Advances in Protein Chemistry: v. 10

ADVANCES IN PROTEIN CHEMISTRY

VOLUME X

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ADVANCES IN PROTEIN CHEMISTRY EDITEDBY M. L. ANSON

KENNETH BAILEY

Cambridge, Massachusetts

University of Cainbridge Cambridge, England

JOHN T. EDSALL Biological Laboratories, Harvard Uni&ersity Cambridge, Massachusetts

VOLUME X

1955 ACADEMIC PRESS INC., PUBLISHERS NEW YORK, N. Y.

Copyright @ 1955, by ACADEMIC PRESS INC. 125 EAST 2 3 STREET ~ ~ NEW YORK 10, N . Y.

All Rights Reserved No part of this book may be reproduced in any form, by photostat, microfilm, or any other means, without written permission from the publishers.

Library of Congress Catalog Card Number, 44-8863

PRINTED I N T H E UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME X

C. E. DALGLIESH, Postgraduate Medical School, Ducane Road, London, England G . H~MOIR,Laboratory of General Biology, University of LiBge, Belgium GERTRUDE E. PERLMANN, The Rockefeller Institute for illedical Research, New York, N . Y . JACINTOSTEINHARDT, Department of Chemistry, Massachusetts Institute o f Technology, Cambridge, Massachusetts BERTL. VALLEE,The Biophysics Research Laboratory of the Department o f Medicine, Harvard Medical School, and Peter Bent Brigham Hospital, Boston, Massachusetts LIONELA. WALFORD, Fish and Wildlife Service, United States Department of the Interior, Washington, D . C. CHARLES G. WILBER,Chemical Corps Medical Laboratories, Army Chemical Center, Maryland

ETHELM. ZAISER,Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts

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CONTENTS CONTRIBUTORS TO VOLUME X . ..

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v

The Nature of Phosphorus Linkages in Phosphoroproteins BY GERTRUDE E. PERLMANN, The Rockefeller Institute for Medical Research, New York, N . Y . I. Intxoductio .......................................... 1 from Phosphoprot,eins. . . . . . . . . . . . . . . . . . . . 2 111. Phosphoproteins and Phosphopeptones. . . . . . . . . . . . . . . . . . . . . . . . . . . 1V. Enzymatic Dephosphorylation of Phosphoproteins.. . . . . . . V. Possible Biological Function of Phosphoproteins. VI. Summary . . . . . . . . . . . . . . . . . . . . ....................................... 26 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism of the Aromatic Amino Acids By C. E. DALGLIESH, Postgraduate Medical School, Ducane Road, London, England I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . ............................. 33 11. Biosynthesis of the Aromatic Amino ............................. 36 111. Degradation of Phenylalanine and Tyrosine to Acetoacetate ; the Principal Route Used by iVIammals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IIIA. Evidence Derived from Inborn Errors of Metabolism.. . . . . . . . . . . . . . . . . . 46 IIJB. Enzymic Experiments on the Normal Pathway in Mammals. . . . . . . . . . . . 55 IV. Tyrosine Degradation by the Catechol Pathway.. ...................... 65 V. Tyrosine Metabolism via Thyroid Hormones and Other Halogenated Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Pathways of Phenylalanine and Tyrosine Metabolism Utilized Princip by Microorganisms. . . . . . . . . . . . . . . . . . . . . . . . . . VII. Tryptophan Degradation by the Kynurenine-Ni VIII. Tryptophan Degradation by the Enteramine-Serontion Pathway.. . . . . . . 103 IX. Routcs for Tryptophan Degradation Used Principally by Microorganisms. 108 X. Tryptophan Metabolism in Plants. Heteroauxin. . . . . . . . . . . . . . . . . . . . . . . . 113 X I . Natural Products Probably Related to the Aromatic Amino Acids.. . . . . . 115 XII. Future Problems.. . . . . . . . . . . . . . . . . . . . . . . .......................... 121 XIII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................. Hydrogen Ion Equilibria in Native and Denatured Proteins BY JACINTO STEINHARDT A N D EwmL M. ZAISER,Department of Chemistry, Massachusetts fnslitute of Technology, Cambridge, Massachusetts I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Acid-Base Diesociations of Native Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Iinreactive Prototropic Groups in Native Prot.eins... . . . . . . . . . . . . . . . . . . . References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

152 153 180

221

...

CONTENTS

Vlll

Fish Proteins BY G . HAMOIR.Laboratory of General Biology. University of Lihge. Belgium I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 I1 Proteins from Skeletal Muscle ........................... 228 I11. Fish Enzymes . . . . . . . . . . . . . . . . .............................. 269 IV Fish Blood Proteins ......................................... 273 V . Fish Protamines., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .274 . V I . Connective Tissue Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 VII . Conclusion: The Comparative Biochemistry of Fish Proteins . . . . . . . . . 279 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

. .

The Sea as a Potential Source of Protein Food BY LIONELA . WALFORD A N D CHARLES G WILBER,Fish and Wildlife Service, United States Department of the Interior, Washington, D C and Chemical Corps Medical Laboratories, Army Chemical Center, Md . I World Protein Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 . 303 11 Proteins in Marine Organisms., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Variations in Protein Conten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 IV . The Biological Value of Mari roteins . . . . . . . . . . . . . . . . . . . . . . . 313 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

.

. .

. .

Zinc and Metalloenzymes BY BERTL . VALLEE,The Biophysics Research Laboratory of the Department of Medicine, Harvard Medical School, and Peter Bent Brigham Hospital, Boston, Massachusetts A . General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 I1. Metalloproteins and Metal-Protein Complexes . . . . . . . . . . . . . . . . . 380 I11. Characteristics of Metalloenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 IV . Characteristics of Metal-Enzyme Complexes . . . . . . . . . . 325 V . Empirical Formulas for Metalloenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 327 VI . Instrumental Methods for the Detection of Metals . . . . . . . . . . VII . References to Metalloenzymes Containing Copper, Iron, an 332 denum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Zinc Metalloproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 V I I I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 TX . Carbonic Anhydrase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 X . Experimental Approach for Studies on the Leukocyte Zinc Protein, Carboxypeptidase, and Yeast Alcohol Dehydrogenase . . . . . . . . . . . . . 337 X I . The Zinc-Containing Protein from Human Leukocytes . . . . . . . . . . . . . 339 . . . . . . . . . . . . . . . . . . . 3-13 XI1. Pancreatic Carboxypeptidase . . . . . . . . . . . . . XI11. Yeast Alcohol Dehydrogenase . . . . . . . . . . . . . . . . . . . . 353 XIV . Coordination Chemistry of Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .370 References.,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 AUTHOR INDEX . . . . . . . . . .

SUBJECTINDEX .........

. . . . . . . . . . . . . . . . . . . . . . . . . .

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:155

lo!)

The Nature of Phosphorus Linkages in Phosphoproteins BY GERTRUDE E. PERLMANN The Rockefeller Institute for Medical Research, New York, N . Y .

CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Phosphoamino Acids Derived from Phosphoproteins. . . . . . . . . . . . . . . . . . . . . . 1. 0-Phosphorylserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 0-Phosphorylthreonine . . . . ...................................... 3. Phosphoarginine . . . . . . . . . . ...................................... 4. 0-Phosphorylserylglutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Phosphoproteins and Phosphopeptones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Casein.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Phosphopeptones from Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Vitellin and Vitellenin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Phosphopeptones from Vitellin and Vitellenin . . . . . . . . . . . . . . . . . . . . . . . . . a . Vitellinic Acid.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Ovotyrines . . . . . . . . . . . . . . . . ................................ 5. P h o s v i t i n , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Enzymatic Dephosphorylation of Phosphoproteins, . . . . . . . . . . . . .. 1. Ovalbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. 2. Casein.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. 3. P e p s i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ V. Possible Biological Function of Phosph ........................ VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ References . . . . ...............................................

1 2 2

3 3 3 4 4 5 6 6 6

6 7 9 11 16 22 25 26

27

I. INTRODUCTION The almost universal occurrence of phosphoproteins and their abundance in embryonic and rapidly growing tissue and in foodstuffs like milk and eggs initiated a study of this type of material more than fifty years ago. Our incomplete knowledge, however, is mainly due to the fact that none of the phosphoproteins thus far isolated satisfies the presently adopted criteria of purity of proteins-they are mixtures. They may have as few as one to two, or as many as thirty to fifty, phosphorus atoms per molecule. Their classification is based entirely on the fact that in contrast to the nucleoproteins, they lack purine and pyrimidine bases and contain the phosphoric acid esterified to an amino acid residue. Their main characteristic is that the phosphate groups are readily hydrolyzed in dilute alkali a t room temperature, e.g., in 0.25 N sodium hydroxide at 25"C., but that they are stable in acid under these conditions (74).

No attempt will be made here to review the extensive literature pertaining to the chemistry of phosphoproteins. 111 this article tlic subject will tie limited to the qucstion of the position of thc phosphorus in the nioleculr and to sonic of thc methods used in eliicidating thtl c*hemicbalnatiirr of thv bonds in whivh this clcment occurs. hicf survey The followiiig plan has hccii adoptcd for this discussioii. of: 1 . The rhemistry of three phosphoainino acids isolated from biological material. 2. The properties of the dipeptide 0-phosphorylserylglutamic acid. 3 . The phosphoproteiiis casein, vitellin, vitellenin, and phosvitin. 4. The phosphopeptones derived from milk and egg proteins. 5. The use of enzymes in the study of the nature of phosphorus bonds in phosphoproteins. 6. Remarks 011 the biological function of these materials.

11. PHOSPHO MINO ACIDSDERIVED FROM PHOSPHOPROTEINS Let us first turn to the question of how the phosphorus is bound to a protein. Both -0-Parid -N-Pesters present themselves as possibilities. Such linkages would involve either alcoholic or aromatic hydroxyls, on the one hand, or free amino groups or the guanido group of arginine, on the other. T o date, only three phosphoamino acids have been isolated from biological materials: 0-phosphorylserine and O-phosphorylthreonine, both representatives of an -0-Pester, and phosphoarginine, which has a -N-P-bond. In addition it has been possible to prepare phosphorylated derivatives of other amino acids, eg., tyrosine, oxyproline (37, 39), glycine, alanine, glutamic acid, leucine, and glycylglycine (98), and the methylester of N-phosphorylphenylalanine (40). 1. 0-Phosphorylserine

This amino acid C3HsOaNP (P = 16.7%) and with a n [a]:3of +7.2 (1) was first obtained by Lipmann and Levene from vitelliiiic acid (44). Lipmann subsequently succeeded in isolating this substance from an acid hydrolyzate of casein (45) and thus established that phosphoserine may occur as B constituent in phosphoproteins. More recently Agren, de Verdier, and Glomset crystallized 0-phosphorylserine (1). The following structure is usually assigned to this amino acid 0

\\

€1O-P-O-CH2

HO

I

/

CH

/ \

NH2

COOH

NATUHE OF PHOSPHORUS LINKAGES IN PHOSPHOPROTEINS

3

Here one has to keep in mind, however, the experiments of Bergmann and Miekeley, who showed that in the case of benzoylserine the acidity of the medium determines whether the benzoyl residue is linked to the hydroxyl or to the a-amino group of the amino acid (9). That a similar situation exists in the case of phosphoserine follows from the work of Plapinger and Wagner-Jaureggl (73). These investigators found tjhat on treatment of the N-diisopropylphosphoryl derivatives of the DL-serine methyl ester with boiiing aqueous hydrochloric acid O-phosphorylserine is formed from the Kcompound. Therefore, the existence of phosphoserine with a -N-P-bond in a native protein is feasible, e.g., as the N-terminal amino acid of L: peptide chain. 2. O-Phosphorylthreonine

The second phosphoaniino acid with mi -0-Plinkage, phosphothreonine C4Hlo06NP(P = 15.5%), [ag4 -7.37, was isolated from aii acid hydrolyzate of casein by de Verdier in 1953 (94). Plapinger and Wagner-Jauregg showed that, as in the case of phosphoserine, migration of the phosphate group from the N to the 0 position also occurs in the case of this compound (73). 3 . Phosphoarginine

CsH160&J4P (1' = 11.5 %) is the only amino acid with a -N-Pbond thus far encountered in biological material (57). Its presence in the muscles of invertebrates suggests that this substance fulfills a role similar to that of creatinephosphate in vertebrates. The function of these two compounds as reservoirs of readily available energy is coufirnied by experimental facts (46). However, it is still uiikiiown whether or not phosphoarginine occurs in phosphoproteins. One of the characteristic features of these three phosphoamino acids is that in contrast to the intact proteins, the phosphate group is stable in 0.25 N sodium hydroxide (72). The N-P bond of phosphoarginine, however, is acid-labile.

4. O-PhosphorylserylglutamicAcid I n 1933, shortly following the discovery of phosphoserine (44), Schmidt, and Levene and Hill isolated from a casein hydrolyzate a dipeptide consisting of serine, glutamic acid, and phosphoric acid (38, 84). I n 1941, Posternak and Pollaczek demonstrated the presence of a free a-amino group in the serine moiety of the molecule and assigned the following structure 1 The possibility of a phosphate migration in phosphoserine from the Nto the 0- position had already been suggested by LinderstrZm-Lang in 1933 (Linderstr$mLang, Ii. (1933). Ergeb. Physiol. u . Exptl. Pharmakol. 36,415.)

4

GERTRUDE E. PERLMANN

to the dipeptide (77) COOH

I I CBZ I CH / \ CH,

0

\\

HO-P-O-CHe

I

/

CH

€10

N 11,

/

\

CO-NII

COOH

Moreover, these investigators showed that the pept ide bond of phosphorylserylglutamic acid was resistant to the action of a dipeptidase from pig intestine. However, after removal of the phosphate group with the aid of kidney phosphatase the dipeptidase readily hydrolyzed the peptide bond. It is thus clear that the phosphate group has a protective action on the peptide linkage, and one can conclude that not only the type of linkage but also the surrounding molecular configuration determines whether an enzyme will act.

111. PHOSPHOPROTEINS AND PHOSPHOPEPTONES Following this discussion of some of the properties of three naturally occurring phosphoamino acids and of the dipeptide, O-phosphorylserylglutamic acid, a few examples of phosphoproteins, i.e., casein, vitellin, vitellenin, and phosvitin, and of the phosphopeptones derived from these materials, will be considered. 1. Casein

Casein, the most important and most thoroughly studied phosphoprotein, accounts for 80 % of the total nitrogen of cow’s milk (56) and about 30% of that of human milk (8). The interest of a great number of investigators in this protein may have been stimulated in part by the ease with which it may be separated from the other milk proteins by acidification to pH 4.6 (26). For a long time casein was considered to be a pure protein (15, 26). As will be discussed in a later section of this chapter, its inhomogeneity was, however, demonstrated as early as 1927 by LinderstrGm-Lang and Kodama (41). Since then it has become clear that the products obtained during fractionation are determined by the method of purification. The different procedures, as well as the most commonly used ones of Warner (95) and of Hipp and co-workers (30), have been reviewed recently by McMeekin (51, 52) and will not be discussed here. Electrophoretic analyses of the acid-precipitated casein (Hammarsten

NATURE OF PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

5

casein) indicate its heterogeneity (54, 95). I n addition, there is in the literature a wide divergence among the values for its molecular weight. Svedberg, Carpenter, and Carpenter (91) dctermined a molecular weight of 75,000 t o 100,000 and a sedirncntation constant of Szo = 5.6 X In contrast, Pedersen (63) reported a value of SZO = 12 X 10-13. The type of buffer used by these two groups of investigators differed, and this may explain the discrepancies in the sedimentation constant. Burk and Greenberg (12), with the aid of osmotic pressure measurements, found a molecular weight of 33,000 for casein in 6.66 M urea. It is apparent, therefore, that this protein undergoes association in the presence of salts. If 33,000 is assumed to be the minimum molecular weight, the acidprecipitated casein-complex with 0.8 % of phosphorus contains eight atoms of this element which are labile in 0.25 N sodium hydroxide a t 25°C. (15). The phosphorus resists the action of purified phosphomonoesterases (3, 79, 85). The fission of a few peptide linkages, however, with the aid of proteolytic enzymes renders some of the phosphate groups accessible to enzymatic hydrolysis (78, 79). In contrast to these findings are reports of Travia and Veronese (93), Lofgren (47), and others, who state that intact casein can be dephosphorylated b y phosphatases of mammalian origin. The findings of these investigators, however, may be due to the fact that crude extracts of spleen or kidney were used as enzyme source and that such preparations were contaminated with proteolytic enzymes. 2. Phosphopeptones from Casein

On treatment of casein with the aid of proteolytic enzymes, phosphopeptones are formed. However, there is a striking variation in the composition of the individual peptones. Posternak (75) isolated from a tryptic digest of cow’s casein a phosphopeptone with 5.9 % phosphorus and 11.9 % nitrogen. Moreover, he was able to demonstrate the presence of glutamic and aspartic acids, isoleucine and serine. This, as well as similar findings on a phosphopeptone from vitellin, led him t o suggest that the phosphorus in casein and vitellin was esterified in part a t least to serine (75, 76). Independently, Rimington and Kay (79) also prepared a phosphopeptone from a partial tryptic hydrolyzate of casein. Their product, however, contained 7.05 % phosphorus and 10.13 % nitrogen. Using the peptictryptic digestion method of Damodaran and Ramachandran (16), Mellander obtained the barium salt of a peptone with 5.35 % phosphorus and 7.05 % nitrogen which contained half of the original casein phosphorus (55). In view of these variations in the composition, i t is clear that there are striking differences in the chemical structures of these products. As will be discussed below, the following facts emerge from the work of two groups of investigators. Rimington and Kay demonstrated that

G

GEltTltUDE E. I’EltLMANN

casein resisted the action of phosphatases. 111 contrast,, hoivever, their phosphopcptonr preparations, which consisted of tcri to twelw amino acid residucs, ~vcrcreadily dcphosphorylattd. Thus, kidiicy phosphat:rw rcmoved all of the pcl~toiic-pbospliorus,mhercas only ti\-o-thirds was libcrated by t,he act ion of boiw phosphatasc (79). ‘l’hcsc investigators cxplain their results oil thc basis of cnzyinc specificity for crrtain types of bonds, e g . , that part of the phosphorus may be present in the configuration of nionoesters, whereas some of it is a different type of linkage (80). In their work, Posternak and Pollaczek draw attention to the fact that both the rrlative position of a phosphate group in the peptide chain arid tlic adjacent amino acids may be the factors determiiiiiig whether or not (*w zymatic hydrolysis occurs. These authors isolated two phosphopeptoncls, each of which contained three phosphate groups. Oiie of these, phosphopeptone I, consisted of ten to eleven amino acid residues, whereas phosphopeptone II had only seven. From phosphopeptorie IT all three phosphate groups are liberated with the aid of kidney phosphatase, whereas this enzyme removes only two of the phosphorus atoms of peptone I (78). Moreover, it is of interest that, as in the case of the dipeptide, O-phosphorylscrylglutamic acid, an intestinal aminopeptidase did not act on these peptones but that after removal of the phosphate groups with the aid of a phosphatase some of the peptide bonds were hydrolyzed by the actioii of the proteolytic enzyme. 3. Vitellin and Vitellenin

Whereas casein has been investigated rather extensively, little is known about the phosphoproteins from eggs, i.e., vitellin, vitellenin, and phosvitin, respectively. Two of these proteins are present in combination with phospholipids representing 25% of the egg yolk solids (2, 14, 62). After removal of the lipids with 80 % ethanol, vitellin and vitellenin are obtained with a phosphorus content of 1% and 0.29 %, respectively. Nothing is known about the homogeneity of these preparations. Since the literature pertaining to the properties of these compounds has been presented in the recent review of Fevold (2l), only a few observations which have a bearing on the present discussion will be recapitulated here.

4. Phosphopeptones from Vitellin and Vitellenin a. Vitellinic A c i d . Levene and Alsberg prepared from vitellin a peptide of high phosphorus content by extraction with 12 % ammonium hydroxide (36). After neutralization with acid and removal of the nitrogenous material with picric acid the copper salt, of vitellinic acid was obtained. This complex contained 9 % to 10% phosphorus and 0.6% iron. The phosphorus was labile in weak alkali at room temperature. b. Ovotyrines. I n coiitrast to the findiiigs of Levene and Alsberg (30)

NATURE O F PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

7

TABLE I CowLposition of Digestion Products 01 V i t e l l i ~ Composition in per cent

a

Compound

N

I’

Fe

Ovotyrine CY Ovotyrine p1 Ovotyrine 8 2 Ovotyrine y

10.87 11.33 10.92 10.70

13.76 12.55 12.09 7.90

None None 3.31 None

Taken from Posternak (761.

digestion of vitellin with the aid of proteolytic enzymes yielded three fragments which were designated as a-,p-, and y-ovotyrine (76). Ovotyrine p could be fractionated into p1 and pz . The composition of these substances is given in Table I. On the basis of the iron content these investigators identified their ovotyrine 02 with the vitellinic acid of Levene and Alsberg. Moreover, they emphasized that the high content of serine in ovotyrine pz (i.e., vitellinic acid) indicated that the phosphorus-containing unit of vitellin was phosphoserine. As discussed earlier this suggestion then led to the isolation of O-phosphorylserine from vitellinic acid (44). I n 1934, Blackwood and Wishart studied the action of pepsin and trypsin on vitellin (11). According to these workers pepsin splits the protein into a phosphorus-poor and a phosphorus-rich fraction. The acid-insoluble portion with 73% of the total phosphorus and a ratio of P/N of 3.65 was resistant t o further action of the enzyme. illthough treatment with trypsin also yielded two fragments, tryptic digestion differed in that the acid-soluble residue contained only 30% of the protein phosphorus and had a ratio of phosphorus to nitrogen of 1.5. I n the light of the work of Hergmann and Fruton a great deal of knowledge has been gained on the specificity of proteolytic enzymes for certain peptide bonds (10). Thereforc, the appareut contradiction of the results of Blackwood and Wishart is not surprising. I n addition it should be pointed out that the crude proteolytic enzyme preparations used by these investigators may well have contained small amounts of phosphatases. Fission of certain pc.ptide linkages, through the action of one enzyme but not by the other, thus may have rendcrcd some of the vitellin-phosphorus accessible to subsequent hydrolysis by phosphatases. 6. Phosvitin

As sho~vnby Mecham arid Olcott (53) 6 % of the total solids of egg yolk consists of the protein phosvitin with 10% of phosphorus, i.e., GO% of the

8

GERTRUDE E. PERLMANN

phosphoprotein-phosphorus of egg yolk is in this fraction. Although homogenous in the ultracentrifuge, phosvitin has at least two eleetrophoretic components. On the basis of osmotic pressure measurements, this protein has a molecular weight of 21,000. I n the presence of magnesium sulfate a value of 38,000 was obtained both with the aid of ultracentrifugation and by osmotic pressure measurements, indicating an aggregating effect of this salt on the protein. Amino acid analyses of phosvitin preparations also indicated inhomogeneity. It is striking, however, that an equal number of P-hydroxyamino acids and of phosphorus atoms were found to be present. The phosphorus is alkali-labile at room temperature. Phosvitin is readily dephosphorylated with the aid of a n acid phosphatase from citrus fruits (5). Bone phosphatase, on the other hand, does not liberate phosphorus. Moreover, the base binding capacity of this protein indicates that all of the phosphate groups are present in the form of monoesters with two dissociable hydroxyls. Although not reported here in detail, a striking feature emerges from the early work on phosphoproteins and peptones. Invariably, amino acid analyses revealed that the portions of the peptide chains which contain the phosphorus-probably esterified to serine-always are rich in leucine, isoleucine, aspartic and glutamic acids (78, 79). As will be discussed later in this article, these observations and some recent results indicate that certain types of amino acid sequences recur in all phosphorus-containing proteins. Another point of interest is the following : in the preceding paragraphs the lability of the phosphate groups of phosphoproteins in 0.25 N sodium hydroxide a t 25°C. has been stressed repeatedly. Although normally esters of phosphoric acid are resistant to the action of dilute alkali, Fond has shown that the monoesters of the glycerophosphates are readily hydrolyzed in this medium (23). As pointed out by Todd (92) the simplest explanation of this phenomerion is the formation of cyclic intermediates, i .e., triesters of phosphoric arid; such compounds are readily hydrolyzed in weak alkali. It, therefore, is feasible that the instability of the phosphoproteins in 0.25 N sodium hydroxide at rooin temperature may be due to the fact that also in these materials the phosphorus may undergo intxamolecular migration through cyclization involving adjacent groups, i.e., -NH2, -OH, or -COOH. The formation of such cyclic compounds which are known to be unstable is favored a t alkaline pH values and would explain the alkali-lability of the phosphate group in the protein in contrast to its stability in the phosphoamirro acids (72). Likewise, it is not improbable that a reverse situation cbxists in the case of an K-P bond. Moreover, it is not unlikely that the lability of an N-P linkage in dilute acid

NATURE O F PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

9

decreases considerably or is lost if the N-phosphorylamino acid residue is present in a peptide bond. From these observations it follows that (1) some of the chemical properties of a phosphoaniino acid may change considerably on incorporation into a peptide or protein, (2) that the adjacent molecular configuration may be responsible for the stability of the phosphate group, and (3) that the acidity of the medium determines whether migration of a phosphoric acid residue from the -0to the -Nposition occurs. As outlined in a previous section in this article N-phosphorylserine and N-phosphorylthreonine, respectively, would represent possible configurations of the N-terminal amino acid of a peptide chain in a native protein.

IV. ENZYMATIC DEPHOSPHORYLATION OF PHOSPHOPROTEINS Considerations of this kind led the author to undertake a n investigation using enzymes t o reveal the chemical nature of phosphorus bonds that may occur in phosphoproteins. This interest came through the accidental observation that a variety of phosphomonoesterases of mammalian origin and from plants will dephosphorylate ovalbumin, a protein with a low phosphorus content. Of course, a prerequisite in the selection of the enzymes for such work is that the dephosphorylation process should not be accompanied by any other enzymatic reactions that might result from the presence of small amounts of impurities in even highly purified phosphatase preparations; in particular, an extensive proteolysis has to be excluded. The phosphomonoesterases that proved most useful in this work, although free of proteolytic impurities, were found to be complex in their behavior toward phosphate esters. As indicated in Table 11, if tested with the aid of low molecular weight substrates, the intestinal (85) and the potato phosphatase (34) act on 0-P and N-P bonds, whereas the prostate enzyme (86) hydrolyzes only 0-P linkages.2 After the discovery of the specificity of two of these enzymes for low molecular weight N-P esters, it was noticed that the intestinal enzyme, although classified in the literature as “alkaline” phosphatase, hydrolyzes N-P bonds both a t p H 5.6 and 9.0, but not at pH 7.0. Since the pH range of 5 to 6 is that of maximum stability of almost all proteins, most experiments were carried out in this pH range. Thus the use of these three enzymes, either alone or in combination with each other, proved to be quite a powerful tool. A less desirable feature, however, also indicated in Table 11, is that these three phosphatases contain ah impurities siiiall amounts of phosphodicsterase and pyrophosphatasc. In contrast t o the monoesterases these eiizyiiic’s require the preaeiice of magnesium ions and act at pTI values 1 IR a recent article Max-hlgiller reported that acid phosphatase from seminal plasma hydrolyzes both 0-P and N-P esters (50).

10

GERTRUDE E. PERLMANN

TABLE I1 Dephosphorylation of Low Molecular U‘eight Phosphate Esters as Function o,f p H ~

Type of Phosphate Bond ~~

0

// -0-P4H

Enzyme

\OH

PfI

Prostate phosphatase Intestinal phosphatase

+

5.6 7.0 5.6 7 .o

-

+ +

5.6

2I

0

0

II

\OH

II

-0-PUP4-

dH

dH

dH

f

f f f

+f + +

0

II

-0-P-0-

-

=t

9 .o

Potato phosphatuse

0

// -N-P4H

+ active, f slightly active, - inactive. //

0

-0-P-OH

: 8-glycerophosphate,serine phosphate. oxyproline phosphate

\OH 0

// -N -P-OH

A

0 : N-(p-chlorophenyl) amidophosphate

\OH

II

-04-0-

A

: p-bis(nitrop1ienyl)phasphate

different from those a t which the moiioesterases are active. Hence their interference with the monoesterase activity could be avoided and their preseiice did not complicate the work. I n addition to phosphomonoesters, the occurrence, in these materials, of 0 0

I

II

linkages such as those of pyrophosphates, -0-P-0-P-0-, phospho0 0 I I I II OH or1 tlicstcrs, -0--1’-0- or -N-P-0-, aiid thc preseticc of thew holds

1

I

I

OH I3 013 i i i cyc*licarraiigelueiil , have to he anticipated. Thus it should be possihlc to deiiioiistrate such structural units with the aid of specific eiizymes which hring about their triLiisforniatioti into niorioesters that are then readily hydrolyzed I)y the wtioii of phosphonioiioc,sterascs. l‘hc purified phosphodiesterasc froiii rat1 lrsiiakc vetioiii, (‘rotulits udainuritms (ST),acting o i i thv -0-I’ 1)oiicl of ti tlic~htt~, slid the crystalliiw pyrophosphzltase f r o ~ n yeast (35) should Ix specific for thrh type of linkages ~nentioiic~l ahovc. Iiere one has to keep i i i n i i i i d , however, that not oiily thc spwifivity of :in c~iieyiiic~ for a. wrtaiii lmid hut also its adjucriit inolrci~larconfiguration may determitic slid modify the riizymatic action. This f:wt has recwitly bccii dcmotistratc>d experimciitally by Dekker (18). ~

NATURE O F PHOSPHOIIUS LINKAGES I N PHOSPHOPROTEINS

11

The author's work has been developed according to these general ideas mid will be presented as follows: 1 . A study of the dephosphorylation of ovalbuiniri, a protriii with the phosphorus in form of monocsttrs. 2 , An investigation of two typicd phosphoproteiiis, a- aiid fl-caseiii, which coiitaiii their phosphorus us moiioestcrs, diesters, and pyrophosphates. 3 . Work on the phosphorus of pepsin and pepsinogen. 1. Oualhumin

That ovalhmin is a phosphorus-containing protein was first demonstrated in 1900 by Osborne and Campbell ( 6 2 ) and was later substantiated

a.

t

Time in hours

,A1

Time in Electrophoretic composition hours

Atoms phosphorus per mole protein Computed Observed

85% AI 14% A9

trace AS 58% At 40%A2 trace A3 47% A1 49% A2 4% Aa 36% AI 58% Az 6%A3 94%Az 6% A3

1.8,

1.82

1.56

1.48

1.43

1.35

1.30

1.20

0.9,

0.9,

FIG.1. Dephosphorylation of ovalbumin with prostate phosphatase as a function of time (taken from PerImann (68)). Each reactmionmisture contained 4.6% ovalbumin and 0.01% enzyme. Electrophoresis was carried out in sodium phosphate buffer of p H 6.8 and 0.1 r/2 for 12,600 seconds a t 6.2 volts per centimeter.

12

GERTRUDE E. PERLMANN

by Sgreusen and collaborators (49). In 1949, Linderstrgm-Lang and Ottesen suggestml (43) that thc failurC2 of the phosphorus content of ovalhumin to correspond to an integral number of atoms per inolecule if the molecular weight were 24,000 could be correlated with the electrophoretic complexity of this protein (48). Thus the major, fast-moving component A1 should contain two phosphates a i d the slower moving protein A2 , one. The hypothesis advanced by the Danish workers mas coilfirmed by the experiments presented in Figs. 1 and 2. As shown in Fig. la, prostate phosphatase releases about 46% of the ovalbumin phosphorus. If the liberation of this element is followed with the aid of electrophoretic analyses (Fig. l b ) , it becomes apparent that the component A1 is transformed into

Time in Electrophoretic composition hours

A1

0 *2

d

85% Ai 14% A2 trace As

Atoms phosphorus per mole protein Computed Observed

1.84

1.82

0.97

12

50% Az 40% Aa

0.50

0.58

lo%*

0

FIG.2. Dephosphorylation of ovalbumin with intestinal phosphatase as a function of time (taken from Perlmann (68)). Each reaction mixture contained 4.6% ovalbumin and 0.006% enzyme. Electrophoresis was carried out in a sodium phosphate buffer of pH 6.8 and 0.1 r/2 for 12,600 seconds a t 6 volts per centimeter.

* Probably

due t o proteolysis.

13

NATURE O F PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

a protein with an electrophoretic mobility similar to that of Az , which has one phosphorus atom per molecule (65). If, on the other hand, either the intestinal phosphatase a t p H 9.0 or the potato enzyme a t p H 5.6 are added t o ovalbumin, the reaction is more complex. The protein is rapidly dephosphorylated until 46 % of the phosphorus is released (Fig. 2a). Here, A1 is again converted into a protein with the properties of A2 . Dephosphorylation, however, eoiitinues and a iicw component, AS, appears which moves more slowly than A2 and is a phosphorus-free ovalbumin (line 4, Fig. 2b). Thus: Proytnte intestinnl or potato) phosphata’se

Oval bumiii (85% A1:2P/mole) (15% At:lP/mole)

Intestinnl or yotnto

,

phosphatltse

2‘

AI loses 1 atom of phosphorus

-42 loses 1 atom of phosphorus

-

At

One point of interest resulting from these experiiiients is the difference in the electrophoretic behavior of A 1 , Az , and A3 . In general, as the p H of a protein solution is increased, various groups within the protein molecule

I

4.0

I

5.0

I

I

6.0 7.0 pH - 0°C.

I

8.0

I

9.0

FIG.3. Mobilities of the ovalbumin component A1 and the dephosphorylated ovalbumin At and A2 as function of pH.

14

GERTRUDE K . PKltLMAITK

lose their protons. Therefore, a coniparisoii of the electrophoretic mobilities of two proteins that are almost identical, except that one is formed from the other by removal of a few charged groups, gives a qualitative picture of the type of groups involved. Morcw-er, as show1 helow, at a given pH the evaluation of the number of groups lost i n the reactiori is possible. In Fig. 3, the electrophoretic niohilities of A, , h2, and A 3 are plotted as a function of pII. 'L'hescl curves diverge until a coilstarit mobility difference of Au = 0.6 X l W 5 is reacbhecl in the p1-I range of 7.0 to 9.0. If this mobility differelwe is corrclated with the haw hidiiig capacity of ovalbumiri (13), Au corresponds to a charigeiu the net charge of -2 (64, 6'3). I t , therefore, can be inferred from these measurements that the phosphate groups of A1 and A:! in the pH raiige of 7.0 to '3.0 are present as nionoesters with two ionized hydroxyls. In the pH range of 4.5 to 5.0, however, the mobilities differ by a value of 0.3 x lW5. Here the removal of each phosphorus is accoiiipaiiied by the loss of one itegzbtive charge only, RS indicated in Fig. 3. That the phosphorus is present as a monoester is further supported by the finding that on pretreatment of ovalbuinin with the phosphodiesterase from snake venom and subsequent iricubatiori a t pH 5.6 with prostate phosphatase, the same amount of phosphorus is released as with this enzyme alone, i.c., 4G %. Moreover, no change in the electrophoretic behavior occurs (72). The presence of a diestm in ovalbuinin haviitg thus been excluded, 011 Iht. basis of electrophoretic aiid enzymatic evidence, the failure of the prostate enzyme to remove the remaining phosphorus may be taken as a11 indicatioii either that the two phosphate groups of A 1 arc csterified to two different amino acid residues of the protein or that the adjacent molecular configuration is different in the two cases aiid renders one of the phosphate groups inaccessible to the action of the prostate enzyirie. In this coiiriection it is of interest that if ovalbumin is digested with pepsin at pH 1.5, followed by treatment with trypsin and chyniotrypsiii at pH 5.0, prostate phosphatasr still fails t o remove more than 46% of the phosphorus. Moreover, 0 1 1 hydrolysis of the phosphorus both of the intact protein and the proteolytic digest with 0.25 N sodium hydroxide at 37"C., liberation of 46% occurs rapidly, followed by a much slower release of the remaining phosphorus. This indicates that even on extensive degradation of the protein to polypeptides, no additional phosphorus becomes more readily accessible to the action of the enzyme, nor does the kinetics of hydrolysis in 0.25 N sodium hydroxide change ( 7 2 ) . On fractionation on starch columns of a partial proteolytic digest such as that described above a peptide fraction was isolated containing aspartic,

NATURE O F PHOSPHORUS LINKAGES I N PHOSPHOPROTEINS

15

acid, glutamic acid, alaniiie, leucine, and seriiie and having about 50 % of the phosphorus of thc digest (68, 7 2 ) . I t thus caould bc c~onc~luded that half of the ovalbumin phosphorus is present :is phosphoserinr. Recently t l i ~orcurreiicbo of phosphoserinc i n ovalhumin has hren confirmed by Iplavin (22). I n addition to phosphosrrinc Flavin s i u from it partial acid hydrolyzatc the follon-ing pcpl ides: hsp.SerP; Asp.(Glu, SerP) ; Asp.(Crlu,Ileu,SerP) ; SerP. (hla,Glu,Ileu); Asp.(Ala,Glu,Ileu,SerP) ; SerP.Ala; aiid possibly Glu. (Ala,SerP).3 After dephosphorylatioii of ovalbumiii with the prost ate phosphatase, SerP.Ala aiid Glu.(Ala,SerP) could no longer be detected in the acid hydrolyzate. He therefore coricludes that the one phosphoric acid residue hydrolyzed by the prostate phosphatase is csterified to seririe and is present in the sequence Glu.SerP.Ala. One point of interest emerges from these experiments, namely, that ovalbumin is the secoiid protein from which phosphoserine has been isolated. As in the case of the dipeptide, SerP.Glu, isolated from casein (38,77) ovalbumiii also contains an amino acid sequence with phosphoserine adjacent t o a dicarboxylic acid. Moreover, the close association of this amiiio acid with aspartic and glutamic acids, isoleuciiie and alariiiie in ovalbumin (22, 68) and casein (78-80) suggests the existence of a systematically recurring sequence in phosphoproteins. The nature of the second phosphorus bond in A 1 is still uncertain. The observations made in this laboratory that the intestinal enzyme a t pH 5.6 hydrolyzes low molecular w i g h t substrates with N-P bonds only would suggest that thc sccoiid phosphate of ovalbumin is ail N-P ester, e.g., that the phosphorus is linked to the guanido group of arginine, t o the e-amino group of lysine, or t o a terminal a-amino group. 111 contrast t o this view based on the onzymatic findings are, however, the results of Flavin, who, in addition to the amino acid sequence Glu.(Ala,SerP), found a small amount of a second phosphoserine-(.oiitainiiig peptide Asp. (Glu,SerP) in the partial acid hydrolyzate of ovalbumin. From this he concludes that the second phosphorus of A1 is also linked to seriiie but that the adjacent amino acids are diffrrcrit. It is of course feasible that such a specific molecular configuration, i.e., Asp.(Glu,SerP), may modify the enzymatic reaction. However, in view of the increasing evidence of amino acid migration and rearrailgenielit of amino acid sequenres during acid hydrolysis (82, 83), caution has to bc exercised in drawing final conclusions as to the 3 The abbreviations for amino acids and the conventions for indicating their sequence in a peptide are those of Sanger and Tuppy (81) ;in a known sequence of amino acid residues, the amino acid symbols are separated by periods; if t h e sequences of amino acids in a peptide are unknown, they are enclosed in brackets and are separated by commas. SerP = 0-phosphorylserine (22).

1G

GERTRUDE E. PEHLMANN

nature of this second phosphorus bond until additional experimental evidence has been obtained. Esterification of the phosphorus to the polysaccharide moiety of the protein, however, can be excluded, since it was possible to isolate from a partial proteolytic hydrolyzate of ovalbumin a peptide fraction that contained all of the carbohydrate but was phosphorus-free (72).

2. Casein Although it has been reported in the literature (3, 79, 85) that casein is resistant to the action of purified phosphomorioesterases from mammaliati tissues, this protein was nevertheless chosen as an example of a typical phosphoprotein for the reasons outlined below: That so-called acid-precipitated casein (Hammarsten casein) is a mixture of several distinct proteins was first demonstrated by the solubility studies of Linderstrgm-Lang and Kodama (41). This led to numerous attempts to fractionate this protein into its components, and in 1929 LinderstrgmLang achieved a separation into three fractions characterized by a phosphorus content of 0.96 %, 0.52 %, and 0.1 %, respectively (42). Mellander, in his electrophoretic stJudies,showed that casein has three electrophoretic components which he designated as a-, p-, and y-casein (54). It was, however, only in 1944 that Warner succeeded in preparing a- and p-casein, proteins with a phosphorus content of 0.99 % and 0.6 %, respectively, the a-protein being present in the original mixture in concentrations of 75% to 80% (95). As can be seen from the electrophoretic patterns shown in Fig. 4, these two proteins, although not homogenous over the entire p H range, were distinct fractions, neither of which was contaminated with the

a.

a

A

Unfractionated Casein 0 a a d

-a

&

Y

d

a-caaein b . 1

d

-a

d-

D 1

C.

-a

a

A

E

p-casein 6

&

-

0

L

d -

FIG.4. Electrophoretic patterns of unfractionated casein, a-casein, and @-casein (taken from Perlmann (69 )). Electrophoresis was carried out i n sodium phosphate buffer of p H 6.8 and 0.1 r/2 for 14,400 seconds a t a potential gradient of 4.95 volts per centimeter.

17

NATURE OF PHOSPHORUS LINKAGES IN PHOSPHOPROTEINS

other (95). Amino acid analyses of the caseins revealed that the rat,io of the basic to the acidic amino acid residues is the same in both cases, namely, TABLEI11 Composition of a-Casein and @-Casein ~~~

Total nitrogen, per cent5 Total phosphorus, per cent= Total cationic groups, equivalents/l06 g . proteina Total anionic groups, equivalents/l06 g. protein" Cationic groups/anionic groups u X lo6 cm.* set.-' volt-lb

a-Casein

@-Casein

15.53 0.99 115 112 1.03 -7.5

15.33 0.61 91 88 1.03 -3.4

Taken from Gordon, Semmet, Cable, and Morria (26).

* Electrophoresis in sodium phosphate buffer, pH 6.8, 0.1 r/2. 1.03 (25). From this it is apparent that a-casein, the protein with the higher phosphorus content and an electrophoret#icmobility which a t p H 6.8 in a sodium phosphate buffer of 0.1 ionic strength is considerably more negative than that of p-casein, might possess a number of phosphate groups as monoesters. Therefore, a phosphomonoesterase should act on this protein but not on &casein. As shown in Table IV, prostate phosphatase liberates 40 % of the a-casein phosphorus but has no effect on the p-protein. On prolonged exposure of unfractionated casein t o the enzyme about 12 % of the total phosphorus is liberated (66). These findings were recently confirmed by Sundararajan and Sarma (90). TABLE IV Action of Prostate Phosphatase on Casein Fractions Each reaction mixture contained 0.5% protein and 0.005% enzyme in sodium cacodylate buffer of p H 6.1 and 0.1 r/2. Phosphorus released by Time of enzyme (per cent Phosphorus Incubation at of total proteincontent, (%) 37°C. (hours) phosphorus) Unfrrtctionutrd Csseiii

0.8

U-Caseiii

1.o

6 24 6

21 $-Casein

0.6

6 24

0 12.5

24 42 0 0

18

GERTRUDE E. PERLMANN

I n experiments in which a- and p-casein are remixed in different proportions, it is noticed that if the relative concentration of the p-casein in tile mixture exceeds 20 % the presence of this protein inhibits the enzymatic action, the degree of inhibition being proportional to the concentration of the 0-protein. These results inay be taken as an explanation for the failure of previous investigators to dephosphorylate unfractionated casein without preceding transformation to phosphopeptones. As in the case of ovalbumin, the dephosphorylation of a-casein is accompanied by a change in the electrophoretic behavior. With the aid of Fig. 5, it is illustrated that the liberation of phosphorus is accompanied by thc appearance of several new componeiits with lower mobilities. The mobility decrements of these components at pH 6.8 are 0.5 X l W 5 or a multiple thereof. If compared with the base binding capacity of a-casein (31) this value corresponds to ;t change in the net charge of -2. This supports the initial assumption that some of the a-casein phosphorus is present in form of monoesters. a - casein

I\

6

s a-

I

6hOUP5 a -

Q

6

d.

c-----+

Fit,. 5 . Tracings ot electrophoretic patterliS of a-Cabeln Iwloic :LMI alter treat ment Kith prostate phosphatase (taken from Pel lmann (6!))) F:lectrophoreqis \\its carried out in sodium phosplintc hultcr o i p I I (j X : i n t i 0 1 1'/2 for 10,800 seconds at 4 75 voIts per centimeter.

10

NATUHE OF PHOSPHORUS LINKAGES IN PHOSPHOPliOTEINH

The observatioii that prostate phosphatasc liheratrs only 40 % of the a-casein phosphorus and doc5 not act on S-casein foreshadowed the exist ence of phosphodiesters and pyrophosphate bonds in these proteins. Thus the action on these materials of enzymes specific for such bonds should cstablish not only the nature of the linkages but also the proportions in which they occur. The results of such stepwise enzymatic analysis can be best followed with the aid of Table V. If a-casein is treated with either the crystalline pyrophosphatase of yeast a t pH 7.0 (35) or with the snake venom diesterase a t pH 8.2 (87), no inorganic phosphorus is released. However, if the diesterase reaction is carried out in weakly buffered solutions a small drop of p H takcs place (71), indicating the exposure of acidic groups. Subsequent incubation of the diesterase-treated a-casein with prostate phosphatase a t pH 6.0 liberates no more phosphorus than in the absence of the diesterase. If, however, prostate and intestinal phosphatase are added, 78% of the a-casein phosphorus is set free. Since the intestinal enzyme at pH 6.0 acts on low

Yo

molecular weight substrates of the type -JS-P-OH but not on 0 I \ // H OH -0-P-OH, it can be inferred from these results that a-casein contains

\

OH diester linkages of the -N-P-0type (71). If pretreatment of a-casein with pyrophosphatase is followed by incubation with the prostate enzyme, approximately 60% of the phosphorus is liberated instead of the 40% with the prostate phosphatase alone. Thus the difference of 20% presumably originates from monoesters of the type 0 0 0

//

II

II

I

I

derived from a pyrophosphate bond -0-P-0-P-0-

-0-P-OH,

\

.

OH OH OH Finally, if a-casein pretreated with the diesterase a t pH 8.0 and the pyrophosphatase a t pH 7.0 is exposed to the action of both the prostate and the intestinal enzynie, all of the phosphorus is liberated. These experiments thus demonstrate that a-casein contains 40% of its phosphorus as mono0 O

//

esters -0-P-OH,

\

OH

40 % as diestcr -0-P-N-,

II

I

and 20 % as pyrophos-

I

OH H

TABLE V Slcpwise Enzyttmtic Dephospho rylation o j cY-CuseinI., : i t i d Fruton, J . H. (1941). :ttlo~ttcesi i i Enzymol. 1, 63. 11. I3lack\vood, d . I t . , :ind Wishart, (;. A l . (1934). R i o c h t m . . J . 28, 550. 12. 13urk, S . E’., :iritl C;reetil)erg, D. &I. (1!130). J . Biol.(‘hetit. 87, 197. 13. Canniiii, I t . Io 16.95u t o 17.1 12.1

17.0

a

Hablio and l’hilipenko (1947) Kolthoff, Leussing, and Lee (1951) I.Luger, Fallsh, and Erlenmeyer (1955)

The constants fo ol,ol-‘dipyridyl were concentration-dependent.

matic inhibition, on the ease of exchange between bound zinc and free zinc or other metal ions, and on the contribution of zinc to the stability of the protein. It may be of interest that zinc chloride alone and zinc cysteinatc do not have any activity toward CGP (Vallee and Coombs, unpublished data). These studies of inhibition of carboxypeptidase support the conclusion that zinc is both a structural and functional component of the enzyme and that i t participates in its catalytic action.

4. Physiological Implications It has been reported that a large fraction of Zn66administered to dogs is excreted in pancreatic juice (Montgomery, Sheline, and Chaikoff, 1943). KO explanation for this finding has been offered. These data make it appear likely that a t least part of this zinc is associated with carboxypcptidase in pancreatic juice. XTII.

ALCOHOLDEHY1)ROGENASE I . Physical Properties

YEAST

Yeast alcohol dchydrogeiiaac was crystallized from brewer's yeast by Segelein and Wulff (1937) and found to be dependent upon DPN for its activity by Anderson (1934). A distinctly different alcohol dehydrogenase was crystallized from horse liver by Bonnichsen and Wassen (1948) and Bonnichsen (1950). The present discussion will be concert led primarily with those structural and functional aspects of the yeast enzyme which

354

BERT L. VALLEE

relate to its characteristics as a zinc metalloenzyme (Vallee and Hoch, 1955a, b). Its physical, chemical, and enzymatic properties have been reviewed and discussed extensively (Schlenk, 1951; Singer and Kearney, 1954; Velick, 1954; Racker, 1955). Data obtained with the mammalian enzyme will be referred to where deemed pertinent, since a completely separate discussion of these two systems is difficult a t best. The enzyme is prepared from dried baker's yeast by extraction, differential heat denaturation, and fractionation with acetone and ammonium sulfate (Racker, 1950; Hayes and Velick, 1954). Thin platelike crystals result. The enzyme is stable in the dry state and in solution a t pH 7 and 0" C. but is destroyed rapidly below pH 4.5 and above pH 8.5. Electrophoresis of a crystalline preparation revealed one major enzymatically active component and a minor inactive one (Negelein and Wulff, 1937). The smaller, inactive constituent varied from 5 % t o 20% of the total protein. Re-examination of the electrophoretic properties of yeast ADH crystals confirmed the presence of one slow, presumably active, and one fast-moving, presumably inactive component. Their relative amounts did not change systematically with recrystallization as shown by area analysis. The percentage of inactive component was a function of age of the solution and duration of preliminary dialysis. Preparations dialyzed for 20 hours a t pH 5 contained as much as 14 % to 25 % of the inactive coinponent, while dialysis for 4 hours showed as little as 6 %. The second component was assumed to be an inactive transformation product of the active enzyme (Hayes and Velick, 1954). The molar absorption coefficient, E , at 280 mp is EN, = 1.89 X mole liter-1/cm.-2. Measurement of the optical density a t 280 and 280 mp measures the degree of removal of DPN and DP N H from the apoenzyme. The O.D.zso/O.D.zeo of a highly purifiedpreparationwas 1.82. The sedimentation constant, S20,w = 6.72 X I&l3 sec-l. Diffusion measurements gave a diffusion coefficient D z o ,=~ (4.70 f 0.03) X lO-' cm.2sec-'. The partial specific volume D is 0.769 ml. per gram. The molecular weight, was cnlculatpd t o t e 150,000 (Hayes and Velick, 1954; Velick, 1954). Pedersen, quoted by Theorell and Bonnichsen (1951a), found a sedimentation constant S20,w = 7.61 X l&13. The molecular weight calculated 011 this basis is about 140,000 (Bonnichsen, 1953). The molecular weight of horse liver ADH was shown t o be 73,000, about half that of the yeast enzyme (Theorell and Bonnichsen, 1951a). Neither of the ADH enzymes is isolated as DPN complexes, but each has a strong and characteristic affinity for the reduced and oxidized forms of the coenzyme (Velick, 1954). The O.D. 280/260 ratio measures protein and nud~ot i dec*omponentsof a particular ADH preparation, and a high ratio of a pure protein denotes removal of DPN or DPNH from the apocnzyme.

ZINC AND METALLOENZYMES

355

Horse liver ADH binds two molecules of DPNH per molecule of ADH between pH 7 and 9; a t pH 10, about one DPNH per one ADH molecule is bound. These data were obtained by taking advantage of the lowering of the absorption maximum of DPNH and its shift from 340 to 325 mp on the addition of mammalian ADH to DPNH (Theorell and Bonnichsen, 1951a). While the velocity of association with the liver enzyme is apparently equal for DPN and DPNH, the ADH-DPNH complex has a dissociation constant, a t equilibrium, of lW7M at pH 7.0, and the calculated dissociation constant for ADH-DPN is 2 X lo+’ M . Thus D P N is bound about 200 times less “tightly” to the mammalian enzyme than is DPNH (Theorell and Bonnichsen, 1951a). It was concluded that both are bound by ADH a t the same locus. The difference in the turnover number of liver ADH for two substrates was explained in part on the basis of the difference in strength of binding between ADH-DPN and ADH-DPNH (Theorell and Chance, 1951). No change in wavelength or absorbance of DPNH a t 340 mp occurs on its combination with yeast ADH. However, Hayes and Velick (1954) demonstrated that a total of 4 moles of DPNH and/or DPN are bound to 1 mole of the yeast apodehydrogenase a t pH 7.8, utilizing an ultracentrifugal separation method (Velick, Hayes, and Harting, 1953; Velick, 1953). The four binding sites appeared to be equivalent. The dissociation constant was 2.6 x 10-4 M for yeast ADH-DPN and 1.3 X 1 e 6 M for yeast ADH-DPNH. The respective Michaelis constants were of the same order of magnitude. DPNH replaces D P N competitively, while the total amount bound remains constant. Evidence that DPN and DPNH compete for the same catalytic site on ADH was also obtained kinetically. These authors assumed the formation of a yeast ADH-acetaldehyde complex with a dissociation constant of 1.8 X lW4 M , a t pH 7.9, 26” C., to explain an observed equilibrium shift (Hayes and Velick, 1954).* It has been suggested that this apparent agreement between kinetic data and theory may be fortuitous, however (Racker, 1955). 2. Chemical Composition a. Basic Composition. The dry protein contains 52.8% C, 6.96% H, 16.54% N, 1.21% S, 0.015% P, 0.0027% Fe, 0 % Cu. The presence of aromatic amino acids has been inferred from the 280 mp absorption band (Negelein and Wulff, 1937). Though the amino acid composition is unknown, Hayes and Velick (1954) concluded that the yeast enzyme has a higher content of aromatic amino acids than the mammalian enzyme, based on the absorption coefficients a t 280 mp. The partial specific volume 3 They also obtained good correspondence between equilibrium and kinetic data by assuming the formation of a ternary complex, ethanol-ADH-DPN (Hayes and Velick, 1954).

BERT L. VALLEE

356

Tmm

1s

Mefal (‘onterbf vJ’ Different (‘rystulline I’eust A D H I-’reparulzvns (All values in pg. of metal per g. of protein) 4 5 2 3 1 Metal

Zinc Magnesium Calcium Aluminum Barium Strontium Lead Cadmium Chromium Iron Copper Moles zinc/molc protein Rloles maguesium/moles protein

Expt . tcxpt. 1Sxpt. Expt . Expt . Expt. Expt. 8-225 !6-156 8-200 3-178 23-91 13-135 23-148

___

___-

1 ,440

,600 130 0 500 28 0 0 0 0 0

296 105 48 20 2 0 0 0 81

1,660 1,180 39 79 11

4

3.3

3.7

45 13 8 80 165 3.8

1.8

0.80

7.3

*

*

,910 95 .,103(143), 127 Gibbs, 11. J , , 215, 216, 219, 220, 222, 227, 226

Gibson, .I. (i.,[ I , 835. M!), 340, 345, 379, S84 Gibson, ( 2 . I I . , 35(28X), I:?/ Gillis, J., 163, 223 Gilvarg, C . , 37(964a), 30(288a, 907), 43, 131, 148, 149 Ginoulhiac, E., 86(292), 88(620), P9(293), 131, 140 Glass, B., 333, 381 Glassman, H. N., 167,174,223 Glasstone, S., 214, 223 Glazer, H . S.,94(294), 181 Gleysteeu, L.F., 165, 167, 168, 172, 225 Glynn, I,. E., 49(205), 52(295), 131 Glomset, J., 2,27 Goldblith, S. A , , 272, 286, 287 Golder, Ti. H., 281, 286 Goppert, R., 228, 283 Goldacre, 1’. J,., 114(296), 132 Goldbloom, A , , 60(611), 140 Goldenberg, RI., 66(297), 162 Goldsmith, G . A , , 80(755), I43 Goldstein, A., 220, 223 Goodall, MrC., 66(298, 299), 132 Gooder, €I., 92(300), 111 ( N O ) , 132 Goodland, It. l,,, 55(789), 57, 60(791), 144 Goodloe, RI. 13., 273,279, 283 Goodman, D. S., 173,223, 320, 380 Goodwin, S., 63(946), 99(946), 14.9 Gorbman, A4.,75(718), 142 Gordon, H. H., 52(5,55, 556, 557), 60 (648a), 138, 141 Gordon, &I., 39(301), 41(302), 132 Gordon, 9. A . , 111(303, 304), 132 Gordon, W. G., 17, 28 Gorini, I,., $25 Gortner, W. A , , 114(305), 132 Goryachenkovo, I+:. V . , X9(102), 01 (306), 126, 132

Gots, J. S., 40(307), 132 Goutarel, It., 119(308), 1% Govan, C. D., Jr., 60(648a), 141 Govier, W. M., 107(278), 131 Grabar, P., 5(3), 16(3), 28 Graham, B. IC., 107(278), 131 Gralcn, N., 194, 223, 253, 284, 331, 383 Granick, S., 320, 333, 379

Graser, G., 85(947), 149 Grnu, C . R . , 59(309), 132 Green, A . A , , 44(861), IOJ(B!)3), / 4 2 , 141, 181, 187, 222 ({repn, D. E., 59(G8, 69), 77, 126, /&‘, 3 l ! l . 323,324,330,333,879, S R / Green, H., 26(58), 29 Green, X. M., 343,344,380 Green, NI. M., 85(310), I36 Greenberg, D. M., 5, 21(12), 28, 57(752, 933), 59(523, 560), 61, 04(523), 137, 138, 146, 148, 174, 222, 223, 251, 285, 319, 322, 343, 381 Greenberg, L. D., 94(311), 132 Greenberg, S., 78(100), 126 Greene, C. W., 228,284 Greenstein, J. P., 65(592), 139, 181, 185, 214,216, 217, 219, 220, 224 Grifliths, R , 54(961a), 149 Gripenberg, J., 116(312), 132 Gros, H., 52, 58, 232 Gros, P., 85(314a), 132 Groschke, A. C., 91(12,315), 124, 132 Gross, J., 74, 75(320, 321), 132 Gross, O., 48, 139 Gross, S. R., 39(856), 1.46’ Grossman, W. I., 83(492), 102, 135, 137 Groves, M. L.,4(30), 18(31), 28, 154, 217, 220, 223 Guba, F., 237,245,257,258,284 Gudaitis, H., 94(898, 899, 900), 148 Guggenheim, E. A., 202,223 Cuggenheim, M., 65, 66(324), 107(325), 132

Gullberg, M. E., 81(371), 133 Gunsalus, I. C., 41(890), 76(326,327), 111 (890, 95i), 132,14r, 149 Gurd, F. R. N., 173,266,320,325,326, 58F Gurin, S., 55,66(328), 132, 144 Gustafson, F. G., 113(329), 132 Gustafsson, K., 100(423), 165 Gustavson, K. H., 277, 278, 279, 284 Gutfreund, H., 218,219,223,267,28? Gyarfas, E C., 363,366,378

H Haan, A. M. F. H., 240,252,284 Haagen-Smit, A. J., 79(551), 80(857), 110(330), 113(496, 497), 132, 137, 138, 147 Haberland, G. L., 76(330a), 132

AUTHOR INDEX

Hackman, R. H., 71(331, 332), 132 Haddox, C. H., 39, 133 Hahn, G., 118(334,335,336,337,338), 133 Haines, W. J., 35(728, 729), 143 Haissinsky, M., 377, 380 Hakala, N. V., 334, 382 Hakim, A. A . , 111(339,310),155 Halawani, A., 60(271), 13f Halikis, D. N., 58(164), 65(161), 158 Hall, C., 329, 380 Hall, I). A., 111(202), 129 Hallman, I,. P., 46(130), 49(130), 167 Halsey, J. T., 34(227), 130 Hamberg, U., 66(245), 130 Hameed, K. A., 104(283), 131 Hamill, R. I,., 121(132), 127 Hamilton, A., 60(588), 139 Hamilton, T. S., 231, 234,286 Hamlin, K. E., 104(341), 133 Hammarsten, O., 4, 28 Hamoir, G., 154, 222, 235, 237, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 277, 280, 281, 284 Handler, P., 101(342, 533), 103(429), 133, 135, 138

Hanke, hI. T., 76(495), 77(495), 137 Hankes, L. V., 80(381), 81(343), 97(344), 133, 134

Hansel, A., 118(335), 133 Hanser, G.,84(117),89(124),106(124), 127 Hanson, H. T., 344,349,350,352,363,383 Hanson, J., 228, 285 Happold, F. C . , 10(167), 76(346), 78(167, 346), 92(300), Il0(28), 111, f.24, 128, 129,138, 193

Hara, >I., 52(852), 146 Hara, R., 364, 367, 380 Hardin, R. I’., 320,333,380 Harington, C. R., 71, 72, 73, 74, 127, 133 Harley-Mason, J., 41, 68(359), 69(112, 113), 70, 91(361), 104(363), 105(165, 360), 107(363), 110(362), 126,187, 128, 133 Harpur, E. R., 60(611), 140 Harris, D. L., 22(27), 26(27), 28 Harris, 1,. J., 110(494), 137 Harris, M . , 153, 154, 160, 161, 165, 166, 167, 168, 169, 172, 177, 178, 199, 223, 235

393

Harris, R. S., 228, 269, 284, 320, 383 Harrison, G. A , , 112(364), 193 Harrison, G. R., 329, 380 Harshman, S., 15(83), 30 Hart, E. B., 334,336,380 Harting, J., 355, 384 Harvey, H. W., 291, 315 Hashimoto, K., 255, 286 Iiasltins, F. A , , 39(301), 40, 12(650), 132, 133, 141

Hasselbach, W., 236, 237, 244, 284 Hastings, A. B., 336,380 Ha ta , M., 231, 288 Hatz, F., 88(941), I49 Haugaard, N., 334, 380 Haurowitz, F., 320,333,380 Hawkins, V. R., 94(294), 131 Hayaishi, O., 78(826), 83(366), 84(743), 85(366), 91 (367), 108(826, 827, 828, 874), 133, 143, 146, 147 Hayes, J. E., Jr., 331, 354, 355, 357, 35S, 363,367, 374,380, 384 Hays, E. E., 116(368), I S 3 Ileden, C. G., 41(686b), I & Hegsted, D. Rf., 100(562), 138 Heidelberger, C., 81(369, 370, 371), 133 Heidelberger, AT., 181, 185, 217, 223 Heilmeyer, L., 196, 223 Heimberg, hI., 88(372), I33 Heinrich, W. D., 94(400), 134 Heinsen, H. A., 67(373), 68(373), 134 Heinzelmann, It. V., 104(821), 145 Heise, It., 66(406), 134 Hellerman, L., 319, 327, 380, 382 HelImann, H., 84(117), 87, 89(376), 98 (376), 99(374), 127, 134, 149 Hellmann, K., 107(79), 226 Hellner, S., 66(245), 130 Helwig, H. L., 254, 285 Hempelman, I,.H., 76(776a), f 4 4 Henbest, H. B., 114(377), 115(107, 377, 452), 126, 134, 135 Henderson, J. AT., 114(378), I34 Henderson, L. M., 80(381), 81(387), 86 (385,387),89(387), 91 (384), 97,98(382, 763), 99(386, 564), 100(564), 110(517), 134, 137, 138, 154 Hendrix, B. M., 185,023 Henrotte, J. G., 241, 252, 253, 285 Henschen, G. E., 23 Henze, M., 75(388), 134

394

AUTHOR INDEX

Hermanns, J,., 104(389),134 Hernler, F., 322, 380 Herriott, R. kl., 22(28), 24, 28, 168, 170, 171, 223 Herter, C. A., 110, 134 I-Ierter, 15., 76(38), 125 Hesse, G . , 107(913),148 Heymanil, €I., 87(140), 127 Heytler, P G., 333, 379 Hibiki, S.,308, 316 Hickman, E. R l . , 35(65), 54(64, 65), 125 Hicks, C. S., 196, 223 Hill, D. W.,3, 15(38), 28 Hill, H. N., !)8(382), 09(564), 100(564), 134, 138 Himsworth, H. l’., 49(295), 52(295), 131 Hingerty, D., 234,283 Hinsvark, 0. N., 109(417),135 Hipp, N. J., 4, 18(31), 28, 154, 217, 220, 223 Hirai, K., 78(391), 134 Hirata, Y., 81(392), 134 Hird, F. J. It., 59(393), 134 Hirs, C. H. W., 53(832), 146 Hirsrh, H. RI., 07(383),134 Hishikawa, M., 94(509), 1.97 Hitchcock, D. C., 198,199,223 Hoagland, C. I,., 101(3!?4),f34 Hoch, F. I,., 320, 323, 324, 325, 328, 331, 333, 339, 340, 341, 342, 345, 354, 356, 358, 360, 361, 362, 364, 365, 366, 367, 369, 380, 384 Hoffman, M., 113(956),149 Hoffman, 0 . D., 4(8), 28 Hogben, L., 48(395), 134 Hogness, D. S., 103(542), 138 Hogness, J. R., 103(738),143 Holden, H. F., 196, 201, 223 Holley, A. D., 115(396),134 Holley, R. W., 115(396),184 Holman, W. I. M., 80(399), 97(397), 103 (397,398), 134 Holmberg, C. G., 322, 330, 380 Holt, L. E., Jr., 54(816a), 80(817), 145 Holt, P. F., 110(401),134 Holton, P., 50(80), 126 Holtz, P., 66(402, 404, 405, 406, 407), 68 (4031, 194 Homer, A., 79(408), 110(409), 112, 113, 134, 135

Honsley, S.,115(51), 185 Hopkins, F. G., 34,35(410), 109(412),110, 131, 135 Hoppe-Seyler, F., 112(413),135 Hoppe-Seyler, G., 111(414), 135 Horecker, B. I,., 44(415), 136, 327, 380 Horwith, M. K., 277, 289 Hoshino, T , 107(416), 136 Houff, W. H., 109(417), 135 Hough. A., 165, 169, 223 Hove, E., 334, 336, 380 Hoyle, I,., 110, 133 Hsia, D. Y. Y., 62(209a), 129 Huebscher, G., 322,381 Huff, J. W., 80(731), 81(732), 88(732), 89 (732), 94(732), 102(181),128, 135, 143 Hughes, D. E., 101, 103(420), 155 Hughes, G. K., 117(422),135 Hughes, T. R., 327,380 Hughes, W. Id., Jr., 173,223,320,323,328, 333, 341, 342, 384 Hultin, T., 100(423), 135 Humphrey, J. H., 106, 135 Hundley, J. M., 81(427), 103(426),135 Hunt, C . R., Jr., 40(307), 192 Hunter, S.F., 59(428), 103(429), 136 Hurt, W. W.,80(430), 135 Hutchings, B. TA., 116(903),148,333,879 Hutchison, W. C., 25(17), 28 Huys, J. V., 253, 285 Huxley, H. E., 228, 285

I Ichihara, K., 60(878), 61, 62,78(502, 654, 879), 89(434), 111(742, 877), 136, 196, 137, 141, 143, 14Y Ihle, J. E. W., 228, 285 Ikawa, &I.,78(593),91(593), 139 Inada, T., 94(503, 504, 505, 506), 137 Inagami, K., 71(431), 86(575), 155] 133 Ingraham, J. L., 78(568,829), 139,lqS Ingraham, L. L., 116(432),136 Inouye, A,, 96(510), i3Y Irai, I., 40(5), 1.94 Irisawa, A. F., 273,285 Irisawa, H., 273,286 Iritani, H., 84(11), 12.4 Irving, H., 376, 377, 380 Ito, F., 98(433), 156 Ito, T., 89(434), 136

395

AUTHOIt INDEX

Itoh, F., 97(572), 08(572), 139 I~vno,J., 79!507), 137 Izquirrdo, ,J. A , , 112(435), 135

J ,J:tckoki~K ~ ~. lA , ,,

78(214), 106(214), 12s Jitckson, A. H . , 104(363), 107(363), fS.9 ,J:lckson, Philip., 299, 315 ,Jackson, It. W., 35(436), 135 Jac-ob, J., 235, 237, 239, 240, 252, 280,283, 285 Jscol,s, B., 313,311,615 J a c o b s ~F. A , , 116(368), 133 Jacobs, hl. B., 304, 312, 315 Jacohsen, C. F., 170, 171, 823, 224 Jacobsen, E., 68(281), 131 Jacoby, T. F., 185, 226 J a k o b y , W. B., 85, 01(438, 439), 94(439), 135 Jacquot, It., 231, 234, 285 .Jnisle, F . ,257, 285 .James, A. T., 116(158), 128 James, W. O. , 117(440), 135, 319,321, 336, 380 Jamieson, G . S., 75(910), 14!l Janisch, H., 66(407), 134 Janot, AX. M., 119(308), 132 .Jaques, I