p e d f o r polyacrylamide gels focusing

&-Fructose

Oxidases. the

study

enzymes

i m m o b i l i z e d on

can

assessed

be

and

by

-of

a

PI of

An

runs

The

which

zymogram

and

isoelectric

c a t a l y s e d by

kinetic allow

behaviour

a

the surface kinetics,

separate and t h e

The m e t h o d h a s b e e n a p p l i e d i n t h e

s t u d y o f s u c r o s e i n v e r s i o n by 8 - & - f r u c t o s e

o x i d a s e i m m o b i l i z e d on

resin.

2-Galactose Oxidases. of

new

reactions

s u p p o r t s .497

experimental

f l u i d - p a r t i c l e mass t r a n s f e r .

A

e x p e r i m e n t a l p r o c e d u r e has been

liquid-phase

porous

The enzyme h a d a

cellobiose oxidase i n

electrophoresis

evaluation o f the internal diffusion,

IRA-93

5.4.

the detection o f

following

.

proposed for

were

O x y g e n was n o t c o n s u m e d n o r was h y d r o g e n p e r o x i d e

p r o d u c e d by c e l l o b i o s e o x i d a t i o n o f c e l l o b i o s e . molecular

potential of however,

glycosidases

!-Galactose

and

oxidase

--

The s t e r i c f a c t o r s i n v o l v e d i n t h e a c t i o n

g-galactose

o x i d a s e have been i n ~ e s t i g a t e d . ” ~

i s s t e r i c a l l y hindered by c e r t a i n types of

branching i n substrate oligosaccharide chains

and w i l l n o t o x i d i z e

550


t h e 4 - g a l a c t o s e r e s i d u e i n t r i s a c c h a r i d e ( 5 ) b u t w i l l i n ( 6 ) . PG a l a c t o s e a s i n ( 7 ) i s not s u s c e p t i b l e t o o x i d a t i o n w i t h !-galactose oxidase u n t i l a f t e r t h e a p p l i c a t i o n of B-Q-2-acetamido-2deoxygalactosidase. a-NeuNGL 2

I 6

B-g-Gal-( 1+3)-!-GalNAcol (5)

B-Q-Gal-(1+3)-g-GalNAcol 2

I 1 ? - L-- F u C

(6)

B-Q-GalNAc-(1+3)-B-g-Gal-(l+3)-~-GalNAcol 2

I 1

a-i-Fuc (7)

48

Carbohydrate Transferases

A review of t h e a f f i n i t y chromatography of g l y c o s y l t r a n s f e r a s e s summarizes t h e use o f b i o s p e c i f i c chromatography t e c h n i q u e s i n t h e ~~ that are p u r i f i c a t i o n of mammalian g l y c o s y l t r a n ~ f e r a s e s . ~Ligands analogues of donor o r a c c e p t o r s u b s t r a t e s have been l i n k e d t o cyanogen b r o m i d e - a c t i v a t e d a g a r o s e f o r use a s a f f i n i t y a d s o r b e n t s . Immobilized l e c t i n s have been employed t o r e c o g n i z e t h e c a r b o h y d r a t e m o i e t i e s o f g l y c o s y l t r a n s f e r a s e and remove them f r o m complex m i x t u r e s . The a p p l i c a t i o n of these methods has p e r m i t t e d e x t e n s i v e p u r i f i c a t i o n of many membrane-bound g l y c o s y l t r a n s f e r a s e s , some t o homogeneity. T h e p o s s i b i l i t y t h a t murein g l y c o s y l t r a n s f e r a s e of E s c h e r i c h i a c o l i may f u n c t i o n a s an exoenzyme t o c l e a v e t h e j u r e i n

551

6: Enzymes s a c c u l u s i n a s y s t e m a t i c f a s h i o n has been i n v e s t i g a t e d . 4 9 9

Two

m o l e c u l a r s p e c i e s o f t h i s h y d r o l y t i c enzyme h a v e b e e n i s o l a t e d and characterized:

one i s a s s o c i a t e d w i t h t h e s o l u b l e f r a c t i o n

o t h e r w i t h t h e e n v e l o p e f r a c t i o n o f r u p t u r e d E.

and t h e

c o l i cells.

The

s o l u b l e enzyme was e m p l o y e d t o d i g e s t m u r e i n s a c c u l i t h a t h a d b e e n uniformly

l a b e l l e d w i t h { 3H)diaminopimelic

acid.

The

analysis

of

t h e r e a c t i o n p r o d u c t i n d i c a t e d t h a t t h e enzyme d i d n o t c l e a v e t h e glycan

c h a i n s random l y

.

To

d e t e r m i n e whet h e r

g l y c o s y l t r a n s f e r ase

from t h e 2-acetamido-2-deoxy-~-glucosyl

r e l e a s e d muropeptide f i r s t

or t h e 1 , 6 - a n h y d r o m u r a m y l e n d s o f t h e g l y c a n c h a i n s ,

t h e {3H)-

d i a m i n o p i me l a t e - l a b e l l e d s a c c u li w e r e f u r t h e r r a d i o l a b e l l e d a t t h e i r

2-acetamido-2-deoxy-~-glucosyl ends w i t h { 14)-Q-galactose galactosyltransferase reaction.

by a Q-

The g l y c o s y l t r a n s f e r a s e r e l e a s e d Q-

g a l a c t o s e - l a b e l l e d X + X’ m u r o p e p t i d e s ( w h e r e X = 2 - a c e t a m i d o - 2 deoxy-~-glucosyl-1,6-anhydro-~-acetylmuramyl-~-alanyl-~-~-glutamylmeso-diaminopimelic o f digestion,

= X-2-alanine)

a c i d , X’

e a r l y d u r i n g t h e course

suggesting exoenzymatic cleavage of t h e glycan chains

preferentially from the 2-acetamido-2-deoxy-~-glycosyl

ends.

The

k i n e t i c s o f t h e a c t i v i t y o f t h e membrane-bound enzyme w e r e f o u n d t o b e i d e n t i c a l t o t h o s e o f t h e s o l u b l e enzyme, molecular species of

indicating that both

g l y c o s y l t r a n s f e r a s e f u n c t i o n as exoenzymes

i n vitro. I n a c t i v a t i o n of

-----mutans

and S .

by

from o r a l Streptococcus

photochemical

oxidation

has

been

C e l l - f r e e Q - g l u c o s y l t r a n s f e r ase of Q-glucose-grown

r e p o r t e d .500 S.

P-glucosyltransferases

sanggL5

m u t a n s AHT was c o m p l e t e l y i n a c t i v a t e d i n t h e p r e s e n c e o f 0.002%

o f M e t h y l e n e B l u e a t 25OC a n d pH 7.0 a f t e r i l l u m i n a t i o n w i t h a 1 5 0 W i n c a n d e s c e n t lamp. v a l u e s (7.0.

T h e r a t e o f i n a c t i v a t i o n was d e c r e a s e d a t pH

i-Histidine

was t h e o n l y a m i n o a c i d r e s i d u e m o d i f i e d

t o a s i g n i f i c a n t e x t e n t , and t h e r a t e s o f o x i d a t i o n o f I - h i s t i d i n e and loss o f enzyme a c t i v i t y c l o s e l y a g r e e d .

Production o f both

w a t e r - i n s o l u b l e and - s o l u b l e

f r o m s u c r o s e by t h e

oxidized

k-glucan f r a c t i o n s

1-glucosyltransferase

inhibited.

preparations

was

significantly

P h o t o - o x i d a t i o n w i t h 0.002% Rose B e n g a l a t pH 7.0

also

induced complete i n a c t i v a t i o n o f t h e Q-glucosyltransferase. These r e s u l t s s t r o n g l y suggest t h a t t h e i m i d a z o l e p o r t i o n o f I - h i s t i d i n e may

function

as

part

glucosyltransferase responsible for

o f

the

active

isoenzymes of

t h e s y n t h e s i s o f (1

+

S.

s i t e s

o f

m u t a n s AHT,

both

Q-

which are

3 ) - and (1 + 6 ) - a - Q - g l u c o s i d i c

linkages.

I t has

been r e p o r t e d

that

the

glucosyltransferases

of


552 S t r e e --------------tococcus mutans ----

have

been r e s o l v e d i n t o t w o components

e s s e n t i a l t o w a t e r - i n s o l u b l e glucan synthesis.501

Collagen-D=-galactotransferase

and

collagen-Q-

g l u c o s y l t r a n s f e r a s e a c t i v i t i e s h a v e been s t u d i e d i n c u l t u r e d human f o e t a l l u n g WI-38

and I M R - 9 0 d i p l o i d f i b r o b l a s t s . 5 0 2

functioned i n concert hydroxylysine membranes, UDP-!-Gal

units

t o

as

found

naturally

i n

collagens,

and c e r t a i n serum g l y c o p r o t e i n s . and UDP-g-Glc

These enzymes

s y n t h e s i z e Q-glucosyl-Q-galactosyl-L-

as g l y c o s e d o n o r s ,

basement

The t r a n s f e r a s e s u s e d c o l l a g e n s and c o l l a g e n -

d e r i v e d p e p t i d e s o r g l y c o p e p t i d e s as g l y c o s e

acceptors,

and w o r k e d

b e s t i n t h e p r e s e n c e o f m a n g a n e s e as a r e q u i r e d d i v a l e n t c a t i o n . Two pH o p t i m a , b e t w e e n pH 6 a n d 6 . 5

a n d b e t w e e n pH 7 . 5

noted for

and t h e s e o p t i m a ,

each t y p e o f

transferase,

i n t h e case o f Q - g l u c o s y l t r a n s f e r a s e , s i z e of

were e v i d e n t r e g a r d l e s s o f

a c c e p t o r employed i n t h e assay.

a c t i v i t y of

and 8, were particularly

About 35% o f t h e t o t a l

e a c h enzyme was f o u n d i n t h e s o l u b l e f r a c t i o n s o f

cell

homogenates, and about 50% of t h e p a r t i c u l a t e f r a c t i o n a c t i v i t i e s c o u l d be r e l e a s e d by m i l d s o n i c a t i o n o r by t r e a t m e n t w i t h T r i t o n X100.

Assessment o f t r a n s f e r a s e a c t i v i t i e s as a f u n c t i o n o f c e l l u l a r

a g e i n g i n c u l t u r e r e v e a l e d t h a t s i g n i f i c a n t d e c r e a s e s i n enzyme l e v e l s o c c u r r e d a s t h e c e l l a p p r o a c h e d s e n e s c e n c e ( l a t e p h a s e II), a n d t h e s e e f f e c t s w e r e r e v e r s e d when t h e c e l l a t t a i n e d s e n e s c e n c e ( p h a s e 111). conditions

A d d i t i o n of known

hydroxylation,

the

glycosyltransferases

a s c o r b i c a c i d t o young c u l t u r e s ,

increase

caused

glycosyltransferases suggested t h a t

t o

no

endogenous

on

effects

the

activities

t o w a r d exogenous a c c e p t o r s . activities

of

under

collagen-peptide of

collagen-hydroxylases

m i g h t n o t be c o - o r d i n a t e l y

regardless o f t h e h y d r o x y l a t i o n events,

the

These r e s u l t s and

r e g u l a t e d and t h a t ,

g l y c o s y l a t i o n of t h e peptide

m i g h t be l i m i t e d t o a s p e c i f i c f r a c t i o n o f i - h y d r o x y l y s i n e r e s i d u e s during the post-translational

m o d i f i c a t i o n of collagen.

Bovine g l y c o p r o t e i n B-E-galactosyltransferase have two metal-binding

h a v e been e s t a b l i s h e d by u s i n g k i n e t i c , structural

s p e c t r o s c o p i c , and a f f i n i t y -

M e t a l s i t e I,

c h r o m a t o g r a p h i c approaches.503 maintaining the

h a s b e e n shown t o

the f u n c t i o n a l properties o f which

sites,

integrity

of

which i s i n v o l v e d i n

the

protein,

must

be

l i g a n d e d p r i o r t o o t h e r s u b s t r a t e s b i n d i n g and p r i o r t o a second m e t a l b i n d i n g t o s i t e 11, w h i c h n- - g a l a c t o s e b i n d i n g .

i s shown t o be a s s o c i a t e d w i t h UDP-

B o t h m e t a l s i t e s can b i n d a v a r i e t y o f

metals.

However,

c a l c i u m and i t s f l u o r e s c e n t a n a l o g u e e u r o p i u m b i n d o n l y t o

s i t e I I.

F 1u o r es c e n t r e s o n a n c e - e n e r gy t r a n s f e r m e a s u r e m e n t s b e t w e e n

553

6: Enzymes

e u r o p i u m i n s i t e I1 and c o b a l t i n s i t e I i n d i c a t e a d i s t a n c e o f 18 0

3 A between t h e two s i t e s .

mercuric-N-dansylcysteine

2

C h e m i c a l - m o d i f i c a t i o n s t u d i e s w i t h 5i n d i c a t e t h a t one ( o f a t o t a l o f t h r e e

e x p o s e d s u l p h y d r y l g r o u p s ) c a n be s p e c i f i c a l l y d a n s y l a t e d and t h a t t h i s s u l p h y d r y l group i s i n or near t h e UDP-a-Salactose-binding site.

Resonance-energy

transfer

measurements

i n t r o d u c e d s u l p h y d r y l group and c o b a l t distance of

2

19

3

dl

between t h i s

i n metal s i t e I

between these p o i n t s ,

give

a

consistent with the

i n t e r p r e t a t i o n t h a t the UDP-a-galactose-binding

site,

which

i s

a s s o c i a t e d w i t h m e t a l s i t e 1 1 , i s l o c a t e d some d i s t a n c e f r o m t h e s t r u c t u r a l m e t a l s i t e ( s i t e 1). A !-galactose

protein),

B-galactosyltransferase (lactose synthetase A

w h i c h t r a n s f e r s Q - g a l a c t o s e f r o m UDP-D-galactose

t o 2-

a c e t a m i d o - 2 - d e o x y - ~ - g l u c o s e , was p u r i f i e d 2 8 6 , 0 0 0 - f o l d t o h o m o g e n e i t y w i t h 40% y i e l d f r o m human p l a s m a by r e p e a t e d a f f i n i t y c h r o r n a t o g r a p h y o n a - l a c t a l b u m i n - S e p h a r ~ s e . ~ ~S~o d i u m d o d e c y 1 s u l p h a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of

t h e p u r i f i e d enzyme

showed a s i n g l e p r o t e i n band w i t h a m o l e c u l a r w e i g h t o f 49,000.

The

enzyme i s a g l y c o p r o t e i n w i t h 11% by w e i g h t c a r b o h y d r a t e w h i c h h a s o n l y 2-acetamido-2-deoxy-glucose-~-asparagine g l y c o p e p t i d e l i n k a g e s . The enzyme showed c h a r a c t e r i s t i c changes i n a c t i v i t y a t d i f f e r e n t alactalbumin concentrations,

i n d i c a t i n g t h a t t h e enzyme i s t h e A

Km v a l u e s f o r t h e s u b s t r a t e s were rnM f o r U D P - Q - g a l a c t o s e and 0.20 m M f o r Mn2+. The

p r o t e i n of lactose synthetase. f o u n d t o be 0.056

a c t i v i t y o f t h e enzyme was n e u t r a l i z e d b y a n t i - e n z y m e a n t i b o d y ,

but

the antibody d i d not neutralize the bovine m i l k Q-galactosyltransferase (A p r o t e i n ) a c t i v i t y . Bovine

a-lactalburnin

enzymatically f u l l y

active,

has

been

highly

dansylated

fluorescent

t o

derivative

on t h e N - t e r m i n a l I - g l u t a m i c a c i d residue.505

give

an

labelled

This fluorescent

d e r i v a t i v e of

a - l a c t a l b u m i n has been c o v a l e n t l y

glycoprotein

B-P-galactosy ltransferase

p i me lirn id a t e.

Resonance - e n e r gy t r a n s f e r

by

crosslinked t o

using

dirnethy 1

me as u r e rnents u s i n g c o b a I t

b o u n d t o t h e t r a n s f e r a s e as t h e a c c e p t o r o f e n e r g y t r a n s f e r f r o m t h e d a n s y l g r o u p on t h e a - l a c t a l b u m i n 32 of

A"

i n d i c a t e t h a t t h e d a n s y l group i s

A model o f the a c t i v e s i t e f r o m t h e c o b a l t on t h e t r a n s f e r a s e . t h e t r a n s f e r a s e and i t s i n t e r a c t i o n w i t h a - l a c t a l b u r n i n i s

proposed on t h e b a s i s of

t h e s e and p r e v i o u s s t u d i e s .

have a l l o w e d an e x t e n s i o n o f s y n t h e t a s e and g i v e g r e a t e r

the active-site

These s t u d i e s

mapping o f l a c t o s e

i n s i g h t i n t o t h e i n t e r a c t i o n o f a-

l a c t a l b u m i n w i t h t h e t r a n s f e r a s e and i t s r o l e i n l a c t o s e s y n t h e t a s e .

554


G1ycoprotein:C-fucosyltransferase from sheep b r a i n , s o l u b i l i z e d and p r e p u r i f i e d by hydrophobic chromatography on e t h y l - a g a r o s e , has been f u r t h e r p u r i f i e d by chromatofocalization.506 T h i s p r o c e d u r e y i e l d e d f o u r isoenzymes. The s o l u b l e & - f u c o s y l t r a n s f e r a s e o f t h e r a t s m a l l - i n t e s t i n a l mucosa p u r i f i e d b y g e l f i l t r a t i o n on S e p h a d e x G-100 h a s b e e n r e s o l v e d i n t o two i s o e n z y m e s F1 ( P I = 4 . 4 7 ) and F 2 ( P I = 4.96) b y i s o e l e c t r i c focusing.S07 The u s e of D E A E - c e l l u l o s e i n s t e a d of S e p h a d e x G-100 l e a d s t o a n o t h e r component F 3 ( P I = 8 . 7 0 ) . K i n e t i c p a r a m e t e r s (5, and ymax)f o r t h e t h r e e isoenzymes were d e t e r m i n e d . The e n z y m a t i c mechanism s e e m s t o be a b i - b i random t y p e f o r t h e t h r e e isoenzymes, and F 3 a p p e a r s a s t h e most a c t i v e s p e c i e s . T h e i s o e l e c t r o f o c u s i n g p a t t e r n s o f 2 - a - i - , 3 - a - i - , and 4-a-Cf u c o s y l t r a n s f e r a s e s from human m i l k serum have been r e p o r ted.’08 The s p e c i f i c i t y of s i a l y l t r a n s f e r a s e has been i n v e s t i g a t e d w i t h r e f e r e n c e t o t h e s i a l y l a t i o n of o v i n e s u b m a x i l l a r y mucin i n v i t r o which was { 1 4 C ) s i a l y l a t e d i n v i t r o u s i n g a p o r c i n e l i v e r c e l l - f r e e p r e p a r a t i ~ n . ’ ~ ~The o l i g o s a c c h a r i d e c h a i n s were c l e a v e d from t h e p r o d u c t g l y c o p r o t e i n by B - e l i m i n a t i o n under r e d u c t i v e c o n d i t i o n s , f r a c t i o n a t e d by g e l f i l t r a t i o n on Bio-Gel P-2, and c h a r a c t e r i z e d by t h i n - l a y e r chromatography. The s t r u c t u r e o f t h e product c h a i n was s t u d i e d by p e r i o d a t e o x i d a t i o n and a n a l y s i s of t h e p e e l i n g p r o d u c t s formed i n t h e B - e l i m i n a t i o n s t e p . I t appeared t h a t { 1 4 C ) s i a l i c a c i d had been i n t r o d u c e d e x c l u s i v e l y t o t h e Q - g a l a c t o s e r e s i d u e s o f Q G a l - ( 1 + 3)-Q-GalNAc d i s a c c h a r i d e u n i t s o c c u r r i n g on t h e mucin a s minor c h a i n s . No i n d i c a t i o n f o r a t r a n s f e r t o 2-acetamido-2-deoxyIn g - g a l a c t o s y l r e s i d u e s on t h i s g l y c o p r o t e i n was o b t a i n e d . agreement w i t h t h i s r e s u l t s i a l y l t r a n s f e r a s e a c t i v i t i e s of p o r c i n e , r a t , human, and c a n i n e l i v e r w i t h B - G a l - ( l + 3 ) - Q - G a l N A c - p r o t e i n a c c e p t o r s w e r e i n v a r i a b l y much h i g h e r t h a n t h o s e w i t h o v i n e submaxillary asialomucin. When t h e a s i a l o m u c i n had been { 1 4 C ) s i a l y l a t e d b y an o v i n e s u b m a x i l l a r y g l a n d c e l l - f r e e p r e p a r a t i o n , a n a l y s i s of t h e p r o d u c t o l i g o s a c c h a r i d e c h a i n r e v e a l e d t h e i n t r o d u c t i o n o f { 1 4 C ) s i a l i c a c i d t o p o s i t i o n C - 6 on t h e 2acetamido-2-deoxy-!-galactose r e s i d u e s . The s p e c i f i c i t y o f t h i s t r a n s f e r was r e f l e c t e d by t h e very high s i a l y l t r a n s f e r a s e a c t i v i t i e s of g l a n d p r e p a r a t i o n s w i t h g-Gal-(l + 3)-Q-GalNAc-protein a s w e l l a s a-GalNAc-protein a c c e p t o r s . Mixed-enzyme e x p e r i m e n t s i n d i c a t e d t h a t t h e d i f f e r e n c e i n l i v e r and g l a n d o v i n e s u b m a x i l l a r y a s i a l o m u c i n s i a l y l t r a n s f e r a s e a c t i v i t i e s was n o t d u e t o t h e p r e s e n c e o f a s p e c i f i c i n h i b i t o r i n t h e l i v e r o r an a c t i v a t o r i n t h e g l a n d . It w i t h T r i t o n X-100

555

6: Enzymes

was c o n c l u d e d t h a t l i v e r s o f man, p i g , r a t , a n d dog c o n t a i n a a - G a l s i a l y l t r a n s f e r a s e which i s involved i n the

(1 + 3)-g-GalNAc-protein

sialylation o f g-glycosidically glycoproteins.

l i n k e d c a r b o h y d r a t e c h a i n s on serum

g-GalNAc-protein

sialyltransferase activity,

which

r i c h l y o c c u r s i n o v i n e s u b m a x i l l a r y g l a n d , h o w e v e r , a p p e a r s t o be lacking from l i v e r tissue. A t e c h n i q u e h a s been d e s c r i b e d by a f f i n i t y c h r o m a t o g r a p h y o f

o r o t a t e p h o s p h o r i b o s y l t r a n s f e r a s e f r o m E s c h e r i c h i a c o li K - 1Z?'

I-Iduronic Acid 2 - S u l p h a t e S u l p h a t a s e s

49

A m o r e s e n s i t i v e assay h a s been d e v e l o p e d f o r t h e I - i d u r o n i c 2 sulphate

sulphatase

which

i s

deficient

i n cases o f

the Hunter

The s u b s t r a t e i s 2 - ( a - C - i d o p y r a n o s y l u r o n i c

syndrome.511 su1phate)-( 1

.+

a c i d 2-

4) - 2 , 5 - a n h y d r o -Q-{ 3 H - l } r n a n n i t o 1 6 - s u l p h a t e

a f t e r incubation,

,

and,

i t i s s e p a r a t e d f r o m t h e p r o d u c t by i o n - e x c h a n g e

r a t h e r t h a n by c h r o m a t o g r a p h y o n a m i c r o - c o l u m n o f Dowex 1 x 2 (Cl') h i g h - v o l t a g e e l e c t r o p h o r e s i s o r ECTEOLA c e l l u l o s e c h r o m a t o g r a p h y . S i n c e t h e b l a n k c o r r e c t i o n i s t h e n much s m a l l e r ,

a shorter

i n c u b a t i o n t i m e c a n be u s e d and c o n v e r s i o n o f t h e s u b s t r a t e r e d u c e d f r o m a p p r o x i m a t e l y 50% down t o l e v e l s w h e r e c o m p l i c a t i o n s r e s u l t i n g f r o m s u b s t r a t e d e p l e t i o n and p r o d u c t i n h i b i t i o n a r e m i n i m a l .

Using

With w h o l e s e r u m t h e a p p a r e n t 5, f o r t h e s u b s t r a t e i s 0.2 n m o l 1-l. a n i n c u b a t i o n t i m e o f 20 m i n , s e r a f r o m h e t e r o t y g o t e s e x h i b i t e d a p p r o x i m a t e l y 35% o f t h e n o r m a l l e v e l s of L - i d u r o n a t e 2 - s u l p h a t e sulphatase carriers, females).

(0.11-0.61, 0.24-2.35,

mean 0.34, mean 0.94,

nmol h'l

n m o l h'l

mg-l

protein for

21

m g - l p r o t e i n f o r 37 n o r m a l

S e r u m a n a l y s e s c a n t h u s be u s e d t o s u p p l e m e n t t h o s e on

h a i r r o o t s i n t h e d e t e c t i o n o f c a r r i e r s o f t h e H u n t e r syndrome. The L - i d u r o n i c a c i d 2 - s u l p h a t e s u l p h a t a s e o f human p l a c e n t a h a s been s e p a r a t e d by ion-exchange and g e l - f i l t r a t i o n c h r o m a t o g r a p h y i n t o t w o components, a l e s s a c i d i c f o r m A and a more a c i d i c f o r m The t w o f o r m s have d i f f e r e n t m o b i l i t i e s on g e l e l e c t r o p h o r e s i s a n d d i f f e r e n t i s o e l e c t r i c p o i n t s ( A PI 5.0, B PI 4.5).

similar

They show t h e same pH o p t i m a i n s o d i u m a c e t a t e b u f f e r a n d

Em v a l u e s

f o r { 3H)disulphated disaccharide substrate.

f o r m i s more heat l a b i l e t h a n t h e B form.

w e i g h t s w e r e f o u n d by g e l f i l t r a t i o n w h i l e s i m i l a r estimated

by

sucrose

gradient

The A

Different molecular

centrifugation.

values were

Neuraminidase

t r e a t m e n t o f t h e t w o f o r m s g i v e s e v i d e n c e t h a t t h e s e enzymes c o n t a i n


556 s i a l i c a c i d residues.

Human u r i n e h a s been shown t o c o n t a i n a n o v e l s u l p h a t a s e w h i c h i s

specific

2-deoxy-2-sulphamido-4-glucopyranoside

for

3-

~ u l p h a t e . O~ f ~t h~ e t h r e e i s o m e r i c s u l p h a m a t e d e r i v a t i v e s , 3-g-,

0-,

and 6 - g - s u l p h a t e

enzymatic

esters,

activity

only t h e 3-g-ester

required that

4The

t h e a m i n o g r o u p be s u l p h a t e d .

S u l p h a t e i s n o t r e l e a s e d ift h e a m i n o g r o u p

i s

free or acetylated.

I t h a d a pH o p t i m u m o f 6.3 a n d was

The e n z y m e was p u r i f i e d 7 0 - f o l d . i n h i b i t e d by

was h y d r o l y s e d .

i n o r g a n i c s u l p h a t e and p h o s p h a t e .

t h i s enzyme s u g g e s t e d t h a t a 3 - g - s u l p h a t e d

The s p e c i f i c i t y

of

2-amino-2-deoxy-~-glucose

m o i e t y may h a v e a r o l e i n t h e p h y s i o l o g i c a l a c t i v i t y o f

or

heparin

heparan sulphate.

50

Arylsulphatases

A s t u d y h a s b e e n made o f l y s o s o m a l - e n z y m e a c t i v i t i e s i n c l u d i n g

A

arylsulphatase disease.60

i n

serum

For further

and

in

leukocytes

chronic

hepatic

d e t a i l s see i n i t i a l c i t a t i o n o f r e f . 6 0 .

A v a r i a n t f o r m o f a r y l s u l p h a t a s e A h a s been i d e n t i f i e d i n human u r i n e derived d i r e c t l y from the r e n a l pelvis. t h e enzyme was f o u n d i n n e p h r o s t o m i c u r i n e , form,

The v a r i a n t

w h i c h i s t h e s o l e component o f a r y l s u l p h a t a s e A i n v o i d e d

urine.514

The

n e p h r o s t o m i c enzyme

enzyme w i t h r e s p e c t

differed

from

t o the k i n e t i c parameters,

the

p o i n t s of t h e voided-urine respectively. and

5,

The i s o e l e c t r i c

a n d n e p h r o s t o m i c e n z y m e s w e r e 4.7

and

The n e p h r o s t o m i c enzyme was more h e a t l a b i l e a t

62.5OC t h a n t h e v o i d e d - u r i n e (130,000)

voided-urine

the isoelectric

p o i n t , h e a t s t a b i l i t y , and i m m u n o l o g i c a l r e a c t i v i t y . 5.3,

form o f

i n a d d i t i o n t o the minor

values

of

s u l p h a t e as s u b s t r a t e were

enzyme. the

Although the molecular weights

two

enzymes

a l m o s t t h e same,

with the

nitrocatechol

1

value o f the

n e p h r o s t o m i c enzyme was 10% t h a t o f t h e v o i d e d - u r i n e enzyme. d e m o n s t r a t e d by v a r i o u s methods, p u r i f i e d voided-urine

enzyme,

I t was

using IgG antibody against the

t h a t the nephrostomic

e n z y m e was

a n t i g e n i c a l l y d i s t i n c t f r o m t h e v o i d e d - u r i n e enzyme. R a b b i t l i v e r a r y l s u l p h a t a s e A i s a g l y c o p r o t e i n c o n t a i n i n g 4.6% carbohydrate g l u c o s e (71, 140,000).515 chains.

c o m p r i s i n g p-mannose

(25),

2-acetamido-2-deoxy-Q-

and s i a l i c a c i d ( 3 r e s i d u e s p e r enzyme monomer m o l .

The p r o t e i n h a s a r e l a t i v e l y h i g h c o n t e n t o f

glycine,and

wt.

E a c h monomer c o n s i s t s o f t w o e q u i v a l e n t p o l y p e p t i d e I-leucine,

&-proline,

I-

and t h e a m i n o a c i d c o m p o s i t i o n i s s i m i l a r t o

557

6: Enzymes that

of

other

known

liver

sulphatases.

The

arylsulphatase A

catalyses the h y d r o l y s i s o f a wide v a r i e t y o f sulphate esters, although i t appears t h a t cerebroside sulphate i s a p h y s i o l o g i c a l s u b s t r a t e f o r t h e enzyme b e c a u s e t h e

Em i

s very low (0.06

The

mM).

turnover r a t e f o r hydrolysis of nitrocatechol sulphate or related synthetic

substrates

i s

much

higher

than

the

rate

with

most

n a t u r a l l y o c c u r r i n g s u l p h a t e e s t e r s s u c h as c e r e b r o s i d e s u l p h a t e , s t e r o i d sulphates,

$-tyrosine

sulphate, or B-Q-glucopyranose

6-

sulphate.

t h e turnover r a t e with ascorbate 2-sulphate

i s

However,

comparable t o t h e r a t e s measured u s i n g most s y n t h e t i c s u b s t r a t e s . These r e s u l t s a r e d i s c u s s e d i n r e l a t i o n s h i p t o s e v e r a l p r e v i o u s l y d e s c r i b e d s u l p h a t a s e enzymes w h i c h were c l a i m e d t o have u n i q u e specificities. A r y l s u l p h a t a s e has been p u r i f i e d 2 1 9 - f o l d species."6

and i m m u n o l o g i c a l a n a l y s i s . weight

f r o m Pseudomonas

The f i n a l p r e p a r a t i o n was homogeneous by e l e c t r o p h o r e t i c

about

51,000,

The enzyme i s a monomer o f m o l e c u l a r

w i t h a Stokes r a d i u s of

f r i c t i o n a l r a t i o o f 1.2,

3.0

x

cm,

and a s e d i m e n t a t i o n c o e f f i c i e n t o f

a

4.1 S.

I t h a s been r e p o r t e d t h a t a r y l s u l p h a t a s e a c t i v i t y i s s t i m u l a t e d by

arylamines

produced.517

and

evidence

number

A

of

for

substrate

arylamines

tryptamine) increased the i n v i t r o a c t i v i t y Pseudomonas sp.

(2.9.

s t r a i n C12B.

I-tyrosine

and

a c t i v a t i o n has

(including of

been

tyramine

arylsulphatase

and from

Amino a c i d a n a l o g u e s o f t h e s e a m i n e s

I-tryptophan)

failed

t o exert

an e f f e c t .

S t i m u l a t i o n o f a c t i v i t y by t y r a m i n e c o u l d n o t be a c c o u n t e d f o r i n t e r m s o f s u l p h o t r a n s f e r a s e a c t i v i t y f o r t h i s p h e n o l , a n d no s h i f t i n the

pH o p t i m u m

tryptamine. enzyme

for

Increased

concentration

concentration.

the

enzyme

occurred

i n

lmax due t o t h e s e a m i n e s but

varied

the

presence

of

was i n d e p e n d e n t o f

significantly

with

substrate

Evidence i s presented which suggests t h a t arylamines

enhance a r y l s u l p h a t a s e a c t i v i t y by f o r m i n g a s a l t l i n k a g e w i t h t h e s u b s t r a t e and r e n d e r i n g i t more s u s c e p t i b l e t o e n z y m a t i c and a c i d catalysed hydrolyses.

The r e c r y s t a l l i z e d t r y p t a m i n e s a l t o f t h e

s u b s t r a t e e x h i b i t e d a reduced a f f i n i t y

for

the

h y d r o l y s e d more r a p i d l y t h a n t h e p o t a s s i u m s a l t ,

enzyme

but

was

which i s n o r m a l l y

e m p l o y e d as t h e a s s a y s u b s t r a t e . The s t i m u l a t i o n o f a r y l s u l p h a t a s e s y n t h e s i s i n P s e u d o m o n a s a e r u g i n o s a by exogenous n u c l e o t i d e s has been r e p o r t e d . 5 1 8

558


2-Acetamido-2-deoxy-Q-glucose 6 - S u l p h a t e S u l p h a t e s

A d e f i c i e n c y of 2-acetamido-2-deoxy-g-glucose 6-sulphate r e q u i r e d f o r heparan s u l p h a t e degradation has been r e p o r t e d i n p a t i e n t s w i t h S a n f i l i p p o d i s e a s e t y p e D.519 Skin f i b r o b l a s t s from t w o p a t i e n t s who h a d s y m p t o m s o f t h e S a n f i l i p p o s y n d r o m e ( m u c o p o l y s a c c h a r i d o s i s 111) a c c u m u l a t e d e x c e s s i v e a m o u n t s o f h e p a r a n s u l p h a t e a n d were u n a b l e t o r e l e a s e s u l p h a t e f r o m 2 - a c e t a m i d o - 2 deoxy-g-glucose 6-sulphate l i n k a g e s i n heparan sulphate-derived oligosaccharides. Keratan sulphate-derived oligosaccharides bearing t h e same r e s i d u e a t t h e n o n - r e d u c i n g e n d a n d 4 - n i t r o p h e n y l 2 acetamido-2-deoxy-Q-glucopyranoside 6 - s u l p h a t e were d e g r a d e d normally. K i n e t i c d i f f e r e n c e s between the s u l p h a t a s e activities of T h e s e o b s e r v a t i o n s s u g g e s t t h a t 2n o r m a l f i b r o b l a s t s were f o u n d . acetamido-2-deoxy-a-glucose 6-sulphate a c t i v i t i e s degrading heparan s u l p h a t e and k e r a t a n s u l p h a t e , r e s p e c t i v e l y , can be d i s t i n g u i s h e d . It is the a c t i v i t y directed towards heparan s u l p h a t e t h a t is d e f i c i e n t i n these p a t i e n t s . The a u t h o r s propose t h a t t h i s d e f i c i e n c y c a u s e s S a n f i l i p p o d i s e a s e t y p e D.

52

Miscellaneous E n z y m e s

Dextransucrase. -- D e x t r a n s u c r a s e f r o m L e u c o n o s t o c m e s e n t e r o i d e s has been p r o d u c e d i n a s e m i c o n t i n u o u s c u l t u r e w i t h s l o w a d d i t i o n o f a concentrated sucrose solution.520 The r e s u l t i n g h i g h a c t i v i t y o f t h e f e r m e n t a t i o n b r o t h a l l o w e d a o n e - s t e p p u r i f i c a t i o n m e t h o d , by g e l - p e r m e a t i o n c h r o m a t o g r a p h y i n 96.4% y i e l d . This procedure r e s u l t e d i n 140-fold purification, with s p e c i f i c a c t i v i t y of 122 U mg-l. The e n z y m e was i m m o b i l i z e d o n t o a n a m i n o - S p h e r o s i l s u p p o r t activated with glutaraldehyde. Preparations with dextransucrase a c t i v i t i e s a s h i g h a s 4 0 . 5 U g - l o f s u p p o r t were o b t a i n e d w h e n l o w s p e c i f i c a r e a s u p p o r t s were u s e d a n d m a l t o s e was a d d e d d u r i n g t h e enzyme c o u p l i n g . D i f f u s i o n a l l i m i t a t i o n s were f o u n d d u r i n g e n z y m e r e a c t i o n , a s s h o w n by a k i n e t i c s t u d y . As a c o n s e q u e n c e o f immobilization, the average molecular weight of dextrans appeared t o increase. I m m o b i l i z e d d e x t r a n s u c r a s e c o u l d be p r o m i s i n g f o r l o w molecular-weight dextran production. C l i n i c a l d e x t r a n was s y n t h e s i z e d when t h e p o l y s a c c h a r i d e s p r o d u c e d i n t h e p r e s e n c e o f The m a l t o s e were u s e d a s a c c e p t o r s o f a s e c o n d s y n t h e s i s r e a c t i o n . m o l e c u l a r - w e i g h t d i s t r i b u t i o n of t h e r e s u l t i n g p r o d u c t i o n was less

559

6: Enzymes

d i s p e r s e d t h a n when c l i n i c a l d e x t r a n was p r o d u c e d by a c i d h y d r o l y s i s

-

-

o f h i g h mo l e c u l a r w e i g h t dex t r an. S t u d i e s have been c a r r i e d o u t on t h e a c c e p t o r s p e c i f i c i t y o f

d e x t r a n s u c r a s e

w h i c h

h a d

been

i s o l a t e d

f r o m

Streptococcus ~ a n g u i s . R ~ a~d ~ i o a c t i v e a c c e p t o r s were e m p l o y e d i n r e a c t i o n s w i t h c o l d s u c r o s e and t h e c o u n t s i n c o r p o r a t e d were t a k e n as a m e a s u r e o f a c c e p t o r a c t i v i t y .

An o r d e r o f r e l a t i v e a c t i v i t y

was f o u n d t o be p o l y s a c c h a r i d e > o l i g o s a c c h a r i d e > g l y c o s i d e > monosaccharide.

An e v a l u a t i o n o f t h e t i m e - c o u r s e o f t h e r e a c t i o n

w i t h methyl a-q-glucopyranoside,

or maltose,

homologous s e r i e s o f o l i g o s a c c h a r i d e s

was f o r m e d f r o m

showed t h a t each.

a

This

s u g g e s t e d t h a t t h e i n d i v i d u a l members o f t h e s e r i e s were r e l a t e d as precursors different

and

products.

The

kinetics

a c c e p t o r s were s t u d i e d .

of

the

reaction

with

A l l a c c e p t o r s s t u d i e d caused an

a c t i v a t i o n o f t h e enzyme and changes i n t h e

5,

f o r sucrose.

The

k i n e t i c c o n s t a n t s o b t a i n e d were a l s o used t o compare t h e v a r i o u s acceptors.

I t has been d e m o n s t r a t e d t h a t a - g - 1 - f l u o r o g l u c o s e

i s a glucosyl

donor f o r d e x t r a n s u c r a s e i s o l a t e d f r o m S t r e p t o c o c c u s ~ a n q = . ~ ~ * The d e t a i l s o f t h e r e a c t i o n have been e s t a b l i s h e d as w e l l as p r o v i n g t h a t t h e mechanism o f 1 - f l u o r o g l u c o s e t r a n s f e r i s c o m p a r a b l e t o t h a t of p - g l u c o s y l t r a n s f e r f r o m s u c r o s e . the reaction i s reported,

A new p r o c e d u r e f o r m o n i t o r i n g

and i s based on t h e measurement o f p r o t o n

f o r m a t i o n u s i n g t h e pH i n d i c a t o r 6 r o m c r e s o l P u r p l e . F-

Production o f

was f o u n d t o be s t o i c h i o m e t r i c w i t h p r o t o n p r o d u c t i o n .

studies

with

the

substrate

indicate

undergoes spontaneous h y d r o l y s i s , presence of

When { 1 4 C ) m a l t o s e a n d a - q - 1 -

f l u o r o g l u c o s e o r a-l-Q-{ 1 4 C ) f l u o r o g l u c o s e observed.

a series

Rate

a-g-1-fluoroglucose

w h i c h is g r e a t l y i n c r e a s e d i n t h e

nucleophilic buffers.

w i t h dextransucrase,

that

of

and m a l t o s e were i n c u b a t e d

oligosaccharide

products

was

The r e s u l t s i n d i c a t e t h a t t h e c - g l u c o s y l m o i e t y o f a-e-1-

f l u o r o g l u c o s e t r a n s f e r r e d t o t h e acceptor.

The n a t u r e o f f o r m a t i o n

of t h e products i s c o n s i s t e n t w i t h a s e r i e s o f precursor-product reactions.

P r o d u c t a n a l y s i s of

the

r e d u c t i o n a n a l y s i s demonstrated t h a t t o t h e non-reducing

end o f

maltose.

saccharides the g-glucosyl

When e i t h e r

by

borohydride

u n i t was added

1-{1 4 C ) f r u c t o s e or

a - Q - l - { 1 4 C ~ f l u o r o g l u c o s e was i n c u b a t e d w i t h enzyme, a r e a c t i o n was o b s e r v e d w h i c h was a n a l o g o u s t o t h e i s o t o p i c - e x c h a n g e r e a c t i o n c a t a l y s e d by t h e enzyme i n t h e p r e s e n c e o f

g-{l4C}fructose

and

sucrose. S t u d i e s on d o n o r - s u b s t r a t e

s p e c i f i c i t y have been p e r f o r m e d w i t h


560 dextrans~crase.~~P ' revious studies

-Proc.

S O C . Exp.

linked

B i o l . Med., 1 9 7 2 , f l u o r i n e atom a t C - 1 o f

activation to permit this dextransucrase.

s e r v e d as

competitive

A comparison o f

importance of

specific

several

for

a t the donorhas been

of

sucrose,

the

natural

p r o v i d e d i n f o r m a t i o n about t h e

changes i n t h e !-glucose

moiety

w i t h regard

S i m i l a r k i n e t i c s t u d i e s were c a r r i e d out and

the

corresponding free

These w e r e f o u n d t o be n o n - c o m p e t i t i v e i n h i b i t o r s ,

and t o b i n d p o o r l y . substrates

exception of

inhibitors t h e sis

l-fluoro-B-Q-sugars

monosaccharides. donor

substrate

a s e r i e s o f l - f l u o r o - a - Q -- s u g a r s

t o b i n d i n g t o t h e enzyme. with

a n a l o g u e t o be a d o n o r

I n k i n e t i c e x p e r i m e n t s , i t has been d e t e r m i n e d t h a t

synthesized. substrate.

glucose

Hehre,

h a v e s h o w n t h a t a n aprovided sufficient

I n order t o study the s p e c i f i c i t y

substrate-binding site, they

G e n g h o f f and E.J.

(D.S.

l40,1 2 9 8 )

The l - f l u o r o - a - ! - s u g a r s i n reactions

w e r e a l s o e x a m i n e d as

w i t h known

l-fluoro-a-!-glucose,

acceptors.

none o f

With the

t h e s e a n a l o g u e s was

active i n t h i s capacity. Several carbohydrates, designed, synthesized,and

i n c l u d i n g sucrose

analogues,

h a v e been

t e s t e d as d e x t r a n s u c r a s e i n h i b i t o r s . 5 2 4

C e r t a i n c a r b o h y d r a t e s c o n t a i n i n g an a m i n o g r o u p w e r e s hown t o be p o t e n t i n h i b i t o r s o f dextransucrase. M u l t i p l e f o r m s o f L e u c o n o s t o c m e s e n t e r o i d e s d e x t r a n s u c r a s e have b e e n s e p a r a t e d by g e l f i l t r a t i o n a n d e l e c t r o p h o r e t i c a n a l y s e s . 5 2 5 Two c o m p o n e n t s o f e n z y m e , h a v i n g d i f f e r e n t a f f i n i t i e s f o r d e x t r a n gel,

were

s e p a r a t e d by a c o l u m n o f

component

was

treated

with

Sephadex

dextranase

e l e c t r o p h o r e t i c a l l y homogeneous s t a t e . m o l e c u l a r w e i g h t o f 64,000-65,000, 17% o f c a r b o h y d r a t e .

The

G-100.

and

The

purified

major to

an

p u r i f i e d enzyme had a

PI v a l u e o f 4.1,

and c o n t a i n e d

H 4 - e d t a showed a c h a r a c t e r i s t i c i n h i b i t i o n on

t h e enzyme w h i l e s t i m u l a t i v e e f f e c t s w e r e o b s e r v e d by t h e a d d i t i o n o f exogenous d e x t r a n t o t h e i n c u b a t i o n m i x t u r e . was s t i m u l a t e d b y v a r i o u s d e x t r a n s a n d i t s

w i t h i n c r e a s i n g c o n c e n t r a t i o n of showed no a f f i n i t y

for

a Sephadex

dextran. G-100

The enzyme a c t i v i t y

Km v a l u e

was d e c r e a s e d

The p u r i f i e d enzyme

g e l and r e a d i l y a g g r e g a t e d

a f t e r p r e s e r v a t i o n a t 4OC i n a c o n c e n t r a t e d s o l u t i o n .

S y n t hases.

--

C o n c e n t r a t i o n s o f ADP-Q-glucose:a-1,4-Q-glucan-4-

g l u c o s y l t r ans f e r a s e ( s t a r c h s y n t h a s e

glucan-6-glycosyltransferase seeds

of

Pisum sativum

have

a n d a - 1 ,4-O_- - g l u c a n : a - l , 4 - Q --

( b r a n c h i n g enzyme) f r o m d e v e l o p i n g been

measured.526

Primed

starch

s y n t h a s e a c t i v i t y i n c r e a s e d f r o m 0 t o 1 4 d a y s a f t e r a n t h e s i s and

561

6: Enzymes d e c r e a s e d by 50% a t 26 days.

C i t r a t e - s t i m u l a t e d s t a r c h synthase

a c t i v i t y was h i g h e s t a t 10 d a y s a f t e r a n t h e s i s , d e c r e a s i n g t o l o w l e v e l s by 22 days. B r a n c h i n g - e n z y m e a c t i v i t y i n c r e a s e d f r o m 8 t o 18 d a y s a f t e r a n t h e s i s and d e c r e a s e d l i t t l e by 26 days.

Two f r a c t i o n s

o f s t a r c h s y n t h a s e w e r e r e c o v e r e d b y g r a d i e n t e l u t i o n f r o m DEAEcellulose of

extracts

fractions differed

from

12- and 1 8 - d a y - o l d

Em f o r

i n primer specificity,

r e l a t i v e amount o f c i t r a t e - s t i m u l a t e d a c t i v i t y .

seeds.

The

two

ADP-Q-glucose, a n d

A m a j o r and

a

minor

f r a c t i o n o f b r a n c h i n g enzyme w e r e o b s e r v e d i n e x t r a c t s f r o m b o t h 1 2 and 1 8 - d a y - o l d

seeds.

Marked d i f f e r e n c e s i n t h e r e l a t i v e a b i l i t i e s

of t h e two branching-enzyme f r a c t i o n s t o s t i m u l a t e phosphorylase and t o b r a n c h a m y l o s e as w e l l as i n pH o p t i m a w e r e f o u n d .

Although

t h e c o n t e n t o f t h e s t a r c h s y n t h a s e and branching-enzyme f r a c t i o n s v a r i e d w i t h s e e d age,

l i t t l e d i f f e r e n c e was s e e n i n t h e p r o p e r t i e s

o f chromatographically s i m i l a r fractions. s t a r c h synthase

and b r a n c h i n g - e n z y m e

T h e r e f o r e , t h e changes i n activity

d u r i n g pea

seed

development r e s u l t e d f r o m changes i n t h e c o n c e n t r a t i o n s o f a few enzyme f o r m s b u t n o t t h e a p p e a r a n c e of d i f f e r e n t enzyme f o r m s . The h o r m o n a l r e g u l a t i o n o f g l y c o g e n s y n t h a s e p h o s p h o r y l a t i o n i n p e r f u s e d r a t s k e l e t a l m u s c l e h a s b e e n i n v e ~ t i g a t e d . ~ ~U’ s i n g t h e r a t hind-limb p e r f u s i o n technique, k i n e t i c evidence t h a t glycogen synthase i s s u b s t a n t i a l l y

phosphorylated i n c o n t r o l s k e l e t a l muscle

a n d t h a t e p i n e p h r i n e c a u s e s f u r t h e r p h o s p h o r y l a t i o n was o b t a i n e d . The p h o s p h a t e c o n t e n t o f

t h e enzyme p u r i f i e d f r o m

control

and

h o r m o n e - t r e a t e d m u s c l e has been measured and a good c o r r e l a t i o n b e t w e e n t h e p h o s p h o r y l a t i o n s t a t e o f t h e enzyme and a c t i v i t y has been found. The a m i n o a c i d s e q u e n c e o f a r e g i o n i n r a b b i t s k e l e t a l m u s c l e g l y c o g e n s y n t h a s e p h o s p h o r y l a t e d by c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e h a s been r e p o r t e d . 5 2 8

Analogues of

t h e amino a c i d sequence

s u r r o u n d i n g s i t e l a d e m o n s t r a t e d t h a t s i t e s l a and l b a r e s e p a r a t e d by o n l y 1 3 a m i n o a c i d s i n t h e p r i m a r y s t r u c t u r e o f g l y c o g e n s y n t h a s e , w h i c h c o m p r i s e s 770 r e s i d u e s . The i n h i b i t o r y e f f e c t s

*

31-B-q-glucan

gated.529

o f p a p u l a c a n d i n B and a c u l e a c i n A o n (1

synthase from

G e o t r i c h u m l a c t i s h a v e been i n v e s t i -

The i n h i b i t i o n i s s p e c i f i c

and o c c u r s n o t o n l y i n v i t r o

but also i n vivo since the addition o f the antibiotics t o a culture leads t o c e l l - f r e e

e x t r a c t s with a p a r t i a l l y i n a c t i v e synthase.

Some c h a r a c t e r i s t i c s o f t h e i n h i b i t i o n a r e d e s c r i b e d . C h i t i n synthase i n t h e s t i p e of

--b i s e ---orus

has

been

isolated

and

the basidiomycete Agaricus i t s

properties

have

been


562 i n v e s t i g a t e d .530

--

Lyases. has

been

Heparan s u l p h a t e p u r i f i e d

by

l y a s e from

repeated

Flavobacterium heparinum

hydroxyapatite

column

c h r o m a t o g r a ~ h y . ~The ~ ~ enzyme was u s e d t o d e g r a d e h e p a r a n s u l p h a t e o c c u r r i n g on t h e s u r f a c e s o f

a s c i t e s hepatoma c e l l s ,

AH66,

and was

f o u n d t o be more e f f e c t i v e t h a n t r y p s i n i n r e m o v i n g h e p a r a n s u l p h a t e from

the

cells.

Furthermore,

on a n a l y s i n g

g l y c o s a m i n o g l y c a n s and

g l y c o p e p t i d e s from t h e enzyme-treated c e l l s and c o n t r o l c e l l s , i t was c o n c l u d e d t h a t h e p a r a n s u l p h a t e was e x c l u s i v e l y p r e s e n t on t h e c e l l s u r f a c e and a c c e s s i b l e t o t h e h e p a r a n s u l p h a t e l y a s e whereas o t h e r c e l l - s u r f ace c o m p l e x c a r b o h y d r a t e s r e m a i n e d i n t a c t . The c h a n g e s i n t h e c a t a l y t i c a c t i v i t y o f N - a c e t y l n e u r a m i n a t e l y a s e h a v e been s t u d i e d as a f u n c t i o n o f t e m p e r a t u r e i n t h e p r e s e n c e

o r absence of

i t s substrates.532

The

l y a s e was f o u n d t o

was s u g g e s t e d t h a t t h i s p r o t e c t i v e e f f e c t

be

and i t

e f f e c t i v e l y p r o t e c t e d a g a i n s t h e a t i n a c t i v a t i o n by p y r u v a t e ,

c a n b e u s e d i n a new

s i m p l e s t e p i n t h e p u r i f i c a t i o n o f t h e enzyme. A m u l t i f u n c t i o n a l enzyme, a c t i v e o n b o t h p o l y s a c c h a r i d e s and

glycosides, P.

has

placenta.533

A

been

isolated

from

molecular weight

the

wood-decay

o f 185,000

fungus

was d e t e r m i n e d w i t h

l o w e r - m o l e c u 1a r - w e i g h t s u b u n i t s c h a r a c t e r ized on SDS po 1y a c r y l a m ide g e l electrophoresis.

An u n u s u a l PI o f 1.8

was e s t a b l i s h e d f o r t h i s

protein.

--

Dehydrogenases.

The g e n e t i c and b i o c h e m i c a l c h a r a c t e r i z a t i o n o f

Q-arabinose

dehydrogenase

reported.534

The enzyme was p u r i f i e d t o h o m o g e n e i t y f r o m w i l d - t y p e

N.

c r a s s a 74-A

from

Neurozeora crassa

and f r o m t w o c o l o n i a l m u t a n t s ,

o f t h e enzyme.

has

been

f o u n d t o c o n t a i n more

The e n z y m e s w e r e c h a r a c t e r i z e d b y m e a s u r e m e n t o f

s e v e r a l k i n e t i c and p h y s i c o c h e m i c a l p a r a m e t e r s and w e r e t h e same i n a l l characteristics

studied.

Immunological studies performed w i t h

enzyme p r e p a r a t i o n s f r o m t h e t h r e e s t r a i n s showed a n t i g e n i c i d e n t i t y and

indicated that

enzyme,

those

colonial

strains

c o n t a i n more n o r m a l

r a t h e r t h a n t h e u s u a l amount o f an a l t e r e d ,

i m p r o v e d enzyme.

Q u a n t i t a t i o n o f t h e enzyme i n c r u d e e x t r a c t s , p e r f o r m e d by s i n g l e r a d i a l immunmodiffusion, the

l e v e l

o f

enzyme

characterization, heterokaryosis,

showed t h a t t h e c o l o n i a l s t r a i n s h a v e t w i c e as

the

performed

and r e v e r s i o n s ,

by

wild-type analysis

of

strain. meiotic

Genetic products,

indicated that the difference i n

p-

a r a b i n o s e d e h y d r o g e n a s e a c t i v i t y d e t e c t e d among t h e t h r e e s t r a i n s i s

563

6: Enzymes p r o b a b l y d e t e r m i n e d by one gene.

D - F r u c t o s e dehydrogenase o f G l u c o n o b a c t e r i n d u s t r i u s has been i s o l a t e d and c h a r a c t e r i z e d f o r t h e e n z y m a t i c m i c r o d e t e r m i n a t i o n o f Q-Fructose

dehydrogenase

p u r i f i e d f r o m t h e membrane f r a c t i o n o f

was

solubilized

g l y c e r o l - g r o w n G.

and

industrius

by s o l u b i l i z a t i o n o f t h e e n z y m e w i t h T r i t o n X - 1 0 0 a n d s u b s e q u e n t f r a c t i o n a t i o n by i o n - e x c h a n g e c h r o m a t o g r a p h i e s . was

tightly

bound

to

a c-type


cytochrome

e x i s t i n g as a d e h y d r o g e n a s e - c y t o c h r o m e

and a n o t h e r

complex.

peptide


was deemed p u r e by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n as w e l l as by g e l f i l t r a t i o n on a Sephadex G-200 column. 140,000)

on

sodium

electrophoresis

The enzyme complex ( m o l .

sulphate

showed t h e p r e s e n c e o f

molecular weights and 19,700

dodecyl

of

67,000

50,800

Only p - f r u c t o s e

dichlorophenolindophenol, or phazine methosulphate. dinucleotide,

n i c o t i n a m i d e adenine

and oxygen d i d n o t f u n c t i o n of

!-fructose

fructose)

was s t a b l e a t pH 4.5

The o p t i m u m pH

The e n z y m e ( K m lO’*rnM

t o 6.0.

2,6-

Nicotinamide

d i n u c l e o t i d e phosphate,

as e l e c t r o n a c c e p t o r s .

o x i d a t i o n was 4.0.

c),

(cytochrome

was r e a d i l y o x i d i z e d

b y t h e enzyme i n t h e p r e s e n c e o f d y e s s u c h as f e r r i c y a n i d e , adenine

gel

t h r e e components h a v i n g

(dehydrogenase),

(unknown f u n c t i o n ) .

wt.

polyacrylamide

for

p-

S t a b i l i t y of the p u r i f i e d

enzyme was much enhanced by t h e p r e s e n c e o f d e t e r g e n t i n t h e enzyme Removal o f d e t e r g e n t f r o m t h e enzyme s o l u t i o n f a c i l i t a t e d

solution.

t h e a g g r e g a t i o n o f t h e enzyme and caused i t s i n a c t i v a t i o n .

Levansucrase.

--

Levansucrase,

t h e p o l y s a c c h a r i d e l e v a n from

which

catalyses

the g-fructosyl

the

synthesis

of

r e s i d u e s o f sucrose,

was i s o l a t e d f r o m t h e c u l t u r e f l u i d o f G l u c o n o b a c t e r o x y d E a n d p u r i f i e d t o a homogeneous s t a t e by c h r o m a t o g r a p h y on h y d r o x y a p a t i t e and g e l f i l t r a t i o n . 5 3 6

According t o t h e data of electrophoresis,

t h e m o l e c u l a r weight of

l e v a n s u c r a s e i s e q u a l t o 58,000.

m o l e c u l e c o n t a i n s 25.86% a c i d i c a m i n o a c i d s , acids, per

and 12.77% a r o m a t i c amino a c i d s ,

mole.

The u l t r a v i o l e t

The enzyme

13.74% b a s i c a m i n o

and one m o l e o f m e t h i o n i n e

absorption spectrum

of

purified

l e v a n s u c r a s e h a s a maximum a t 280 nm a n d a m i n i m u m a t 254 nm.

By

measuring

by

the

l e v a n s u c r a s e of

i n i t i a l G.

rates

of

the

reactions

catalysed

oxydans i n t h e p r e s e n c e o f { 1 4 C } s u c r o s e ,

i t was

e s t a b l i s h e d t h a t t h i s enzyme c a t a l y s e s a r e a c t i o n o f h y d r o l y s i s o f sucrose,

a

reaction

of

synthesis

of

levan,

and

a reaction

of

exchange o f t h e l a b e l l e d Q - g l u c o s e h a l f o f s u c r o s e w i t h f r e e Qglucose. The i n i t i a l r a t e s o f l i b e r a t i o n o f E - g l u c o s e a n d p -

5 64


f r u c t o s e d u r i n g t h e t r a n s f r u c t o s y l a t i o n r e a c t i o n were d e t e r m i n e d . levan t o the r e a c t i o n m i x t u r e a s a p r e c u r s o r of p o l y s a c c h a r i d e s y n t h e s i s l e a d s t o a d o u b l i n g of t h e i n i t i a l r a t e of l i b e r a t i o n of l a b e l l e d p_-fructose and an i n c r e a s e i n t h e y i e l d o f l e v a n from 41 t o 69%. I t was shown t h a t t h e a d d i t i o n of low-molecular-weight

P e c t i n e s t e r a s e s . -- T e c h n i c a l p e c t i c m u l t i e n z y m e p r e p a r a t i o n s , P e c t i n e x U l t r a and Rohament P , were chromatographed on an a n a l y t i c a l s c a l e u s i n g m e d i u m - p r e s s u r e l i q u i d c h r o m a t o g r a p h y on a g l y c o l m e t h a c r y l a t e r i g i d m a c r o r e t i c u l a r g e l , S p h e r o n 1 0 0 0 , and i t s i o n e x c h a n g e d e r i v a t i v e s . 4 4 7 A c o m b i n a t i o n o f i s o c r a t i c and l i n e a r g r a d i e n t e l u t i o n ( w i t h g r a d i e n t s i n i o n i c s t r e n g t h o r p H ) was employed and f r a c t i o n s were monitored by measurements o f absorbance Conditions (4285 and A2541, c o n d u c t i v i t y , pH, and enzyme a c t i v i t y . f o r r a p i d s e p a r a t i o n s of p e c t i c enzymes a r e e l a b o r a t e d . The r e s u l t s i n d i c a t e t h e p o s s i b i l i t i e s of s e p a r a t i n g t h e t e c h n i c a l l y u n d e s i r a b l e p e c t i n e s t e r a s e a c t i v i t y from t h e o t h e r enzyme a c t i v i t i e s , and o f a more d e t a i l e d b i o c h e m i c a l i n v e s t i g a t i o n of t h e s e enzymes, i m p o r t a n t f o r t h e food i n d u s t r y . The aerobic b a c t e r i a associated w i t h s o f t r o t i n onions ( A l l i u m c e p a ) w e r e i s o l a t e d and i d e n t i f i e d a s a V i b r i o s p e c i e s , M i c r o c o c c u s e p i d e r m i d i s , Eudomonas c e p a c i a , an A c i n e t o b a c t e r s p e c i e s , Xanthomonas s p e c i e s , B a c i l l u s polyrnyxx, and B a c i l l u s m e g a t e r i ~ m . ~W~i t~h t h e c u p - p l a t e assay method, no p e c t i n h y d r o l a s e could b e d e t e c t e d from any of t h e s e i s o l a t e s when they were c u l t u r e d i n p e c t i n medium, b u t l y a s e and p e c t i n e s t e r a s e s w e r e d e t e c t a b l e . Onion t i s s u e c u l t u r e s showed p e c t i n h y d r o l a s e a c t i v i t y f o r P . c e p a c i a and B. polymyxa and l y a s e and p e c t i n e s t e r a s e a c t i v i t i e s f o r a l l of t h e i s o l a t e s , u s u a l l y a t h i g h e r l e v e l s o f a c t i v i t y than I n both c u l t u r e t h o s e of t h e p e c t i n medium c u l t u r e f i l t r a t e s . m e d i a , V i b r i o s p e c i e s showed t h e h i g h e s t l y a s e and p e c t i n e s t e r a s e activities. I n t h e v i s c o m e t r i c t e s t , a l l of t h e i s o l a t e s achieved a t l e a s t a 50% d e c r e a s e i n v i s c o s i t y f o r l y a s e e n z y m e , w i t h M . e p i d e r m i d i s and V i b r i o s p e c i e s r e c o r d i n g v i s c o s i t y d e c r e a s e s a s h i g h a s 8 3 % . The a b i l i t y t o c a u s e s o f t r o t i n o n i o n b u l b s was d e m o n s t r a t e d b y P. c e p a c i a and Xanthomonas s p e c i e s . Benzoic a c i d a t a c o n c e n t r a t i o n o f 0.8 mg m 1 - l c a u s e d t o t a l s u p p r e s s i o n of enzyme p r o d u c t i o n whereas sodium b e n z o a t e a t t h i s c o n c e n t r a t i o n reduced p e c t i n e s t e r a s e p r o d u c t i o n b y 71% and l y a s e p r o d u c t i o n by 72%. The p o s s i b l e use of t h e s e p r e s e r v a t i v e s i n t h e c o n t r o l o f s o f t r o t i n o n i o n s i s noted.

6: Enzymes

565

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1,

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575

6: Enzymes 430 431 432 433 434 435 436 437 438 4 39 440 441 442 443 444 44 5 446 44 7 44 8 449 450 4 51 452 453 454 455 4 56 457 458 459 460 461 462 463 464 465 466 467 468 469 4 70 471 472 473 474 4 75

J. Biochen. ("b 0 1 , 1981, 89, 963. J.Perraudin, Y.?mze, J . V i G e n t e l l i , and J.Leonis, Comp. Biochem. Physiol., P t B, 1980, 65, 127. K.Bel1, H.A.McKensie, V.Muller, and D.C.Shaw, M o l . C e l l . Biochem., 1980, 29, 3. V. Sidhan and S. Gurnani, Agric. B i o l . Chgn., 1981, 45, 1817. B.W. Matthews, S.J. Remington, M.G. G r i i t t e r and W.F. Anderson, J. M o l . B i o l . , 1981, 147, 545. m u s h i m a , Y. Murata, N. Nishikido, G. Sugihara and M.Tanaka, Bull. Chem. Sec. Jpn., 1981, 54, 3122. P.J. S t e i n and K. C r a i g Heehn, Biochem. Biophys. R e s . C o m E . , 1980, 95, 547. T. Imoto, T. Om and H. Yamada, J. Biochen. ("bkyo),1981, 90, 335. A. Shrake and J.A. Rupley, Biochemistry, 1980, 19, 4044. Y. Yang and K. Hamaguchi, J. Biochen. (Tokyo), 1980, 88, 829. Y. Yaw, S. Kurarrtitsu and K. Hamguchi, J. Biochem. (Tokyo), 1981, 89, 1357. A. Masaki, T. Fdcamizo, A. Otakara, T. T o r i k a t a , K. Hayashi and T. Imoto, J. Biochgn. ("bkyo), 1981, 90, 527. E.P. Savel'ev, G.I. P e t r o v , Z.F. Shmakova and S.A. Bitko, Biochemistry (Engl. Transl.), 1980, 5, 247. B. Szewczyk and T. Skorko, Biochim. Biophys. A c t a , 1981, 662, 131. G. Kleppe, E. Vasstrard and H.B. Jensen, Eur. J. Biochm., 1981, 119, 589. H. Sjijstrom, 0. Nor&, L. C h r i s t i a n s e n , H. Wacker and G. Semenza, J. B i o l . Chen., 1980, 255, 11332. M. Sat0 and A. Kaji, A ric. B i o l . Chem., 1980, 44, 1345. J. Gen. Microbiol. , 1980, 120, 295. G.S. Coleman, D.C., - aS D. Mikeg, J. S e d l s k o v a , L. Rexov&Benko-v& J. Chromatoqr. , 1981, 207, 99. Y. Itoh, K. Izaki and H. Takahashi, Agric. B i o l . Chem., 1980, 44, 1135. S.K.C. Obi and G.M. Umezurike, 1. Ehvir. Microbiol., 1981, 42, 585-589. Rex~vd-Benk~vdand M. .M -okd ~ Res. I 1981, 98, 115. G.A. Tucker, N.G. Robertson and D. G r i e r s o n , Eur. J. Biochem., 1980, 119. S.C. Lee and C.A. W e s t , Plant Physiol., 1981, 67,640. S.C. Lee and C.A. W e s t , Plant Physiol., 1981, 67, 633. P. Magro, P. Dilenna, P. Marciano and C. P a l l a v i c i n i , J. Gen. Microbiol., 1980, 120, 105. M. Sat0 and A. Kaji, Agric. B i o l . Chem., 1980, 44, 717. J. Yamada, Agric. B i o l . Chen., 1981, 45, 747. J. Y m d a , Carbohydr. Res., 1981, 90, 153. J. Yamada, Agric. B i o l . Chen., 1981, 45, 1269. A. H e r s m v i c s , A. Quaroni, B. Bugge and K. Kirsch, Biochem. J., 1981, 197,

E.

112,

5ll.

E. Bar-Guilloux, D. Robic and J.-E. Courtois, Biochimie, 1980, 62, 719. S. Rajendran and M. Muthu, Experi e n t i a , 1981, 37, 886. J. C o m t a t and J.P. J o s e l e a u , Carboh d r . Res., 1981, 95, 101. P. B i e l y , M. Vrganskdand 2. KrdtkqIyEur. J. Biochem., 1980, 112, 375. P. Biely, M. V&anovd and 2. K r d w , Eur. J. Biochem., 1981, 119,565. and M. V&anskd, Eur. J. Biochen., 1981, 559. P. Biely, 2. -tJ@ P.A.D. R i c k a r d and T.A. Laughlin, Biotechnol. Lett., 1980, 2, 363. D.B. Wankhede, K.R. Vijayalakshmi and M.R.R. R a o , Carbohydr. Res., 1981, 96, 249. H. Yoshioka, S. Chavanich, N. Nilubol and S. Hayashida, Agric. B i o l . Chem., 1981, 45, 579. D. m r a d , Biotechnol. Lett., 1981, 2, 345. P.A.D. Rickard and S.P. P e i r i s , Biotechnol. Lett., 1981, 3, 39. P.A.D. Rickard, M.I. Rajoka and J.A. Ide, Biotechnol. Lett., 1981, 2, 487. Z. K r d w and P. Biely, Eur. J. Biochem., 1980, 112, 367. K. Nakanishi and T. Yasui, Agric. B i o l . men., 1980, 44, 1885. 2729. K. Nakanishi and T. Yasui, Aqric. B i o l . Chem., 1980, A.V. Ananichev, I.V. Ulezlo, A.M. Egorov, A.M. Bezborodov, E.V. Berezin,

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44,


576 476 477 478

Biochemistry, 1980, 45, 753. M.A. Van K e u l e n , K. V e l l e n g a a n d G.E.M. JOOSten, B i o t e c h n o l . Bioenq., 1 9 8 1 , 23, 1437. C.-S. Gong, L-F. Chen, M.C. F l i c k i n g e r , L.-C. C h i a n g a n d G.T. T s ~ o , A p p l . Envir. Microbiol., 1981, 41, 430. T. Kume, H. Watanabe, S. Aoki a n d T. S a t o , Agric. B i o l . Chem., 1 9 8 1 , 45,

1311.

T. Kume, H. Watanabe, M. T a k e h i s a a n d T. S a t o , Agric. B i o l . Chem., 1981, 45, 1351. 480 T. Kasumi, K. H a y a s h i , N. Tsumura a n d T. T a k a g i , Agric. B i o l . Chem., 1981, 45, 1097. 481 T. Kasumi, K. H a y a s h i , N. TsUmUra a n d T. T a k a g i , Agric. B i o l . Chem., 1981, 45, 1087. 482 T. Kasumi, K. Hayashi and N. T m u r a , Agric. B i o l . Qlem., 1981, 45, 619. 483 B. Larsen and H. Grasdalen, Carboh dr. Res., 1981, 92, 163. 484 K. I z u m o r i , S. S u g i m o t o a n d B. k A g r i c . B i o l . Chem., 1980, %(1), 223. 48 5 0. Valentovd, M. Marek, F. g v e c , J. gtamberg a n d 2. V c d r & k a , B i o t e c h n o l . Bioenq., 1981, 23, 2093. 486 W. Hartmeier, S t a r c h , 1981, 33, 97. 487 S. P i l l a i , Biochan. J., 1981, 193, 825. 1981, 23, 1037. 488 E. Sada, S. Katoh and M. T e r a s h h , Biotechnol. Bioen 489 Ya.A. A l e k s a n d r o v s k i i , L.V. B e z h i k i n a a n d Yu.V. Rozionov, B i o c h e m i s t r y , 1981, 46, 593. 49 0 V. L i n r k , P. Benei?, F. Hovorka a n d 0. H o l e z e k , B i o t e c h n o l . Bioenq., 1981, 23, 1467. 49 1 V. L i n e k , P. Beneg, J. S i n k u l e , 0. H o l e z e k and V. Malq, B i o t e c h n o l . Bioenq., 1980, 22, 2515. 49 2 B. Solomon, N. L o t a n a n d E. K a t c h a l s k i - K a t z i r , J. Chromatogr., 1 9 8 1 , 215, 121. 49 3 S. Hayashi and S. Nakamura, Biochim. Biophys. A&, 1981, 657, 40. 494 E. N a p c h i , M. Nishikimi and K. Yagi, J. Biochen. (Tbkyo), 1981, 90, 33. 49 5 R. J e n n e s s , E.C. B i r n e y a n d K.L. Ayaz, Comp. Biochem. P h y s i o l . , 1980, 67, 479

c

.,

49 6 497 49 8 499 500 501 50 2 503 504 505 506 50 7 508 5 09 510

511 512 513 514 515

195.

R.F.H.Dekker, J. e n . Microbiol., 1980, 120, 309. L. Tom and D. Gaudioso, Can. J. Biochan., 1980, 58, 667. J.E. Sadler, T.A. Beyer and R.L. H i l l , J. Chrmtogr., 1981, 215, 181. E.H. Beachey, W. Keck, M.A. D e P e d r o a n d U. S c h w a r z , Eur. J. Biochem., 1981, 116, 355. T. Koga and M. I m u e , C a r b h y d r . Res., 1981, 93, 125. K. Fukushima, R. Motoda, K. Takada a n d T. I k e d a , FEBS L e t t . , 1981, 128, 213. HH. Carnicero, A.M. Adaamany and S. Ehglard, Arch. Biochem. Biophys., 1981, 210, 678. E.T. O'Keeffe, R.L. H i l l and J.E. Bell, Biochemistry, 1980, 19, 4954. Y. F'ujita-Yamaguchi and A. Yoshida, J. Biol. Chan., 1981, 256, 2701. E.T. O'Keeffe, T. Mordick and J.E. Bell, Biochemistry, 1980, 19, 4962. P. B r q u e t and P. Louisot, Biochimie, 1981, 63, 803. A, M a r t i n , M.C. B i o l , E. A r r a m b i d e , M. R i c h a r d a n d P. L o u i s o t , B i o c h i m i e , 1981, 63, 241. C. C-Mulet, J. Badet, J.P. Cartron, FEBS. L e t t . , 1981, 126, 123. D.H. van d e n E i j n d e n , M.L.E. Bergh, B. D i e l e m a n a n d W.E.C.M. S c h i p h o r s t , Hoppe-Seyler's 2, Physiol. Chan., 1981, 362, 113. G. Dodin, FEBS. L e t t . , 1981, 134, 20. I.M. A r c h e r , P.S. Harper a n d F.S. Wusteman, C l i n . Chim. Acta, 1981, 107. I.G. Leder, Biochan. Biophys. Res. (3m'mun., 1980, 94, 1183. P. D i Natale and L. R o n s i s v a l l e , Biochim. Biophys. A c t a , 1981, 661, 106. T. I s h i b a s h i , A. M a r u , Y. I m a i , A. M a k i t a a n d I. T s u j i , Biochim. Biophys. A c t a , 1980, 616, 218. A. Waheed and R.L. van E t t e n , Biochim. Biophys. A&, 1980, 2,92.

112,

6: Enzymes

577

516 J.R. George and J.W. F i t z g e r a l d , J. B a c t e r i o l . , 1981, 145, 1428. 517 J . R . George and J.W. F i t z g e r a l d , J. Bacteriol., 1981, 147,69. 518 J.W. F i t z g e r a l d , RB. Kellogg and G.J. S t e w a r t , FEMS Microbiol. Lett., 1981, 11, 93. F i g u r a , W. G i l b e r g a n d W. F u c h s , 519 H. Kresse, E. P a s c h k e , K.V. Proc. Natl. Acad. S c i . USA - B i o l . Sci., 1980, 77, 6822. 520 P. Monsan and A. Lopez, Biotechnol. Bioenq., 1981, 23, 2027. 5 21 M.M. M a t t h e w s , C.L. F u t e r m a n , V.K. P a r n a i k a n d S.M. J u n g , Arch. Biochem. Biophys., 1981, 208, 278. 522 S.M. Jung and R.M. Mzyer, Arch. Biochem. Biophys., 1981, 208, 288. 523 T.J. Greir and R.M. Mayer, Arch. Biochen. Biophys., 1981, 212, 651. Doyle, 524 S. T h a n i y a v a r a n , S. S i n g h , C.M. Maynard, K.G. T a y l o r a n d R.J. Carbohydr. R e s . , 1981, 96, 134. s. A c t a , 1980, 614, 46. 52 5 M. Kobayashi and K. Matsuda, Biochim. B i o 526 G.L. Matters and C.D. & y e , P h y t o c h a n i s g 1980, 20, 1805: 52 7 J.-L. C h i a s s o n , J.H. Aylward, H. Shikama a n d J.H. E x t o n , FEBS. L e t t . , 1 9 8 1 , 127, 97. 528 P.J. P a r k e r , A. A i t k e n , T. Bilham, N. Embi a n d P. Cchen., FEBS. L e t t . , 1981, 123, 332. 529 P. P a e z , R. VXOM, I. G a r c i a - M a , A. W a n , FEBS. L e t t , 1981, 129, 249. 530 G.D. Craig, D.A. W o o d and K. G u l l , FEMS Microbiol. Lett., 1981, 10, 43. 531 Y. Ohkubo, I. F u n a k o s h i and I. Yamashina, J. Biochem. (Tokyo), 1981, 89, 161. 532 F.N. Kolisis, T.G. S o t i r o u d i s a n d R E . E v a n g e l o p o u l o s , FEBS. L e t t . , 1980, 121. 533 K.E. W o l ter , T.L. H i g h l y a n d F.J. Evans, Biochem. Biophys. R e s . Commun., 1980, 97, 1499. 534 A. Carrasw, G. P h c h e i r a and T. Ureta, J. B a c t e r i o l . , 1981, 145, 164. 535 M. Ameyama, E. S h i n a g a w a , K. M a t s u s h i t a a n d 0. A d a c h i , J. Bacteriol., 1981, 145, 814. 536 fi.E l i s a s h v i l i , Biochemistry (Engl. Transl. 1, 1980, 45, 14.

7

Glycolipids and Gangliosides BY I. M. MORRISON 1 Introduction

Various aspects of glycolipids are reviewedin the second of a twovolume book on cell membranes and viral envelopes.’ The structure and function of plant lipids occupy Volume 4 of the comprehensive treatise on the biochemistry of plants.2 A review on the role of lipids in biological membranes has appeared .3 2 Analytical and General Methods 1.r.

photoacoustic spectroscopy has been used to study skin Using CHC13-MeOH (2: 1 ) extracts, the results were similar to those obtained by i.r. transmission spectroscopy,but only l o - * times the amount of material was required. The phenol-sulphuric acid method for the estimation of sugars in lipids has been modified by keeping the products at 100°C for 5 More reproducible results were obtained. A dry-column method for the quantitative extraction and simultaneous separation of lipid classes has been developed.6 The tissue sample, anhydrous sodium sulphate,and diatomaceous earth are ground together and packed in a column. Total lipid or lipid classes are then eluted by isocratic or sequential elution, respectively. A two-stage, one-dimensional t.1.c. method has been developed for the separation of lipid classes.’ Lecithin was found to influence the chromatography of steroid glucosiduronates .8 It was concluded that hydrogen bonds were formed between the phosphodiester group of lecithin and the hydroxy groups of the steroid conjugates. D-Galactose oxidase is able to oxidize the hydroxymethyl group of lipids containing 9-galactose or 2-acetamido-2-deoxy-Dg a l a c t o ~ e .These ~ sugar residues can then be labelled by reduction with radiolabelled borohydride. Conditions are described for the most efficient production of product. Methylation techniques for the structural analysis of

lipid^.^

7: Glycolipids and Gangliosides

579

glycolipids have been reviewed. It has been shown that particles, previously observed in freeze-fracture replicas of mixed phospholipid dispersions, occur in dispersions of mixed ~ - 3 - ~ - g a l a c t o p y r a n o s y l d i a c y l - g l y c e r o l s l. 1 These particles correspond to lipid micelles sandwiched within a membrane bilayer.

3 Gangliosides The gangliosides GM1 and GD have been specifically oxidized at la the +position of the sphingosine moiety with 2,3-dichloroo f Triton X-1 00.l 2 5,6-dicyanobenzoquinone in the presence Subsequent reduction with borohydride restored the ganglioside to its original form, and if a radiolabel was used 99% of the radioactivity was incorporated. Proof of the reaction was obtained by reoxidation when the label was removed. The interaction of gangliosides with Ca2+ ions and some polar head-group requirements for the establishment of particular interactions with phosphatidylcholine have been studied in monolayers at the air-0.145M NaCl interface. l 3 The gangliosideCa2+ interaction depended on the position occupied by the neuraminosyl residues in the oligosaccharide chain. Favoured interactions o f polyneuraminosylgangliosides with phosphatidylcholine may result from a configuration which allows a partial matching of two oppositely orientated electrical vectors contributed by the zwitterionic phosphocholine group and particular neuraminosyl groups. The gangliosides G D ~and GT were biosynthesized as secondary la products from lactosylceramide and GM1,respectively.l 4 By a series of reactions involving periodate oxidation, borohydride reduction, and hydrolysis, the linkage between the two neuraminic acid residues was identified as (2+8). 'H n.m.r. spectroscopy has demonstrated that N-acetyl-aacid is the first product released by the D-neuraminic neuraminidases of both Clostridium perfringens and Arthrobacter ureafaciens when hydrolysing neuraminic acid-containing carbohydrate chains. l 5 Spectrofluorimetric techniques have been used to study various The studies ganglioside and ganglioside-lecithin dispersions. l 6 indicate that the ultimate and/or penultimate carbohydrate moieties of the neutral sugar backbone of gangliosides and the

580


topographical difference in the locations of the neuraminic acid linkage influence the integrity of the membranes including the hydrophobic region. The micellar properties of gangliosides in aqueous solutions have been investigated by quasi-elastic light - scattering The scattered intensity data allowed the measurements. l 7 derivation of an upper limit to the critical micelle concentration and the evaluation of the molecular weight of the micelle from GMl and GDla. Aqueous dispersions of G M 1 and Triton X-100, in different proportions, have been analysed for some physicochemical properties and their susceptibility to ;-galactose oxidase. l8 Differences in the molar ratio gave well defined transitions between different micellar species as measured by the physicochemical methods, and these transitions were also evident in the kinetics of the !-galactose oxidase. The properties of these micelles were further studied using light-scattering methods. The ;-galactose oxidase acted very poorly on homogeneous G *1 micelles,but at fixed G M , concentrations the rate was increased by increasing Triton X-100 concentrations. The formation of statistical micelle aggregates was followed by inhibition of the ?-galactose oxidase activity. H and 13C n.m.r. spectroscopy, laser-light scattering,and differential scanning microcalorimetry have been used to study the influence of GH, and G D l a on the structural and thermotropic properties of sonicated small 1,2-dipalmitoyl-a-~-phosphatidylcholine vesicles.” Since the average dimensions of all vesicles were the same, differences in the temperature dependence of heat capacity in the presence of the gangliosides were attributed to differences in interaction of the hydrophobic parts of the molecules. The higher content of C20 sphingosines in G D l a as compared to GM was considered to be one possible source of 1 difference. Trineuraminosylgangliosides are spontaneously incorporated into 1,2-dipalmitoyl-a-~-phosphatidylcholine vesicles and, since the ganglioside, on incorporation, is susceptible to neuraminidase action, it is concluded that incorporation occurs only on the outer face of the bilayer.*l Calorimetric studies have indicated that the ganglioside stabilizes the vesicular structures by inhibiting the fusion of small vesicles that occurs below the phase-transition temperature. Cholesterol enhanced the binding of thyrotropin to 1,2-dipalmitoyl-a-~,~-phosphatidylcholine liposomes containing G, 1


581

ganglioside but not those containing the gangliosides G, and 1 The role of gangliosides in the binding and action of GDla' thyrotropin has been re-evaluated by comparing thyrotropin binding with cholera-toxin binding in a cloned line of normal rat thyroid cells and neuraminidase-treated cells . 2 3 The results indicate that more complex gangliosides do not serve as a component of the thyrotropin receptors nor are they involved in the transmission of the hormone signal across the cell membrane of these cells. Gangliosides that bind cholera toxin have been detected by the direct binding of 1251-labelled toxin on t.l.c.24 The method is and should be applicable to other sensitive to 70 fmol G M ~ carbohydrate-binding materials. The preparation of a receptorspecific affinity chromatographic matrix for the large-scale purification of cholera toxin has been described.25 The ganglioside G M ~is hydrolysed to lyso GM,, which is coupled, via stabilized Schiff's bases, to porous silica beads. The amount of toxin bound was proportional to the amount of lyso G M ~used. Ganglioside affinity filters have been used to identify toxigenic strains of Clostridium botulinum types C and D but could be used on a general procedure for identifying botulinal toxin and toxigenic Antibodies to the gangliosides G D ~ and G M ~ have been prepared and their properties determined.27 The antibodies to G, were 3 highly specific while those to G M 1 reacted with many other A periodate-oxidized gangliosides and their derivatives. itself. derivative of GM, cross-reacted almost as readily as G, 1 The specificity of limulin and wheat-germ agglutinin towards neuraminic acids has been studied using lipid vesicles containing GM3. 2 8 Limulin binds specifically to E-glycoloyl derivatives of G but the hydroxy group at C-4 and the carboxy group must also 3, be free. The side chain is not involved in binding. Wheat-germ agglutinin only binds when the hydrophilic tail is removed. The acetamido group but not the carboxy group is involved. The ganglioside GM,, incorporated in a planar lipid bilayer, interacts specifically with Ricinus toxin.29 A fourteen-fold increase in conductance was observed. An interspecies comparison of the brain ganglioside patterns from fish, amphibians, birds,and mammals has been studied by twodimensional t. 1 .c .30 Over 30 components were detected in mammalian neural samples. Differences were observed between the vertebrate classes,but greater similarity exists between the lower and higher

**

582


classes than had previously been noted. Various surfactants have been shown to be able to replace the requirements f o r the activator protein in the hydrolysis of G M ~ ganglioside by the B-~-2-acetamido-2-deoxy-hexosidase A from human liver.31 In the presence of saturating concentrations of the activator, the enzymatic hydrolysis of the substrate was further stimulated. The surfactants had no effect on the enzymatic 2-acetamido-2-deoxy-B-Dhydrolysis of 4-methylumbelliferyl glucose. A model for this reaction is discussed. A mononeuraminosylganglioside has been shown to be the antigen of a monoclonal antibody that is specific for cells from carcinoma of the human colon.32 The surface distribution of the ganglioside G, on human blood 1 cells and the effect of endogenous GM, and neuraminidase on cholera-toxin surface labelling have been demonstrated by an immunocytochemical study.33 Neutrophils were the most heavily labelled blood cells while lymphocytes, erythrocytes,and platelets were only labelled to a limited extent. The labelling was significantly altered on neuraminidase treatment. The lymphocytes of human peripheral mononuclear cells have been activated by oxidation with periodate .34 The neuraminosyl group was converted to a 7-carbon analogue, but the L-fucose and !-galactose residues were also oxidized. The response to periodate was reduced 5 50% by pretreatment with neuraminidase. The gangliosides from human peripheral blood lymphoctyes and neutrophils have been isolated and their structures determined by mass spectrometry and glycosidase hydrolysis results.35 The three were found in both types with ( 1 ) being the structures ( 1 ) - ( 3 ) major ganglioside in lymphocytes and ( 3 ) the major ganglioside in (2) may be a leukocyte-specific neutrophils. Structure glycosphingolipid. Large gangliosides with the general structure (4) were also isolated. The gangliosides in human leukocytes have been characterized and differences were observed between types of leukocyte .36 More ganglioside was present in normal leukemic cells than in granulocytes, while the complex gangliosides were more abundant in myelogenous cells than lymphocyte cells. Concanavalin A was shown to increase the incorporation of labelled 2-amino-2-deoxy-~-glucose into the gangliosides . 3 7 Thus, associated with cellular ganglioside synthesis is not only division but also occurs within a short time of lymphocyte activation.

583

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Anti-asialoganglioside sera has been shown to cross-react with human blood-group N antigen .38 An immunocytochemical study of the surface ganglioside GM in 1 haemic cell lines of normal human bone marrow has been carried out similarly to ref. 33.39 The extent of incorporation was related to specific cell types and to their stage of maturation. The ganglioside patterns of horse, donkey,and mule brains have been deter~nined.~' Forebrains contained the highest concentration with the brain stern of the mule having the lowest value. The N-glycoloylneuraminic acid content was (3% of the total neuraminic acid. G M l , GDla, and G D l b were the major gangliosides. The gangliosides from equine kidney and spleen have been characterized as haematoside, GM2, G M ~ ,and G D , ~4.1 G D ~was found in the kidney and G D l b in the spleen. The relative proportions of 1-acetyl and N-glycoloyl neuraminic acids were also determined. Liposomes, composed of gangliosides from bovine brain and dipalmitoyllecithin, have been studied by e.s.r. spectro~copy.~~ At temperatures above the transition temperature ( 4 7 ° C ) the gangliosides diffuse freely in the lipid matrix,but at 19'C, in the gel state, the gangliosides cluster together. Gangliosides from bovine brain have been modified by attaching biotin hydrazide to the aldehydo group after periodate oxidation.43 The modified gangliosides were incorporated into mature rat thymocytes,and the results suggested that gangliosides may be involved in transmembrane communication during lymphocyte stimulation. A sulphated ganglioside has been isolated from bovine gastric mucosa, and its structure (5) was elucidated by partial acid hydrolysis, specific glycosidase degradation, periodate oxidation, and methylation analyses.44 71% of the neuraminic acid was the N-acetyl derivative. A sulphated ganglioside from bovine gastric mucosa had the structure (6) confirmed by the usual procedure^.^^ 65% of its neuraminic acid was the 1-acetyl derivative. A non-specific lipid-transfer protein, purified from bovine liver, will stimulate the transfer of the ganglioside GM, and neutral glycosphingolipids from phosphatidylcholine vesicles to erythrocyte ghosts .46 E-Glycoloylhaematoside and a ganglioside, containing N-glycoloyl-neurarninic acid and having an oligosaccharide chain identical to that of erythrocyte neuraminosylparagloboside, have been isolated from both normal and leukemic bovine blood were identified in lymphocytes. 47 Three other gangliosides


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leukemic lymphocytes. The gangliosides have been isolated from bovine optic nerve and were found to be GM (1291, G D (~9 8 1~, G D l b ( 9 7 1 , G T ( 8~0 1 ~, G D ~ 1 (311, and Gq, (121, the concentrations being ug g-l wet tissue.48 Together they accounted for 97% of the total neuraminic acid present. The three L-fucogangliosides ( 7 ) - ( 9 ) have been isolated from porcine nervous tissue.49 A l l three were found in dorsal-root ganglia and spinal cord but only ( 7 ) and ( 9 ) in the forebrain. The chemical analyses were confirmed by mass-spectral analysis. The specificity of the neuraminosyltransferase from bovine liver microsomes has been determined.50 The transfer was solely to C-3 of E-galactose residues with no transfer to 2-acetamido-2deoxy-;-galactose residues. Eight gangliosides have been identified as constituents of ovine testis, with GMg, GD3, GMl,and GD , respectively, being the la most abundant.5 1 A new solvent system has been developed for t.1.c. of the gangliosides of rat ~ e r e b e l l u m . ~The ~ separation takes 1 h and virtually no tailing occurs. The considerable heterogeneity of this material was demonstrated by identifying 28 separate gangliosides. 2-Acetamino-2-deoxy-~-glucose has been shown to be incorporated 2-3 times higher into undernourished rats than normal rats.53 This enhanced incorporation was found in all brain areas. The turnover of the gangliosides was slower in young undernourished rats, relative to the controls, but greater in older rats. Microsomal gangliosides in rat brain have a higher initial incorporation of 2-acetamido-2-deoxy-~-mannose than synaptosomal g a n g l i ~ s i d e s . ~ ~ The gangliosides G T l b and G q l b were more highly labelled than other gangliosides,but a decrease in incorporation was observed on treatment with the convulsant pentylenetetrazol. The role of neuraminosyl compounds on choline uptake by synaptosomes has been studied.55 Neuraminidase treatment of synaptosomal fractions brought about a reduction in the uptake of choline. The activation of (Na+, K+) ATPase by the ganglioside G M ~has been shown to follow biphasic kinetics.56 The break is at 50nM G,l with a stable complex below that value and a loosely bound one above it. It was suggested that the activation was a specific phenomenon not related to the amphiphilic and ionic properties of the ganglioside but due to a modification of the membrane lipid

587


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n U h

I

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environment around the enzyme. The highest degree of labelling in homogenized neuronal perikarya from rat brain with f3Hl-5-acetylneuraminic acid has been found in subcellular fractions that also showed the highest specific activities for several ganglioside glycosyltransferases.57 The neuraminic acid-containing glycoconjugates remained associated with the membrane after all treatments tried except sodium deoxycholate extraction. Some of the results suggested that the gangliosides change their accessibility to added enzymes in the process of transport through the axon and incorporation into the synaptic membrane. An increased threshold for electrical stimulation to induce somatosensory-evoked potentials in peripheral nerves followed the transection and rejoining of the nerve in rats.58 The amount of this increase was diminished by treatment with gangliosides s o it was concluded that gangliosides enhance the rate of nervous regeneration. (UDP-2-acetamid0-2-deoxy-~-galactose-G~~-2GM2-Synthase acetamido-2-deoxy-D-galactosyltransferase) has been studied in Golgi-rich fraction from rat liver.59 Many requirements of this enzyme were demonstrated along with its physical properties. Methods for the chemical detection of gangliotetraosylceramide, the rat T lympohocyte macrophage-associated antigen, have been reported along with its cellular distribution.60 The glycolipids of murine lymphocyte subpopulations have been investigated. A neuraminosyl derivative of asialo GM,, which is sensitive to neuraminidase treatment, is increased in concentration in splenic lymphocytes.61 Asialo G M , is a normally occurring lipid and not an acid-hydrolysed product of G M ~ . The total glycolipids from thymocytes of leukemic A K R / J murine thymus have been shown to be greater than those of non-leukemic thymocytes.62 Of the individual gangliosides, GM3, GM2, GDla,and G D l b contents were higher while GM and G, were lower. Parallel 1 1 with these findings were the observations that two neuraminosyltransferases were also at an elevated level. During cells from mice into the differentiation of leukemia MI macrophages, the ganglioside content has been shown to change markedly.63 A several-fold increase in GM content (confirmed by lb neuraminidase treatment) was noted with a concomitant decrease in the dineuraminosylganglioside fraction. The glycolipid content of a murine cell line ( J L S - V 9 ) and its ouabain-resistant mutant clone ( J L S - V g OR) have been compared.64 The ganglioside, G M ~ ,content of


589

the mutant cells was 1.14 times that of the parent cells but, whereas 98% of the neuraminic acid in the parent cells was in the N-acetyl form, only 69% of that in the mutants was in the same form The ganglioside pattern of rabbit brain has been studied by twodimensional t .1 .c. and a ~ t o r a d i o g r a p h y . ~ ~Numerous minor components, representing previously uncharacterized gangliosides, were identified, several of which were novel L-fucose-containing gangliosides. The gangliosides of rabbit thymus have been analysed by a ganglioside mapping procedure.66 The G M gangliosides and the for 76.6 and 23.3%, combined G, and G , species accounted respectively, of the lipid-bound neuraminic acid. The gangliosides (10) and ( 1 1 ) accounted for 38.4 and 31.3%,respectively,of the total fraction while G D 3 (11.8%) and G M ~( 1 7 % ) accounted for the remainder. The GD3 and 62% of GM3 were in the N-glycoloyl form. The lacto-N-neotetraose backbone of ( 1 0 ) and ( 1 1 ) has been shown to be tissue specific for thymus with 70% of all thymus gangliosides possessing it.67 In addition the N-glycoloyl derivative of neuraminic acid represents 90% of that species. The mononeuraminosyl gangliosides of rabbit skeletal muscle have been analysed with G M representing 73% of the fraction.68 A 3 novel ganglioside, 5-acetylneuraminosyl lacto-N-noroctaosylceramide (12),constituted 5% of the fraction. Addition of gangliosides has induced a statistically significant increase in the number of neurites in the outgrowth zone of guinea-pig spinal ganglia.69 However, only a slight increase in neurite length was observed. Three novel gangliosides (13)-(15) have been isolated from frog fat body and their structures confirmed by the usual methods. 7 0 During phylogeny and early ontogeny of higher vertebrates the number of ganglioside fractions was reduced especially with regard to the more polar ~pecies.~'Arranging the different ganglioside fractions subsequent to their paths of formation, it has become apparent that in elasmobranch fish and embryonic chicken the third pathway is used whereas in the adult chicken there is a switch to the first and second pathway. The results are taken as further evidence of the recapitulation principle according to Haeckel's biogenetic rate. An unusual pattern of myelin gangliosides has been observed in avian central nervous system. 7 2 The total concentration was

.


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considerably higher than in other mammalian species,with GM3 being present in concentrations similar to G M ~ . A new t.1.c. system has been used to separate complex gangliosides from embryonic chicken brain. 73 Ten fractions were detected, eight of them also being present in the brain of rays. Six of these were similar to the gangliosides GT39 GT2’ GTlc9 G Q l c , GPlc,and GPlb identified in cod brain. Four of these have been partially characterized and they contain neuraminic acid: sphingosine ratios of 4: 1 , 5: 1 , 6:1, and 7: 1. 7 4 Mild neuraminidase treatments yield G T and ~ G ~ D transiently ~ ~ but G M ~finally. During brain development the amounts of the forms with ratios of 6 and 7 decreased. The brain gangliosides of the ice fish Trematomus hansoni have an extremely high content of neuraminic acid which confers a high degree of polarity on the neuronal membrane and maintains their temperature^.^^ At least four gangliosides functionality at low were detected which were more highly substituted than the pentaneuraminosyl ganglioside G, 1 Two novel gangliosides have been isolated and characterized from the sea urchin Strongylocentrotus intermedius.76 The first had the structure ( 1 6 ) and the second was similar except that it contained a sulphate ester group at C-8 of the terminal neuraminic acid residue. The neuraminidation of gangliosides has been studied during maturation of cultured neurons.77 During the first 4 h GM and GD 3 3 are produced, after 12 h GD and GD are produced,and finally la lb and G T l b are produced. GD3 does not appear to be incorporated G M ~ into the membranes formed during the period of synaptogenesis in the same way as GDla and polyneuraminosylgangliosides. The ganglioside contents of SV40-transformed Balb/c3T3 cells (SV3TC cells) and concanavalin A-selected SV3T3 revertant cells have been determined and compared with untransformed cells. 7 8 There was a decrease in the concentration of higher gangliosides in transformed and revertant cells, but the content of GM3 in revertant cells was higher than in transformed or original cells. GM3 may have a role in growth control.

.

4 Animal GlvcoliDids A new solvent system coupled with

been

developed

a porous silica-gel column has for separating glycolipids containing mono- to


593

tetrakaidecasaccharide chains in 60 min. 79 The separation and microdetection ( 1 0 pmol) of oligosaccharide chains of glycolipids have been achieved on high-performance cellulose t.1.c. and autoradiofluorography .80 Differential scanning calorimetry and X-ray diffraction of anhydrous and hydrated N-palmitoyl-D-galactosylsphingosine have provided evidence for a complex polymorphic behaviour and interconversions between stable and metastable structural forms.8 The anhydrous glycolipid exhibits three lamellar crystal forms below 143°C and a liquid-crystal form between 1 4 3 and 180°C. The 360 MHz ' H and 90.5 MHz 13C n.m.r. spectra of Q-galactosylceramides have been recorded in DMSO and show a 4 C l configuration of the sugar ring.82 In contrast to an aqueous solution, the hydroxymethylene group at C-6 can freely rotate A series of glycolipids has been around the C-6 : C-5 bond. analysed by 360 MHz I H n.m.r. s p e c t r o ~ c o p y .The ~ ~ resonance of all H-1 and H-2 as well as most H-3 and H-4 and some H-5 protons could be assigned with the aid of spin-decoupling difference spectroscopy. Regularities were observed in the type, anomeric configuration, site of glycoside linkage,and sugar residues which could be used for the structure of more complex glycolipids. Mass spectrometry has been used for the sequencing of the oligosaccharide chain of a dodecaglycosylceramide.84 This is the largest saccharide conclusively identified. Mass spectrometry, after t.l.c., has been used to identify the structures of glycolipid mixtures from a variety of species.85 The anomalous thermotropic phase-transition behaviour of 1 , 2 distearoyl-p-galactolipids has been investigated .86 The penetration of melittin and myelin basic protein into glycosphingolipid monolayers depends on the lipid polar head group, the protein concentration, and the initial surface pressure.87 The interaction leads to a modification of the surface properties of both the glycosphingolipid and the protein. Glycolipid synthetases have been measured in a two-phase scintillation assay carried out in a small The method should be suitable for many assays but the specific enzyme determined in this investigation was ceramide : UDP-E-glucose q-glucosyltransferase. has been derivatized for use as the D-Galactosylcerebroside ligand in affinity chromatography by ozonolysis of the sphingosine The ant i -gdouble bond followed by oxidation. 89

'


5 94

galactosylcerebroside antibodies purified on this materia.1 were highly specific. Proteins can be covalently bound to liposomes after periodate oxidation of the glycosphingolipids in the vesicle membrane. The method has potential for the antibody-mediated targeting of vesicles to cells. The synthetic glycolipids lactosyland melibiosylphosphatidylethanolamine can be incorporated into unilamellar l i p o ~ o m e s . ~ Both are agglutinated by Ricinus communis lectin but only the latter by Banderiaea simplicifolia isolectin I. Efficient fusion of phospholipid vesicles with monolayer cultures of eukaryotic cells has been accomplished by attaching glycolipid-containing vesicles to the cell surface by using a lectin displaying binding for both the cell surface and the glycol ipid Single bilayer vesicles of glycosphingolipids and phosphatidylcholine have been prepared and their physical Incubations with high-density properties determined.9 3 lipoprotein-3 resulted in the transfer o f the glycolipid, a process which did not occur in the absence of phosphatidylcholine. The complex has been further characterized,and only 69% of the lipid is transferred from the vesicles.94 The lipid compositions of axolemma-enriched fractions and myelin from human brains have been determined . 9 5 D-Galactolipid accounted for 25.8% of the lipid in the axolemma fractions, 83% of this being cerebrosides. The q-galactosylceramidase of human brain is activated by phosphatidylserine but the G M , ganglioside 8-P-galactosidase is not activated . 9 6 A review of sulpho-2-galactosylceramide has suggested that this glycolipid possesses multifunctional properties in biological membranes. The characteristic deposits of glycolipid in tissues from patients with Fabry’s disease have been studied by electron microscopy.9 8 Fourier transformation for the analysis of the distribution periodicity of the inclusions showed that, in synovial membrane and muscle, the inclusions had a monocrystalline character while in the other tissues they were amorphous or polycrystalline. The crystalline morphology of the trihexoside ceramide inclusions characteristic of Fabry’s disease have been Image processing further examined especially in synovial tissue

.

.”

confirmed

the

membrane-like

structure

of

trihexoside ceramide


595

inclusions and corroborated the proposed classification. (Gaucher's storage material) have been D-Glucosylcerebrosides shown to stimulate the release of lymphocyte activating factor from macrophages. l o o Other ceramides had no effect. The importance of these observations to Gaucher's disease was discussed. The complete structure of a trisaccharide ( 1 7 ) from a patient l3 C n.m.r. with mannosidosis has been established by spectroscopy.lol One of the features of a patient with mannosidosis has been excessive gingival hyperplasia with storage oligosaccharides . ' 0 2 The trisaccharide ( 1 7 ) was of D-mannose-linked the predominant material in the urine, but tetraand pentasaccharides were more abundant in gingiva. The neutral glycolipid compositions of human gastric and colonic cancer tissues have been compared with those from normal The cancerous tissues had elevated levels of tissues. Io3 lactosylceramide and 4-fucoglycolipids. The 4-fucoglycolipids contained both 3-0- and 4-g-substituted residues o f 2-acetamido2-deoxy-P-glucose. Similar studies have confirmed these results, especially those of the L-fucoglycolipids, and further showed that the levels of sulpho-E-galactolipids were also elevated. lo4 Blood group A-active glycolipids were present in cancer tissue from two patients with blood group 0 but not in the associated healthy tissue. The liver and kidney of a patient with I-cell disease have been di- and tri-hexoside found to contain elevated levels of This was associated with a B-P-galactosidase ceramide. lo5 deficiency. It was concluded that the B-8-galactosidase deficiency in I-cell disease is a more specific phenomenon rather than a secondary inhibition as found in mucopolysaccharidoses. Evidence has been presented that Niemann-Pick disease, type C, is caused by the deficiency of an activating factor stimulating sphingomyelin and P-glucocerebroside degradation in the spleen of these patients. Io6 The glycolipids o f the meconium of a human 0 Le (a-b+) secretor have been characterized by structural and immunological The dominating species were E-glucosyl- ( 1 5 % ) and methods . I o 7 (30%). Of the higher saccharides, the lacto Ij-galactosyl-ceramide (30% of total weight) was represented by series lactotetraosylceramide and H-active, Lea-active, and Leb-active Similar studies, involving more glycolipids of the lacto series. detailed separations and characterization methods, have suggested

596


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-

h c

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597


that meconium is a rich source of glycolipids which allows the chemical fingerprinting of the blood-group type in individuals.' O 8 The, non-acidic glycosphingolipids of human cord blood erythrocytes from blood groups 0 and A have been characterized.lo9 Lactosylceramide and globoside were the major components. Increased amounts of penta- and hexa-glycosylceramides, which lacked L_-fucose substituents, were present and could be A and H biosynthetic precursors of ordinary blood - group glycosphingolipids. The glycosphingolipids of human plasma lipoproteins have been reviewed with specific interest in the way the different glycolipids exchange between the lipoproteins and erythrocytes. l o The identification of a blood-group Leb-active glycosphingolipid in plasma by m.s. and n.m.r. spectroscopy has suggested a new method of chemical blood-typing of human individuals.' A series of papers has been published on the immunochemistry o f the Lewis blood-group system. In the first, the complete structures of the oligosaccharide chains of the Lea-, Leb-, and H-type 1-active glycolipids have been established as (18)-(201, respectively. 1 1 * The second and third papers consider the m.s. analysis113 and 'H n.m.r. spectroscopy,ll 4 respectively, which have been used to establish these structures. Neutral glycosphingolipids have been isolated from the hairy cell leukemia and comprise cells of a patient with hairy lactosylceramide,and two higher glycolipids of g-glucosylceramide, the globo series which are similar to those found in human lymphocytes and chronic lymphocytic leukemia cells. The glycolipid pattern may be useful in classifying leukemias of uncertain origin. A high incidence of an autoantibody against the neutral glycolipid asialo G p l l has been observed in patients with systemic lupus erythematosus. l 6 Since no activity was shown against other glycolipids or gangliosides it was suggested that the antibody played a role in the pathogenesis of such neurological disorders. The glycosphingolipids of normal human lymphocytes have been separated by h.p.1.c. The major component was lactosylceramide with others being detected in smaller amount^."^ Purified B- and T-cell fractions had a similar complement of glycolipid but the B-cells contained significantly more of each component. Similar studies have been carried out on human peripheral blood lymphocytes and the lymphoid cells of a patient with B-cell

'

-

'

598

''


chronic lymphocytic leukemia. The glycol ipids were the same as those in ref. 114. A blood-group A-specific tetrasaccharide (21) has been isolated and characterized from human urine.ll9 The sugar had A activity almost as great as that of the most active oligosaccharide isolated from soluble blood-group A substance. The alkyl-q-galactolipids from calf brain have been separated by h .p. 1 .c . 1 2 0 The two oligosaccharides B-~-mannopyranosyl-(l+4)-2-acetamido2-deoxy-;-glucose and 42-g- 6-D-mannopyranosylchitobiose accumulated in the kidney of a goat with mannosidosis.12' The amounts represented three times those found in the brain,but while lesions of the brain caused profound neurological disorders functional impairment of the kidney was not evident. Using an epithelial cell line derived from monkey kidney, it has been shown that E-galactosylcerebroside is present in the cytoplasm in close association with colchicine-sensitive microtubule-like subcellular structures.' The distribution of glycero-g-glucolipids in the mucous barrier of dog stomach fundus, body, and antrum has been investigated. 1 2 3 The content of neutral glycero-8-glucolipid in the antral portion was higher than that in the other two areas,but the content of sulphated glycolipid was even higher. The localization of q-galactosylcerebroside in distal tubules, ascending limbs of Henle's loops and collecting tubules of kidney, periportal bile ducts and hepatic parenchyma of liver, and bronchioles and alveoli of lungs has been demonstrated by an immunological method in these organs from hamsters.l 2 4 The existence was further confirmed by chemical analysis. The presence of SV40-specific glycolipid antigens in the plasma membrane, the nuclear membrane,and probably other inner membranes of SV40-transformed cells from hamsters has been indicated by immunofluorescence methods. 2 5 Incubation of NIL 2 c l cells, a cloned hamster cell line, with an equilibrium mixture of radioactive UDP-P-glucose and UDP-9-galactose has suggested that this is a useful method for determining the glycolipid composition of cultured cells. 26 Using the same cell line, the sequential glycosylations of endogenous glycosphingolipids have been studied using nucleotide sugars. 27 Differences were noted in the sequential glycosylation between cells harvested from confluent cultures and sparse cultures.

'

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-

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2 $1

599

600


Furthermore, there was an indication that two distinct pools of each glycolipid were present. Precursor-product relationships do not exist among the main pool of each glycolipid. of UDP-q-galactosylceramide: The specific localization P-galactosyltransferase in heavy-myelin and membrane fractions of rat brain at an early age when myelination is just beginning has been suggested as a site for the myelination process.128 The turnover of glycolipids in the adult rat brain has been studied vivo by determining the half-life of the carbohydrate portion. 12’ These half-lives were much shorter than that of the total carbon pool, Since lactosylceramide had the shortest half-life, it was suggested that this glycolipid may serve as a branch point for the biosynthesis of cerebral gangliosides in vivo and not as a degradation product of more complex molecules. In various rat neural tumours, the contents of lactosylceramide were up to 80 g kg-l compared with normal neural tissue values of 10 g kg-l.130 The changes in lipid-bound surface carbohydrates during cell differentiation have been studied in the small intestine of rats. 1 3 ’ The g-glucosylceramide content increased 2-3 fold. There was little change in many of the complex glycolipids. In similar studies, the content of trihexosylceramide has been shown to increase up to about 13 days after p a r t ~ r i t i 0 n . l ~ ~ A novel sulphoglycosphingolipid ( 2 2 ) has been isolated from rat kidney and characterized by the usual methods. 3 3 The clearance of D-glucocerebrosidase by rat liver has shown the presence of two distinct enzyme forms with different recognition characteristics, 3 4 Neuraminosyl, q-galactosyl, and 2-acetamido-2-deoxy-~-glucosyl residues are important in the uptake of the enzyme. Modification of the enzyme to expose some of these residues results in increased delivery to specific cell and types. The disaccharides cx-~-mannopyranosyl-(l+2)-~-mannose a-E-mannopyranosyl-(l+3)-g-mannose have been synthesized from dolichyl-D-mannosylphosphate and g-mannose by rat liver membrane systems. 1 3 5 The proportion of each product was dependent on the assay conditions. and dolichyl The formation of dolichylphosphate-D-glucose diphosphate oligosaccharides in rat spleen lymphocytes has been studied. 136 It was observed that the phospho-oligosaccharide populations contain far fewer products which had been E-glucosylated than the dolichyldiphosphate oligosaccharides from


60 1

which they are derived, suggesting that the p-glucosyl units inhibit the reaction. Sulpho-D-galactosylacylalkyl glycerol is enriched in a plasma membrane fraction from adult rat testis.137 By comparing 11 separate tissues from rats, the presence of this lipid has been shown to be restricted to the testis.'38 Several novel sulpholipids were detected during this study. L-Fucose-terminated glycoconjugates are recognized by pinocytosis receptors on macrophages where the uptake of 6-P-glucuronidase is blocked. 3 9 An anti-glycosphingolipid antibody has been prepared and purified and has been used to demonstrate the localization of asialo G M ~ on the tips of the surface membrane of rat ascites hepatomas, 4 0 Analogues of ceramides which inhibit ;-glucocerebroside synthetase in mouse brain have been in~estigated.'~' The most potent compound tested was 2-decanoylamino-3-morpholino-1-phenylpropanol. An examination of the effects suggested that the active region of the enzyme contains four active sites, one of them being an anionic moiety that binds the 9-glucose residue in an activated form. The concentrations of myelin-characteristic D-galactolipid in organotype cultures of newborn mouse cerebellum have been determined by h . p.l.~.'~~ These lipids are detectable at 9 days and increase steadily in concentration until the explants are more than 3 weeks old. The g-galactosylceramide sulphotransferase activity of the cerebrum and cerebellum of normal and jimpy mice has been measured during postnatal development. 1 4 3 The activity was related to net sulphatide synthesis and was 25-505 lower in the brain parts of the mutants when myelination was taking place in the controls. The effective antibody in an antiserum having anti-natural killer cell activity and raised against mouse brain tissue is directed against the cell-surface glycolipid asialo G M .144 ~ The distribution of the same glycolipid in the small intestine of the mouse has been determined using an immunofluorescence staining method.145 Clear staining o f the brush border and basolateral membranes of epithelial cells, cell membranes of cryptic cells,and some secretory granules was observed. q-Glucosylceramide, on intravenous injection in mice, is stored predominantly in the liver and has a half-life of 3.5 days.146 High levels of this lipid were found in the bile of one patient


602

and the liver of two patients with a biliary obstruction,which suggests a relationship between the biliary excretion of this lipid and Gaucher's disease. The glycolipid on the surface of natural killer cells from mouse spleen has been identified as asialo G, 147 1' A method has been developed for the extraction of carbohydrate-defined Ia antigens from murine spleen and serum cells.14* The antigen appeared to have a glycolipid structure with the antigenic activity residing in an oligosaccharide unit containing more than seven monosaccharide residues. Two marker glycolipids of alloantigen-activated murine T-lymphocytes have been partially characterized as a-E-galactosyl6-IJ-galactosyl-P-glucosylceramide and the same ceramide with a 2-acetamido-2-deoxy-~-~-glucosyl residue at the non-reducing terminal position. 149 A monoclonal antibody, directed to the stage-specific embryonic antigen in mice, has been shown to react with a branched glycosphingolipid similar in structure to Ii antigen. 150 Two related sublines of the mouse lymphoma L5178Y, which were selected for their different susceptibility to natural killer cell-mediated lysis, have different glycolipid patterns. 15' The ~ was not sensitive line contains a high level of asialo G M which detected in the resistant line. However, there was no correlation between asialo G M ~ content and sensitivity in sublines. Modification of the surface of distearoylphosphatidylcholine vesicles with synthetic glycolipids has dramatically affected the rate of uptake of these vesicles by murine peritoneal The high rate of uptake of 6-amino-6-deoxy-Pmacrophage. 15' mannose-modified vesicles is inhibited by cytochalin B and chloroquine but not by colchicine, indicating that the mechanism is phagocytosis. These vesicles remain intact after phagocytosis as aggregates of fused and intact vesicles surrounded by a single bilayer membrane structure. Several blood-group-type glycolipids from the small intestine of an individual rabbit have been identified by select-ion monitoring. 5 3 The dominating species was a hexaglycosylceramide group B-type sequence, while a second with a blood hexaglycosylceramide having a blood-group A-type sequence was also found. into lipid-linked The incorporation of [ 2-3H]-g-mannose is stimulated by oligosaccharides of rabbit mammary gland

-


603

hormones.154 An I-active ceramide decasaccharide (23) has been isolated and chacterized from rabbit erythrocyte membranes.155 The homologous series isomaltose to isomaltoheptaose can be coupled to stearylamine by reductive amination using cyanoborohydride ion. 1 5 6 The stearyl oligosaccharides from isomaltotriose to the heptasaccharide, when incorporated into rabbit liposomes, were agglutinated in the presence of specific antibodies and were lysed if complement was also present. As the disaccharide conjugate was not agglutinated, it may not protrude far enough from the surface to react with the antibody. The specificities and the sizes and shapes of the antibody combining sites in the above systems have been investigated by quantitative precipitin and precipitin-inhibition studies.157 The sizes of 15 antisera have been determined and all had combining sites in the range isomaltotriose to isomaltohexaose. One of the antisera to stearylisomaltopentaose has been separated into IgM and IgG fractions, and the antibody binding sites were studied.158 Both were similar to unfractionated antibodies with sites complementary to isomaltotetraose. Another antiserum had two site sizes, one for isomaltopentaose and the other for isomaltotetraose. The specific antibodies elicited in rabbits against D-glucosylceramide did not cross-react with E-galactosylceramide or ~ u 1 p h a t i d e . l ~ ~ They did react with the natural hapten present in red-blood-cell membranes of sheep. Very low levels of glycolipid have been found in chick embryonic brain yet nearly adult levels were reached by the 5th day. 160 The deposition of mono-D-galactosyldiglyceride in the cerebrum was unique in that all the secretion occurred between the 16th day of embryonic life and the 5th day after hatching, a period characterized by a high rate of myelination. The spermatozoa of the fresh-water bivalve Hyriopsis schlegelii have a complex spectrum of neutral glycolipids. Two of the minor components (24) and (251,which accounted for

8

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Table 7 continued

EC No.

G 1u t a raldehyde-me d i a t e d r e a c t i o n

c h l o r i d e membrane

Adsorption on porous p o l y v i n y l

w i t h n y l o n 6 6 t o f o r m a membrane

Glutaraldehyde-mediated r e a c t i o n

Entrapment i n p o l y a c r y l a m i d e g e l

t i o n w i t h c e l l u l o s e beads

G l u t a raldehyde-me d i a t e d r e a c -

t i o n w i t h P t gauze

glutaraldehyde-me d i a t e d reac-

C o - i m m o b i l i z e d w i t h c a t a l a s e by

coated glass microbeads

Adsorption on polyethyleneimine-

v i n y l a t i o n o f t h e enzyme

a n d mode o f c o u p l i n g

M a t r i x o r macromolecule coupled

analysis

o f enzymic a c t i v i t y A c t i v e i m m o b i l i z e d enzyme

,

study o f mechanical c o n t r o l

A c t i v e i m m o b i l i z e d enzyme,

o n l y when m e c h a n i c a l l y s t retched

Membrane w i t h enzyme a c t i v i t y

t h e a c t i v e i m m o b i l i z e d enzyme

Study o f t h e r m a l s t a b i l i t y o f

i n serum

f o r measurement o f q - g l u c o s e

u t i1i z i n g chem i 1um i n e sce n c e

I m m o b i l i z e d enzyme s e n s o r

i n fermentation broths

t r o d e f o r ;-glucose

O x y g e n - s t a b i l i z e d enzyme e l e c -

A c t i v e i m m o b i l i z e d enzyme

Use o f p r o d u c t

723

722

721

716

70 0

698

6 97

Ref.

2

2

2

2.

3

s

&

s

m

c1

o\

w

4

3.2.1.31

1.2.1.12

6-E-Glucuronidase

G l y c e r a l d e h y d e 3-

g i l i s cells

Kluyveromyces

Inulinase

fra-

dehydrogenase

3a-Hydroxysteroid

-

3.2.1.7

1.1.1.50

phosphate dehydrogenase

EC No.

Enzyme

Table 7 continued

or macromolecule coupled

Enbrapment i n a l g i n a t e g e l

A d s o r p t i o n on D E A E - c e l l u l o s e

activated cellulose

R e a c t i o n w i t h cyanogen b r o m i d e -

a c t i v a t e d agarose


with alkylamine glass

Glutaraldehyde-mediated r e a c t i o n

membrane

asymmetric a c e t y l c e l l u l o s e

w i t h t h e p o r o u s s i d e o f an

and mode o f c o u p l i n g

Matrix

Ref.

continu-

lactose t o ethanol

Continuous conversion o f

syrup from i n u l i n

ous p r o d u c t i o n o f f r u c t o s e

i m m o b i l i z e d enzyme,

Study o f p r o p e r t i e s o f a c t i v e

3a-hydroxysteroids

Continuous f l o w a n a l y s i s o f

i m m o b i l i z e d enzyme

Study o f p r o p e r t i e s o f a c t i v e

h.p.1.c.

g a t e s p r i o r t o a n a l y s i s by

Analysis o f glucuronide conju-

enzyme

i t y o f active immobilized

618

726

47 6

168

167,

725

S t u d y o f p r o p e r t i e s a n d s t a b i l - 724

ment o f E - g l u c o s e i n human serum o r w h o l e b l o o d

enzyme e l e c t r o d e f o r m e a s u r e -


w 4

3 ~

p5

a

0

i;.

2

zs

R

3

$

??

1.1.1.27

L a c t a t e dehy d r o -

L y s o z yme

Luciferase

3.2.1.17

-

-

L e u c o n o s t o c mesen-

teroides c e l l s

-

La c t o p e r o x i dase

ge na se

EC No.

E nz yme

Table 7 continued

a c t i va t e d a ga r o se

R e a c t i o n w i t h cyanogen bromide-

d i a l y s i s membrane

l i n k i n g w i t h albumin onto a

Glutaraldehyde-me d i a t e d cross-

cellulose f i l t e r

Entrapment i n a g a r on an a c e t y l -


or with co n ca na V a l in A - a ga r o s e

interaction with a n i o n i c d e t e r ge n t s

Study o f

creatine kinase a c t i v i t y

Microscale analysis of

serum

o f I - p h e n y l a l a n i n e i n human

lactate electrode f o r analysis

Used i n c o n j u n c t i o n w i t h a

a c t i v e immobi 1i z e d enzymes

enzyme immobi 1i z a t i on Comparison o f p r o p e r t i e s o f

for

S t u d y o f new s u p p o r t

p a c k e d i n h.p.1.c. columns R e a c t i o n w i t h cyanogen bromide-


artichoke tubers Study o f p r o p e r t i e s o f a c t i v e

e t h a n o l from J e r u s a l e m

Repeated b a t c h p r o d u c t i o n o f


A d s o r p t i o n o n PTFE p a r t i c l e s

Adsorption on alumina



171

729

99

170

72 0

727

619

Ref.

$4

23

L

oL1

w

4

-

3.6.1.22

NAD+ pyrophosphatase

Nocardia e r y t h r o p o l i s o r opaca c e l l s

3.2.1.18

-

-

-

E C No.

Neuraminidase

Naringinase

Methanosarcina barkeri c e l l s Mycobacterium p h l e i cells

Enzyme

Table 7 continued

A d s o r p t i o n on DEAE-cellulose o r s i l i c a entrapment i n p o l y acrylamide g e l

Adsorption on p h o s p h o c e l l u l o s e

R e a c t i o n w i t h cyanogen bromidea c t i vated aga r o s e

w i t h alkylamine g l a s s

Adsorption on DEAE-cellulose o r s i l i c a , e n t r a p m e n t i n polyacrylamide g e l G 1 u t a r a 1de h yde -me d i a t ed r e a c t i o n

reaction w i t h gelatin-agarose Entrapment i n a l g i n a t e g e l

d i t h i o )pro p i ona te-me d i a t ed

-N-S u c c i nim i d y 1-3 -( 2- py r i d y 1-

Matrix o r macromolecule coupled and mode o f c o u p l i n g

3

730

6 21

620

410

w 4

0

g.

g5

$

2 g.

E

k

En

Ref. k

Desi a 1 y l a t i on o f a n g i o t e n s i n172 c o n v e r t i n g enzyme D e s i a l y l a t i o n o f human 173 platelets Enzyme r e a c t o r f o r p r o d u c t i o n 4 95 o f n i co t i nam i d e mono nucl eo t i d e L i v i n g immobilized c e l l s f o r 621 steroid transformations

Act i ve i m m o b i 1i z e d e nz ym e

Development of a new method f o r p r e p a r a t i o n o f immunoadso r be n t s Conversion o f methanol t o methane L i v i n g immobilized c e l l s f o r steroid transformations

Use of p r o d u c t

3

R

? 0

philus cells Papain

Pachysolen tanno-

n u c l ea s e

M ic r o co c ca 1 e n d o-

Nuclease P

Enzyme

Table 7 continued

3.4.22.2

-

3.1.31.1

-

EC No.

carbodi-

Ti4+-complexation

c r o s s l i n k i n g w i t h 4-amino-

T e r e p h t h a 1ic d i az i d e -me d i a t e d


R e a c t i o n w i t h cy ano gen b r om i d e

e t h y l - agarose

Reaction with succinylamino-

Entrapment i n a l g i n a t e g e l


R e a c t i o n with cyanogen bromide-

r e s i n s y&

Reacti o n with ion-exchange

complex

reaction with Ti4+-cellulose

anion-exchange c e l l u l o s e , o r

imide-mediated r e a c t i o n w i t h

activated cellulose,




Study o f p r o p e r t i e s o f a c t i v e i m m o b i l i z e d enzyme

enzyme i n a c o l u m n r e a c t o r

Use o f a c t i v e i m m o b i l i z e d


- -xylose Q

Production o f ethanol from


a c i d h y d r o l y s i s by a c t i v e

Study o f k i n e t i c s o f n u c l e i c

A c t J. ve i m m o b i 1iz e d e nz ym e



467

175

174

622

732

7 31

47 7

Ref.

3

3

2

a-

&

-

4 P 0

Pepsin

enzyme

Penicillium duponti

3.4.23.1

-

3.5.1 .ll E n t r a p m e n t i n g e l a t i n

w i t h 6-aminohexyl-agarose

G 1u t a r a 1de h y de -me d i a t e d r ea c t ion

l i n k i n g onto collagen

G1 u t a r a 1de h yde - m e d i a t e d c r o s s -

e l e c t r o l y t e complexes

t i o n w i t h w a t e r - so 1ub 1e po 1y-

Cyan u r i c c h l o r i de-me d i a t e d r e a c -

mide g r a f t c o p o l y m e r s

t i ve s o f a 1g i na t e- p o l y a c r y 1a-

Reaction w i t h hydrazide deriva-

r e a c t i o n w i t h CM- c e l l u l o s e

methacrylate esters o r

Reaction with r e a c t i v e poly-

activated cellulose

R e a c t i o n w i t h 4 -be n z o q u i no ne

benz y l - c e l l u l o s e

-

o r macromolecule coupled


P e n i c i l l i n amidase

Matrix

EC No.

Enzyme

Table 7 continued

F(ab’)2

fragments f o r

Digestion o f IgG t o obtain



M i c h a e l i s-Me n t e n c o n s t a n t

f o r determining the

V a l i d a t i o n o f m o d i f i e d method

enzyme immo b i l i z a t i o n

S t u d y o f new s u p p o r t f o r




176

7 35

734

6 80

733

693

471

Ref.

P

4

0

2.

$

R,

Q

s

$

PP n

l a t a chroma t o pho r e s -

Rhodopseudomonas c a p s u -

cells

Rhizopus n i g r i c a n s

ficans c e l l s

Pseudomonas d e n i t r i -

cells

-

-

-

-

1.11.1.7

Perox i d a s e

Pseudomonas dacunhae

EC No.

__

E nz yme

~~

T a b l e 7 continued

hyde-crosslin king

-

l i n k i n g w i t h albumin and with/

G 1 u t a r a 1de h y de -me d i a t e d c r o s s

agar g e l

Entrapment i n p o l y a c r y lamide o r

Entrapment i n a l g i n a t e g e l

Entrapment i n k-carrageenan g e l

w i t h a l k y l a m i n e g l a s s beads

625

624

6 23

736

7 00

I n v e s t i g a t i o n o f t h e i n f l u e n c e 626 o f immobilization on l i g h t -

p r o ge s t e r o n e

lla-Hydroxylation o f

Living immobilized c e l l s

Living immobilized c e l l s


f o r measurement o f serum

g- g l u c o se

a f i l m f o l l o w e d by g l u t a r a l d e G 1 u t a r a l d e h y d e -me d i a t e d r e a c t i o n

u t ili z in g c h em i1um ine s ce n c e

I m m o b i l i z e d enzyme s e n s o r

o f enzyme i m m o b i l i z a t i o n

crosslinkable prepolymer i n t o

Copolymerization w i t h a photo-

i n g v i n y l a t i o n o f t h e enzyme

e r e n t p o l y s a c c h a r i d e s f 0 1 1 ow-

512

411

D e v e l o p m e n t o f a new m e t h o d

Graft polymerization t o d i f f -

Ref.


i n t r a v e n o u s use


Reaction with chitosan



Carbohydrate Chemistry v.15 - Part II

Carbohydrate Chemistry v.14 - Part II

Carbohydrate Chemistry v.21-Part I