Current Topics in Membranes and Transport, Volume 08

Current Topics in Membranes and Transport Volume 8

Advisory Board

Robert W . Berliner I . S. Edelman I . M. Glynn Franpis Morel Shmuel Razin Aser Rothstein H . J . Schatzmann Stanley G. Schultz Philip Siekevitz Daniel C. Tosteson

Contributors

William J . Adelman, Jr. Robert J . French R. P . Garay P. J . Garrahan R. Kinne M . A. Moscarello Rivka Panet D. Rao Sanadi

Current Topics in Membranes and Transport

VOLUME 8

Edited by Felix Bronner Department of Oral Biology llniversity of Connecticut Health Center Farmington, Connecticut and Arnort Kleinzeller Department of Physiology [Jniversity of Pennsylvania School of Medicine Philadelphia, Pennsylvania

1976

Acodernic Press

New York

San Froncisco

London

A Subsidiary of Harcourl Brace Jovanovich, Publishers

COPYRIGHT 0 1976, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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LIBRARY OF CONGRESS CATALOG CARD NUMBER:70- 11709 1 ISBN 0- 12-153308-5 PRINTED IN THE UNITED STATES O F AMERICA

List of Contributors, vii Contents of Previous Volumes, ix

Chemical and Physical Properties of Myelin Proteins M. A. MOSCARELIJO

I. Introdurtion, 1 11. Ihcephalitogenic Protein of Myelin, 3 111. IV. V. VI. VII.

Physical Structure of Myelin Basic Protein, 7 l’roteolipid Protein Fraction of Myelin, 10 Other Protein Fractions Prepared from Whole Brain, White fut,ter, or Myelin, I Conclusions on the Nature of Myelin l’roteins, 18 Localization of Proteins i n Myelin, 21 Referencaes, 24

The Distinction between Sequential and Simultaneous Models for Sodium and Potassium Transporf 1’. J. GARRAHAN AND R. P. GARAY

I. Introduction, 29 11. Cation-Binding Sites of the Xu Pump, 31 111. Experimental Evidence for Simultaneous Existence of Inner and Outer CationBinding Sites, 40 IV. Intermediate Stages in Hydrolysis of ATP by the Na Pump, 68 V. Mechanism of Ion Transport, 77 References, 91 Soluble and Membrane ATPases of Mitochondria, Chloroplasts, and Bacteria: Molecular Structure, Enzymatic Properties, and Functions RIVKA PANET AND TI. RAO SANADI

I. Introduction, 99 11. Mitochondria1 Coupling Factor, 100 111. Oligomycin-Sensitive ATPase, 118 IV. Chloroplast Coupling Factor, 126 V

CONTENTS

Vi

V. Bacterial ATPase, 141 VI. General Conclusions and Perspective, 149 References, 150 Competition, Saturation, and Inhibition-Ionic Interactions Shown by Membrane Ionic Currents in Nerve, Muscle, and Bilayer Systems ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR.

I. 11. 111. IV. V.

Perspectives, 161 Saturation Phenomena, 166 Blocking and Competition, 169 Models and Analyses, 189 Concluding Remarks, 198 References, 200

Properties of the Glucose Transport System in the Renal Brush Border Membrane R. KINNE

I. Introduction, 209 11. Isolation and Characterization of Plasma Membranes from Proximal Tubular Epithelium, 211 111. Interaction of D-Glucose with Isolated Renal Plasma Membranes, 218 IV. Interaction of Phlorizin with Isolated Renal Plasma Membranes, 233 V. Molecular Characteristics of the Sugar Transport System in the Brush Border Membrane, 245 VI. Conformational Response of the Glucose Transport System, 251 VII. Relation of Renal Glucose Transport System to Enzymes Interacting with Carbohydrates, 256 VIII. One or Several Glucose Transport Systems in the Brush Border Membrane?, 257 IX. Summary and Conclusions, 259 References, 259 Note Added in Proof, 267 Subject Index, 269

Numhers in parentheses indicate the pages on which the authors’ contributions begin. Lahoratory of Biophysics, IRP, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Education and Welfare, Marine Biological Laboratory, Woods Hole, Massachusetts (161)

William J. Adelman, Jr.,

Laboratory of Biophysics, IRP, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Department of Health, Education and Welfare, Marine Biological Laboratory, Woods Hole, Massachusetts (161)

Robert J. French,

Departamento de Quimica Bioldgica, Facultad de Farmacia y Bioquimica, Buenos Aires, Argentina (29)

R. P. Gamy,

Departamento de Quimica Bioldgica, Facultad de Farmacia y Bioquimica, Buenos Aires, Argentina (29)

P. J. Garrahan,

R. Kinne,

Max-Planck-Institut fur Biophysik, Frankfurt, Germany (209)

M. A. Morcarello,

Research Institute, The Hospital for Sick Children, Toronto,

Canada (1) Department of Cell Physiology, Boston Biomedical Research Institute, Boston, Massachusetts (99)

Rivka Panet,*

Department of Cell Physiology, Boston Biomedical Research Institute, Boston, Massachusetts (99)

D. Roo Sanadi,

* Present address: Department of Nuclear Medicine, Hadassah Haspital, Jerusalem, Israel. vii

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Contents of Previous Volumes Volume 1

Some Considerations about the Stnicture of Cellular Membranes MAYNARD M. DEWEYAND LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichia coli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function ASER ROTHSTEIN Molecular Architecture of the Mitochondrion DAVID13. MACLENNAN Author Index-Subject Index Volume 2

The Molecular Basis of Simple Diffusion within Biological Membranes W. R. LIER AND W. D. STEIN The Transport of Water in Erythrocytes

ROBERTE. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCE AND M. MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDERTZAGOLOFF Mitochondria1 Compartments: A Comparison of Two Models HENRYTEDESCHI Author Index-Subject Index ix

X

CONTENTS OF PREVIOUS VOLUMES

Volume 3

The Na+, K+-ATPase Membrane Transport System : Importance in Cellular Function ARNOLDSCHWARTZ, GEORGEE. LINDENMAYER, AND JULIUS C. ALLEN Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function ANTHONY MARTONOSI The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow across Neural Membranes W. J. ADELMAN, JR. AND Y. PALTI Properties of the Isolated Nerve Endings GEORGINA RODR~GUEZ DE LORESARNAIZ AND EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells : I n Vitro Studies J. D. JAMIESON The Movement of Water across Vasopressin-Sensitive Epithelia RICHARD M. HAYS Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm WILLIAMR. HARVEY AND KARLZERAHN Author Index-Subject Index Volume 4

The Genetic Control of Membrane Transport CAROLYN W. SLAYMAN Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMARJAIN Regulation of Sugar Transport in Eukaryotic Cells HOWARD E. MORGAN AND CAROLF. WHITFIELD Secretory Events in Gastric Mucosa RICHARDP. DURBIN Author Index-Subject Index Volume 5

Cation Transport in Bacteria: K+, Na+, and H+ FRANKLIN M. HAROLD AND KARLHEINZ ALTENDORF Pro and Contra Carrier Proteins; Sugar Transport via the Periplasmic Galactose-Binding Protein WINFRIEDBoos

CONTENTS OF PREVIOUS VOLUMES

xi

Coupling and Energy Transfer in Active Amino Acid Transport ERICHHEINZ The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption : Theory and Applications to the Reptilian Bladder and Mammalian Kidney WILLIAni A. BRODSKY AND THEODORE P. SCHILB Sodium and Chloride Transport across Isolated Rabbit Ileum A N D PETER F. CURRAN STANLEY G. SCHULTZ A Macromolecular Approach t.3 Nerve Excitation ICHIJI TASAKI AND EMILIO CARBONE Subject Index Volume 6

Role of Cholesterol in Biomembranes and Related Systems MAHENDRA KUMAR JAIN Ionic Activities in Cells A. A. LEV AND W. McD. ARMSTRONG Active Calcium Transport and Ca2+-ActivatedATPase in Human Red Cells II. J. SCHATZMANN The Effect of Insulin on Glucose Transport in Muscle Cells TORBEN CLAUSEN Recognition Sites for Material Transport and Information Transfer HALVOR N. CHRISTENSEN Subject Index Volume 7

Ion Transport in Plant Cells E. A. C. MACROBBIE H+ Ion Transport and Energy Transduction in Chloroplasts A. DILLEYAND ROBERTT. GIAQUINTA RICHARD The Present State of the Carrier Hypothesis PAULG. LEFEVRE Ion Transport and Short-circuit Technique S. REHM WARREN Subject Index

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Chemical and Physical Properties of Myelin Proteins M . A . MOSCARELLO Research Institide The Hospital for Sick Children Toronto, Canada

Introduction . . . . . . . . . . . . . . . . . . 1 3 Encephalitogenic Protein of Myelin . . . . . . . . . . . . Physical Structure of Myelin Basic Protein . . . . . . . . . 7 Proteolipid Protein Fraction of Myelin . . . . . . . . . . . 10 Physical Studies . . . . . . . . . . . . . . . . . 13 V. Other Protein Fractions Prepared from Whole Brain, White Matter, or Myelin . . . . . . . . . . . . . . . . . . . . 17 VI. Conclusions on the Nature of Myelin Proteins. . . . . . . . . 18 VII. Localization of Proteins in Myelin . . . . . . . . . . . . 21 References . . . . . . . . . . . . . . . . . . . 24

I. 11. 111. IV.

1.

INTRODUCTION

The word protein is derived from the Greek word proteios, meaning primary, the fundamental material. Until recently, models of membranes have ignored this primary role of proteins, but emphasis on the lipid components has been widespread. The myelin membrane, which consists of approximately 70-75010 lipid and 25-30010 protein, has always been considered as a bilayer of lipid with protein interacting with the charged groups of the lipids on the cxtcrnal surfaces (Danielli and Davson, 193435). This is the Danielli-Davson model based on the original work of Gortrr and Grrndel (1925) with cbrythrocytcs. Although it was generally agreed that the membranc proteins must be important, they were simply placed on the outside surface of the bilaycr. Thc more recent model of Singer and Nicolson (1972) emphasizes the nature of membrane proteins, some of which are visualized as being partially embedded in the lipid matrix, while others pass through the lipid. 1

2

M. A. MOSCARELLO

A single lipid bilayer appears to be a relatively fixed structure, impermeable to most water-soluble substances. On the other hand, large protein molecules, which can undergo many conformational transitions, might provide the needed flexibility required of a fluid mosaic membrane. It has become of central importance to the understanding of membrane structure and function to isolate and characterize membrane proteins. In the past, most membrane models were derived from data obtained from electron microscopy and X-ray diffraction studies. The lack of information concerning the number, nature, and characteristics of the various proteins in the membrane resulted in a simplistic model in which the protein was somehow stuck to the charged groups of the bilayer. An excellent critique of the methods used and interpretations made was published by Korn (1966). The possibility of artifacts arising from fixation techniques was carefully documented. In recent years, the primary structure of the encephalitogenic protein of myelin has been elucidated (Westall et al., 1971; Carnegie, 1971; Eylar, 1972). A “proteolipid” fraction consisting of several proteins and lipids soluble in chloroform-methanol has been known since 1951 (Folch Pi and Lees, 1951). A highly hydrophobic protein has recently been isolated from the myelin fraction soluble in acidified chloroform-methanol, purified (Gagnon et al., 1971), and some of its properties studied (Moscarello et al., 1973). Thus, on the one hand, a highly basic protein (the encephalitogenic or A1 protein) well suited for interactions with negatively charged phosphoryl groups was isolated from myelin. On the other hand, a totally different protein, the hydrophobic protein, ideally suited for interacting with the hydrocarbon chains of lipids and other apolar molecules was isolated from the same membrane. Both these very different proteins coexist in the myelin membrane along with other proteins still to be isolated. It is unlikely that these proteins coexist in a single 25-A layer of protein outside the bimolecular lipid leaflet as concluded by Finean (1953) and others from X-ray diffraction studies. The proteins must form an integral part of the membrane structure. The role they play must be determined by the properties of the proteins themselves. A highly charged protein, such as the basic protein of myelin, would be expected to take part in ionic interactions with the polar groups of lipids; the hydrophobic protein, on the other hand, would be expected to interact with fatty acid chains. Such interactions with different domains in the membrane must in some way reflect the functional role different proteins play in the membrane. The lipids, however, are usually associated with each other in the form of a bilayer structure. It is for this reason that the protein must be responsible for providing the “structural steel” of which membranes are made. It is proposed to discuss what is currently known

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

3

about myelin proteins with a view to showing that different proteins interact with the lipid in different ways, largely determined by the chemical and physical characteristics of the proteins themselves. Evidence from such studies will be used to show how these proteins may be responsible for the structural steel of the myelin membrane.

II. ENCEPHALITOGENIC PROTEIN OF MYELIN

Known as the basic protein of myelin (Kies, 1965) , the encephalitogenic protein, or the A, protein (Eylar et al., 1971), it comprises about 30% of the myelin protein. It is readily extracted from whole brain under a variety of conditions including buffers a t pH values from 3 to 4 and from isolated myelin by dilute mineral acid (Lowden et al., 1966). Because of these two features, i.e. , availability in rather large amounts and ease of extraction into aqueous media, it was possible to study it by conventional methods of protein chemistry (Eylar el al., 1969). The isolated material was shown to have potent biological activity. It induced experimental allergic encephalomyelitis (EAE) in the guinea pig a t concentrations of less than 5 pg per animal (Eylar, 1972). With the isolation of this protein, the earlier observations on EAE by RobozEinstein el al. (1962) and Kies et al. (1965) , who described EAE in a crude homogenate of brain, were extended significantly. It soon became obvious that a protein with such potent biological activity should be subjected to extensive physical and chemical studies in a number of laboratories, with a view to elucidating its complete structure and possibly defining its mechanism of action. The basic protein has been isolated in several laboratories (Carnegie, 1971; Lowden et al., 1966; Eylar et al., 1969; Itoboz-Einstein et al., 1962; Tomasi and Kornguth, 1967). The isolation from spinal cord (RobozEinstein et al., 1962) involved preliminary extraction in acetone-petroleumether followed by isolation of the protein with 5 1 0 % KCI. Other procedures have employed defatting the tissue with chloroform-methanol (Kies et al., 1965; Tomasi and Kornguth, 1967; Nakao et al., 1966; Wolfgram, 1965; Carnegie et al., 1967; Kibler and Shapka, 1968) prior to extraction of the protein. Basic protein has also been isolated from human central nervous system myelin by dilute mineral acid (Lowden et al., 1966). The isolation and purification of this protein was followed by structural studies that culminated in the elucidation of the complete amino acid sequence. The first significant chemical finding was the report (Lowden et al., 1966) that cystine was absent from the primary structure. The molecule was, therefore, not held together by disulfide bonds and, conse-

4

M. A. MOSCARELLO

quently, consisted of a single polypeptide chain. Eylar et al. (1971) isolated 27 tryptic peptides and 16 peptic peptides to establish the complete sequence of the 170 amino acid residues of the bovine spinal cord protein. No general periodicity of the basic residues was observed; the distribution appeared to be random. Several unusual features are noteworthy. The arginine residue at position 107 is present in both monomethylated and dimethylated derivatives. Not far from the methylated arginine residues is a proline-rich sequence, Pro-Arg-Thr-Pro-Pro-Pro, which may induce a sharp bend in the molecule, although other conformations are possible when the models of this region are constructed. If a sharp bend were present at this site near the center of the molecule, it may help to explain the axial ratio of 1O:l found by viscosity studies (Eylar et al., 1969). The Pro-ProPro sequence is not commonly found in proteins. It has been reported in rabbit immunoglobulin IgG (Smyth and Utsumi, 1967) in which it forms the hinge region. The threonine residue of this sequence has been reported by Hagopian and Eylar (1969) to be the site of glycosylation with N-acetylgalactosamine and an enzyme from submaxillary glands forming a GalNAc-0-Thr linkage. Only the deglycosylated natural acceptor of the enzyme served as an acceptor. Eylar et al. (1971) proposed that the A1protein (basic protein) may be glycosylated during myelin synthesis and deglycosylated when it becomes a constituent of the completed myelin membrane. If this were so, the glycosylated protein should be present in the Schwann cell. However, it has not as yet been isolated from this source. The structural determinants required for encephalitogenic activity vary from species to species. In the guinea pig, the sequence around the single tryptophan residue appears to be necessary for activity. The sequence of (Arg) . this region was shown to be Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys The C-terminal lysine could be replaced by arginine. An extensive study of this region was made by Westall et al. (1971). Using the solid-phase peptide synthesis technique (Merrifield, 1963), several peptides were prepared, each differing from the natural peptide by a single residue. Each peptide was tested for disease-producing activity in the guinea pig. As a result of these studies, it was concluded that the Trp - - -Gln-Lys (Arg) residues were essential. Activity was lost if the terminal Lys was removed. Replacement of the terminal Lys by Ile also resulted in loss of activity. Replacement of Gln by Ile and of Trp by Phe or Val produced inactive peptides. The evidence in favor of the view that the Trp region represents the major encephalitogenic site in the guinea pig can be summarized as follows: (i) the synthetic peptides have approximately the same activity on a mole/mole basis as the complete protein; (ii) modification of the Trp

CHEMICAL AND PHYSICAL PROPERTIES OF MYELIN PROTEINS

5

region with 2-hydroxy-5-nitrobenzyl bromide (Eylar et al., 1970), which attacks Trp exclusively, yields an inactive peptide; (iii) proteins isolated from several species including man, monkey, dog, rabbit, guinea pig, rat, mouse, and horse, all of which are equally encephalitogenic in the guinea pig, have preserved the Trp- - -Gln-Lys (Arg) sequence. This region is altered in the chicken and turtle proteins, but these are nonencephalitogenic. I n the chicken, the essential Gln residue is replaced by His (Eylar et al., 1974). Although the requirements for encephalitic activity in the guinea pig appear to be well defined, other requirements may be necessary for activity in the rabbit. An encephalitogenic peptide (Kibler et al., 1969) of 43 residues derived from another region of the molecule has been shown to be encephalitogenic in the rabbit but not in the guinea pig (Carnegie, 1969). Neither of the above two peptides, i.e., the one active in guinea pigs and the one active in rabbits, is active in the monkey (Eylar et al., 1972). This prptidc drrived from residues 117-170 (Iiibler et al., 1969) was produced by thc BNPS-skat ole [2- (2-nitrophenylsulfcnyl) -3-mcthyl-3’-bromo-indoleninc] reaction that cleaves the peptide chain a t a tryptophanyl residue. Thrrrforr, the Trp region, csscntial for activity in the guinra pig, is not essrntial for activity in the monkey. The main disease-inducing site cffrctive in the monkey resides in the carboxy terminal of the molecule (Eylar et nl., 1974). Since the region responsiblc for EAE activity varies from species to species, it appears unlikely that genrralizations of structural requirements for encephalitogenic activity can be made from the above studies, and it is reasonable to conclude that a large family of sequences may possess disease-inducing properties. I n discussing the possible viral etiology of multiple sclerosis, which may be similar to EAE, Westall (1974) speculates that the virus may bring about an appropriate change in a nucleic acid base which could result in an altered sequence. This sequence, in turn, would confer encephalitogenic activity on the protein. Other antigenic sites have been localized to different parts of the molecule (Borgstrand and Kallen, 1973). The regions that induce transformation of lymph node cells from immunized rabbits are located in regions 1 4 3 , 44-116, and 117-170. The Trp region was not active in these experiments. The complete amino acid sequence of the encephalitogenic, basic or A1 protein, is shown in Table I. No marked differences between the human myelin basic protein and those of other species have been observed. As found for the bovine protein, the N-terminal residue is blocked with an acetyl group (Carnegie, 1971). A deletion of His-Gly at residues 10 and 11 has been reported. The tryptophan nanopeptide responsible for EAE activity in the guinea pig

6

M. A. MOSCARELLO

TABLE I AMINOACIDSEQUENCEOF BOVINE A 1 PROTEIN' ~

~~~

~~

N-Ac-Ala-Ser-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-~u Ala-Ser-Ala-Ser-Thr-Met-Asp-His-Ala-Arg-His-Gly-Phe-Leu-Pro-Arg-His Arg-Asp-Thr-Gly-Ile-Leu-Asp-Ser-Leu-Gly-Arg-Ph~Phe-Gly-Ser-Asp-Arg-Gly-AlaPro-Lys-Arg-Gly-Ser-Gly-Lys-Asp-Gly-His-His-Ala-Ala-Arg-Thr-Thr-His-Tyr-GlySer-Leu-Pro-Gln-Lys-Ala-Gln-Gly-His-Arg Pro-Gln-Asp-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val Thr-Pro-Arg-Thr-Pro-Pro-Pro-Ser-Gln-Gly-Lys-Gly-Arg-Gly Leu-Ser-Leu-Ser-Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-Pro-Gly Phe-Gly-Tyr-Gly-Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Leu-Lys-GlyHis-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys Ile-Phe-Lys-Leu-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met

Ala-Arg-Arg-COOH From Eylar (1972).

is identical to the bovine sequence except for the replacement of the Cterminal Lys in the bovine protein for an Arg in the human. Other encephalitogenic determinants (residues 1-21 and 45-88) have been found, but these are 500-1000 times less active than the principal encephalitogen about the tryptophan region. Central nervous system myelin of rodents, of the suborders Myomorpha and Sciuromorpha, such as the rat, contain two basic proteins (Martenson et al., 1970). One is similar to that of other species in amino acid composition and size (18,400 daltons) ; the other is smaller by 4000 daltons. The smaller rat basic protein differs from the larger in having a 40 amino acid piece missing in the C-terminal half of the molecule. The complete sequence has been reported recently by Dunkley and Carnegie (1974). Carnegie (1971) has put forward an interesting proposal concerning a possible neuroreceptor role for the basic protein. It is based on the postulated binding site for 5-hydroxytryptamine (Smythies et al., 1970), formed by two molecules such as tryptophan, located one above the other, so that 5-hydroxytryptamine is held in position by the bonding energy from two a-cloud interactions. This allows it to intercalate between the two molecules. Carnegie suggests that the encephalitogenic determinant can accommodate the 5-hydroxytryptamine molecule, with the indole ring sandwiched between Phe 113 and Trp 115, held by a hydrogen bond to the peptide chain and an electrostatic interaction with Glu-118. Experimental evidence for such a binding site has as yet not been obtained. Although the major features of the primary structure of myelin basic protein have been elucidated, several problems remain unresolved. The

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

7

protein has been shown to be a suitable substrate for phosphorylation by B protein kinase (Carnegie et al., 1973; Miyamoto and Kakiuchi, 1974). The latt,er authors showed that myelin basic protein was phosphorylated by a cyclic AMP-dependent protein kinase from the brain. The phosphorylated amino acids were shown to be serine and threonine. Phosphorylation was found to take place both in vitro and in vivo. Miyamoto and Kakiuch postulated that the phosphorylation was in a dynamic state with dephosphorylation. Steck and Ape1 (1974) have shown that 14-day-old rat myelin was a better substrate for protein phosphorylation than adult myelin. They concluded that the basic protein was more accessible to the enzyme in young animals than in mature myelin. No biological role was postulated for the phosphorylated derivative.

111.

PHYSICAL STRUCTURE OF MYELIN BASIC PROTEIN

The elucidation of the complete amino acid sequence of basic protein from several species is a necessary prerequisite for understanding its secondary structure and its role in the structure of the myelin membrane. Unfortunately, studies of the secondary structure are not as advanced as the chemical studies. Some physical measurements (viscosity, optical rotary dispersion, and circular dichroism) were made early. These have led to the interpretation that myelin basic protein is not structured but has an open conformation. It has been argued that the open conformation, with a large number of basic residues (lysine, histitline, and arginine constitute 25% of all residues), is ideally suited for interacting with the phosphoryl groups of the lipid bilayer. That this conclusion may be premature will become evident from the data presented below. Some of the physicochemical properties of this protein were studied by Eylar and Thompson (1969). On the basis of viscosity studies, they determined an axial ratio of 1 : l O and concluded that the shape of the molecule was that of a prolate ellipsoid. Choa and Einstein (1970) found an axial ratio of 1:17. Optical rotary dispersion studies by Eylar and Thompson (1969), Choa and Einstein (1970), and Palmer and Dawson (1969) established that little helical structure was present in the molecule. However, both the high intrinsic viscosity and the optical rotary data are consistent with a highly ordered protein than has a specific tertiary structure and is relatively devoid of a-helical or P structures. In a recent study, Epand et al. (1974) showed that the protein had a nonrandom structure in solution. Intrinsic viscosity studies confirmed the axial ratio of 1 : l O found by Eylar and

8

M. A. MOSCARELLO

Thompson (1969). Circular dichroism studies in 0.2 M acetic acid and 0.1 M sodium hydroxide were very similar but different from the spectra in 6 M guanidine hydrochloride, a denaturing solvent. Moreover, no drastic temperaturedependent changes were observed in the spectra over 0-80°C. The radius of gyration, calculated from low-angle X-ray scattering measurements, was found to be 39 + 2 d. This corresponds to a prolate ellipsoid with an axial ratio of 10: 1. Sedimentation velocity data correspond to a prolate ellipsoid of axial ratio 12, corroborating the viscosity data. Therefore, X-ray scattering, viscosity, and sedimentation velocity are in good agreement with a prolate ellipsoid model for the basic protein of myelin with an axial ratio 1O:l and of dimensions 150 X 15 d. These studies indicate that the molecule is highly structured. Other recent data support the view that the basic protein of myelin is a nonrandom highly structured molecule. Circular dichroism spectra were run from 250-190 nm at pH values of 1.68, 11.3, and 12.1. At pH 1.68 and at 25"C, very low ellipticity values were observed at 220 nm. This indicates little or no helical structure. Below this wavelength, a large decrease in ellipticity occurred, with a minimum at 200 nm. When the molecule was heated to 85"C, its ellipticity at 220 nm decreased. This indicates an increase in helical content. Also, the ellipticity at 200 nm was less at 85°C than at 25"C, indicating a less random structure. The spectra a t pH 11.3 and 25°C showed little evidence of structure throughout the scan, with low ellipticity values. Heating to 85°C decreased the ellipticity, which was especially marked at 200 nm. At pH 12.1 and 25"C, some helical structure was evident at 220 nm. The helical content increased on heating to 85"C, similar to the observations at pH 1.68. The ellipticity at 200 nm was large at 25°C and decreased considerably on heating to 85°C. Support for the circular dichroism data has been obtained from surface tension measurements. The application of this technique to the study of the conformation of proteins has been described recently (Neumann et al., 1973). The technique was successfully applied to the study of the basic protein of myelin (Moscarello et al., 1974). The surface tension ( yLv) was studied as a function of temperature at different pH values from 2.2 to 12.5. The significant features are as follows: (i) the surface tension at 20°C decreased with increasing pH value-at pH 2.23, the surface tension was 46.1 ergs/cm2, whereas it was 41.6 erg/cm2 at pH 12.5; (ii) there was little evidence of phase transitions at intermediate pH values (3.7, 6.9, and 9.9) ; (iii) phase transitions occurred at extremes of pH, i.e., pH 2.2, 11.4, and 12.5; (iv) the low-temperature transition at 40°C was observed at low pH (pH 2.2), whereas the high-temperature transition at 80"-85"C was observed at high pH values.

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

9

The decrease in surface tension a t 20°C from 46.1 ergs/cm2 a t low pH to 41.G a t high pH is probably the result of the decrease in charge of the protein as the pK is approached. The decrease in charge is reflected in an increase in hydrophobicity so that more protein goes into the surface. The lack of phase transitions a t intermediate pH values is consistent with a highly ordered, rigid molecule. The conclusion from the optical, viscosity, X-ray, and surface tension data must be that the molecule is highly structured and nonrandom. This conclusion can be emphasized because the literature is full of references to the “open,” random conformation of this protein. I n fact, a whole theory of myelin breakdown has been proposed on the basis of an open conformation (Roboz-Einstein, 1972). It is argued that the basic nature of the protein, its open conformation and its external position in the myelin membrane, would render this molecule susceptible to attack by proteinases. Only the basic nature of this protein has been confirmed to date. The structure appears to be rigid and its position in the membrane has not been determined. Data from this laboratory, which support the nonexposed position of this prot,ein, are discussed in Section VII. The above observations may be pertinent to an observation first reported by Martenson and Gaitonde (1969) in their studies with basic protein from bovine white matter. When basic protein was subjected to polyacrylamide gel electrophoresis at acid pH values, a single component, was observed. However, when electrophoresis was done a t high p H values (pH lO.G), multiple components were detected. Isolation of these various components revealed that they had the same encephalitogenic activity and identical amino acid compositions (Martenson el al., 1970). A partial explanation for this heterogeneity was suggested by Baldwin and Carnegie (1971) as due to variable methylation of the arginine residues. However, Deibler and Martenson (1973) have shown that all components contain monomethylarginine and symmetrical dimethylarginine in approximately 4: 1 ratio. From our surface tension studies with basic protein, it was shown that phase transitions did not occur below pH 10, but do occur above this pH. A possible explanation for the heterogeneity observed a t high p H may he the presence of different conformational forms of the protein. The different conformations may migrate more slowly on acrylamide gels a t low than a t high pH values. The use of the myelin membrane for the studies of membrane structure has many advantages over the use of other membranes. At this stage in our understanding of membrane components and their assembly, the relatively simple structure of myelin represents almost a model system.

10

M. A. MOSCARELLO

With the elucidation of the structure of the basic protein, attention was focused on the interaction of this protein with lipids. Basic protein was shown to bind avidly to certain lipids resulting in large changes in the circular dichroism spectrum (Anthony and Moscarello, 1971s). More recently, basic protein was shown to interact with acidic phospholipids and sulfatides yielding an interesting lamellar phase. This phase contained two lipid bilayers in its unit cell: one contained mainly the phospholipids with the hydrocarbon chains in liquidlike conformation, and the other contained mainly the sulfatides, with at least one fraction of the chains stiff and hexogonally packed. The segregation of the lipids was brought about as a result of the interaction with basic protein. The double bilayer in the unit cell closely resembled myelin (Mateu et al., 1973). This interesting preliminary result suggests that the protein has “organized” the lipid so that the unit cell contained two bilayers with similar spacing to that of myelin. The data support the view expressed here concerning the central and possible primary role of proteins in the organization of this biological membrane.

IV.

PROTEOLIPID PROTEIN FRACTION OF MYELIN

The term proteolipid was proposed by Folch Pi and Lees (1951) to describe the unusual properties of proteinaceous material from the brain, soluble in mixtures of chloroform-methanol. The soluble material was shown to consist largely of lipids but also contained a significant amount of protein. Lipoproteins had been known for some time to be composed of protein with a small amount of lipid and thus to be water-soluble. The term proteolipid was used to distinguish chloroform-methanol-soluble material from water-soluble material. The term, therefore, has historical significance. A complete review of the historical background can be found in Folch Pi and Stoffyn (1972). The material soluble in chloroform-methanol has been shown to contain several proteins (Gagnon et al., 1971; Miyamoto and Kakiuchi, 1974; Chan and Lees, 1974); therefore, proteolipid as originally isolated is not a homogeneous protein. In fact, lyophilized myelin can be dissolved in chloroform-methanol. It is important to recognize this fact because the literature of the last 10 years is full of references to “proteolipid protein” and “proteolipid apoprotein,” creating the misleading impression that the term refers to a homogeneous protein. In the absence of evidence for homogeneity, it is necessary to use caution in interpreting data on amino acid composition, analytical ultracentrifugation, end-group analyses, or

11

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

optical measurements. For this reason, I have referred to proteolipid as the proteolipid protein fraction of myelin. The various methods available for the preparation of the proteolipid protein fraction include dialysis in organic solvents and Sephadex LH-20 chromatography. The methods reviewed by Folch Pi and Stoffyn (1972) have not yielded a single material. Therefore, the proteolipid of different workers may vary in composition, depending on the method of preparation. Some of the properties of this protein fraction have been reported by Folch Pi and his collaborators. The amino acid composition of their material is shown in Table I1 and compared to the purified proteins of Gagnon et al. (1971) and Nussbaum et al. (1974). A glance at the amino acid analyses indicates several similarities between the proteolipid proteins isolated from bovine brain by Folch Pi's group and the protein isolated from human myelin by Gagnon et al. (1971) and Nussbaum et al. (1974) from the rat brain. Differences in overall composition are noted in aspartic TABLE I1 AMINOACIDCOMPO5ITION OF RR.4IN WHITE MATTER,PROTEOLIPID FRACTION, AND PROTEINS PURIFIED FROM PROTEOLIPID PROTEIN FRACTION OF MYELIN"

Amino acid

Bovine proteolipid apoproteinb

Human myelin protein (N-2)C

Rat brain P7d

Aspartic Tlireonine Serine Glutamic Proline Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

4.2 8.5 8.5 6.0 2.8 10.3 12.5 6.9 4.0 1.9 4.9 11.1 4.6 7.9 4.3 1.8 2.6

5.4 8.2 5.9 6.4 3.1 10.4 10.6 6.8 2.9 1.6 4.9 11.2 4.2 8.3 4.7 2.6 2.9

5.5 8.5 6.0 6.5 3.0 11.0 12.0 8.5 4.0 1.5 6.0 12.0 5.0 8.5 5.0 2.5 3.0

a

Residue per 100.

* Folch Pi and Stoffyn (1972). Gagnon et al. (1971). Nussbaum et al. (1974).

12

M. A. MOSCARELLO

acid, serine, alanine, cystine, and histidine. Some of the differences may be attributed to species variation. A definite statement can be made only when the preparation of Folch Pi is obtained in higher purity. Gagnon et al. (1971) used several methods to monitor the purity of their preparation. In addition to sedimentation velocity, they used equilibrium ultracentrifugation. A single component was demonstrated on polyacrylamide gels using both the system of Takayama et al. (1964) and that of Weber and Osborn (1969) at different pH values. In a recent study of the Folch Pi apoprotein, Nicot et al. (1973) were able to isolate two major components, one of molecular weight 25,100 and the other 20,700. The two components were isolated by preparative electrophoresis. Amino acid analysis showed some differences in composition; notably threonine, glutamic, serine, and leucine. Minor differences were found in several other amino acids. Because the preparation was obtained by extracting bovine white matter and not purified myelin, the authors were not able to rule out contamination with a nonmyelin proteolipid. Chan and Lees (1974) have studied bovine white matter and myelin preparations in polyacrylamide-sodium dodecyl sulfate (SDS) gels with and without urea. Multiple bands were observed for white matter proteolipid. On indirect evidence, they concluded that the multiple bands represented different states of aggregation of a monomeric unit of molecular weight 5000. A homogeneous proteolipid apoprotein P7 has been reported by Nussbaum et al. (1974). Myelin was prepared from rat brain and extracted with chloroform-methanol. On preparative electrophoresis, they obtained three proteolipid proteins; P7 accounted for about 60%. The amino acid analysis reported by them is shown on Table 11. Differences in amino acid composition with that of Gagnon et al. (1971) were found in alanine, valine, cystine, and isoleucine. They reported a total of 219 residues for P7, whereas Gagnon et al. (1971) reported 223 residues for N-2. These differences in composition and total number of residues may reflect a species difference. By using an automatic Edman degradation technique, Nussbaum et aZ. ( 1974) have reported an N-terminal sequence after performic oxidation of the protein. The sequence as reported was Gly-Leu-Leu-Glu-CysSOs-

CysSO~-Ala-Arg-CysSO~-Leu-Val-Gly-Ala-Pro-Phe-Ala-X-Leu-Val-Ala - -.

This sequence must be interpreted with caution since the yields reported by them for each turn of the sequencer are very low, often less than 10%. An interesting structural feature of this group of proteins is the presence of about 2% fatty acid, apparently covalently linked to the protein. The first report of the presence of fatty acids was made by Wood et al. (1971)

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

13

who stated that glycerol was not found in either acid or alkaline hydrolyzates of the protein. Neither phosphorus nor sphingosine was detected by careful analysis. Attempts to remove the fatty acids, shown to be C16and C l ~ : lby several methods, were unsuccessful. These included Soxhlet extraction procedures and the charcoal method of Chen (1967) for the removal of fatty acids from serum albumin (Gagnon et al., 1971). It was concluded that the fatty acid was probably covalently bound to the peptide chain. About 3 months after the initial report of Wood et al. (1971), a similar study was published by Stoffyn and Folch Pi (1971). They showed that 2.2-3.0% fatty acids, which could not he removed by extensive dialysis with chloroform-methanol, remained associated with their proteolipid apoprotein. Choline, sphingosine, inositol, and hexosamine were not detected, and glycerol was less than 0.03%. The fatty acids were converted to their methyl esters after alkaline hydrolysis, and reaction with diazomethane and sodium borohydride reduction converted them to their corresponding alcohols. They concluded that the fatty acids were esterified to the peptide chain, probably with hydroxy amino acids. The role of fatty acids covalently bound to protein is not understood. They would certainly contribute to the hydrophobicity of the protein, important in interactions with lipid hydrocarbon chains of the bilayer. A similar, highly hydrophobic protein has been isolated from endoplasmic reticulum by MacLennan et al. (1972) as part of the ATPase complex. It has been demonstrated to contain 2% fatty acid also. They concluded that the fatty acid was responsible for the hydrophobic nature of their protein.

Physical Studies

Sherman and Folch Pi (1971) studied the rotary dispersion and circular dichroism of brain proteolipid protein. Their water-soluble preparation showed a helical content that varied between 16 and 40%, depending on the preparation. Using optical rotary dispersion (ORD) , they were unable to detect a random coil (characterized by a trough a t 205 nm). By circular dichroism (CD) , the protein was shown to be highly a-helical in chloroform-methanol but of low helicity in water. The N-2 protein isolated from normal human myelin by Gagnon el al. (1971) was studied by a number of physical techniques. The conformation of their protein was found to be very flexible. Anthony and Moscarello (1971b) prepared a water-soluble form of the protein by dissolving it in phenol-acetic acid-water (3: 1 :1) containing 2 M urea, followed by

14

M. A. MOSCARELLO

dialysis against decreasing concentrations of acetic acid. If the dialysis was begun with 50% acetic acid, followed by 25, 10, 5% and then water, an a-helical circular dichroism spectrum was obtained with minima at 210 and 220 nm. If the dialysis from phenol-acetic acid-water solution of the protein was started with 50% acetic acid containing 2 M urea in the first step, followed by 15, 10, 5% acetic acid and water, the circular dichroism spectrum was that of a /3 conformation with a minimum at 216 to 217 nm. The conformation obtained in either dialysis procedure was not affected by performic acid oxidation or by the presence of mercaptoethanol during dialysis. The conformation was not affected by varying the pH from 1.5 to 6.0. More detailed studies on the conformational flexibility of this protein (N-2) were reported by Moscarello et al. (1973). The ORD parameter b P 2 was calculated from the spectra run between 420 and 300 nm. The data for the two conformations are shown in Table 111. In the formation of the /3 form, the first step in the dialysis procedure following the removal of the phenol results in a conformation with low bO2l2,However, if phenol and urea were removed together with 25% acetic acid, the bO2l2value was -330, corresponding to about 50% helix. The subsequent stages in dialysis do not affect the bO2l2values. The a-helical and /3 conformations were confirmed by infrared analysis. The a-helical form showed a major infrared peak at 1650 cm-' and a smaller peak at 1630 cm-' representative of a-helical and /3 conformations, respectively. In the case of the /3 form, the major peak was at 1630 cm-l, with a smaller peak at 1650 em-'. The shoulder at 1690 cm-l in the /3 form TABLE I11 MOFFITT PARAMETER bozle OF N-2

DURING

VARIOUSSTAGES OF DIALYSIS

bf" or-Helical f o m

Dialysis stage ~

+ 2 M urea

50% Acetic acid

-440 -

25% Acetic acid

-330

10% Acetic acid

-320 -320 -320

Phenol-acetic acid-water (3: 1 :1) 2 M Urea in 50% acetic

5% Acetic acid

Water

9,

Form

~

-440 -110

-200 -200 - 160 - 180 - 185

15

CHEMICAL AND PHYSICAL PROPERTIES OF MYELIN PROTEINS

is attributed to the antiparallel /3 form (Miyazawa and Blout, 1961), The infrared spectrum of the protein in phenol-acetic acid (2 :l) showed a sharp peak at 1650 cm-l, representing n large amount of helix. In summary, the protein is highly helical in the presence of phenol. Removal of both phenol and urea simultaneously results in a preparation with about 50% helix. Equilibrium ultracentrifugation was carried out in water, 98% formic acid, and in 0.01 M sodium phosphate buffer containing 0.5% SDS. The data are shown in Table IV. In formic acid and SDS, the molecular weights obtained were 28,000 and 24,000, respectively. These values probably represent the molecular weights of the monomer. The a-helical form with a molecular weight of 86,000 consists of three subunits, whereas the /3 form, with a molecular weight of about 500,000, consists of many. The a-helical form was readily converted to the 0 form by heating. At 25"C, the protein was largely a-helical with minima in the CD a t about 220 and 210 nm. When heated to 9O"C, the spectrum changed to a /3 type with a single minimum at 217 nm. A small recovery was obtained on cooling to 25°C. The conversion to the /3 form was accomplished in the heating phase and not in the cooling one. The change in [ O ] 2 2 2 with temperature was studied. No change was observed until about 40"C, after which a steady decrease in [0]222 was recorded with increasing temperature. The broadness of the transitions in the temperature range from 40" to 90°C suggested that it was not highly cooperative or that the enthalpy of transition was small. Recently, the two conformations have been studied by electron microscopy after negative staining. The photographs are shown in Fig. 1. The a-helical form is much smaller than the ,8 form and corresponds to a molecular weight of less than 100,000. TABLE IV EQUILIBRIUM ULTRACEN.TRIFUGATION OF THE PROTEIN I N W A T E R , FORMIC ACID, AND SODIUM DODECYL SULFATE Sample a

Form (water)

@ Form (water)

Protein in 98a/, formic acid Protein in 0.01 M sodium phosphate buffer (pH 7.2) containing 0.5% SDS

Molecular weight 86,000 -500,000 28,000 24,000

FIQ.1. Electron micrographs of N-2 stained with 1% uranyl acetate. (A) The a-helical form; (B) the 0 form. X160,OOO; bar = 500 = 1 cm.

CHEMICAL AND PHYSICAL PROPERTIES OF MYELIN PROTEINS

V.

17

OTHER PROTEIN FRACTIONS PREPARED FROM WHOLE BRAIN, WHITE MATTER, OR MYELIN

A number of other workers have used a variety of methods to prepare chloroform-methanol-soluble proteins from whole brain or white matter. The relationship of any of these preparations to that of Folch Pi and Lees (1951) has not been determined. Wolfgram (1966) extracted a protein into acidified chloroform-methanol that differed from that of Folch Pi and Lees (1951) in being trypsindigestible. Recently, Wiggins el al. (1974) have resolved this proteolipid fraction into at least three different proteins, one of which, denoted WI, may be homogeneous. Braun and Radin (1969) prepared a water-soluble bovine brain myelin protein by modification of the method of Tenenbaum and Folch Pi (1966) and studied the interaction with lipids. They found that anionic lipids formed insoluble complexes that were stabilized by divalent cations. Nonionic lipids formed nonprecipitating complexes. Soto et al. (1969) fractionated proteolipids from cat gray and white matter by chromatography on an organophilic dextran gel (Sephadex LH-20) , similar to the method of Mokrasch (1967). Barrantes et al. (1972) prepared proteolipid proteins from bovine cerebral cortex using chromatography on Sephadex LH-20 and elution with NIN-dimethylformamide. Pasquini and Soto ( 1972) extracted proteolipids from bovine brain in n-butanol-water systems. Uda and Nakazawa (1973) prepared proteolipid from bovine brain with chloroform-methanol and then treated this material with ethanol-ther to extract sphingomyelin. The residue was dialyzed against chloroform-methanol. Nussbaum and Mandel ( 1973) extracted total brain proteolipids from whole brain of mutant mice in chloroform-methanol and then partitioned the protein between phases. Proteolipids have also been extracted from isolated myelin by the method of Gonzalez-Sastre ( 1970). The chloroform-methanol-soluble material was treated with 0.1 M KC1 to precipitate the basic protein. Eng et al. (1968) used several extraction procedures, including neutral salt and Triton X-100 and neutral salt extraction to obtain protein fractions from myelin. The soluble material was further fractionated with organic solvents. Several groups of workers (Mehl and Halaris, 1970; Eng et al., 1971) have isolated water-insoluble proteins by electrophoresis in acrylamide gel blocks with the aid of phenol-acetic acid-water solvents. Finch and Moscarello (1972) have reported the isolation of a protein fraction from normal human myelin with mercaptoethanol. The fraction is not homogeneous and appears to resemble the fraction of Wolfgram (1966) and the one recently reported by Wedege (1973), which has been obtained in homogeneous form. An interesting structural feature of the material of Finch et al. (1971) was the presence of the amino acid citrulline in covalent

18

M. A. MOSCARELLO

bond, which could be released by proteolytic digestion. It would be interesting to know if the homogeneous protein reported by Wedege (1973) contains citrulline. Agrawal et al. (1972) demonstrated the presence of a minor protein component on polyacrylsmide gels migrating between myelin basic protein and proteolipid in extracts of rat brain and white matter from bovine, dog, and rabbit brains. They called this material DM-20 and consider it to be a unique myelin component. This latter conclusion remains to be substantiated.

VI.

CONCLUSIONS ON THE NATURE OF MYELIN PROTEINS

In this article, I have reviewed what is known about the chemical and physical properties of myelin proteins. The basic protein of myelin is best understood, since the complete amino acid sequence of this encephalitogenic protein from the myelin of a number of animal species has been elucidated. A protein isolated from the chloroform-methanol-soluble fraction has also been purified in at least two laboratories. In our own laboratory, we have called it N-2 for convenience because it was the second Azsopeak of four, isolated from Sephadex LH-20 when a chloroform-methanol extract of normal human myelin was applied. Nussbaum et al. (1974) have reported the isolation of a similar protein from rat brain, which they called P7. Obviously at some point these trivial names must be replaced by descriptive names. Folch Pi and his collaborators have been in favor of using the name “proteolipid” protein for the chloroform-methanol-soluble material. As I have tried to point out in the foregoing, this fraction is not homogeneous. In fact, the basic protein of myelin will dissolve in chloroform-methanol in the presence of large amounts of myelin lipids (Gonzalez-Sastre, 1970). The main objection to the use of the term proteolipid is that it does not refer to a defined chemical entity. As pointed out above, different workers have isolated proteolipid protein by a variety of methods with no evidence that they all have isolated the same material. Although the name is useful, it must be clearly understood that it does not refer to a specific protein of defined chemical composition in contrast to the basic protein of myelin. It is my feeling that each of the proteins, isolated from the chloroformmethanol soluble material, should be given appropriate names that reflect the chemical composition. This point has been emphasized for myelin basic protein. It has been suggested that our protein N-2 be called lipophilin to describe its tendency to associate closely with lipids (D. Papahadjopolous, private communication).

CHEMICAL AND PHYSICAL PROPERTIES OF MYELIN PROTEINS

19

The two proteins that we have discussed, the myelin basic protein and the hydrophobic protein, must play very different roles in the maintenance of the integrity of the membrane. On the one hand, the basic protein with its rigid structure may have some function in maintaining a fixed structure since it would be less susceptible to denaturing conditions. On the other hand, the role of the hydrophobic protein (N-2) must be different. Because it is more flexible conformationally, it may be associated with functional aspects of myelin. An interesting view of the biological function of proteins has been put forward recently by Nickerson (1973). He proposes a concept of multistability. A protein is defined as multistable if it can be shown to have more than one most stable conformation. Multistability would permit a protein to function in different conformations depending on environmental circumstances, e.g., an enzyme that carries out a certain function when it is in a particular conformation may carry out the same reaction in a different conformation by altering its K , value. Such changes in K , values have been observed for pyruvate kinase of crab muscle and lactic dehydrogenase of rainbow trout as a mechanism of temperature compensation. Boos and Gordon (1971) have shown that the galactose-binding protein from Escherichia coli, responsible both for binding galactose and for its transport through the membrane, can exist in two conformational states-one with a high affinity for galactose and the other with a low affinity. They speculate that when galactose is bound a t the surface, the protein conformation is changed so that the resulting difference in surface charge allows the protein to move through the membrane. The second conformation has a lower affinity for galactose than the first so that the sugar is released on the inside of the membrane. The proteins of the sarcoplasmic reticulum have been shown to include ATPase of molecular weight 102,000 as the major protein; two other proteins of molecular weight 55,000 and 44,000 that are the high Ca2+binding protein and calsequestrin, respectively; several minor proteins; and a proteolipid (MacLennan, 197.5). The function of this proteolipid, which exists in a 1 :1 ratio with ATPase, is not known. It may have a structural role, forming a nucleus around which other proteins and lipids may bind. It may act as ionophore, transferring Cn2+ across the membrane. This latter role is especially attractive since the conformational flexibility described earlier for X-2 may be a characteristic of this protein as well, although this has not yet been established. Rhodopsin, an integral component of the disc membranes of the vertebrate rod outer segment of the eye, is a highly hydrophobic protein and similar in many respects to N-2. It has 235 residues and a molecular weight of 30,000, compared to 223 residues and a molecular weight of 25,000-

20

M. A. MOSCARELLO

28,000 for N-2. It contains 126 neutral residues compared to 124 in N-2 (calculated from the data of Heller, 1968). Although the role in visual excitation is not defined, it may store transmitter substance that can be released by photons of light, This role would be mediated by the conformational state of the protein. Conformational flexibility would appear to be important in this protein also (Chen and Hubbell, 1973). By freeze fracture studies (Chen and Hubbell, 1973; Vail et al., 1974), it has been shown to partition in lipid bilayers in a manner similar to N-2. Cytochrome b6 is a membrane protein of the endoplasmic reticulum of mammalian cells. In liver microsomes, it forms an important part of the system for the conversion stearoyl-CoA to oleoyl-CoA. It has been shown to exist in two domains-a hydrophilic and a hydrophobic one (Robinson and Tanford, 1975). The hydrophobic domain can produce a hydrophobic environment acting as a nucleus for the formation of micelles. Part of the molecule has an important enzymatic function, whereas the other part may function in organizing the membrane. Semlicki forest virus contains spikes protruding from the surface. These spikes are part of a membrane glycoprotein and can be cleaved by proteolytic digestion. When the spikes are gone, the virus is noninfectious; the remaining protein in the membrane is highly hydrophobic (Uterman and Simons, 1974). As in the case of cytochrome b6, the hydrophobic core appears to be important in anchoring the protein in the membrane. Although the above-mentioned hydrophobic proteins have been shown to possess a variety of functions related to enzymatic activities (ATPase and cytochrome b 6 ) , visual excitation (rhodopsin) , and viral infectivity (Semlicki forest virus), no functional role has been assigned to N-2. A possible role in energy transduction is mentioned in the following. Conformational changes in the protein components of the membrane may be important during some phase of the passage of an impulse. During the passage of an impulse, there is a net heat production in rabbit nerve of the order of 2 pcal/gm/impulse (Ritchie, 1973). Some mechanism must be available for the removal of this heat. If myelin is insulating the nerve, then the heat must be transmitted through the axonal membrane and through the myelin sheath in some way. A possible mechanism for the dissipation of heat may be a conformational change in a protein. The protein must be able to exist in more than one conformational state at any one time and these states must be interconvertible. The basic protein of myelin is too rigid a structure, since it has been shown to undergo conformational changes only at extremes of pH. However, the hydrophobic protein has been shown to be conformationally flexible. The absorption of heat by the molecule may result in a shift of equilibrium from one state to another that may help to dissipate the heat.

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

VII.

21

LOCALIZATION OF PROTEINS IN MYELIN

I n the lipid bilayer model of the myelin membrane developed from electron microscopic and X-ray diffraction data, the protein is considered to occupy an external position on either side of the lipid bilayer. A number of observations have forced us to reexamine this model to give to the protein components more defined and important roles. Napolitano et al. (1967) have reported electron micrographs of the myelin membrane after extraction of 98% of the lipids in chloroformmethanol and acidified chloroform-methanol. The micrographs showed the same multilamellar structure with 170-A spacings found in unextracted myelin. The authors concluded that the protein was responsible for maintaining the three-dimensional structure of the membrane. An earlier study by Shanthaveerappa and Bourne ( 1962) showed electron micrographs of optic nerve myelin in which a radial component was visualized. These “lines” were considered bridges between the layers. Akers and Parsons ( 1970) have discussed these observations and they considered the interesting possibility that protein bridges may be stabilizing the membrane structure. The above-mentioned observations support the view that the proteins of myelin may be inserted into the bilayer, in whole or in part, as has been proposed for rhodopsin in the retina. Studies in our laboratory have shown that the hydrophobic protein of myelin (N-2) can be inserted into lipid vesicles. I n a recent study, Vail el al. (1974) have shown that freeze fractures of liposomes membranes showed smooth fracture surfaces, whereas liposomes into which the hydrophobic protein (N-2) from human myelin had been incorporated showed particulate fracture surfaces. It was concluded that this protein was partially exposed and partially buried in the liposome membrane. These freeze fracture micrographs are shown in Fig. 2. A detailed study of this system by Papahadjopoulos et al. (1975) has shown that N-2 binds strongly to phospholipids irrespective of surface charge, the presence of cholesterol, or of double bonds on the fatty acyl chains. The buoyant density of the resulting lipoprotein membranes was intermediate between that of the pure lipids and proteins. The presence of N-2 in the membranes increased the permeability to sodium by 2-3 orders of magnitude. We concluded that N-2 was incorporated into the lipid bilayer and was partially exposed and partially buried. The myelin basic protein has been considered to be ideally suited for interacting with the phosphoryl groups of the lipid a t the surface of the myelin membrane. However, little evidence is available concerning the localization of this protein in myelin. Herndon et al. (1973) were unable to visualize the basic protein of myelin using immunoelectron microscopic

FIG.2. (1)Freeze fracture of a liposome. Note that the fracture surfaces are smooth without particles. (2) Freeze fracture of liposomes after the incorporation of a hydrophobic protein extracted from human myelin. Note the particles are present on the fracture surfaces. Total magnification, X75,OOO; bar = 0.1 pm.

CHEMICAL A N D PHYSICAL PROPERTIES

OF

MYELIN PROTEINS

23

techniques. They concluded that the antigenic determinants of the basic protein are occluded in vivo. A different approach to the localization of myelin proteins was reported recently (Wood and Moscarello, 1975) using a nonpenetrating reagent, 4,4’-diisothiocyano-2,2’-ditritiostilbenesulfonate ( DID5L3H). This reagent has been used by Cabantchik and Rothstein (1972) to label membrane proteins of the red cell. It is presumed to react with exposed amino groups of proteins, forming a covalent linkage. The myelin membrane was labeled with DIDS-”, and the basic protein and the hydrophobic protein (N-2) were isolated. After a %minute exposure to the reagent, the specific activity of N-2 was 30 times greater than that of basic protein. Water shock or sonication treatments of myelin did not affect the specific activities of the isolated proteins. When the isolated proteins were reacted with DIDS-3H the specific activity of basic protein was 10 times greater than that of K-2, the reverse of the situation found when the membrane was labeled. Therefore, we concluded that the basic protein was not accessible to the reagent when it formed a part of the myelin membrane. The basic protein could either be buried inside the bilayer or else it could be near the surface but occluded from the hydrophilic environment as a result of interactions with other membrane components such as phospholipids. Recently, Poduslo and Braun (1975) have shown that basic protein was not labeled by lactoperoxidase1251reaction when applied to the intact myelinated nerve bundle. Cyanogen bromide fragments of N-2 were prepared and resolved into a t least four Azso peaks on hydroxylopatite. The specific activity of one fragment was G times greater than the others implying that this fragment was more exposed to the hydrophilic surface than other parts of the protein. A similar study was done with myelin isolated from cases of multiple sclerosis (Wood et al., 1975). The specific activities of the basic protein and the hydrophobic protein isolated from chronic multiple sclerosis myelin were similar to the normals. By contrast, the specific activity of the basic protein isolated from acute multiple sclerosis myelin was about 400% higher than that of the basic protein from either normal or chronic multiple sclerosis material. The specific ttctivity of K-2 from the acute case was only 50% of that of N-2 isolated from chronic multiple sclerosis or from normal myelin. It was concluded that the arrangements of proteins in isolated chronic multiple sclerosis myelin was not markedly altered in comparison to that in isolated normal myelin. However, the arrangement of proteins in acute multiple sclerosis myelin appeared to be considerably different from that of the other two myelins. The experimental work on the arrmgement of proteins in myelin is just beginning. It is clear from this early work that the proteins are interacting

24

M. A. MOSCARELLO

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CHEMICAL A N D PHYSICAL PROPERTIES

OF MYELIN PROTEINS

25

Danielli, J. F., and Davson, H. (1934-35). A contribution of the theory of permeability of thin films. J . Cell. Comp. Physaol. 5 , 495408. Deibler, G. E., and Martenson, 11. E. (1973). Chromatographic fractionation of myelin basic protein. Partial characterization and methylarginine contents of the multiple forms. J . Biol. Chem. 248, 2392-6. Dunkley, P. R., and Carnegie, P. R. (1974). Amino acid sequence of the smaller basic protein from rat brain myelin. Biochern. J . 141, 243-261. Eng, L. F., Chao, F. C., Gerstl, B., Pratt, I)., and Tavastsjevna, M. G. (1968). The maturation of human white matter myelin. Fractionation of the myelin membrane proteins. Baochemistry 7, 4455-4465. Eng, L. F., Bond, P., and Gerstl, B. (1971). Isolation of myelin proteins from disc acrylamide gels electrophoresed in phenol-formic acid-water. Neurobiology 1, 58-63. Epand, R. M., Moscarello, M. A., Zirenberg, B., and Vail, W. J. (1974). The folded conformation of the encephalitogenic protein of the human brain. Biochemistry 13, 1264-1267.

Eylar, E. H. (1972). The structure and immunologic properties of basic proteins of myelin. Ann. N . Y . Acad. Sci. 195, 481-91. Eylar, E. H., and Thompson, M. (1969). Allergic encephalomyelitis: The physicochemical properties of the basic protein encephalitogen from bovine spinal cord. Arch. Biochem. Biophys. 129, 46&479. Eylar, E. H., Salk, J., Beveridge, G., and Brown, L. (1969). Experimental allergic encephalontekutus : An encephalitogenic basic protein from bovine myelin. Arch. Biochern. Biophys. 132, 3 H 8 . Eylar, E. H., Caccam, J., Jackson, J., Westall, F., and Robinson, A. B. (1970). Experimental allergic encephalomyelitis : Synthesis of disease-inducing site of the basic protein. Snence 168, 1220-3. Eylar, E. I%.,Brostoff, S., Hashim, G., Caccam, J., and Burnet, P. (1971). Basic A1 protein of the myelin membrane. The complete amino acid sequence. J . Bzol. ChenL. 246, 5770-5784.

Eylar, E. H., Brostoff, S., Jackson, J . J., and Carter, H. (1972). Allergic encephalomyelitis in monkeys induced by a peptide from the A1 protein. Proc. Natl. Acad. S’ci. U . S . A . 69, 617-9. Eylar, E. H., Jackson, J. J., Bennett, C. D., Kniskern, P. J., and Brostoff, S. W. (1974). The chicken A1 protein. Phylogenetic variation in the amino acid sequence of the encephalitogenic site. J . Biol. Chem. 249, 3710-6. Finch, P. R., and Moscarello, M. A. (1972). A myelin protein fraction extracted with thioethanol. Brain Res. 42, 177-187. Finch, P. R., Wood, D. D., and Moscarello, M. A. (1971). The presence of citrulline in a myelin protein fraction. FEBS (Fed. Evr. Biochcin. SOC.)Lett. 15, 145-148. Finean, J. B. (1953). Further observations on the structure of myelin. Exp. Cell Res. 5, 202-215. Folch Pi, J., and Lees, M. B. (1951). Proteolipides, a new type of tissue lipoproteins, their isolation from the brain. J . Biol. Chenz. 191, 807-817. Folch Pi, J., and Stoffyn, P. J. (1972). Proteolipids from membrane systems. Ann. N . Y . Acad. Sci. 195, 86-107. Gagnon, J., Finch, P. It., Wood, D. D., and Moscarello, M. A. (1971). Isolation of a highly purified myelin protein. Biochemistry 10, 4756-4763. Gonzalez-Srtstre, F. (1970). The protein composition of isolated myelin. J . Neurochem. 17, 1049-56.

Gorter, E., and Grendel, F. (1925). Bimolecular layers of lipoids on chromocytes of blood. J . Exp. Med. 41, 439443.

26

M. A. MOSCARELLO

Hagopian, A., and Eylar, E. H. (1969). Glycoprotein biosynthesis: The purification and characterization of a polypeptide: N-acetylgalactosaminyltransferase from bovine submaxillary glands. Arch. Biochem. Biophys. 129, 515-524. Heller, J. (1968). Structure of visual pigments. I. Purification, molecular weight, and composition of bovine visual pigment. Biochemistry 7, 2906-2913. Herndon, R. M., Rauch, H. C., and Einstein, E. R. (1973). Immuno-electron microscopic localization of the encephalitogenic basic protein in myelin. Zmmunol. Commun. 2, 163-172. Kibler, R. F., and Shapira, R. (1968). Isolation and properties of an encephalitogenic protein from bovine, rabbit and human central nervous system tissue. J. Biol. Chem. 243, 281-286. Kibler, R. F., Shapira, R., McKneally, S., Jenkins, J., Selden, P., and Chou, F. (1969). Encephalitogenic protein : Structure. Science la, 577-580. Kies, M. (1965). Chemical studies on an encephalitogenic protein from guinea pig brain. Ann. N . Y . Acad. Sci. 122, 161-70. Kies, M., Thompson, B. E., and Alvord, E. C. (1965). The relationship of myelin proteins to experimental allergic encephalomyelitis. Ann. N . Y . Acud. Sci. 122, 148-60. Korn, E. D. (1966). Structure of biological membranes. The unit membrane theory is reevaluated in light of the data now available. Science 153, 1491-1498. Lowden, J. A., Moscarello, M. A., and Morecki, R. (1966). The isolation and characterization of an acid-soluble protein from myelin. Can. J.Biochem. Physiol. 44,567-,577. Martenson, R. E., and Gaitonde, M. K. (1969). Comparative studies of highly basic proteins of ox brain and rat brain. Microheterogeneity of basic encephalitogenic (myelin) protein. J. Neurochem. 16, 889-898. Martenson, R. E., Deibler, G. E., and Kies, M. W. (1970). Rat myelin basic proteins: Relationship between size differences and microheterogeneity. J. Neurochem. 17, 1329-1330. Mateu, L., Luzzati, V., London, Y., Gould, R. M., Vossberg, F. G. A., and Olive, J. (1973). X-ray diffraction and electron microscope study of the interacJions of myelin components. The structure of a lamellar phase with a 150 to 180 A repeat distance containing basic proteins and acidic lipids. J. Mol. Biol. 7 5 , 697-709. MacLennan, D. H. (1975). Resolution of the calcium transport system of sarcoplasmic reticulum. Can. J. Biochem. 53, 251-261. MacLennan, D. H., Yip, C. C., Iles, G. H., and Seeman, P. (1972). Isolation of sarcoplasmic reticulum proteins. Cold Spring Harbour Symp. Quant. Biol. 37, 469-477. Mehl, E., and Halaris, A. (1970). Stoichiometric relation of protein components in cerebral myelin from different species. J. Neurochem. 17, 659-668. Merrifield, R. B. (1963). Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. SOC.85, 2149-2154. Miyamoto, E., and Kakiuchi, I. (1974). I n vitro and in vivo phosphorylation of myelin basic protein by exogenous and endogenous adenosine 3', 5'-monophosphatedependent protein kinases in brain. J. Biol. Chem. 249, 2769-2777. Miyazawa, T., and Blout, E. R. (1961). The infrared spectra of polypeptides in various conformations: Amide I and I1 bands. J. Am. Chem. SOC.83, 712-719. Mokrasch, L. C. (1967). A rapid purification of proteolipid protein adaptable to large quantities. Lije Sn'. 6, 1905-1909. Moscarello, M. A,, Gagnon, J., Wood, D. D., Anthony, J., and Epand, R. M. (1973). Conformational flexibility of a myelin protein. Biochemistry 12, 3402-3406. Moscarello, M. A., Katona, E., Neumann, W., and Epand, R. M. (1974). The ordered

CHEMICAL A N D PHYSICAL PROPERTIES OF MYELIN PROTEINS

27

structure of the encephalitogenic protein from normal human myelin. Biophys. Chem. 2, 290-295. Nakao, A., Davis, W. J., and Einstein, E. R. (1966). I. Isolation and Characterization. Basic proteins from the acidic extract of bovine spinal cord. Biochim. Biophys. Acta 130, 163-170. Napolitano, L., Le Baron, F., and Scaletti, J. (1967). Preservation of myelin lamellar structure in the absence of lipid. A correlated chemical and morphological study. J . Cell Biol. 34, 817-826. Neumann, A. W., Moscarello, M. A., and Epand, R. M. (1973). The application of surface tension measurements to the study of conformational transitions in aqueous solutions of poly-blysine. Biopolymers 12, 1945-1957. Nickecson, K. W. (1973). Biological functions of multistable proteins. J . Theor. Biol. 40, 507-515. Nicot, C., Le Neuyen, T., Le Pretre, M., and Alfsen, A. (1973). Study of Folch-Pi apoprotein. I. Isolation of two components, aggregation during delipidation. Biochim. Biophys. Acta 322, 109-123. Nussbaum, J. L., and Mandel, P. (1973). Brain proteolipids in neurological mutant mice. Brain Res. 61, 295-310. Nussbaum, J. L., Rouayreng, J. F., Mandel, P., Jolles, J., and Jolles, P. (1974). Isolation of terminal sequence determination of the major rat brain myelin proteolipid P7 apoprotein. Biochem. Biophys. Res. Cotnmirn. 57, 1240-1247. Palmer, F. B., and Dawson, K.hf. C. (1969).The isolation and properties of experimental allergic encephalitogenic protein. Biochern. J . 111, G29-636. I’apahadjopoulos, D., Vail, IV.J., and Moscnrello, 3f. A . (1975). Interaction of a purified hydrophobic protein from niyelin with phospholipid membranes: Studies on ultrast,ructure, phase transitions and permeability. J . Mernbr. Biol. 22, 143-164. Pasquini, J. M., and Soto, E. F. (1972). Extraction of proteolipids from nervous tissue with n-BUTANOL-WATER. Life Sci. 11, Part 11, 433443. Poduslo, J. F., and Braun, P. (1975). Topographical arrangement, of membrane proteins in the intact myelin sheath. J . Biol. Chew. 250, 1099-1105. Ritchie, J. M. (1973). Energetic aspects of nerve conduction. The relationship between heat production, electrical activity and metabolism in progress. Biophys. Mol. Biol. 26, 147-187. Robinson, N. C., and Tanford, C. (1975). The binding of deoxycholate, trit,on X-100, sodium dodecyl sulfate, and phosphatidycholine vesicles to cytochrome b5. Biochemistry 14, 369-377. Roboz-Einstein, 13. (1972). Basic protein of myelin and its role i n experimental allergic encephalomyelitis and multiple sclerosis. Handh. Nwrochent. 7, 107-122. Roboz-Einstein, E., Robertson, D., Dicaprio, J., and Moore, W. (1962). The isolation from bovine spinal cord of a homogeneous protein with encephalitogenic activit>y. J . Neurochem. 9, 353-61. Shanthaveerappa, T. R., and Bourne, G. 1%. (1962). Radial bands in the optic nerve myelin sheat,h. Nature (London) 196, 1215-17. Sherman, G., and Folch-Pi, J, (1971). On the type of linkage binding fatty acids present in brain white matter proteolipid apoprotein. Biochem. Biophys. Res. Commun. 441, 157-161. Singer, S. J., and Nicolson, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175, 720-731. Smyth, D., and Utsumi, S. (1967). Structure a t the hinge region in rabbit immunoglobulin-G. Nature (London) 216, 332-335.

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The Distinction between Seauential and Simultaneous Models for Sodium and Potassium Transport P. J . GARRAHAN A N D R. P. GARAY Departamento de Quimica Bioldgica Facultad de Farmacia y Bioqimicu Buenos Aires, Argentina

I. Introduction. . . . . . . . . . . . , . . . . . 11. Cation-Binding Sites of the Na Pump . . . . . . . . . . A. General Properties , . . . . . . . . . . . . . B. Distinction between Affinity of Cation-Binding Sites and Reactivity of Cationsite Complexes . . . . . . . . . . . . C. Interactions among Cation-Binding Sites of the Na Pump . . . D. Are Cation-Pump Complexes in Equilibrium with Free Cations? . E. Inner and Outer Cation-Binding Sites . . . . . . . . . F. Relation between Inner and Outer Cation-Binding Sites. . . . 111. Experimental Evidence for Simultaneous Existence of Inner and Outer Cation-Binding Sites . . . . . . . . . . . . . . A. Biochemical Evidence. . . . . . . . . . . . . . B. Kinetics of Cation Fluxes . . . . . . . . . . . . IV. Intermediate Stages in Hydrolysis of ATP by the Na Pump . . . . A. Phosphorylation and Dephosphorylation of the Pump . . . . B. Apparent Uncoupling between Partial Reactions and Cationic Fluxes C. Meaning of Apparent Uncoupling between Partial Reactions and Cationic Fluxes . . , . . . . . . . . . . . . V. Mechanism of Ion Transport . . . . . . . . . . . . . A. Na Pump as a V System. . . . . . . . . . . , . B. Are Changes in Affinity of the Cation-Binding Sites Necessary for Active Transport?. . . . . . . . . . . . . . C. Other Transport Systems . . . . . . . . . . . References . . . . . . . . . . . . . , , .

.

1.

. . .

29 31 31

. . . . .

33 34 33 37 38

. .

40 40 44

.

. . .

68 69 72

. . .

76 77 77

. . .

84 90 91

.

INTRODUCTION

The active transport of Na and I< driven by the Na pump is one of the most important energy-transducing functions of cell membranes. Although 29

30

P. J. GARRAHAN AND R.

P. GARAY

a large amount of experimental data about this system is now available, no coherent scheme has yet been evolved to integrate these data into a plausible molecular mechanism for the coupling of the hydrolysis of ATP to the active movement of cations. We do not intend in this article to propose a detailed hypothesis to account for this phenomenon. Rather we will try to analyze the available experimental evidence to see if it can be used to place restrictions on the possible molecular mechanisms of active transport. It is hoped that these restrictions, by circumscribing the possible modes of operation of the Na pump, will help to reach the goal of elucidating its mechanism. To do this we will concentrate on one particular problem within the gencral question of the mechanism of active transport, namely, the relation between the inward-facing and the outward-facing, cation-binding sites of the Na pump. One of the salient features of the Na pump is that all the cation movements it is able to drive have strict cation requirements at both the inner and the outer surfaces of the cell membrane. This almost certainly means that cation movements only take place if inward-facing and outward-facing sites of the pump have a definite state of occupation by cations. It is obvious, therefore, that the nature of the linkage between inner and outer sites is a problem that has to be considered by any hypothesis accounting for tho mechanism of active transport. In the second section of this paper, we intend to define with precision the concept of cation-binding sites and to characterize the interactions between sites and with cations. The section ends with a statement of the two alternative explanations that have been proposed to account for the linkage between inner and outer sites: (i) these sites are alternative states of the same set of sites (sequential models) or (ii) they are two physically distinct and independent sets of sites that coexist in each transport unit (simultaneous models). We think that available experimental evidence rather strongly favors the idea of the simultaneous existence of inner and outer cation-binding sites. A critical analysis of this evidence is undertaken in the third section. Although structural and biochemical data are taken into account, the main weight of our reasoning is placed on the results of experiments on the kinetics of cation fluxes. In this respect we are convinced of two facts, which we would like to state clearly before detailing, in the corresponding section, the reasons of our belief. 1. If, as it seems likely, (a) free cations are in equilibrium with the cation-pump complexes and (b) the rate equation for cation fluxes can be expressed as a function of external cations only, times a function of internal cations only, then, not only the simultaneous existence of inner and outer sites may be considered as having been demonstrated, but also only a

31

SEQUENTIAL AND SIMULTANEOUS MODELS FOR Na AND K TRANSPORT

particular class of the set of simultaneous models is able to account for the behavior of the Na pump. 2. Since i t can be demonstrated that only a particular class of simultaneous models gives rate equations with separation of intra- and extracellular variables, lack of separation of variables is not a n argument against simultaneous, or in favor of sequential models, but only against a particular class of simultaneous models. The fourth section of this papcr deals with the biochemical aspects of the Na pump, namely, with the intermediate steps and the partial reactions of the hydrolysis of ATP catalyzed by this system. Most experiments performed on this subject have been interpreted within the conceptual framework of sequential schemes of transport. Our aim in this section is to show that the present knowledge of the mechanism of the hydrolysis of ATP by the Na pump is not incompatible with thc postulates of simultaneous models for act.ive transport. The last section is the most speculative one of this paper. In it we take for granted the simultaneous schcmc devcloped from the equilibrium treatment of the kinetics of cation fluxes, and, in order to further circumscribe the possible mechanisms of active transport, confront this scheme with tho available information on the interaction between cation-binding and metabolite-binding sites and on the degrccs of freedom of mcmbranebound proteins. The picturs that emerges from this confrontation seems to indicate that, in the operation of the Na pump, intcractions in reactivity predominat,e over interactions in affinity and that cation translocation probably takes place by some sort of “internal transfer” mechanism. This article does not intend to present a comprehensive survey of the copious literature on active transport of Na and I ADP (+ EDTA) eADPb (+ Mg'+) cADPb (+ Caz+) SHDP" IDP SHTPe ATP ADP

ADP (+ Mg*+) ADP ADP

Ki

bM) 0.28

11

0.5 0.5 42

> 10 >40

-

0.92d 2.04d 16.6' 4.Y

Technique Gel filtration Ultrafiltration Fluorescent titration Fluorescent titration Absorbance difference spectrum titration Competitive ultrafiltration Competitive ultrafiltration

-

Equilibrium dialysis Ammonium sulfate precipitation Ultrafiltration Ammonium sulfate precipitation

Kz

W)

47 30 43 100-200 -

Technique Gel filtration Steady-state kinetics Ultrafiltration Estimated from gel filtration

-

.-2oo(i

Estimated from steady-state kinetics

-2000

Estimated from steady-state kinetics Steady-etate kinetics Steady-state kinetics

-

650 220

-

-

-

-

-

-

The experimental conditions are described in Hilborn and Hammea (1973). rADP is 3-&~-ribofuranosylimidazo(2,l-i)purine-5'-diphosphate. SHDP and SHTP are 6-mercapto-9-p-~-ribofuranosylpurine-5'-d~phosphate and -5'-triphosphate, respectively. dFrom Hilborn and Hammea (1973). 'From Sanadi et al. (1971). From Zalkin et d.(1965). b

113

MEMBRANE ATPasss

man et al., 1960), the binding of nucleoside diphosphate is also very specific. ADP and 3-~-~-ribofuranosylimidazo ( 2 ,1 4 ) -purine-5’-diphosphate (EADP) are the only diphosphates that bind to F1 in nearly equivalent amounts, whereas IDP, UDP, and AMP bind very little (see Table 11). Different nucleoside diphosphates compete with ADP for the binding, but their dissociation constant is higher (Hilborn and Hammes, 1973). Thus, it appears that the tight binding site (site 1) measured by Hilborn and Hammes is very specific, whereas the weak site (site 2) is less specific toward nucleoside diphosphates (see Table 11). Harris el al. (1973) have claimed that F1 in both soluble and membrane-bound form contains 3 tightly bound molecules of ATP and 2 of ADP per molecule. The nucleotides are not removed from the enzyme by standard physical techniques such as repeated precipitation, treatment with activated charcoal, or filtration through Sephadex and anion exchange resins, but they are removed by perchloric acid denaturation or cold inactivation in the presence of nitrate. Warshaw et al. (1967) also noticed that the bound ADP in Factor A is released by heat inactivation. In a recent paper, Rosing et al. (1974) found that adenine nucleotides (AdN) are released from F1 in parallel with cold activation. They suggest that the first product of cold inactivation still contains bound nucleotides but in a form removable by ammonium sulfate precipitation. They have proposed the following scheme for cold inactivation:

irreversible

00

subunits - AdN

F1 - AdN 30’

subunits

+ AdN

Although their proposal is attractive, more supporting data are needed. 7. DIVALENT CATIONS

Membrane-bound as well as soluble F1 requires divalent cation for maximal activity. The purified F1-ATPase activity shows complete dependency on Mgz+,but, in contrast to the ATPase activity of the mitochondrial membrane, other divalent cations such as Mn2+, Go2+,and Fe2+ could replace Mgzf with varying degrees of effectiveness (Pullman et al., 1960; Schatz et al., 1967; Selwyn, 1967). Stimulation of ATPase by D N P was observed only in the presence of Mg2+ (Pullman et al., 1960; Selwyn, 1967). The lack of specificity toward cations is true also for the yeast F1 (Schatz et al., 1967; Sone et al., 1969). Adolfscn and Moudrianakis (1973) found that the optimal concentration for Mg2+ is not dependent on ATP concentration, but the ATP concentrations that they used were below the K,.

114

RIVKA PANET A N D D. RAO SANADI

C. Molecular Properties

1. MOLECULAR WEIGHTAND SEDIMENTATION COEFFICIENT

Factor F1 from beef heart is globular with S20,wof 11.9 S (Penefsky and Warner, 1965; Schatz et al., 1967; Forrest and Edelstein, 1970) ; rat liver F1 has a sedimentation coefficient of 12.1-12.9 S (Lambeth and Lardy, 1971; Catterall and Pedersen, 1971) ; and the yeast enzyme is 11.6-13.0 S (Schatz et al., 1967; Tzagoloff and Meagher, 1971; Sone et al., 1969). The molecular weight of beef heart F1was first estimated to be 284,000 (Penefsky and Warner, 1965; Kagawa, 1969; Forrest and Edelstein, 1970), but Lambeth et al. (1971) obtained a value of 360,000 by gel filtration and high-speed sedimentation. The revised molecular weight is very similar t o that of rat liver F1 (384,000) by gel filtration and sedimentation on sucrose gradient (Catterall and Pedersen, 1971), or it is 360,000 by sedimentation equilibrium (Lambeth and Lardy, 1971). The yeast F1 has also roughly the same molecular weight, namely, 340,000 (Tzagoloff and Meagher, 1971). It appears that all F1 preparations from different sources are of nearly the same molecular size. Electron micrographs of mitochondria1 ATPase from several sources reveal spherical particles approximately 90 A in diameter with five to six symmetrically arranged subunits (Kagawa and Racker, 1966a; Racker and Horstman, 1967; Schata et al., 1967). 2. SUBUNIT STRUCTURE OF F1

In contrast to previous reports that F1has ten to twelve (Penefsky and Warner, 1965) or six (Datta and Penefsky, 1970; Forrest and Edelstein, 1970) identical subunits, electrophoresis in the presence of phenol-urea (Tzagoloff et al., 1968a; Sanadi et al., 1971) or SDS showed that the subunit composition is considerably more complex. Senior and Brooks (1970,1971) showed that the phenol-urea gels were prone to artifacts. From the summary of the results from different laboratories using SDS gel electrophoresis (Table 111),it is seen that F1 preparations from different sources have a uniform subunit structure, consisting of five to six distinct subunits. The similarities in the subunit structure of F1and Factor A has also been demonstrated (Sanadi et al., 1971). Subunits 1 and 2 are in the highest concentration and nearly equal in amount. They are close in molecular weight and have only a slight difference in amino acid composition (Knowles and Penefsky, 1972; Brooks and Senior, 1972). Knowles and Penefsky separated F1 subunits in bulk and determined their molecular weights by four different methods: (1) SDS gel electrophoresis, (2) amino acid

TABLE I11 SUBUNITSTRUCTURE OF MITOCHONDRIAL ATPase

FROM

DIFFERENTSOURCES' Subunit:

Source

Method

1

2

3

1. SDS electrophoresis 2. Gel filtration in 6 M guanidine 3. Amino acid composition 4. Equilibrium sedimentation

59,400

54,000

33,000

53,200

50,800

53,300

4

5

6

13,600

-

-

33,000

17,300

11,350

5,570

49,000

33,160

16,100

-

5,850

54,800 55,000

50,300 52,000

30,000

17,500 12,500

8,000

-

53,000 53,000 65,000

50,000 60,000

25,000 28,000 36,000

12,500 12,500

58,500

54,000

38,500

31,000

Beef heart Knowles and Penefsky, (1972b)

Capaldi (1973) R a t liver Senior and Brooks (1971) Lambeth and Lardy (1971) Catterall and Pedersen (1971)

-

10,500 8000-9000 -

7,500 -

Yeast Tzagoloff (1971a) a

Values shown are the molecular weights.

12,000

-

116

RIVKA PANET A N D D. RAO SANADI

analysis, (3) gel filtration, and (4) equilibrium sedimentation. In the determinations by the last two methods, high concentrations of guanidine or urea were present together with DTT (Knowles and Penefsky, 1972a) to keep the subunits in solution. The values by these methods were very similar. Subunit 1 with a molecular weight of 53,000 obtained by Lambeth and Lardy (1971) might be unresolved subunits 1 and 2. With subunit 4 the variations among different reports are larger: by SDS gel electrophoresis, values between 12,500-13,500 daltons were obtained with beef heart and rat liver, whereas the subunit 4 of yeast FI was much higher in molecular weight (see Table 111). Knowles and Penefsky (1972b) noted higher molecular weights for subunit 4 (16,000-1 7,500) with their other measurements compared to their SDS gel measurements. The molecular weight determination by SDS gel electrophoresis mobility is less accurate for the smaller peptides and might, thus, explain these variations. 3. STUDIESON

THE

ACTIVESITEOF F1 (AND CF1)

Soluble beef heart mitochondria1 ATPase contains a total of eight sulfhydryl groups and two disulfide bonds per 360,000 gm. Two of the sulfhydryl groups are freely accessible to iodoacetate or Ellman’s reagent (Senior, 1973). The ATPase activity is remarkably resistant to reagents that block sulfhydryl groups in proteins such as N-ethylmaleimide, mercurials, and Ellman’s reagent (Pullman et al., 1960; Senior, 1973). Penefsky (1967) studied the effect of low iodine on F1 activities and found that loss of ATPase activity was accompanied by disappearance of -SH groups. The iodinated F1 lost almost entirely the ability to bind adenine nucleotides but retained coupling activity with N particles. The inhibition of ATPase activity by iodine was not affected by subsequent treatment with - S H compounds such as DTT or mercaptoethanol. Thus, it would appear that IZ reacted with other groups, such as tyrosine (Penefsky, 1967), at the active center of the ATPase. The recent paper of Hilborn and Hammes (1973) shows that the -SH derivative of ADP, 6-mercapto9D-ribofuranosyl purine-5-diphosphate (SHDP), which is known to form disulfides with protein - S H groups, failed to label F1, in agreement with the conclusion that no - S H is present at the ATPase active center. It was shown by Senior (1973) that tetranitromethane and iodoacetate inactivated the F1-ATPase rapidly, and the inactivation was prevented by ATP. When the inactivation by tetranitromethane was complete, in the presence of ATP, a total of 9 to 10 tyrosine residues were nitrated; ATP reduced the amount of inactivation by half and the amount of tyrosine residues nitrated to 6-7. Based on the preceding results, Senior (1973)

MEMBRANE ATPases

117

suggested that tyrosine may be involved in the binding and hydrolysis of ATP. Ferguson et (11. showed that NBD-Cl, which is known to react with tyrosine and -SH groups (Gosh and Whitehouse, 1968), inactivated beef heart Fl-ATPase (Ferguson et al., 1974). From spectral properties of the reaction product, they suggested that the modified group is tyrosine and not 4 H . Bacterial ATPase (Nelson et al., 1974) as well as CFI (Deters et al., 1975) were also found to be inactivated by NBD-Cl. Xelson el al. and Deters et al. claimed that since the inhibition was completely reversed by DTT, the NBD-C1 reacts with -SH at the active site. They observed that the spectral data were not in agreement with a NRD-sulfur derivative but consistent with NBD-tyrosine; nevertheless, they claim that the microenvironment of the chromophore on the protein might change its spectral properties. From its chemistry, NBD-C1 is known to be more reactive with -SH than with tyrosine (Gosh and Whitehouse, 1968), but the spectral properties of NBD bound to ATPases from beef heart, chloroplast, as well as bacterial ATPase are consistent with NBD-tyrosine. The suggestion that -SH group is functional a t the active site contrasts with the finding that the ATPase is resistant to reagents that block -SH groups. It is, thus, necessary to examine how DTT relieves NBD-Cl inhibition. If, indeed, NBD-Cl reacts with -SH in ATPase, it should be possible to demonstrate this directly by -SH titration. D. Summary

I n the last few years, there has been some progress in unraveling the molecular and enzymatic properties of F1. The subunits of the enzyme have been isolated and their molecular weights and amino acid composition determined. Some additional information has become available on the amino acid(s) a t the active site. Isolation of the subunits of F1 holds promise for the elucidation of their role, although the major difficulty may be that they have been isolated in the presence of urea to keep them in solution. If they are denaturated, other methods may have to be devised for their isolation. It is still to be proved unequivocally that tyrosine is, indeed, the amino acid a t the active site and not -SH. It is not known on which subunit it is located. Studies with specific inhibitors, such as aurovertin, Pullman inhibitor, quercetin, and AMP-PNP show clearly that F1 may have two catalytic sites specialized, respectively, for ATP synthesis and ATP utilization. This raises the interesting possibility that the two opposite pathways could be controlled separately. This hypothesis might lead to the identification of two different active groups functional in opposite directions.

118

RIVKA PANET AND D. RAO SANADI

111.

OLIGOMYCIN-SENSITIVE ATPase

The OS-ATPase complex is firmly associated with the inner mitochondrial membrane and could be “solubilized” only by surface-active reagents. Detergents dissociate lipid-protein complexes and, therefore, different detergents produce complexes varying with respect to their phospholipid content. Tzagoloff has published excellent reviews on the properties of this complex (Tzagoloff, 1971a; Tzagoloff, 1973). A. Enzymatic Properties

1. OLIGOMYCIN SENSITIVITY

The OS-ATPase, isolated either from beef heart mitochondria (Kagawa and Racker, 1966a, b; Tzagoloff et al., 1968a, b; Kopaczyk et aZ.,1968) or from yeast (Tzagoloff, 1969a; Tzagoloff and Meagher, 1971), is sensitive to oligomycin, rutamycin, trialkyltin chloride, and DCCD, but FI is resistant. The oligomycin-binding site is probably located in the OS-ATPase complex. Beef heart mitochondria are more sensitive to oligomycin than yeast mitochondria. Twenty times higher concentrations of oligomycin are needed to inhibit the yeast mitochondria than the beef heart enzyme. The yeast OS-ATPase also is less sensitive to rutamycin than the beef heart enzyme reflecting the sensitivity of the mitochondria (Tzagoloff, 1969a). It has been reported that when yeast F1 is bound to beef SMP, the reconstituted hybrid ATPase is inhibited by oligomycin at the same concentrations that inhibit beef ATPase (Schatz et al., 1967). The degree of oligomycin sensitivity depends on the phospholipid composition (Bulos and Racker, 1968b) and amount (Tzagoloff, 1969a) of the complex (see also Sections 111, A, 4 and 111, C). OS-ATPase isolated from oligomycin-resistant yeast mutants could still be inhibited by oligomycin but the concentration required to give 50% inhibition increased thirty-fold compared to the wild-type OS-ATPase. This again reflects the sensitivity of the intact wild-type and mutant yeast cells (Broughall et al., 1973). The OS-ATPase complex isolated with Triton X-100 from yeast (Tzagoloff and Meagher, 1971) is inhibited at higher concentrations than the complex isolated by deoxycholate extraction (Tzagoloff, 1969a), presumably due to the presence of the detergent. The same phenomenon appears when an excess of phospholipid is added to the OS-ATPase complex, more oligomycin being needed for inhibition due to competition by excess lipid (Kagawa and Racker, 1966b; Tzagoloff, 1969a). Compounds DCCD, rutamycin, and tri-n-butyltin chloride (which are all

MEMBRANE ATPaser

119

believed to act at the same site or very close to each other in the overall scheme of ATP synthesis; see Fig. 1) inhibit OS-ATPase from beef heart (Tzagoloff et al., 1968b) or yeast (Tzagoloff, 1969a; Tzagoloff and Meagher, 1971). All the F1 inhibitors including aurovertin, Pullman inhibitor (Tzagoloff el al., 1968b) and antiserum against F1 (Tzagoloff and Meagher, 1971) inhibit the OS-ATPase also. 2. COLDSTABILITY The OS-ATPase is stable at 0°C in contrast to the cold lability of Fl (Tzagoloff et al., 1968b; Kopaczyk et al., 1968). Factor F1isolated from the complex shows the usual cold lability (Tzagoloff et al., 1968b; Kopaczyk et al., 1968).

3. NUCLEOTIDE AND DIVALENT CATIONSPECIFICITY The purified OS-ATPase complex exhibits enzymatic properties similar to those of SMP ATPase; it cleaves nucleoside triphosphates other than ATP. GTP and ITP are hydrolyzed at rates 26 and 17%, respectively, compared to that of ATP, whereas other triphosphates are cleaved at less than 50/, of the rate with ATP (Tzagoloff et aZ., 1968b; Kopaczyk et aZ., 1968). Thus, its specificity to triphosphate is more restricted than that of F1, which cleaves all triphosphates except CTP (see Section 11, B, 5). The OS-ATPase is stimulated by Mg2+ in the same manner as F1-ATPase; cation Mg2+could be substituted by Mn2+ (80%) or Go2+ (3oyO), whereas Ca2+ is almost ineffective. FrhTPase, on the other hand, cleaves ATP well when Mg2+is substituted by Ca2+ (Tzagoloff et al., 196813).

4. PHOSPHOLIPIDS AND THEIR POSSIBLE ROLE Phospholipids are essential components of the OS-ATPase complex and they markedly stimulate ATPase activity (Kagawa and Racker, 1966a; Bulos and Racker, 196813; Kopaczyk et al., 1968; Tzagoloff, 1969a), whereas F,-ATPase activity is not affected by phospholipids. The degree of activation by phospholipids depends on the intrinsic amount of phospholipids already bound to the particular preparation. Kagawa and Racker’s CFo-Fl preparation has very low phospholipids and almost no ATPase activity; activity could be induced by adding phospholipids (1966b). Kopaczyk et al. (1968) purified OS-ATPase with roughly 10% phospholipids from beef heart mitochondria and found that it could be activated up to ten-fold by adding phospholipids. Saturation was obtained with 30% phospholipids. The yeast OS-ATPase isolated using deoxycholate and salt precipitation is also markedly (ten-fold) stimulated by adding phospho-

120

RIVKA PANET AND D. RAO SANADI

lipids (Tzagoloff, 1969a). The beef heart OS-ATPase isolated by Tzagoloff et al. (1968b) has 30% phospholipids by weight and is not stimulated further by phospholipids. The OS-ATPase isolated from yeast mitochondria by a new purification using Triton X-100 (Tzagoloff and Meagher, 1971) is not stimulated by adding phospholipids although it contains only 10% phospholipids (in addition to the detergent present in the complex). It would appear that detergents are able to substitute for phospholipids in the OS-ATPase (Kagawa and Racker, 1966b). Kopaczyk et al. (1968) suggest that the activation of the ATPase by detergent is achieved by making the small amount of phospholipids in the complex more effective. The phospholipid requirement of the OS-ATPase is not specific. Different preparations of phospholipids from different sources, as well as fatty acids or detergents, could induce activity in the complex. In a recent paper, Cunningham and George (1975) showed that acidic phospholipids such as cardiolipin, phosphatidylglycerol, and phosphatidic acid activate the OS-ATPase to higher levels than do the neutral phospholipids such as lecithin and phosphatidylethanolamine. They measured the apparent K , for activation of different phospholipids and showed that it varied from 30 to 100 p M . No correlation was found between the relative affinity of the enzyme for phospholipids and the V , value (Cunningham and George, 1975). The oligomycin sensitivity of the complex, on the other hand, appears more dependent on the nature of the phospholipids (Kagawa and Racker, 1966b; Kopaczyk et al., 1968; Tzagoloff, 1969a). Cunningham and George (1975) could show that oligomycin is a competitive inhibitor with respect to all phospholipids tested except phosphatidylethanolamine and phosphatidylglycerol. It was proposed that ergosterol might have an important role in producing oligomycin sensitivity (Broughall et al., 1973). However, Griffiths and Houghton (1974) showed that there is no correlation between oligomycin resistance and the ergosterol concentration of yeast cells, yeast mitochondria, or mitochondria1 ATPase. Isolation of the proteolipid subunit that binds DCCD might give more information about the site of DCCD and oligomycin binding and the role of the lipid in the inhibition (see Section 111, C).

B.

Molecular Properties

1. MOLECULAR WEIGHTAND SIZE The OS-ATPme complex tends to aggregate if the bile salts are removed. By adding phospholipids to the complex, the amorphous aggregates are

MEMBRANE ATParer

121

converted to vesicular membranes. The fact that low concentrations of Triton X-100 maintain the yeast complex in a dispersed form has made it possible to estimate its molecular size (Tzagoloff and Meagher, 1971). The sedimentation of the complex and of F1 was carried out in a sucrose gradient containing 0.2% Triton. The s2Olw of the yeast complex was found to be 15.3 S and that of F1, 12.1 S. Knowing the phospholipid content and the specific volume of phospholipids, Tzagoloff and Meagher arrived a t a molecular weight of 520,000 for the complex and 340,000 for F1. Despite the limitations of this measurement, it shows that the complex is not very much larger than Fl. Electron microscopy revealed that the complex from yeast is an oval particle with dimensions of 100 X 150 A (Tzagoloff and Meagher, 1971 ; Kopaczyk et al., 1968). Tzagoloff proposed that a small unit, roughly 50-70 A, is attached to Fl to give the oval shape to the complex. 2. SUBUNIT STRUCTURE OF OS-ATPase The OS-ATPase isolated from beef heart (Tzagoloff et al., 1968a; Kopaczyk et al., 1968) and yeast mitochondria (Tzagoloff, 1969a; Tzagoloff and Meagher, 1971) consists of phospholipid and eight or nine different peptides of varying molecular weight. The protein subunits consist of three functional components: (1) F1; and ( 2 ) a membrane factor that is esscmtial for the oligomycin sensitivity; ( 3 ) a small prptide that has been claimed to act as a link between F, and the mrmbrane factor called OSCP (oligomycin sensitivity conferring protein) or F,. a. Factor 1 . The five subunits of Fl and OSCP are components, as shown by gel electrophoresis of beef heart (Tzagoloff et al., 1968a; MacLennan and Tzagoloff, 1968; MacLennan et al., 1968; Capaldi, 1973) and yeast (Tzagoloff and Meagher, 1971) complexes (see also Section 11,C, 2 ) . Extraction of submitochondrial particles with NaBr, which is known t o solubilize F1-ATPase, removes subunits 1 and 2 of the complexes (the major subunits of F1) completely; 3, 4, and 8 only partially; and subunits 5, 6, 7, and 9 that are specific to the complex are not extracted a t all (Tzagoloff and Meagher, 1971). b. OSCP. Compound OSCP is a peptide that, together with a membrane fraction, is essential for expression of oligomycin sensitivity. It is claimed to be the stalk connecting F1to the membrane piece seen in electron micrographs. It has been isolated from the beef heart OS-ATPase complex (MacLennan and Tzagoloff, 1968) and from SMP (MacLennan and Asai, 1968). The OSCP has been extracted from OS-ATPase by dilute ammonium hydroxide and purified to near homogeneity (MacLennan and Tzagoloff, 1968). Bulos and Racker (1968a) isolated from beef heart mitochondria a factor, which they denoted F, or FO1, with properties similar to those of

122

RIVKA PANET A N D D. RAO SANADI

OSCP. Later the F, was shown to be very crude (Senior, 1971) by SDS gel electrophoresis. It has also been purified from yeast mitochondria (Tzagoloff, 1970). The yeast OSCP appears almost identical with beef heart OSCP, although they are not fully interchangeable in hybridization experiments. The term OSCP can lead to misunderstanding; it does not confer oligomycin sensitivity to F, alone but only to a complex of F1and a membrane fraction (MacLennan and Tzagoloff, 1968; Bulos and Racker, 1968a). The complex has much less ATPase activity than the F1 used to prepare the complex. There is evidence indicating that the hydrolytic site in the complex is probably different from the active site of F1. The OSCP also stimulates the P,-ATP exchange activity of ammonia particles and phospholipid particles (MacLennan and Tzagoloff, 1968). Seen in the electron microscope, OSCP has a cylindrical shape, 50-55 A in length, 30-35 A in diameter, and tends to form tetrads (MacLennan and Asai, 1968). It is a strongly basic protein with an isoelectric point of 9.3. Its molecular weight is 18,000 by SDS gel electrophoresis (MacLennan and Asai, 1968; Senior, 1971). It may be identical with subunit 7 of OS-ATPase according to Tzagoloff and Meagher (1971), or to subunit 6, according to Capaldi (1973). The amino acid analysis of OSCP shows 17 lysine and 9 arginine residues per 18,000 molecule weight and only 1.3 histidine (Senior, 1971). c. The Membrane Factor. In addition to F1 and OSCP, the OS-ATPase complex of yeast or beef heart contains three or four other peptide subunits that together have been named the membrane factor (Tzagoloff, 1973) because of its low solubility in aqueous media and its solubilization with detergents (Tzagoloff and Meagher, 1971; MacLennan et al., 1968). The membrane factor is essential for the conferral of oligomycin sensitivity as was shown by the reconstitution of ATPase activity of mitochondrial membranes depleted of F1and OSCP (Tzagoloff, 1970; Bulos and Racker, 1968a). Table IV shows the subunits of yeast and beef heart OS-ATPase compared to F1 subunits. The five subunits of F1 and OSCP are present in both complexes together with at least three more subunits constituting the membrane factor. The molecular weights of the subunits of the membrane factor are quite similar in yeast (Tzagoloff and Meagher, 1971) and beef heart (Capaldi, 1973) (Table IV) . By double-labeling experiments, Tzagoloff et al. (1972) showed that subunits 1, 2, 3, 4, and 8 are common to F1and to OS-ATPase, whereas subunits 5, 6, 7, and 9 are present only in the complex. Subunit 7 is OSCP. Tzagoloff has shown that subunits of the membrane factor are synthesized by the mitochondrial protein-synthesizing system (Tzagoloff, 1969b; 1971b; Tzagoloff and Meagher, 1972; Tzagoloff et al., 1972). The studies on

TABLE IV SUBUNIT STRUCTURE OF OS-ATPase, F,,AND Yeast (Tzagoloff and Meagher, 1971)

Subunit No.

PROTEIN SYNTHESIZED BY MITOCHONDRIA~

Beef heart (Capaldi, 1973)

F1

OS-ATPaseb

Fi

OS-ATPaseb

58,500 54,000 38,500 31,000

58,500 54,000 38,500 31,000 29,000 22,000 18,500 12,000 7,500

55,000 52,000 30,000

55,000 52,000 30,000 29,000 20,000 19,000 12,500 10,000 8,000

-

12,000 a

THE

Values shown are the molecular weights. OSATPase, oligomycin-sensitiveATPase.

-

12,500

-

8,000

Mitochondria1 product Tzagoloff and Akai (1972)

(45,000)

29,000 21,000 12,000 7,000

Thomas and Williamson (1971) 48,000 33,000 28,000

15,000 11,000

124

RIVKA PANET AND D. RAO SANADI

the biosynthesis of mitochondrial ATPase have been reviewed recently (Tzagoloff, 1973) and will not be repeated here. We shall limit ourselves only to the structure of OS-ATPase complex, which can be understood better in the light of the experiments on its biosynthesis. By specificlabeling of the products of mitochondrial synthesis, Tzagoloff and Akai (1972) showed that five subunits are synthesized within yeast mitochondria (see also Table IV). The labeled products have molecular weights of 45,000 (major peak), 29,000, 21,000, 12,000, and 7,800. The 45,000-dalton subunit appears to be a polymeric form of the smallest subunit (7800 daltons) . By pretreatment with organic solvents or depolymerization with SDS under alkaline conditions, the 45,000-dalton subunit undergoes reduction in size and is converted to the 7800-dalton subunit (Tzagoloff and Akai, 1972). Thus, the authors have proposed that the major product of mitochondrial protein synthesis is a small peptide that resists depolymerization under the standard conditions used in SDS gel electrophoresis. This smallest peptide was shown in a later paper (Sierra and Tzagoloff, 1973) to be identical with subunit 9 of OS-ATPase complex. It was purified and its amino acid composition determined. It has an extremely large amount of nonpolar residues which may account for its solubility in organic solvents. In Table IV it may be seen that the four proteins synthesized by mitochondria in vitro (excluding the 45,000-dalton protein which is a polymer of the smallest subunit) have molecular weights close to the molecular weights of the membrane factor (Tzagoloff and Akai, 1972) present in the OS-ATPase complex. They could be identical with subunits .5, 6, 8, and 9. Subunit 8 probably contains two peptides of roughly the same molecular weight-one from F1 and one from the membrane factor. Except for subunit 9, which was shown to be the major product of mitochondrial protein synthesis and identical with the smallest subunit of the complex, it remains t o be established firmly that the other three minor proteins synthesized by mitochondria in vitro are identical with the three in the OS-ATPase complex. Thomas and Williamson (1971) found that five subunits are synthesized by the yeast mitochondria (see Table IV), some with molecular weights different from those reported by Tzagoloff. They noticed that the low molecular weight band is more sensitive to inhibition of mitochondrial synthesis than the others. In contrast to Tzagoloff’s results showing that one major product results from mitochondrial protein synthesis in yeast, isolated rat liver mitochondria synthesized different types of proteins (Kadenbach and Hadvary, 1973; Burk and Beattie, 1973). In agreement with Tzagoloff, Kadenbach and Hadvary (1973) and Burk and Beattie (1973) also found proteolipid

MEMBRANE ATPares

125

synthesis by rat liver niitochoridria. The 40,000-dalton protrolipid (Burk and Beattir, 1973) might also hr a polymeric form of Tzagoloff’s subunit 9. Capaldi (1973) has noted that beef hrart CFo-Fl (Iiagawa and ltackrr, 1966a, b ) contains a 45,000-dalton subunit that, is absent in Tzagoloff’s (1969a) beef heart complex. When th r rrlationship bct w e n th r polymrric and the monomeric forms of the mitochondria1 product is hettcr undrrstood, much of this ineonsistcncy and confusion might bc rrsolvrd. C. The Oligomycin and Dicyclohexylcarbodiimide Site of Action

The mechanism of ATPase inhibition by oligomycin and related compounds is still not understood. The insensitivity of F1to oligomycin suggests that the binding site of oligomycin resides in one or more of the protein subunits associated with OS-ATPase. Kagawa and Racker (1966b) found that radioactive rutamycin interacts with CFo and not, with F,. Bulos and Racker (1968a) showed that exposure to heat or trypsin produced loss of oligomycin sensitivity. They showed also that the DCCD site of action is in the submitochondrial particles, which are resolved in F1 and OSCP and contain the membrane factor. Later, more depleted SMI’ resolved in F,, FO1( = OSCP), and FCz were shown to interact with DCCD (Knowles et al., 1971). Experiments with yeast mutants resistant to oligomycin also showed that the resistance is controlled by the membrane factor. Hybrids of F1 either from oligomycin-resistant mutants or from the wild type produce an oligomycin-sensitive complex only with the wild-type membrane (Criddle et al., 1973). I n the last few years, there has been considerable progress in identifying more specifically the membrane subunit that interacts with DCCD. It seems that a low molecular weight protein with proteolipid characteristics (soluble in chloroform-methanol) synthesized by mitochondria is the site of DCCD action, and it has been suggested that this proteolipid also binds oligomycin (based on the assumption that the two inhibitors act a t the same site) (Cattel et al., 1971; Stekhoven et al., 1972). Cattel et al. (1971) purified a labeled proteolipid of 10,000 daltons from beef heart mitochondria which had been treated with labeled DCCD. The DCCD-binding protein was shown t o be a component of beef OS-ATl’ase complex by Stekhoven et al. (1972) who reported a molecular weight of 14,000. This proteolipid isolated from beef heart mitochondria or from the OS-ATPase complex appears to be similar to Tzagoloff’s subunit 9 (see Section 111, B, 2, c), but this remains to be shown directly. Cattel et al. (1971) found that cardiolipin is the major lipid in the purified proteolipid fraction using Sephadex LH2.

126

RIVKA PANE1 AND D. RAO SANADI

D. Summary

Considerable progress has been achieved in the last few years in defining the molecular properties of OS-ATPase. The OS-ATPase subunits were separated and their molecular weights were determined. On the other hand, there has been little progress in understanding the mechanism of oligomycin inhibition. The proteolipid shown to bind DCCD (and presumably also oligomycin) may be an important clue to explaining the mechanism of oligomycin inhibition. The oligomycin-resistant yeast mutant provides a good tool for the study of the mechanism of oligomycin action. Since no differences were found in subunit mobilities with the mutant on SDS gel and in lipid composition, the lesion would appear to be a small change that could not be detected by conventional methods. The isolation of the specific proteolipid that binds DCCD in the wild type from the oligomycin-resistant mutant should provide better understanding of the mechanism of DCCD inhibition. IV.

CHLOROPLAST COUPLING FACTOR 1

A. Reaction Catalyzed by Chloroplast Coupling Factor 1

1. COUPLING ACTIVITY AND ATP SYNTHESIS

Coupling factor 1 from spinach chloroplasts isolated by washing the chloroplasts with EDTA (Avron 1963) or by acetone extraction (Vambutas and Racker, 1965; McCarty and Racker, 1966) stimulates cyclic photophosphorylation in the presence of phenaeine methosulfate (PMS) (Avron, 1963) and noncyclic photophosphorylation in the presence of ferricyanide (Avron, 1963; Vambutas and Racker, 1965; McCarty and Racker, 1966) as electron acceptor. McCarty and Racker (1967) established the identity of the factors isolated by the two different methods (Avron, 1963; Vambutas and Racker, 1965). Using subchloroplast particles (SCP) sonicated in the presence of phospholipids or washed with EDTA, they showed that both the EDTA extract of Avron and their CFI (Vambutas and Racker, 1965) stimulated cyclic and noncyclic photophosphorylation. Chloroplasts are capable of forming a limited amount of ATP without illumination by changing the pH from acid (pH 4.0) to base (pH 8.0) (Jagendorf and Uribe, 1966). This reaction is inhibited by a specific antiserum against CFI (McCarty and Racker, 1966) which shows that CFI takes part in ATP synthesis by chloroplasts. The ATP-stimulated H+

MEMBRANE ATParer

127

uptake by chloroplasts in the dark is also inhibited by antiserum against C F I (McCarty and Racker, 1966). Direct evidence of the involvement of CF1 in H+ uptake by chloroplasts was produced by Lynn and Straub (1969a,b) who found that CF1 stimulated light-dependent H+ uptake by chloroplasts using ferricyanide or ubiquinone as electron acceptors. 2. I-’,-ATP EXCHANGE

One striking difference between oxidative phosphorylation and photophosphorylation systems is that the former is reversible. High rates of ATPase, P,-ATP exchange, and ATP-dependent, reversed electron transport can be readily observed in mitochondria1 particles. Attempts to demonstrate similar reactions in chloroplasts, on the other hand, have been unsuccessful. Special treatments are needed in order to activate the ATPase and P,-ATP exchange in chloroplasts. The P,-ATP exchange could be demonstrated in chloroplasts under some of the conditions that induce also ATPase (Carmeli and Avron, 1966). Brief illumination of chlorop1:rsts in the presence of high concentrations of D T T induced P,-ATP exchange activity which was linear for at least 20 minutes in the dark. The results were confirmed by McCarty and Racker (1968) who found that by increasing DTT concentration up to 20 mM, the light could be omitted and high rates of I-’,-ATP exchange could be induced in chloroplasts. The reaction in the presence of light may represent reversal of overall oxidative phosphorylation. Cation Mg2+ and PMS (Carmeli and Rvron, 1966) or pyocyanine (McCarty and Racker, 1968) that are needed for photophosphorylation are essential for the light-triggered P,-ATP exchange. Photophosphorylation inhibitors and uncouplers inhihit the light (and DTT) -triggered P,-ATP exchange. Atebrin, octylguanidine, and phlorizin (Carmeli and Avron, 1966), Dio-9, and NH4+ (McCarty and Racker, 1968) all inhibit the exchange if they are present during the exposure to light and DTT. From the foregoing results, it has been proposed that light exposes a high-energy intermediate to DTT and/or causes some conformational changes in the enzyme(s), inducing the reverse reactions and P,-ATP exchange. The role of CF1 in the P,-ATP exchange was examined by McCarty and Racker, who found that the light-DTT-induced exchange in chloroplasts is inhibited by an antiserum against CF1 (1968). Removal of CF1 from the chloroplasts that were illuminated in the presence of DTT, by washing with EDTA, inhibited the P*-ATP exchange. I t has not been shown yet that added CF1 stimulates or induces an exchange in deficient chloroplasts. Although the evidence is indirect, it could be concluded from these results

128

RIVKA PANET AND D. RAO SANADI

that CF1 bound to the chloroplast membrane participates in the Pi-ATP exchange. 3. Ca2+ AND Mgz+-ATPase ACTIVITY Chloroplast factor 1 could be isolated in a latent form with no ATPase activity and then its ATPase activity could be induced. This is similar to the latent ATPase or Factor A of mitochondria (Andreoli et al., 1965; Warshaw et al., 1967). Alternatively, the ATPase activity could be first induced on the membrane and then the active CF1-ATPase could be removed from the membrane. Illumination of the chloroplasts in the presence of --SH compounds activates the ATPase as well as the Pi-ATP exchange (Marchant and Racker, 1963; Hoch and Martin, 1963; Petrack et al., 1965; McCarty and Racker, 1968). ATPase activity could also be induced in the latent form of CF1 by incubating it with -SH compounds (McCarty and Racker, 1968) or by trypsin or heat treatment (McCarty and Racker, 1968; Vambutas and Racker, 1965; Farron, 1970; Farron and Racker, 1970). a. ATPase Activation by DTT. Extraction of chloroplasts with EDTA after illumination and DTT treatment yielded a soluble active ATPase (McCarty and Racker, 1968). On the other hand, extraction of untreated chloroplasts gave CF1 with low-ATPase activity that could be increased by incubation with DTT. Other -SH compounds such as thioglycerol and /3-mercaptoethanol similarly unmasked the ATPase of CF1 but were less effective than DTT (McCarty and Racker, 1968). The DTT-CFI isolated from activated chloroplasts, or isolated in a latent form and then activated, retains its coupling activity (see below). The DTT-CF1 binds to deficient chloroplasts, and by binding to the membrane its ATPase is inhibited. b. Heat and Trypsin Activation of CF1-ATPase. ATPase could be induced in the isolated CFl by a short period of heating in the presence of ATP which protects against complete inactivation (Vambutas and Racker, 1965; Bennun and Racker, 1969; Farron, 1970). Heat activation is also a property of F1-ATPase (see Section 11, A, 1 ) . Farron and Racker (1970) showed that treatment of chloroplasts with N-ethylmaleimide (NEM) or iodoacetamide prevents the heat or trypsin activation of CF 1-ATPase. When the enzyme was previously activated by heat or trypsin, its catalytic activity was not affected any further by NEM treatment. It may be concluded that some - S H group(s) are involved in the activation of the hydrolytic site but are not essential for the catalytic activity. By heat or trypsin activation, the enzyme loses its ability to rebind t o deficient chloroplasts (McCarty and Racker, 1968; Vambutas and Racker, 1965) and as a result loses its coupling activity (Vambutas and Racker, 1965).

MEMBRANE ATParer

129

This effect is different from the stimulatory effect of heat-activated Factor A on urea SMP (Andreoli et ol., 196.5). Heat treatment of CF1 in the presence of digitonin maintains its coupling activity. The presence of digitonin is essential during the heating (Nelson et al., 1972a). The heat activation of CF,-ATPase may explain the ten-fold increase in ATPase rates between 22" to 37°C observed by Karu and Moudrianakis (1969). They isolated the enzyme in a latent form with low-ATPase activity and, by incubation a t 37"C, apparently measured two phenomena-heat activation plus the normal higher rate at higher temperatures. Vambutas and Racker (1965) first showed that incubation of SCP with trypsin stimulated Ca2+ATPase. Later it was shown (McCarty and Racker (1968) that this trypsin treatment of the SCP caused also detachment of CFI from the chloroplast membrane. McCarty and Racker actually measured the activity of the soluble enzyme. For maximal ATPase activation in SCP, prolonged exposure and relatively large amounts of trypsin are needed (Vambutas and Racker, 1965). Lynn and Straub (1969a) showed that brief incubation of chloroplasts with trypsin results in marked activation of Mg-ATPase if the chloroplasts were previously illuminated. The light probably induces some conformational changes in the chloroplasts that expose the hydrolytic site of CF, for trypsin action (as well as for the DTT effect) and resultant ATPase activation. Anti-CF1 inhibited the aforementioned activity of the chloroplasts (Lien and Racker, 1971). McEvoy and Lynn (1973) showed that the incubation of CF1 with trypsin under conditions that activate the latent ATPase caused selective digestion. The a subunit was the one most susceptible to the trypsin digestion, whereas the p subunit was stable. When they incubated chloroplasts with trypsin following illumination and then removed the CF1 from the chloroplasts, the CF1 had all the normal subunits seen in the untreated CF1 as judged by gel electrophoresis. It seems from the above results that the effect of trypsin on CF, is different from its effect on CF1 attached to the chloroplast membrane. B. Enzymatic Properties

1. SPECIFICITY TOWARD DIVALENT CATIONS

Unlike Fl which requires Mg2+ for its activity either in the soluble or the membrane-attached form, soluble CF1 is preferentially activated with Ca2+, whereas the chloroplast membrane-bound ATPase requires Mg2+ (Vambutas and Racker, 1965; McCarty and Racker, 1968; Karu and Moudrianakis, 1969) ; Mg2+ inhibits the Ca2+dependent ATPase. A controversy arose and confusion ensured when Vambutas and Racker (1965) found that

130

RIVKA PANET AND D. RAO SANADI

heat or trypsin treatment of chloroplasts induced high Cazf-dependent ATPase activity, whereas the Mg2+-ATPase was much lower (Vambutas and Racker, 1965; Karu and Moudrianakis 1969). One should remember that photophosphorylation is Mgz+dependent and is inhibited by Ca2+. Treatment of chloroplasts with heat or trypsin, which is known to solubilize CF1 from the membrane, activates Ca2+-dependent ATPase (Vambutas and Racker, 1965), whereas illumination of chloroplasts in the presence of DTT activates Mg2+-dependentactivity (Carmeli and Avron, 1966; McCarty and Racker, 1968) and retains the CFI on the chloroplast membrane. Moreover, it was found that when the CF1-Mg2+-ATPase is extracted from chloroplasts previously illuminated in the presence of DTT, its soluble ATPase activity was about 6 times higher with Ca2+than with Mg2+ (McCarty and Racker, 1968). When latent CF1 is exposed to heat, trypsin digestion, or DTT, the resulting ATPase is Caz+-dependent under the normal assay conditions. Other divalent cations such as Ni2+, Mg2+, Mn2+, Co2+, and Sr2+ (at 10 mM) were less than 3% as effective as Caz+. The ATPase activity of the soluble enzyme is inhibited approximately 50% by 0.3 mM Mg2+ (Vambutas and Racker, 1965; Karu and Moudrianakis, 1969). The finding that brief exposure of preilluminated chloroplasts to trypsin yields Mg2+-dependentATPase (Lynn and Straub, 1969a) might merely indicate that under these mild conditions the CF1 is still attached to the chloroplasts membrane, but this theory remains to be proved. Nelson et al. (1972a) examined the assay conditions for the Mg2+-dependent ATPase and showed that under the right conditions, it is as high as the Ca2+dependent activity. The presence of carboxylic acids and optimal Mgz+ concentration were the main factors determining the rate of Mg2+ATPase activity. At low pH (pH 6.0), sodium maleate accelerated the Mg2+-dependent ATPase of CF1 up to 30 times higher, where@ at high pH (pH 8.0), bicarbonate was more effective than maleate as an activator. The optimal Mg2+concentration is 8 mM at low pH and only 2 mM at the high pH. Higher concentrations of Mg2+inhibited the reaction. The Mgz+-dependent ATPase activated by the carboxylic acids was comparable to the Ca2+-dependent activity. Activation of Mg2+-ATPase by carboxylic acids is in agreement with the finding that photophosphorylation is accelerated by these acids. 2. SPECIFICITY TOWARD NUCLEOTIDES

CF1-ATPase is most active with ATP; GTP and ITP were hydrolyzed at 25% of the rates compared to the rate with ATP, whereas CTP and UTP were ineffective (Vambutas and Racker, 1965).

MEMBRANE ATParer

131

The K , for ATP was found to be 0.4ri m M a t 37°C and 0.82 mM a t 22°C for the Ca2+-dependent activity (Karu and Moudrianakis, 1969) and 0.11 m M for the h!Ig2+-ATPase (Nelson et al., 1972a). Sodium maleate increases the VIn of the Mg2+-ATPase thirty-fold but decreases the affinity of CF1 to ATP by increasing the K , by a factor of 10 (Nelson et al., 1972a). The Mg2+-dependent activity showed the same nucleotide specificity as Ca*+-ATPnse; ATP was hydrolyzed 6 times more rapidly than GTP, ITP, or U T P (Nelson et al., 197%). ADI’ inhibits both Ca*+- (Vambutas and Racker, 1965; Nelson et al., 1972a) and Mg*+-ATPase (Nelson et al., 1972a). The inhibition of CF1-ATPase by ADI’ is specific; other nucleoside diphosphates such as IDP, GDP, and C D P are weak inhibitors (Vambutas and Racker, 1965). The ADP inhibition of CF1-ATPase surprisingly is not simply competitive with ATP; ADP altered the K,, and the V,,,,, for ATP. It changes also the saturation curve of ATP from hyperbolic to sigmoid shape, and the apparent reaction order from 1.0 to 2.3 (Nelson et al., 1972a). This interesting observation indicates that ADP has an allosteric effect on the CF1 enzyme.

3. BINDING OF NUCLEOTIDES BY CF1 Roy and Moudrianakis (1971a) found two ADP-binding sites on their 13 S coupling factor from spinach chloroplasts (which is analogous to CF1) with dissociation constants of 2 X 10-6M and 3.5 X 10-SM. The binding of ADP is slow and reaches maximum aftcr 1 to 2 hours. To release the bound ADP, perchloric acid, urea, or formamide are needed. The binding of ADP, like the inhibition of ATI’ase by ADP, is quite specific; other diphosphates do not compete with ADP. After binding ADP to CF1, nucleotides ADP, AMP, and ATP were detected as bound products on the enzyme. The AMP, although bound when it is produced from ADP, could not be bound directly to CFI even at high concentrations. Roy and Moudrianakis showed with double-labeling experiments that the enzyme is able to carry out a stoichiometric transphosphorylation reaction on its surface. They proposed that the ,&phosphate of one ADP is transferred to the P-phosphate of another ADP molecule in an adenylate kinase type of reaction. Whereas the AMP and ATP produced from ADP are loosely bound to the enzyme and could be recovered by gel filtration, ADP is tightly bound t o CFI, and only drastic treatments promote its release. They showed also that by illumination of chloroplasts, 3H AMP is converted to bound ADP and could be recovered in the CF1 isolated from these chloroplasts. The authors proposed that ADI’ is photosynthetically generated from

132

RIVKA PANET A N D D. RAO SANADI

AMP, Pi, and the energy derived from illumination. They claimed that ADP is the high-energy intermediate of photophosphorylation, and ATP and AMP are formed by an adenylate kinase-like reaction. Evidence has been presented that this preparation is free from contamination by the classic adenylate kinase. They showed also that the formation of ADP-CF1 complex is dependent on photoinduced electron transport, AMP, and Pi. The reaction is sensitive to arsenate and sulfate. The bound ADP exchanged with exogenally added ADP. Roy and Moudrianakis (1971b) propose the interesting mechanism shown in Fig. 2 for photophosphorylation. Yamamoto et al. (1972) found that ADP binding to the Rhodospirillum rubrum chromatophore is very tight, and there was no exchange between bound ATP and free ADP. The ADP is photophosphorylated to ATP. In addition to tightly bound ADP, they reported also a loosely bound ADP; binding is claimed to be oligomycin-sensitive. Consistent with the proposal of Roy and Moudrianakis, Forti et al. (1972) showed that isolated CF1 carries out incorporation of inorganic phosphate into the phosphate of ADP. The experiment involved addition of ADP and Pi to their purified to the @-phosphateof ADP. During CFI, which led to incorporation of 32Pi this incubation, ATP is also formed with the label in the @-phosphate.

+ AMP + Pi

1. MembraneSCF

//photoinduced

’I-+

ADP

2.

3.

Membrane * CF

electron transport

+ HzO ADP

/ADp MembraneaCF

\

ADP

I 4.

/AMP Membrane. CF

\ 5. MembranemCF

Membrane-CF

ATP

1 + ATP + AMP

+ AMP + Pi + ADP

light

Membrane .CF

+ AMP + ATP + HzO

FIG.2. Mechanism of ATP formation in chloroplasts. Roy and Moudrianakis (1971b).

MEMBRANE ATParer

133

However, they could not detect 32Piincorporation into the y-phosphate of ATI’. Their interpretation of the data is t,hat CFI preserved its high-energy state during the purification procedure. Their proposal for the photophosphorylation mechanism is very similar to that of Roy and Moudrianakis (see Fig. 2 ) . 4. COLDLABILITY

The ATPase activity of CF, is cold-labile whether the hydrolytic activity is induced by trypsin, heat (McCarty and Racker, 1966), or by DT T (McCarty and Racker, 1968). The coupling activity of CF1 is also coldlabile. It dissociates into inactive subunits in the cold, as shown by gel electrophoresis (McCarty and Racker, 1966). ADP and ATP protect CF1-ATPase from the cold inactivation (just as for F1-ATPase) . Untreated CFI acquires cold stability on binding to deficient subchloroplast particles. Lipids obtained from chloroplasts also confer some degree of cold stahility. However, the same subchloroplast particles do not confer cold stability to the heat-activated CF,-ATI’ase. I t would seem that heat- or trypsintreated CF,-ATPase loses its capacity to bind to the chloroplast membrane (Rennun and Racker, 1960). Cold inactivation of the enzyme renders the solution cloudy. Some degree of reactivation could be produced by dilution and warming. Addition of glycerol to the dilution medium to prevent nonspecific aggregation restores up to 65% of the original activity (Lien et aE., 1972). 5. INHIBITORS OF CF1-ATPase

a. Dio-9 and Phlorizin. McCarty et nl. (1965) found that Dio-9 inhibited oxygen evolution accompanying ferricyanide reduction in coupled chloroplasts in the presence of ADP, P,, and Mg2+.Only electron transport that is tightly coupled to photophosphorylation is inhibited by Dio-9. Chloroplasts uncoupled by NH4+ are not inhibited by it. Dio-9 inhibits both cyclic and noncycric photophosphorylation, but NH4+ relieves the inhibition (McCarty et aE., 1965). The site of 130-9 inhibition is believed to be the reaction(s) catalyzed by CFl based on the following findings: (1) Phosphorylation accompanying an acid-base transition is inhibited by Dio-9 (McCarty et al., 1965) (for the role of CFI in this phosphorylation, see Section IV, A) ; ( 2 ) Dio-9 inhibits membrane-bound ATPase that has been activated by DTT in the same manner as it inhibits the light-DTTtriggered Pz-ATP exchange; ( 3 ) Dio-9 inhibits the soluble ATPase activity of CF I induced by trypsin or by D T T (McCarty and Racker, 1968); ( 4 ) Dio-9 inhibits the Ca-ATPase more than the Mg-dependent activity

134

RIVKA PANET AND D. RAO SANADI

(Nelson et at., 1972a). The maximal inhibition of CF1-ATPase activity by Dio-9 is only SO%, whereas photophosphorylation is inhibited up to 80-90%. In general, Dio-9 is a more potent inhibitor of the particulate ATPase than soluble CF1-ATPase (McCarty and Racker, 1968). Based on the model of Dio-9 inhibition, McCarty and Racker (1968) proposed that CF1 catalyzed the last step of photophosphorylation, namely, the transphosphorylation to ADP: X P ADP + X ATP. This same reaction has been proposed for F1 in mitochondria. Phlorizin, a muscle phosphorylase inhibitor, inhibits cyclic and noncyclic photophosphorylation, as well as coupled electron transport, in the presence of Pi and ADP (Izawa et al., 1966). As in Dio-9 inhibition, uncouplers of photophosphorylation relieve phlorizin inhibition of the electron transport. In the absence of Pi and ADP, phlorizin does not inhibit electron transport in chloroplasts (Izawa et al., 1966). Like Dio-9, phlorizin inhibits DTTlight-induced Pi-ATP exchange and the ATPase of chloroplasts (Carmeli and Avron, 1966). Although more information is needed on the effect of phlorizin on isolated CF1, it would seem that it inhibits at the same site as Dio-9 does, which is the terminal reaction of photophosphorylation catalyzed by CFl. b. The CF1-ATPase Specific Inhibitor. Chloroplasts, in contrast to mitochondria, have no ATPase activity and catalyze no Pi-ATP exchange or reversed electron flow, under the conditions that permit photophosphorylation to occur. It appears, therefore, that in chloroplasts one of the phosphorylation steps is not readily reversed. It was suggested by several groups that in mitochondria the specific inhibitor of F1 modifies it in such a manner that reversed reactions with ATP are prevented, but the forward reactions with ADP are able to proceed (see Section 11, B, 4). Based on the preceding suggestion, attempts were made to isolate a specific CF1 inhibitor from isolated CF1, but these were unsuccessful. Moreover, no differences were detected between native CF1 and the heat-activated CF1-ATPase in amino acid composition (Farron, 1970). This finding implies that by inducing ATPase in CF1no protein is separated from CFI, but it is possible that an inhibitor is inactivated by the heat but not removed from CF1. Later, it was shown that, indeed, such an inhibitor exists firmly bound to CF1 and could be removed by heat treatment only in the presence of digitonin, followed by fractionation with Sephadex G-200 (Nelson et al., 1972b). The inhibitor is probably a subunit of CF1, as in the case of FI. In F1, it is readily dissociated, whereas the CF1 inhibitor is very hydrophobic and soluble only in the presence of urea or detergent. Heat, trypsin, and incubation with DTT probably remove the inhibitor from the active site of

-

+

+

MEMBRANE ATPases

135

CF1-ATl’ase. Like the mitochondria1 ATPasc-specific inhibitor (Pullman and Monroy, 1963), the CF, inhibitor is sensitive to trypsin, thus, explaining the ATPase activation by trypsin digestion. The inhibitor isolated by Nelson et al. (197%) was identified with the smallest of the five subunits of CF,; its estimated molecular weight is 13,000. It is specific to chloroplast A’I‘l’ase and does not inhibit other ATI’ases . c. Eflect of Photophosphorylation Uncouplers on CFl-ATPase. Few compounds are known that uncouple photophosphorylation. Among them are NH4+, which is the most popular, carhonylcyanide-m-chlorophenylhydr:tzone (CCP) , and n-butyl-3,5-diiodo-4-hydroxybenzoate. The NH4+ increases photohydrolysis of ATP in chloroplasts in the presence of lipoic acid (Petrack et al., 1965) up to 0.74mM and, in this concentration range, it inhibits ATP synthesis by chloroplasts. In 3 mM concentration, NH4+ inhibits photohydrolysis and photosynthesis of ATP (Petrack et al., 1965). The other uncouplers (CCP and n-butyl-3,5-diiodo-4-hydroxybenzoate) stimulate photohydrolysis to the same extent as NH4+. Increasing the concentration of all the uncouplers inhibits photohydrolysis. Vambutas and Racker (1965) showed that trypsin- and heat-activated CF,-ATPases are inhibited by the uncouplers NH4+, CCP, and n-butyl3,5-diido-4-hydroxybenzoate. They measured only the effect of high uncoupler concentration that inhibited also the ATPase of chloroplasts (McCarty and Racker, 1968). Since the ATPase was already activated, it is likely that the activation of ATPasc :it lower uncoupler concentrations was not seen. The inhibition of CF1-ATPase by uncouplers was no more than 50% compared to almost 100% inhibition of ATI’ photohydrolysis by chloroplasts (McCarty and Racker, 1968). d . Eflect of DCCD on Photophosphorylation and ATPase. It was found that the light-DTT-induced Mg2+-ATl’ase of chloroplasts is inhibited by DCCD in concentrations that inhibited photophosphorylation. However, the Ca2+-dependent ATI’ase activity of soluble CFI is resistant to DCCD (McCarty and Racker, 1967). C. Molecular Properties

1. MOLECULAR WEIGHTAND SIZE

The sedimentation value for CF1 measured in the analytical ultracentrifuge was 13.8 S in both the latent and active ATPase form. The molecular weight by this method wits 325,000 or 358,000 (Farron, 1970), which is very similar to that of F1 from different sources (see Section 11, C, 1 ) . The purified CF1 is a sphere, roughtly 90 A in diameter (Moudri-

136

RIVKA PANET AND D. RAO SANADI

anakis, 1964; Vambutas and Racker, 1965). By electron microscopy, these

90-A spheres appear attached to the outer membrane of SCP (Vambutas and Racker, 1965; Lien and Racker, 1971), and the resolved particles contain only few of the membrane-attached spheres. Silicotungstate, which solubilizes F1 from mitochondria, also removes CF1 from SCP. The treatment removes the 90-& spheres from the SCP (Lien and Racker, 1971). 2. SUBUNIT STRUCTURE OF CF1 In the presence of 5 M guanidine-HC1, the 325,000-dalton enzyme is dissociated into subunits of 62,000 daltons, whereas by amino acid analysis the minimal molecular weight is 28,000 (Farron, 1970). As in F1, the subunit structure of CF1 is more complicated. The existence of nonidentical subunits was first shown by immunoelectrophoresis which revealed the existence of at least two antigenically distinct subunits (Lien et al., 1972). By disc electrophoresis in the presence of SDS, the CF1 revealed two, major, slow-moving bands and three more rapidly moving bands that had not been seen in all the preparations of CF1. The molecular weights of the for the two major bands, and five subunits were 59,000(a) and 56,000 37,000 (y), 17,500 (a), and 13,000 (e), for the three minor bands (Nelson et al., 1973). A similar pattern was obtained by McEvoy and Lynn (1973) with their preparation of CF1 (Table V) . A comparison of the CF1subunits with the subunits of F1 is shown in Table V, the similarity is quite remarkable. Using urea and mercaptoethanol to dissociate the enzyme, Nelson et al. (1973) separated the five subunits of CF1 by DEAE cellulose chromatography. They determined their amino acid composition and prepared antibodies against each one of the five CF1 subunits, which provides a useful

(a),

TABLE V

MOLECULAR WEIQHTSOF CFI

Subunit 1 2 3 4 5 6

AND

Fl SUBUNITS

Nelson el al. (1973)

McEvoy and Lynn (1973)

Brooks and Senior (1971)

59,000 56,000 37,000 17,500 13,000

62,000 57,000 38,000 21 ,000 14,000

53,000 50,000 25,000 12,500 10 ,500 7,500

-

MEMBRANE ATParer

137

tool to study their role. They were able to extract the two low molecular weight subunits from the CF1using pyridine and to purify the CF1 inhibitor from the extract. The smallest subunit, 13,000 daltons, has been identified as a potent inhibitor of CFl-ATPase (Nelson et al., 1973), resembling the inhibitor of mitochondria1 ATPase (see Section 11, B, 4).

3.

I’OSSIBLE

ROLEOF CF1 SUBUNITS

The role of CF1 subunits has been examined using specific antibodies prepared against each one of the five subunits. a. The 6 and t Subunits. Nelson el al. (1973) found that CFl, after extraction with pyridine to remove the two smallest subunits, retained activity with only three subunits ( a , 0, y). Both the ATPase and the coupling activity were unaffected by removing the two smallest subunits. They proposed that the latter may have a regulatory role but are not essential for CF1 activity (Nelson et al., 1972b, 1973). b. The a, 0, and y Subunits. Nelson et al. (1973) have examined the role of these three subunits using specific antibodies to each one of them. Their results may be summarized as follows: (1) antibodies against the a and y subunits strongly inhibited photophosphorylation and light-triggered Mg-ATPase in chloroplasts when added separately; (2) anti-a inhibited also the stimulation of Hf uptake by AT]’ into chloroplasts; ( 3 ) the antibodies against a and 0 agglutinated subchloroplast particles; (4) none of the antibodies inhibited CF1-ATPase when added singly. However, a combination of a and y together inhibited the ATPase to the same extent as anti-native-CF,. From these results they conclude that the a and y subunits are involved in the coupling activity of CFI. The E subunit appeared to be the ATPase inhibitor which might play a regulatory role, but more data are needed to confirm this assumption. Considerable caution is warranted in interpreting data or activity changes obtained with antibodies to individual subunits. It seems quite likely that an antibody to a subunit could modify the activity of the complex by inducing configurational alterations in the entire complex although the subunit is not directly involved in the activity. In dealing with coupling activity, it has to be recognized that some of the subunits may be present in the membrane and capable of associating with the complementary subunit, although unavailable to the antibody. In a recent paper, Deters et al. (1975) claim that the a and 0 subunits are involved in CFL-ATPase activity (and not a and y as claimed by Nelson et al., 1973), based on the following effects of trypsin. a. They treated CFl with trypsin (free of chymotrypsin) and obtained a preparation that, according to amino acid analysis, appeared to contain a

138

RIVKA PANET AND D. RAO SANADI

mixture of a and j3 subunits, although they could not always separate the two. Anti-CF1 inhibited the activity of the trypsin-treated CF1 but antibodies against a and j3 subunits did not inhibit the ATPase activity, casting doubt on their conclusion that intact a and j3 subunits were present in the trypsin-treated preparation. b. They showed that NBD-Cl, which inhibits the ATPase of CF1 and of the trypsin-treated preparation, was incorporated into the B subunit of the enzyme. It seems a little premature to make any conclusions regarding the role of CF1 subunits from the above results, and probably the system is not so simple. McEvoy and Lynn (1973) have examined the effects of trypsin on the subunits of CFl and on ATPase activation, and found by SDS gel electrophoresis that the highest molecular weight subunit (a)is more susceptible to trypsin, whereas the j3 subunit is stable. The three minor subunits were almost unaffected. The mobility of proteins in SDS gel electrophoresis is not the most sensitive method to detect the small changes that could occur by tryptic digestion, and thus one cannot reliably conclude from their results that the hydrolytic site is on the a subunit. The Mg2+-ATPase, activated by exposure of chloroplasts to trypsin and recovered by EDTA washing, showed a normal pattern on SDS gel electrophoresis. According to Nelson et al. (1973), trypsin causes inactivation of the isolated CF1-ATPase inhibitor, but this has not been shown with the inhibitor attached to CF1. 4. THE--SH CONTENT OF CFl

A total of twelve -SH groups were titrated per mole CF1 after reduction. Only eight -SH groups out of the twelve are present if the reduction is omitted. Similar results were obtained with F1 (see Section 11, C, 3). The CF1-ATPase activity is not affected by blocking its -SH groups as in F1. Once the ATPase activity is induced by trypsin or heat, its catalytic activity is not affected by incubation with N-ethylmaleimide (NEM) or iodoacetamide (Farron and Racker, 1970). The -SH groups are probably involved in the ATPase activation of CFI, isolated or attached to the membrane (see Section IV, A, 3, a). The distribution of the -SH groups in the five subunits WM studied by McEvoy and Lynn (1973). After reduction with mercaptoethanol, they incubated the CF1 with NEM-W. The alkylated CFl was then analyzed by SDS gel electrophoresis for distribution of radioactivity. They found 9.75 -SH per mole CF1 (mol w t 325,000) in the a subunit, 3.25 in the B subunit, and only 1.35 in the y subunit. Based on the finding that photophosphorylation catalyzed by chloroplasts is inhibited by NEM only in light but not in the dark (McCarty

MEMBRANE ATPorer

139

et al., 1972), McCarty and Fagan (1973) showed that light markedly enhanced the radioactive NEM incorporation into the CF1 of chloroplasts previously incubated with unlabeled NEM in the dark. The CFl isolated from these treated chloroplasts showed that only the y subunit (out of the five subunits) contained most of the radioactivity. According to Nelson et al. (1973), the y subunit was one of the two subunits involved in CFl activity (see the previous section). D. Effect of light on Conformational Changes in Chloroplast Coupling Factor 1

There is considerable evidence in the literature suggesting that light causes conformational changes in the CF1 attached to illuminated chloroplasts. Lynn and Straub (1969a) showed that by illumination of chloroplasts, even a brief exposure t,o a small amount of trypsin activated the Mg-ATPase. This is in contrast to the long incubation with trypsin needed to activate ATPase in the dark (Vambutas and Racker, 1965; Lynn and Straub, 1969a). The ATPase activation by trypsin in illuminated chloroplasts is blocked by uncouplers, suggesting that trypsin may be acting on an energized “intermediate” or an energized state (Lynn and Straub, 1969a). lZyrie and ,Jagendorf (1971) have carried out experiments designed to look for conformational changes in chloroplasts associated with energy coupling. Their finding may be summarized as follows. 1. Chloroplasts were illuminated briefly in the presence of tritiated water, MgZ+,ADP, P,, and the electron carrier pyocyanine; then CF1 was removed from the membrane by EDTA washing in the presence of ATP to prevent cold inactivation. The protein isolated from illuminated chloroplasts was labeled, but that from chloroplasts kept in dark was not. The labeling of CF1 by tritium was very low despite the high specific radioactivity in the medium. 2. Tritium uptake occurred during illumination in the presence of pyocyanine (cyclic phosphorylation) or ferricyanide (noncyclic phosphorylation). It occurred also during the acid-base transition. They concluded that tritium uptake by CF1 is concomitant with the formation of a highenergy intermediate or state in the chloroplasts. 3. The ADP and P,, when added together, decreased the CF1 tritiation, but there was no effect when they were added separately, indicating that phosphorylation is not needed for CF, labeling. 4. Uncouplers (such as NH4+, CCP, hexylamine) and electron-flow inhibitors inhibited CF1 tritiation.

140

RIVKA PANET AND D. RAO SANADI

5. Dio-9 and phlorizin did not inhibit the incorporation of tritum into CFl in the presence or absence of ADP and Pa. They proposed that parts of the protein become exposed on illumination, get tritiated, and subsequently refold into regions of the molecule inaccessible to solvent hydrogen. From the foregoing results, it seems that the conformational state undergoing labeling occurs prior to the entry of Pi into the sequence, since it is not needed for the labeling. The labeling is also independent of the transphosphorylation to ADP since Dio-9 and phlorizin do not affect it. From the effect of uncouplers, it would appear that the conformational change permitting labeling is coupled to electron transport. The hypothesis that a conformational change is produced by light in CFI is consistent with the ideas about similar conformational changes in F1, based on the fluorescence of the F1-aurovertin complex. Also, ADP, ATP, and Pullman inhibitor all seem to produce conformational changes in F1 (on the membrane or in the isolated state), as indicated by their effect on the fluorescence of the F1-aurovertin complex (for details, see Sections 11, B, 4, b and c). E. Summary

Recently, the subunits of CF1 have been separated and their molecular weight and amino acid composition determined, but their specific role in ATPase or coupling is uncertain. There is general agreement that the subunit is a specific CF1-ATPase inhibitor. The confusion is regarding which subunit(s) are involved in the coupling activity, and which in the ATPase. The experiments with antibodies against each subunit might provide some clues when isolated CF1 is used and its activity without the membrane is examined. With membrane-bound CFI, however, antibody interaction could yield misleading results since accessibility to antigen could be impaired by the membrane. Labeling of the enzyme with the specific ATPase inhibitor could give direct information on the regulation of the active site of the enzyme. In a similar manner, the /3 subunit was labeled by NBD-C1 (Deters et al., 1975), and most likely it is the subunit carrying the active center of ATPase but this still needs to be confirmed. The isolation of CF1 preparations lacking one or more subunits would be a useful approach for determining their role (see Futai et al., 1974 and Section V, C) . The inhibitor of CFI by NBD-C1 has promise of yielding information on the location of the active center of CF1 and on the nature of the active group reacting with it.

141

MEMBRANE ATPases

The experiments demonstrating conformational changes in CFI during photophosphorylation are consistent with the recent hypothesis of Boyer (1973).

V.

BACTERIAL ATPace

This section deals with soluble ATPases from different bacteria, their enzymatic and molecular properties, comparison with the ATl’ases from mitochondria and chloroplasts, and their role on the membrane. Other aspects of bacterial energy conservation have been reviewed recently (Harold, 1972; Abrams and Smith, 1974; Cox and Gibson, 1974; Kovac, 1974). A. Reactions Catalyzed by Bacterial ATPase and the Use of Mutants

1. OXIDATIVEPHOSPHORYLATION A N D TRANSHYDROGENATION

It is now accepted that the bacterial RTPase is analogous to the ATPase in mitochondria and chloroplasts and that it catalyzes the terminal step in oxidative phosphorylation. Indeed, Rogin et al. (1970) have shown that coupling factors of mammalian and bacterial origin could be interchanged although the activities in the assay system were quite low. It was shown that ATPase isolated from Escherichia coli stimulated ATP-driven energydependent transhydrogenase reactions in particles deficient in ATPase (Bragg and Hou, 1972). Mutants defective in oxidative phosphorylation are just beginning to be used for defining the precise role of the ATPase and for analyzing other aspects of energy conservation. Direct evidence for the participation of ATPase in bacterial oxidative phosphorylation comes also from the recent isolation of E. coli mutants with greatly reduced levels of membrane ATPasc activity (Bultin et al., 1971; Cox et a!., 1971; Kanner and Gutnick, 1972a, b ; Scharier and Haddock, 1972; Rosen, 1973; Bragg and Hou, 1973). These mutants, although capable of oxidizing lactate, fail to couplo such oxidation to the phosphorylation of ADP (Bultin et al., 1971). The inhibition of bacterial ATPase by Dio-9 (Harold et al., 1969) and by azide (Munoz et al., 1969; Johansson et al., 1973; Hanson and Kennedy, 1973) also support the conclusion that this enzyme catalyzes the phosphorylation of ADP to ATP. Both Dio-9 and azide are known to inhibit soluble and membrane-bound yeast F1 (see Section 11, B, 4). Dio-9 inhibits also CF1 (see Section IV, B, 5). Vesicles from the mutants of E. coli with reduced levels of ATPase (Bultin et al., 1971; Kanner and Gutnick, 1972a;

142

RIVKA PANET AND D. RAO SANADI

Bragg and Hou, 1973) cannot carry out the ATP-dependent transhydrogenase reaction as in the parent strain. The vesicles, however, retain their ability to catalyze the transhydrogenase driven by respiration (Kanner and Gutnick, 1972a). These findings attest to the presence of the primary energy-transducing reaction coupled to respiration in the mutants, but the ability either to make ATP or to utilize ATP for energy-dependent reactions is lost. 2.

ROLEOF ATPase

IN

ACTIVETRANSPORT

ATPase is present in E. coli, grown either aerobically or anaerobically, and in Streptococcus faecalis which does not ordinarily derive metabolic energy from aerobic oxidative phosphorylation. I n both cases and in others as well, it appears to be the single major ATPase as judged by purification and inhibition studies (Hanson and Kennedy, 1973). The role of this ATPase in the transport of K+, amino acids, and phosphate was first suggested by the finding that an inhibitor of membrane-bound ATPase, DCCD, inhibits glycolysis-dependent uptake of K+ ions (in exchange for intracellular Hf or Na+) , alanine, and phosphate. Furthermore, Dio-9 and chloroheximide inhibit bacterial ATPase as well as net uptake of K+ by exchange for Na+ and Hf (Harold et al., 1969). It was proposed that the ATPase mediates between the ATP synthesized by the cytoplasm and the membrane to communicate energy for the membrane functions, such as active transport. The finding that the amount of membrane ATPase in S. faeculis nearly doubles when the organism is grown at K+ levels low enough to limit the growth rate, and that in these cells the rates of net K+ and *Rb uptake is increased during glycolysis (Abrams and Smith, 1971) support the idea that the bacterial ATPase plays a role in cation transport. Experiments of Pavaslova and Harold (1969) indicated that the E. coli ATPase might function in the active transport of galactosides also. They reported that in cells grown anaerobically, thiomethylgalactoside accumulation was blocked by uncouplers of oxidative phosphorylation, although ATP was present at normal levels and could be used for other reactions. Direct evidence for the involvement of ATPase in galactoside transport comes from the finding that mutants of E. coli lacking the Mg2+-Ca2+activated ATPase could not catalyze the active transport of galactosides (Scharier and Haddock, 1972; Rosen, 1973). In summary, it seems that the bacterial ATPase, like FI, catalyzes the last step of oxidative phosphorylation and is involved in the ATPdriven transhydrogenase activity and in the active transport of K+, amino acids, inorganic phosphates, and galactosides.

MEMBRANE ATParcr

B.

143

Enzymatic Properties

1. ACTIVATIONOF ATPase

The ATPase activity from different bacteria could be unmasked by trypsin (Ishikawa, 1966; Munoz et al., 1969; Milton et al., 1972), heat (Adolfsen and Moudrianakis, 1971b), or DNP (Gurraia and Peck, 1971). Many preparations exhibit considerable activity without the need for any unmasking (Abrams, 1965; Evans, 1969; Evans, 1970; Gross and Coles, 1968; Guarraia and Peck, 1971; Harold et al., 1969), presumably because the activation is produced during the isolation. In one case it is reported that ATPase can be isolated from E. coli in two forms-one needs trypsin activation and the other does not (Milton et al., 1972). The bacterial ATPase is cold-labile, like F1 and CFI, whereas the membrane-bound enzyme is cold-stable (Ishida and Mizushima, l969a, b ; Evans, 1970; Mirsky and Barlow, 1971) (see also Tahlt. V I ) . 2. SPECIFICITY TOWARD NUCLEOTIDEY

All purified ATPases isolated from different bacteria cleave purine nucleoside triphosphates with different degrees of effectiveness (see Tables VI and VII) (Abrams, 1965; Mirsky and Barlow, 1971; Hanson and Kennedy, 1973). The K,,, reported for Mg2+-ATP is in the range of 0.1 to 2.5 mM (see Table VI). The ATl’ases isolated from different hacteria are all inhibited competitively by ADP, with a K i= 0.7 mM for the S. faecalis ATPase (Schnebli and Abrams, 1970) (see also Table VI), and noncompetitively by Pi (Schnebli and Abrams, 1970; Hanson and Kennedy, 1973). CATIONS 3. MONO-AND DIVALENT Bacterial ATPase is completely dependent on divalent cations for its activity. Cation Mg2+ is the common activator (see Table VI), but Ca2f could replace Mg2+ for the ATPase from E. coli, Alcaligenes faecalis, Micrococcus lysodeikticus, and Bacillus megaterium. In the last two bacterial ATPases, the Ca2+-dependent activity is much higher than the Mg2+ATl’ase, reminiscent of CFI-ATPase. In these cases, Mg2+inhibited the Ca2+-ATPase (Munoz et al., 1969). The concentration curve for activation by Ca2+described by Munoz el al. (1969) with the M . lysodeikticus ATPase is sigmoidal, whereas with Mg2+it is bell-shaped. The ATPase isolated from Rhodospirilluin rubrum chromatophores is activated by Ca2+ alone, and Mg2+, Mn2+, CoZ+, or Zn2+ inhibit the Ca2+-dependent activity. The effects are similar to those seen with CFI (Johansson et al., 1973).

d

P P

TABLE

VI

COMPARISON OF ENZYMATIC AND MOLECULAR PROPERTIES OF ATPase Characteristics

E. coli

S . faeculis

MgZ+ or Mn2+, Activation by cations Mg2+ or Caz+, no activation by Na+ Caz+ ineffective or K+

M . lysodeikticus

ATP, GTP, ITP; ADP inhibits

K, for ATP is 0.1

ADP inhibits

Inhibitors

Nitrate, C1-, acetate, azide, Pi

Die9 and chlorohexidine

Azide

Cold 1ab;lity

Cold labile

No activation is needed

A . faecalis

Mg", Caz+, Cdz+; K+ stimulates at low ATP

ATP, GTP, UTP, K, for ATP is 2.5 mM

-

B. megaterium CaZ+ or less so Mg2+

ATP, GTP, ITP, K, for Mg-ATP is 0.29 mM

Latency of activity

DIFFERENT BACTERIA

CaZ+ and less Mg2+, Mg*+inhibits Caz+ ATPase, high Na-or K+ inhibit

Specificity toward nucleotides

-

FROM

mM

-

$ 3

3> Z

P

P

Cold stable Activation by trypsin

P

Cold labile Activation is needed

0

S value and molecular weight

S

Subunits

60,000 (a), 56,000 (19, 36,000 (Y), 13,000 (6) or 58,000 (a), 52,000 (81, 31,000 (Y), 31,000 (a), 20,000 (f)

EM shows hex-

Evans (1970); Davies and Bragg (1972); Hanson and Kennedy (1973); Bragg and Hou (1973); Futai et al. (1974)

Abrams (1965); Abrams and Baron (1967); Harold et al. (1969); Redwood et al. (1969); Shnebli et al.

Source

12.9; 365,000-390,000 Z = ~

S*

= 13.4;

s m = 14-13

385,000

agonal array

(1970)

>

2 100,000, 379,000

S%

= 13

z W

D

rn Z

EM shows 9O-.k

E M shows 100-d spere with central subunit encircled by 6 additional units. Two subunits: 62,000 and 60,000

Two subunits: 68,000 and 65,000 in equal proportion

Yamashita and Ishikawa (1965); Munoz et al. (1968, 1960) ; Milton et al. (1972)

Ishida and Adolfsen and Mizushima Moudrianakis (1969a,b); hlirsky (1971b, 1973) and Barlow (1971, 1973)

sphere

3 -0

2 2

146

RIVKA PANET AND

D.

RAO SANADI

TABLE VII SPECIFICITY OF E. Coli ATPase FOR METALSA N D NUCLEOTIDES~ Metal MIX* Mg* Mg* Mg* Mg" Ca2+ Co2+ Ni2+ Mn* None 0.

Nucleotide ATP GTP ITP UTP CTP ATP ATP ATP ATP ATP

Relative rates 100 61 33 11

3 108 19 14 5 0

Data from Hanson and Kennedy (1973).

Monovalent cations such as Kf or Na+ are known to activate membrane-bound ATPase (Hafkenscheid and Bonting, 1969; Adolfsen and Moudrianakis, 1971a). On removing the enzyme from the membrane, Kf or Na+ activation is lost (Hafkenscheid and Bonting, 1969; Davies and Bragg, 1972). High concentrations of Naf or K+ (100 mM) were found, in one case, to inhibit the ATPase (Munoz et al., 1969). Activation of ATPase by K+ occurs with the intrinsic ATPase activity, before activation by trypsin, and might represent a more natural property of the enzyme (Adolfsen and Moudrianakis, 1973).

4. INHIBITORS OF BACTERIAL ATPase Like CF1 and yeast F1, bacterial ATPase is inhibited by Dio-9 (Harold et al., 1969). Azide, which is known t o inhibit the mammalian and yeast FI, also inhibits bacterial ATPase (Munoz et al., 1968; Hanson and Kennedy, 1973). Oligomycin inhibits neither the membrane-bound nor the soluble bacterial ATPase (Sato et al., 1971). On the other hand, DCCD is a potent inhibitor of membrane-bound S. faecalis ATPase, but does not inhibit the soluble enzyme (Harold and Barda, 1969). The fact that the bacterial enzyme is released easily from the membrane provides a good tool for the study of the site of action of DCCD. Harold and Barda (1969) showed that when they released the ATPase from the previously inhibited S. faecalis membrane, the ATPase activity was restored. Conversely, sensitivity to DCCD was restored by reconstituting the ATPase membrane complex from the solubilized active ATPase and the depleted membrane.

MEMBRANE ATParer

147

From these results they concluded that the DCCD inhibits the bacterial ATPase indirectly by reacting with a membrane component(s) with which the enzyme is associated rather than with the enzyme itself. Similar results have been obtained with F1 and CF1 (see Sections 111, C and IV, €3, 5). The DCCD-binding membrane component(s) is probably a hydrophobic protein, since hydrophobic carbodiimides were more potent than the water-soluble carbodiimides (Abrams and Baron, 1970). The inhibition of a soluble ATPase preparation from E. coli by DCCD (Evans, 1970) might be explained by the fact that Evans solubilized the enzyme using detergent, which can extract membranous components. Abrams et al. (1972) isolated by direct selection S. jaeculis mutants resistant t o DCCD, and by reconstitut,ion experiments they showed that the sensitivity t o DCCD requires membrane ATPase. They showed that when the complex was reconstituted with mutant ATPase, mutant nectin (Baron and Abrams, 1971) , and wild-type-depleted membranes, the enzyme was normally sensitive to DCCD. However, when mutant membranes were used for reconstitution, the attached ATPase was insensitive to DCCD, indicating that the mutation affecting DCCD binding was on the membrane and not in the ATPase or nectin (Abrams et ul., 1972). Purified ATPase isolated from E. coli was found to he inhibited by NBD-Cl; this inhibition is reversed by DTT, and the diazole was found preferentially associated with the 0 subunit of the enzyme (Nelson et ul., 1974). From this finding the author concluded that there are -SH group(s) at the active site of E. coli ATPase, which is contrary to what has been accepted for Fl- and CF1-ATPases (for more details, see Section 11, C, 3 ) . C. Molecular Properties

Structurally the ATPase preparations from different bacteria closely resemble the F1 and CF1. I,n the electron microscope the enzyme appears nearly spherical with 100 A diameter, but it contains a hexagonal array of six subunits arranged around a central subunit (Munoz et al., 1968; Redwood et al., 1969; Schnebli et al., 1970) (see also Table VI). The enzyme is a highly acidic protein (Abrams and Baron, 1967). The sedimentation constant is in the range of 12.9 to 15.0 S for different preparations, and the molecular weight 360,000-385,000. The bacterial ATPase has been reported to contain five (Bragg and Hou, 1972; Bragg et al., 1973; Futai et al., 1974) or only four (Hanson and Kennedy, 1973; Nelson et al., 1974) nonidentical subunits. The preparation of Hanson and Kennedy (1973) consisted of only four subunits but had a low specific ATPase activity. Recently, Nelson et al. (1974) purified this enzyme to a

148

RIVKA PANET A N D D. RAO SANADI

homogeneous state with the highest specific activity reported so far and found also only four subunits. Preparations of E . coli ATPase with only four subunits lost their coupling activity, but the five subunit preparations retained the coupling activity. Futai et al. (1974) showed that the four and the five subunit enzymes have the same ATPase activity, but only the five subunit enzymes could stimulate ATPdriven transhydrogenase in deficient membranes. They suggested that the 6 subunit, which is missing in the four subunit preparation, is required for the binding of the enzyme to the membrane. The two large subunits (aand p ) appear to be sufficient for the ATPase activity of the soluble enzyme since three preparations of bacterial ATPase have been reported with only these two subunits (Schnebli et al., 1970; Milton et al., 1972; Mirsky and Barlow, 1973). Nelson et al. (1974) were able to convert by trypsin digestion the four-subunit ATPase to one with two subunits (a and p ) with retention of its activity. D. Comparison of Bacterial ATPase with FI and CF,

Bacterial ATPase resembles F1 and CF1 in its morphology and enzymatic and molecular properties. They all have latent ATPase activity that needs to be activated; their activity is cold-labile and DCCD-insensitive in the soluble form, and cold-stable and DCCD-sensitive when bound to the membrane. From an evolutionary standpoint, it is not surprising that the terminal ADP phosphorylation enzyme complex is quite similar to that in mammals, plants, and microorganisms. E. Summary

Despite a late start, studies on bacterial ATPase are quite advanced. The role of the enzyme in active transport and oxidative phosphorylation has been established using mutants lacking this enzyme. The use of different mutants offers a powerful tool in studying the role of the ATPase, its enzymatic and molecular properties, and its role in energy-linked reactions. At present, it would seem that the a and/or /3 subunits are needed for ATPase activity since some preparations of bacterial ATPase were reported to contain only these two subunits. But it has yet to be settled whether one or both are needed. The finding that the j3 subunit gets labeled by NBD-C1 (Nelson el al., 1973) probably locates the active site of the ATPase, but additional evidence would be needed before it can be accepted fully.

149

MEMBRANE ATPares

Very important is the finding of Futai et al. (1974) that preparations lacking the 6 subunit have no coupling activity, whereas preparations with this small subunit have full ATPase and coupling activities. Mutants resistant t o DCCD could be used to identify the membrane components that are needed for this sensitivity.

VI.

GENERAL CONCLUSIONS AND PERSPECTIVE

I n the intact organelle (or membrane system in bacteria) , the electrontransport process and ATP production are tightly coupled. Oxidation does not seem to occur unless the energy is utilized purposefully. There is evidence that the (nonphosphorylated) energized state can also be coupled directly t o processes such as membrane transport. Once ATP is formed, it does not seem t o be broken down (resulting in ATPase activity) unless the energy is utilized for a specific function. Thus, the high ATPase activity of membrane fractions derived from mitochondria are clearly artifacts. They are useful artifacts for designing experiments to understand some aspects of the mechanism of action of the enzyme system. The information obtained by examining the ATP (and other nucleotide) binding, regulation of the binding properties, and identification of the subunits concerned with the binding and their regulation, are bound to be of immense value. Progress on this aspect may be fast since the standard methods in enzymology and protein chemistry can be applied readily. However, the hydrolytic step or process is of little value, and, in fact, may be a hindrance, in understanding the mechanism of coupling of oxidative energy to ATP synthesis. To the best of our knowledge, the coupling process occurs in a hydrophobic environment where the hydrophobic site seen in F1 may not even exist, or if it exists, it may not be accessible to water and may well have quite a different conformation. In this connection, an interesting question to explore in the future may be the relationship between the different forms of the ADP phosphorylation enzyme, namely, FI, Factor A (with its low ATPase activity), and the membrane-bound state. Techniques for the fractionation of hydrophobic membrane proteins are currently being developed, and there is reason to be optimistic that rational methods may soon become available. However, the more challenging problem of how t o study these interactions in the hydrophobic membrane phase shows little progress. The next phase in research on ADP phosphorylation enzyme complexes will no doubt deal with the questions: How does the membrane ATPase “pick up” energy from the electron-transport complexes? What are the subunits acting in the coupling process? and

150

RIVKA PANET AND D. RAO SANADI

How is ATP produced? Current detailed mechanistic studies on how ATP is broken down may conceivably have but little bearing on this synthet,ic process and should be interpreted with caution. ACKNOWLEDGMENT This work was supported by Grant No. GM 13641 from the National Institutes of Health. REFERENCES Abrams, A. (1965). The release of bound adenosine triphosphatase from isolated bacterial membranes and the properties of solubilized enzyme. J . Biol. Chem. 240,3675-3681. Abrams, A., and Baron, C. (1967). The isolation and subunit structure of streptococcal membrane adenosine triphosphatase. Biochemistry 6, 225-229. Abrams, A., and Baron, C. (1970). Inhibitory action of carbodiimides on bacterial membrane ATPase. Biochem. Biophys. Res. Commun. 41, 858-862. Abrams, A., and Smith, J. B. (1971). Increased membrane ATPase and K+ transport rates in Streptococcus fuecalis induced by K+ restriction during growth. Biochem. Biophys. Res. Commun. 44, 1488-1495. Abrams, A., and Smith, J. B. (1974). Bacterial membrane ATPase. I n “The Enzymes” (P. Boyer, ed.), Vol. 10, pp. 395-429. Academic Press, New York. Abrams, A., Smith, J. B., and Baron, C. (1972). Carbodiimide-resistant membrane adenosine triphosphatase in mutant of Streptococcus fuecalis. I. Studies of the mechanism of resistance. J . Bio2. Chem. 247, 1484-1488. Adolfsen, R., and Moudrianakis, E. N. (1971a). Kinetics characterization of oxidative phosphorylation in Alcaligenes faeca2is. Biochemistry 10,434-440. Adolfsen, R., and Moudrianakis, E. N. (1971b). Purification and properties of two coupling factors of oxidative phosphorylation from Alcaligenes faeculis. Biochemistry 10, 2247-2253.

Adolfsen, R., and Moudrianakis, E. N. (1973). Roles for metal ions in the hydrolysis of adenosine triphosphate by the 135 coupling factors of bacterial and mitochondria1 oxidative phosphorylation. Biochemistry 12, 2926-2933. Andreoli, T. E., Lam, K. W., and Sanadi, D. R. (1965). Studies on oxidative phosphorylation. A coupling enzyme which activates reversed electron transfer. J . Biol. Chem. 2@, 2644-2653. Asami, K., Junti, K., and Ernster, L. (1970). Possible regulatory function of a mitochondrial ATPase inhibitor in respiratory chain-linked energy transfer. Biochim. Biophys. Acts 205,307-31 1. Avron, M. (1963). A coupling factor in photophosphorylation. Biochim. Biophys. Actu 77,699-702.

Baron, C., and Abrams, A. (1971). Isolation of a bacterial membrane protein, nectin, essential for attachment of adenosine triphosphatase. J . Biol. Chem. 246,1542-1544. Bennun, A,, and Racker, E. (1969). Partial resolution of the enzymes catalyzing photophosphorylation. Int,eraction of coupling factor 1 from chloroplasts with components of the chloroplast membrane. J . Biol. Chem. 244, 1325-1331. Bertina, R. M., Schrier, P. I., and Slater, E. C. (1973). The binding of aurovertin to mitochondria and its effect on mitochondrial respiration. Biochim. Biophys. Actu 305, 503-518.

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Bogin, E., Higashi, T., and Brodie, A. F. (1970). Interchangeability of coupling factor from bacterial and mammalian origin. Bioch,em. Biophys. Res. Commun. 38,47&490. Boyer, 1’. D., Cross, R., and Mnnsen, W. (1973). A new concept for energy coupling in oxidative phosphorylation based on a molecular explanation of the oxygen exchange reactions. Pror. Nall. Acad. Sci. [J.S.A. 70, 2837-2839. Bragg, 1’. D., and Hou, C. (1972). Purification of a factor both aerobic-driven and ATP-driven energy-dependent transhydrogenases of Escherichia coli. FEBS (Fed. Eur. Biochem. Soc.) Lett. 28, 309-312. Bragg, 1’. D., and Hou, C. (1973). Reconstitution of energy-dependent transhydrogenase in ATPase-negative mutants of Escherichiu coli. Biorhem. Biophys. Res. Comniun. 50, 729-737. Bragg, P. D., Davies, 1’. L., and Hou, C. (1973). Effect of removal or modification of subunit polypeptides on the coupling factor and hydrolytic: activities of the Ca++ and hlg++-activated adenosine triphosphatase of Escherichia coli. Arch. Biochem. Hiophys. 159, 664-670. Brooks, J. C., and Senior, A . E. (1971). Studies on the mitochondrial oligoniycininsensitive ATPase. The relationship of the specific protein inhibitor to the ATPase. Arch. Hiochem. Biophys. 147, 467470. Brooks, J. C., and Senior, A. E. (1972). Methods for purifications of each subunit of the mitochondrial oligomycin-insensitive adenosine-triphosphatase. Biochemistry 11, 46754678. Broughall, J. M., Griffiths, D. I