ACADEMIC PRESS RAPID MANUSCRIPT REPRODUCTION

Johnson Research Foundation Colloquia

Energy-Linked Functions of Mitochondria Edited by Britton Chance 1963

Rapid Mixing and Sampiing Techniques in Biochemistry Edited by Britton Chance, Quentin H. Gibson, Rudolph H. Eisenhardt, K. Karl Lonberg-Holm 1964

Control of Energy Metabolism Edited by Britton Chance, Ronald W. Estabrook, John R. Williamson 1965

Hernes and Hemoproteins Edited by Britton Chance, Ronald W. Estabrook, Takashi Yonetani 1966

Probes of Structure and Function of Macromolecules and Membranes Volume I Probes and Membrane Function Edited by Britton Chance. Chuan-pu Lee. J. Kent Blasie 1971

Probes of Structure and Function of Macromolecules and Membranes Volume II Probes of Enzymes and Hemoproteins Edited by Britton Chance, Takashi Yonetani, Albert S. Mildvan 1971

Biological and Biochemica! Oscillators Edited by Britton Chance, E. Kendall Pye. Amal K. Ghosh, Benno Hess 1973

Alchohol and Aldehyde Metabolizmg Systems Edited by Ronald G. Thurman. Takashi Yonetani, John R. Williamson. Britton Chance 1974

Alcohol and Aldehyde Metabolizing Systems Volume II Enzymology and Subcellular Organelles Edited by Ronald G. Thurman, John R. Williamson, Henry R. Drott, Britton Chance 1977

Alcohol and Aldehyde Metabolizing Systems Volume III Intermediary Metabolism and Neurochemistry Edited by Ronald G. Thurman, John R. Williamson, Henry R. Drott. Britton Chance 1977

Frontiers of Biological Energetics Volume I Electrons to Tissues Edited by P. Leslie Dutton, Jack S. Leigh, Antonio Scarpa 1978

Frontiers of Biological Energetics Volume II Electrons to Tissues Edited by P. Leslie Dutton, Jack S. Leigh, Antonio Scarpa 1978

Frontiers of

Biological Energetics Volume I: Electrons to Tissues

Edited by P. Leslie Dutton Jack S. Leigh

Antonio Scarpa

Johnson Research Foundation and

Department of Biochemistry and Biophysics University of Pennsylvania Philadelphia, Pennsylvania

A C A D E M I C PRESS New York San Francisco London

A Subsidiär}' of H a r c o u r t Bruce Jovanovich, Publishers

Contents

Contributors for Volumes I and II P*reface

P a r t 1 E l e c t r o c h e m i c a l I n t e r a c t i o n s

Vectorial Electron, Hydrogen, Proton, and Oxyanion Conduction in Chemiosmotic Reaction Mechanisms Peter M i t c h e l l

Energetic Advantage of Ion Countertransport in Chemiosmotic Conversion G r e g o r i o Weber

Tunneling Processes in Bacteriophotosynthesis and Vision P . M . Rentzepis

On the Efficiency of Electron Transfer Reactions in Proteins A . W a r s h e l a n d R. Weiss

Kinetics of Photochemical Electron Transfer Reactions i n V i v o and in V i t r o W. W. P a r s o n , C. C. Schenck, R. E . B l a n k e n s h i p , D . H o l t e n , M . W. W i n d s o r , a n d C. V. Shank

Structural Organization of the Redox Groups in a Bacterial Photosynthetic Reaction Center Complex D . M . T i e d e , J . S. L e i g h , a n d P . L . D u t t o n

Geometrical Relationships between the Cytochrome c-Cytochrome Oxidase Mitochondrial Redox Pairs Jane V a n d e r k o o i , J . S. L e i g h , C h a r l e s S. O w e n , Peter G l a t z , a n d H a y w o o d B l u m

Excited and Ionic States of Dimeric Chlorophyll Derivatives. Biomimetic Modelling of the Primary Events of Photosynthesis M i c h a e l R. W a s i e l e w s k i

vii

Covalently-Linked Porphyrin Quinone Complexes as R C Models Josephine L . Y. K o n g a n d P a u l A . L o a c h

Protein Dynamics, Potential Regulation, and Redox Coupled Conformational Changes in Cytochromes c F . R. Salemme

Electron Transfer by Cytochromes: Mechanisms and Problems M i c h a e l A . C u s a n o v i c h

Cytochromes c2—An Evolutionary Family R i c h a r d E . D i c k e r s o n a n d T s u n e h i r o T a k a n o

Evolutionary Changes of the Herne C Electronic Structure in Cytochromes c K u r t Wüthrich, R e g u l a M . K e l l e r , a n d Sidney L . G o r d o n

Respiratory Proteins of Some Extremely Thermophilic Bacteria James A . F e e , K a r e n L . F i n d l i n g , A l i s o n Lees, a n d T a t s u r o Y o s h i d a

Alternative Cyanide-Insensitive Respiratory Chain in P a r a c o c c u s d e n i t r i f i c a n s M i c h e l e F . H e n r y a n d P a u l e t t e M . V i g n a i s

Specification of and Regulation by the Cytochrome B Region of the Mitochondrial Genome in Yeast H e n r y R. M a h l e r , D e b o r a h K . H e n s o n , a n d D o n a l d H . M i l l e r

Biosynthesis and Intracellular Translocation of Mitochondrial Proteins: Cytochrome c and the Carboxyatractyloside Binding Protein R i c h a r d Z i m m e r m a n n , H a r o l d K o r b , a n d W a l t e r N e u p e r t

Labelling of Complex III with 3 5 S Diazobenzenesulfonate. Orientation of this Electron Transfer Segment in the Mitochondrial Inner Membrane Randy L . B e l l , Jeanne Sweetland, B e r n d L u d w i g , a n d R o d e r i c k A . C a p a l d i

Thermodynamic and EPR Properties of Hydrogen Carriers in the Succinate-Cyt. C Reductase Segment of the Respiratory Chain T o m o k o O h n i s h i , J . C. S a l e r n o , T. M a i d a , C. A . Yu, S. N a g a o k a , a n d T. E . K i n g

Contents

Mitochondrial Ubiquinone Proteins Tsoo E . K i n g , L . Yu, S. N a g a o k a , W. R. W i d g e r , a n d C . A . Yu 174

Thermodynamic and Functional Heterogeneity among the Ubiquinones of Rhodopseudomonas s p h a e r o i d e s K e n - I c h i r o T a k a m i y a , Roger C. P r i n c e , a n d P . L e s l i e D u t t o n 183

Electron and Proton Transfer in Succinate-Cytochrome c Reductase Segment of the Respiratory Chain J . C. S a l e r n o , T. M a i d a , H . B l u m , a n d T. O h n i s h i 191

A Single Ubiquinone Plays a Central Role in Electron Flow Through the Ubiquinone-Cytochromes b-c2

Oxidoreductase Roger C. P r i n c e , W i l l e m H . v a n den B e r g , K e n - I c h i r o T a k a m i y a , C. L i n d s a y B a s h f o r d , a n d P . L e s l i e D u t t o n 201

Effects of Ethanol and Salicylhydroxamic Acid on the Interactions of the Ubisemiquinone Pair with Centre S-3 in Plant Mitochondria A n t h o n y L . M o o r e , D a v i d O. H a l l , a n d H e i n z Rupp 210

Involvement of the 4 4 Protein" in Redox-Coupled Protonation Events of the Quinone Acceptor-Complex in Bacterial Photosynthetic Reaction Centers C. A . W r a i g h t 218

Pigment Interaction and Picosecond Electron Transfer in Bacterial Reaction Centers V. A . Shuvalov 227

Recent Developments on the "Primary" Electron Acceptors in Photosystem I B a c o n K e , V l a d i m i r A . Shuvalov, a n d E d D o l a n 234

Redox Potential Dependence of Electron Transport and Variable Fluorescence in Photosystem I M . C . W . Evans, P . H e a t h c o t e , A . Telf er, a n d J . B a r b e r 241

Flash-Induced Volume Changes in Purple Membrane Suspensions D o n a l d R. O r t a n d W i l l i a m W. P a r s o n 249

Establishments of Electrochemical Gradients: General Views and Experiments on Purple Membrane Benno Hess a n d D i e t r i c h K u s c h m i t z 257

X Contents

Site Specific Interaction of Protons Liberated from Photosystem II Oxidation with a Hydrophobie Membrane Component of the Chloroplast Membrane L . J . P r o c h a s k a a n d R. A . D i l l e y 265

Calibration of Flash Induced pH Changes inside Thylakoids and Kinetic Resolution of Proton Ejection and Consumption Wolfgang J u n g e , A l l i s o n M c G e e r , a n d Winfried Ausländer 275

P a r t 2 E l e c t r o n s , P r o t o n s , a n d E n e r g y

Formation of the Proton Gradient Across the Chloroplast Thylakoid Membrane in Relation to A T P Synthesis W. 5. C h o w , S. W. T h o r n e , a n d N . K . B o a r d m a n 287

The Molecular Mechanism of Light Activated Proton Transport in Purple Membranes of H a l o b a c t e r i u m h a l o b i u m A a r o n L e w i s 297

Coupling of Electron Transfer and Proton Translocation in Purple Photosynthetic Bacteria H i r o y u k i A r a t a a n d M i t s u o N i s h i m u r a 307

Contributions from Both Electron Transport and Ion Transport to the Decay of the Carotenoid Shift After Flash-Activation of Chromatophores J . B . J a c k s o n , J . A . G r e e n r o d , N . K . P a c k h a m , a n d K . M . Petty 316

Inhibition of Electron Transport in RPS. Capsulata by a Ubiquinone Analogue J o h n R. B o w y e r a n d A n t h o n y R. Crofts 326

Vectorial Oxidoreductions: The Ferrous lron Oxidase Complex of T h i o b a c i l l u s f e r r o o x i d a n s and the Nitrate Reductase Complex of E s c h e r i c h i a c o l i W. J o h n I n g l e d e w , J o h n C. C o x , R o b e r t W. Jones, a n d Peter B . G a r l a n d 334

Electron Translocating Function of Cytochrome Oxidase Peter M i t c h e l l a n d Jennifer M o y l e 342

Generation of Electrochemical Proton Gradient by Mitochondrial Cytochrome c Oxidase. Clarification of Some Controversial Experiments Märten Wikströ'm a n d K l a a s K r a b 351

Control Mechanisms for Proton Conduction in the Mitochondrial H + -ATPase A n g e l a P a n s i n i , F . G u e r r i e r i , a n d S. P a p a 359

Contents x i

The Mechanism of Transmembrane A / X H + Generation by Cytochrome c Oxidase S. P a p a , F . G u e r r i e r i , M , L o r u s s o , F . C a p u a n o , G. I z z o , a n d D . Boffoli 367

The Three Proton Pumps of the Mitochondrial Respiratory Chain G. F . Azzone, T. Pozzan, F . D i V i r g i l i o , a n d V. M i c o n i 375

Proton Stoichiometry of Mitochondrial Electron Transport, A T P Hydrolysis, and ATP-Dependent Reverse Electron Flow A l b e r t L . L e h n i n g e r , B a l t a z a r Reynafarje a n d Adolf A l e x a n d r e 384

Protonmotive Stoichiometry of Redox and ATPase Systems Peter M i t c h e l l , J e n n i f e r M o y l e , a n d Roy M i t c h e l l 394

Membrane Potential, Phase Transitions, and Coupling in Mitochondria H a g a i R o t t e n b e r g 403

Further Studies on the Membrane Potential of Giant Mitochondria Using Microelectrodes C h a r l e s B o w m a n , B r u c e L . Maloff, a n d H e n r y Tedeschi 413

Can Energy Coupling Occur in the Mitochondrial Membrane in the Absence of Transmembrane Gradients? D a n a M . Scott a n d B a y a r d T. Storey 422

The Energy Balance of Oxidative Phosphorylation K . v a n D a m , R. P . Casey, R. v a n der M e e r , A . K . G r o e n , a n d H . V. Westerhoff 430

Effects of Anions on the Reaction between Cytochrome c and Cytochrome c Oxidase N e i l Osheroff, W. H . K o p p e n o l , a n d E . M a r g o l i a s h 439

Proton Translocating ATPase Subunit Structure and Pump, Gate, and Channel Activities N o b u h i t o Sone, Masasuke Y o s h i d a , H a j i m e H i r a t a , a n d Yasuo K a g a x v a 450

ATP-Synthesis Induced by a pH-Gradient Imposed Across a Collagen Film Bearing an ATPase-ATP Synthase C a t h e r i n e G o d i n o t , B r u n o B l a n c h y , P i e r r e R. C o u l e t , a n d D a n i e l e C. G a u t h e r o n 459

Site Directed Modifications of B F r A T P a s e From E . c o l i . Comparison of Binding Properties of Bacterial and Mitochondrial Fj with Respect to Aurovertin, D C C D , and E E D Q M i c h e l S a t r e , R i c h a r d P o u g e o i s , J o e l L u n a r d i , A n n e - C h r i s t i n e D i a n o u s , G e r a r d K l e i n , M i r e i l l e Bof, a n d P i e r r e V. V i g n a i s

Solubilization of Coupling Factor 1 From Chloroplast Thylakoids Alters Its Interactions with Nucleotides R i c h a r d E . M c C a r t y , R o n a l d P . M a g n u s s o n , a n d R a y m o n d W o n g

3 '-Esterified Adenine Nucleotides as Extrinsic Probes of the Energy Coupling Device Günter Schäfer

New Results Reveal Subunit Catalytic Cooperativity in F {

ATPase R. Lee H u t t o n , D a v i d D . H a c k n e y , a n d P a u l D . B o y e r

Reconstitution of Oligomycin- and Dicyclohexylcarbodiimide-Sensitive Mitochondrial ATPase from Isolated Components B i r g i t t a N o r l i n g , E l z b i e t a G l a s e r , a n d L a r s E r n s t e r

Complex V: Composition and Molecular and Enzymic Properties Yves M . G a l a n t e , L u c i a n o F r i g e r i , a n d Youssef Hatefi

Biochemical Characterization of the Electrogenic Proton Pump of the Neurospora Plasma Membrane B a r r y J . B o w m a n , F r a n c i s B l a s c o , a n d C a r o l y n W. S l a y m a n

Measurement of the Protonmotive Force in Amine Containing Subcellular Organelles R. G. J o h n s o n , A . S c a r p a , a n d L . Salganicoff

Enzymic Modification of Gastric Transport ATPase G e o r g e Sachs, E d d R a b o n , a n d G a e t a n o Saccomani

Chloride Transport in Gastric Cells and Microsomes A n n i c k S o u m a r m o n a n d Efraim Racker

Membrane Potential and C l ~ Transport Properties of Primary Glial Cultures from Rat Brain H . K . K i m e l b e r g , S. B i d d l e c o m e , a n d R. S. B o u r k e

Contents . x i i i

The Interaction of M g + + and pH in Chloroplast Processes G e o r g e H o c h a n d S a l i l Bose 573

P a r t 3 New I n s t r u m e n t a l A p p r o a c h e s t o C e l l u l a r B i o p h y s i c s

Electron Spin Echo Spectroscopy and Photosynthesis J . R. N o r r i s , M . C. T h u r n a u e r , M . K . B o w m a n , a n d A . D . Trifunac 581

Use of the Molecular Microprobe to Record Raman Spectra of a Single Mitochondrion and a Fiber of Calf Thymus D N A F r a n A d a r 592

Normal Coordinate Models for Herne Raman Spectra: Uses and Limitations L . R i m a i a n d I . Salmeen 600

Added Precision in 5 7Fe Mossbauer Spectrometry of Proteins W. F . F i l t e r , W. R. D u n h a m , R. M . P o l i c h a r , a n d R. H . Sands 608

Dynamics of the Local Iron Environment by the Selective Excitation Double Mossbauer Technique (SEDM) B . B a l k o , E . V. M i e l c z a r e k , a n d R. L . B e r g e r 617

Exchange Interaction in Spinach Ferredoxin Determined by Electron Paramagnetic Resonance H a y w o o d B l u m , J . S. L e i g h , T o m o k o O h n i s h i , a n d J . C. S a l e r n o 625

Precise EPR Measurements on Small Protein Crystals R. A . L i e b e r m a n , W. R. D u n h a m , J . A . F e e , a n d R. H . Sands 634

How Sensitive is the EPR of Herne to a Perturbed Environment? C. P . S. T a y l o r a n d B . R. Sreenathan 644

X-Ray Absorption Studies of Metalloproteins P . E i s e n b e r g e r , B . M . K i n c a i d , a n d /? . G. S h u l m a n 652

Core Expansion vs. Coming in Porphyrins; Resonance Raman Discrimination between Five- and Six-Coordinate High-Spin Fe"1

Hernes Thomas G. S p i r o , J o h n D . Stong, a n d P a u l Stein 660

Dynamics of Non-Linear Electric Field Effects L e o D e M a e y e r a n d F r e d d y Paulussen 671

Herne Protein Reactions at High Pressure and Low Temperature L a u r a E i s e n s t e i n a n d H a n s F r a u e n f e l d e r

Calorimetric Studies of the Heat of Respiration of Mitochondria I c h i r o M a t s u o k a , T a k a h i d e W a t a n a b e , a n d T a k a o N a k a m u r a

The Measurement of AH and the k k 0 n " Rate Constant of the Reactions of C 0 2 and 2,3-DPG with Deoxyhemoglobin by Thermal Stopped Flow R. L . B e r g e r , B . B a l k o , P . B o w e n , a n d R. P a u l

Studies of Stable Metal-Nucleotide Complexes Interacting with Myosin Subfragment J . F . E c c l e s t o n a n d D . R. Fr entkam

Intracellular Enzyme Activity N o b u t o m o I t a d a , L a r a i n e Peiffer, a n d R o b e r t E . F o r s t e r

E . c o l i Unadenylated Glutamine Synthetase: Elucidation of the Catalytic Cycle and the Role of Some Feedback Inhibitors S. G. Rhee, P . B . C h o c k , a n d E . R. Stadtman

High Voltage Microscopy of Cells and Membranes in the Hydrated State D o n a l d F . P a r s o n s

Mitochondrial and Sarcoplasmic Reticulum Contents i n S i t u : Electron Probe Analysis A . P . S o m l y o , H . Shuman, a n d A . V. Somlyo

Augmentation of Tissue Water Proton Spin-Lattice Relaxation Rates by i n V i v o Addition of Paramagnetic Ions P a u l C. L a u t e r b u r , M . H e l e n a M e n d o n q a D i a s , a n d A n d r e w M . R u d i n

Anomalous X-Ray Scattering Studies for the Determination of the Location of Redox Centers in Membranes—A Feasibility Study James Stamatoff, Peter E i s e n b e r g e r , G e o r g e B r o w n , James Pachence, L e s l i e D u t t o n , J o h n L e i g h , a n d K e n t B l a s i e

Magnetic Resonance Studies of the Mechanism of R N A Polymerase from E . c o l i A . S. M i l d v a n , P . S t e i n , R. K o r e n , a n d B . Bean

Contents

3 1 P N M R of Brain Tissue Under Normal, Hypothermie, and Freeze-Trapped Conditions B . C h a n c e , Y. Nakase, M . B o n d ,

J . S. L e i g h , J r . , a n d G. M c D o n a l d 779

Visualization of Dynamic Spatial Structures in Oscillating Cell Free Extracts of Yeast A r n o l d B o i t e u x a n d Benno Hess 789

Contributors of Volumes I and II

Hiroshi Abe, Osaka University Medical School, First Department of Internal Medicine, Osaka, Japan

Kazuhiko Adachi, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Fran Adar, Department of Biochemistry and Biophysics, University of Pennsyl­vania, School of Medicine, Philadelphia, Pennsylvania 19104

N . Akaike, Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77550

Karl E. Akerman, Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10, SF 00170 Helsinki 17, Finland

Adolfo Alexandre, Johns Hopkins University, School of Medicine, Department of Physiological Chemistry, Baltimore, Maryland 21205

Olusoji Amire, Department of Chemistry, University of Ibadan, Ibadan, Nigeria S. G . Angelos, Jr., Department of Chemistry, University of Southern Califor­

nia, University Park, Los Angeles, California 90007 E. Antonini, Istituto di Chimica, Facolta di Medicina, Universita di Roma e

Centro di Biologia Molecolare, Rome, Italy Hiroyuki Arata, Department of Biology, Kyushu University, Fukuoka 812,

Japan Toshio Asakura, Department of Pediatrics, Children's Hospital of Philadelphia,

Philadelphia, Pennsylvania 19104 Winfried Ausländer, Max-Volmer Institut, Technische Universität Berlin,

Strasse des 17 juni 135, 1 Berlin 12, Federal Republic of Germany George Austin, Section of Neurosurgery, Loma Linda University School of

Medicine, Loma Linda, California 92354 Angelo Azz i , Universitität Bern, Med-Chem. Institut, Bühlstrasse 28, 3012

Bern, Switzerland G. F. Azzone, Instituto di Patologia Generale, Via Loredan, Padova, Italy Gerald T. Babcock, Department of Chemistry, Michigan State University, East

Lansing, Michigan

x v i i

AT/7/ Conthbutors o f Volumes I and II

B. Balko, Laboratory of Technical Development, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014

Michael Barany, Department of Biological Chemistry and Research Resources Center, University of Illinois Medical Center, Chicago, Illinois 60612

Donald Barber, School of Biological Sciences, University of East Anglia, Nor-wich NR4 7TJ, England

J. Barber, Imperial College, Department of Botany, London, United Kingdom Clyde H . Barlow, Department of Biochemistry and Biophysics, Johnson Re­

search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

C. Lindsay Bashford, Department of Biochemistry and Biophysics, Johnson Re­search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

B. Bean, Institute of Cancer Research, Fox Chase, Philadelphia, Pennsylvania 19111

John G . Beetlestone, Department of Chemistry, University of Ibadan, Ibadan, Nigeria

Helmut Beinert, Enzyme Institute, University of Wisconsin, Madison, Wiscon­sin 53706

Randy L . Bell , Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

Nils-Olof Bengtsson, Department of Chemistry, Section of Physiological Chemistry, University of Umea, S-901 87 Umea, Sweden

Robert L . Berger, Building 10, Room 5D-20, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014

S. Biddlecome, Division of Neurosurgery, Albany Medical College, Albany, New York

R. Bisson,.Istituto di Patologia Generale, Universita di Padova, Padova, Italy Bruno Blanchy, Universite Claude Bernard-Lyon 1, 69621 Villeurbanne,

France R. E. Blankenship, Department of Biochemistry, J405 Health Sciences Build­

ing, SJ-70, University of Washington, Seattle, Washington 98195 Francis Blasco, Department of Human Genetics/Physiology, Yale University,

New Häven, Connecticut 06510 Kent Blasie, Departments of Chemistry and Biochemistry/Biophysics, Univer­

sity of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Haywood Blum, Department of Biochemistry and Biophysics, Johnson Re­search Foundation, University of Pennsylvania, Medical School, Philadel­phia, Pennsylvania 19104

Contributors of Volumes I and II x i x

W . E. Blumberg, Bell Telephone Laboratories, Murray Hi l l , New Jersey 07974 N . K . Boardman, CSIRO, P.O. Box 225, Dickson, A . C . T . 2602, Australia David F. Bocian, California Institute of Technology, Division of Chemistry and

Chemical Engineering, Pasadena, California 91125 Mireille Bof, Laboratoire de Biochimie, Departement de Recherche Fondamen­

tale, C E N - 85X, 38041 Grenoble, Cedex, France D. Boffoli, Instituto di Chimica Biologica, Facolta di Medicina e Chirurgia,

Universita di Bari, 70126 Bari, Italy Arnold Boiteux, Max-Planck Institut für Ernährungsphysiologie, D-46

Dortmund, Rheinlanddamm 201, West Germany M . Bond, Department of Biochemistry and Biophysics, Johnson Research

Foundation, University of Pennsylvania, School of Medicine, Philadel­phia, Pennsylvania 19104

Walter D. Bonner, Jr., Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Salil Bose, Department of Biology, University of Rochester, Rochester, New York 14627

R. S. Bourke, Division of Neurosurgery, Albany Medical College, Albany, New York

A . Boveris, Instituto de Quimica Biologica, Universidad de Buenos Aires, Buenos Aires, Argentina

P. Bowen, Laboratory of Technical Development, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014

Barry J. Bowman, Department of Human Genetics/Physiology, Yale Univer­sity, New Häven, Connecticut 06510

Charles Bowman, Department of Biological Sciences, State University of New York at Albany, Albany, New York 12222

M . K. Bowman, Chemistry Division D-200, Argonne National Laboratories, Argonne, Illinois 60439

John R. Bowyer, Department of Physiology and Biophysics, University of Il­linois, Urbana, Illinois 61801

Paul D. Boyer, Department of Chemistry and Molecular Biology Institute, Uni­versity of California at Los Angeles, Los Angeles, California 90024

Arthur S. Bri l l , Department of Physics, University of Virginia, Charlottesville, Virginia 22901

F. J. Brinley, Jr., Department of Physiology, University of Maryland, School of Medicine, Baltimore, Maryland 21201

A. M . Brown, Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77550

XX Contributors of Volumes I and II

George Brown, Stanford Synchrotron Radiation Laboratory, Stanford, Califor­nia

Truman R. Brown, Bell Laboratories, Murray Hi l l , New Jersey 07974 Mark Bruckner, Department of Biochemistry and Biophysics, Johnson Research

Foundation, University of Pennsylvania, School of Medicine, Philadel­phia, Pennsylvania 19104

Gary W. Brudvig, California Institute of Technology, Division of Chemistry and Chemical Engineering, Pasadena, California 91125

M . Brunori, Institute of Biochemistry, Faculty of Medicine, Citta Universitaria, 00185 Rome, Italy

C. Tyler Burt, Department of Biological Chemistry and Research Resources Center, University of Illinois Medical Center, Chicago, Illinois 60612

Gerhard Buse, R W T H Aachen, Abt. Physiologische Chemie, Meiatener Strasse 211, D-5100 Aachen, West Germany

Roderick A. Capaldi, Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

F. Capuano, Instituto di Chimica Biologica, Facolta di Medicina e Chirurgia, Universita di Bari, 70126 Bari, Italy

R. P. Casey, Universiteit van Amsterdam, Lab. voor Biochemie, B . C . P . Jarn­sen Institut, Plantage Muidergracht 12, Amsterdam-C, Netherlands

Nicholas Chacos, Department of Biochemistry, Southwestern Medical School, University of Texas, Dallas, Texas 75235

Joseph M . Chalovich, Department of Biological Chemistry and Research Re­sources Center, University of Illinois Medical Center, Chicago, Illinois 60612

Samuel H . P. Chan, Department of Biology, Syracuse University, Syracuse, New York 13210

Sunney I. Chan, California Institute of Technology, Division of Chemistry and Chemical Engineering, Pasadena, California 91125

Britton Chance, Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadel­phia, Pennsylvania 19104

P. B . Chock, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014

W. S. Chow, CSIRO, Division of Plant Industry, Canberra, A . C . T . , Australia Leland C. Clark, Jr., Children's Hospital Research Foundation, Cincinnati,

Ohio 45229 Carol Coan, Laboratory of Physiology and Biophysics, University of the

Pacific, San Francisco, California 94115

Contributors o f Volumes I and II

Fredde Cohen, Department of Physiology and Biophysics, Health Sciences Center, State University of New York at Stonybrook, Stonybrook, New York 11794

L . B . Cohen, Department of Physiology, Yale University, School of Medicine, New Häven, Connecticut 06510

S. M . Cohen, Bell Laboratories, Murray Hi l l , New Jersey 07974 A . Colosimo, Istituto di Chimica, Facolta di Medicina, Universita di Roma e

Centro di Biologia Molecolare, Rome, Italy Pierre R. Coulet, Universite Claude Bernard-Lyon 1, 69621 Villeurbanne,

France John C. Cox, Department of Biochemistry, Medical Sciences Institute, Univer­

sity of Dundee, Dundee DD1 4 H N , Scotland Anthony R. Crofts, Department of Physiology and Biophysics, University of

Illinois, Urbana, Illinois 61801 Michael A . Cusanovich, Department of Chemistry, University of Arizona, Tuc-

son, Arizona 85721 Morris J. Danon, Department of Biological Chemistry and Research Resources

Center, University of Illinois Medical Center, Chicago, Illinois 60612 A . Darszon, Departments of Physics and Biology, University of California at

San Diego, La Jolla, California 92037 P. H . Davis, Department of Physics and Center for Biological Macromolecules,

State University of New York at Albany, Albany, New York 12222 D. W. Deamer, Department of Zoology, University of California, Davis,

California 95616 Gilbert J. DeLeeuw, Department of Biochemistry and Biophysics, Johnson Re­

search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Leo De Maeyer, Max-Planck Institute für Biophysikalische Chemie, D-3400 Göttingen-Nikolausberg, West Germany

J. A . den Hollander, Bell Laboratories, Murray Hi l l , New Jersey 07974 Anne-Christine Dianous, Laboratoire de Biochimie, Departement de Recherche

Fondamentale, C E N - 8 5 X , 38041 Grenoble, Cedex, France M . Helena Mendonqa Dias, Centro de Quimica Estrutural, Universidade de

Lisboa, Instituto Superior Teenico, Lisbon, Portugal Richard E. Dickerson, Chemistry Department, California Institute of Technol­

ogy, Pasadena, California 91125 James Dilger, Department of Physiology and Biophysics, Health Sciences

Center, State University of New York at Stonybrook, Stonybrook, New York 11794

.Y.Y/7 Contributors o f V o l u m es I and II

Richard A . Dilley, Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

F. DiVirgilio, Instituto di Patologia Generale, Via Loredan, Padova, Italy Ed Dolan, Charles F. Kettering Research Laboratory, Yellow Springs, Ohio

45387 W. R. Dunham, Institute of Science and Technology, Biophysics Research Di­

vision, University of Michigan, Ann Arbor, Michigan 48109 P. Leslie Dutton, Department of Biochemistry and Biophysics, Johnson Re­

search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

W. A . Eaton, Laboratory of Chemical Physics, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014

J. F. Eccleston, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Moises Eisenberg, Department of Physiology and Biophysics, Health Sciences Center, State University of New York at Stonybrook, Stonybrook, New York 11794

Peter Eisenberger, Bell Laboratories, Murray Hi l l , New Jersey 07974 George Eisenman, Department of Physiology, School of Medicine, Center for

the Health Sciences, Los Angeles, California 90024 Laura Eisenstein, Department of Physics, University of Illinois, Urbana, Illinois

61801 S. Walter Englander, Department of Biochemistry and Biophysics, University of

Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104 Lars Ernster, Department of Biochemistry, Arrhenius Laboratory, University of

Stockholm, Stockholm, Sweden Ronald W. Estabrook, Department of Biochemistry, Southwestern Medical

School, University of Texas, Dallas, Texas 75235 M . C. W. Evans, Department of Botany and Microbiology, University College,

London W C IE 6BT, England James A . Fee, University of Michigan, Institute of Science and Technology,

Biophysics Research Division, Ann Arbor, Michigan 48109 Hubert Felle, Department of Physiology, Yale University School of Medicine,

New Häven, Connecticut 06510 F. A . Ferrone, Laboratory of Chemical Physics, N I A M D D , National Institutes

of Health, Bethesda, Maryland 20014 Francis G . Fiamingo, Department of Physics, University of Virginia, Charlot-

tesville, Virginia 22901

Contributors of Volumes I and II x x i i i

M . Filipkowski, Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadel­phia, Pennsylvania 19104

W . F. Filter, Institute of Science and Technology, Biophysics Research Divi­sion, University of Michigan, Ann Arbor, Michigan 48109

Karen L . Findling, Institute of Science and Technology, Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109

S. Fleischer, Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235

Robert E . Forster, Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Hans Frauenfelder, Department of Physics, University of Illinois, Urbana, Il­linois 61801

Jeffrey C. Freedman, Department of Physiology, Yale University School of Medicine, New Häven, Connecticut 06510

Terrence G . Frey, Department of Biochemistry and Biophysics, Johnson Re­search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Luciano Frigeri, Scripps Clinic and Research Foundation, Department of Biochemistry, La Jolla, California 92037

David G . Gadian, Department of Biochemistry, University of Oxford, Oxford O X l 3QU, England

Yves M . Galante, Scripps Clinic and Research Foundation, La Jolla, California 92037

Peter B . Garland, Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4 H N , Scotland

Pamela B . Garlick, Department of Biochemistry, University of Oxford, Oxford, O X l 3QU, England

Daniele C. Gautheron, University of Claude Bernard-Lyon 1, 69621 Villeur-banne, France

Elzbieta Glaser, Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, Stockholm, Sweden

Peter Glatz, Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadel­phia, Pennsylvania 19104

Thomas Glonek, Department of Biological Chemistry and Research Resources Center, University of Illinois Medical Center, Chicago, Illinois 60612

Catherine Godinot, Universite Claude Bernard-Lyon 1, 69621 Villeurbanne, France

xxiv Contributors o f Volumes I and II

Sidney L . Gordon, Institut für Molekularbiologie und Biophysik, E T H -Zürich-Honggerberg, CH-8093 Zürich, Switzerland

B. I. Greene, Department of Chemistry, University of Pennsylvania, Philadel­phia, Pennsylvania 19104

J. A . Greenrod, Department of Biochemistry, University of Birmingham, Bir­mingham B15 2TT, United Kingdom

Colin Greenwood, University of East Anglia, School of Biological Sciences, Norwich N R 4 7TJ, England

Ferdinand Greitschus, Max Planck Institut für Systemphysiologie, D-4600 Dortmund, West Germany

A . Grinvald, Department of Physiology, Yale University, School of Medicine, New Häven, Connecticut 06510

A . K . Groen, Universiteit van Amsterdam, Lab. voor Biochemie, B . C . P . Jarn­sen Institut, Plantage Muidergracht 12, Amsterdam-C, Netherlands

Markus Güggi, Max-Planck Institut für Systemphysiologie, D-4600 Dortmund, West Germany

F. Guerrieri, Instituto di Chimica Biologica, Facolta di Medicina e Chirurgia, Universita di Bari, 70126 Bari, Italy

H . Gutweniger, Istituto di Patologia Generale, Universita di Padova, Padova, Italy

David D . Hackney, Department of Chemistry and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90024

Bunji Hagihara, Department of Biochemistry, Osaka University Medical School, Osaka, Japan

David O. Hall, Department of Plant Sciences, King's College, London, En­gland

Don A . Hampton, Alabama Power Company, Birmingham, Alabama 35291 Wesley Harden III, Harrison Department of Surgical Research, University of

Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104 L . J. Harding, Computing Center, University of Michigan, Ann Arbor, Michi­

gan 48109 Alden H . Harken, Harrison Department of Surgical Research, University of

Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104 John Haselgrove, Department of Biochemistry and Biophysics, Johnson Re­

search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Youssef Hatefi, Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037

Norio Hayashi, First Department of Internal Medicine, Osaka University Medi­cal School, Osaka, Japan

Contributors of Volumes I and II xxv

P. Heathcote, Department of Botany and Microbiology, University College, London W C IE 6BT, England

Michele F. Henry, C N R S , DRF/Biochemie C E N - G 85X 38041 Grenoble, Cedex, France

Deborah K . Henson, Department of Chemistry, Indiana Universi ty, Bloomington, Indiana 47401

L . Herbette, Departments of Chemistry & Biochemistry/Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Benno Hess, Max-Planck Institut für Ernährungsphysiologie, Rheinlanddamm 201, D-4600 Dortmund 1, West Germany

Hajime Hirata, Jichi Medical School, Tochigi-ken, Japan 329-04 George Hoch, Department of Biology, University of Rochester, Rochester,

New York 14627 R. M . Hochstrasser, Department of Chemistry, University of Pennsylvania,

Philadelphia, Pennsylvania 19104 Joseph F. Hoffman, Department of Physiology, Yale University School of

Medicine, New Häven, Connecticut 06510

J. Hofrichter, Laboratory of Chemical Physics, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014

D. Holten, Department of Chemistry, Washington State University, Pullman, Washington

Jens Höper, Max-Planck Institut für Systemphysiologie, D-4600 Dortmund, West Germany

H . P. Hopkins, Department of Chemistry, Georgia State University, Atlanta, Georgia

Cheng-Schen Huang, Department of Biochemistry, School of Medicine, Wayne State University, Detroit, Michigan 48201

R. Lee Hutton, Department of Chemistry and Molecular Biology Institute, Uni­versity of California at Los Angeles, Los Angeles, California 90024

James S. Hyde, Department of Biochemistry, Medical College of Wisconsin, Milwaukee County Medical Complex, Milwaukee, Wisconsin 53226

Vito Iacobazzi, Department of Biochemistry, University of Bari, Via Amendola 165, Bari, Italy

Masao Ideda-Saito, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Giuseppe Inesi, Laboratory of Physiology and Biophysics, University of the Pacific, San Francisco, California 94115

x.xv i Contributors of Volumes I and II

W. John Ingledew, Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4 H N , Scotland

Toshiro Inubushi, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Nobutomo Itada, Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

G. Izzo, Instituto di Chimica Biologica, Facolta di Medicina e Chirurgia, Uni­versita di Bari, 70126 Bari, Italy

J. B. Jackson, Department of Biochemistry, University of Binningham, Bir­mingham B15 2TT, United Kingdom

Arco Y . Jeng, Department of Radiation Biology and Biophysics, University of Rochester, Rochester, New York 14642

Frans F. Jöbsis, Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710

Michael Johnson, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, England

Robert G. Johnson, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Robert W. Jones, Department of Biochemistry, Medical Sciences Institute, Uni­versity of Dundee, Dundee DD1 4 H N , Scotland

Elizabeth Joyce, Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadel­phia, Pennsylvania 19104

Wolfgang Junge, Max-Volmer Institut, Technische Universität Berlin, Strasse des 17 juni 135, 1 Berlin 12, Federal Republic of Germany

Ronald Jutzy, Section of Neurosurgery, Loma Linda University School of Medicine, Loma Linda, California 92354

H . R. Kaback, Roche Institute of Molecular Biology, Nutley, New Jersey 07110

Gregory J. Kaczorowski, Laboratory of Membrane Biochemistry, Roche Insti­tute of Molecular Biology, Nutley, New Jersey 07110

Yasuo Kagawa, Jichi Medical School, Tochigi-ken, Japan 329-04 Takenobu Kamada, First Department of Internal Medicine, Osaka University

Medical School, Osaka, Japan Hilde Kanamuller, Department of Biochemistry and Biophysics, Johnson Re­

search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Sunao Kawano, Department of Biochemistry, Southwestern Medical School, University of Texas, Dallas, Texas 75235

Contributors o f Volumes I and II .x.xvii

Bacon Ke, Charles F. Kettering Research Laboratory, Yellow Springs, Ohio 45387

Regula M . Keller, Institut für Molekularbiologie und Biophysik, E T H -Zürich-Hönggerberg, CH-8093 Zürich, Switzerland

Manfred Kessler, Max-Planck-Insti tüt für Systemphysiologie, D-4600 Dortmund, West Germany

Ezzatollah Keyhani, Laboratory for Cell Biology and Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

Jacqueline Keyhani, Laboratory for Cell Biology and Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

H . K . Kimelberg, Division of Neurosurgery, Albany Medical College, Albany, New York 12222

B. M . Kincaid, Bell Laboratories, Murray Hi l l , New Jersey 07974 Tsoo E. King, Department of Chemistry and Laboratory of Bioenergetics, State

University of New York at Albany, Albany, New York 12222 Gerard Klein, Laboratoire de Biochimie, Departement de Recherche Fondamen­

tale, C E N 85X, 38041 Grenoble, Cedex, France Amira Klip, C . H . Best Institute, University of Toronto, Toronto, Ontario,

Canada M 5 G 1L6 Josephine L . Y . Kong, Department of Chemistry, Northwestern University,

Evanston, Illinois 60301 W. H . Koppenol, Department of Biochemistry and Molecular Biology, North­

western University, Evanston, Illinois 60201 Harold Korb, Physiologisch-Chemisches Institut der Georg-August-Universität

Göttingen, 34 Göttingen, Humboldtallee 7, West Germany R. Koren, Institute for Cancer Research, Fox Chase, Philadelphia, Pennsyl­

vania 19111 Arthur Kowalsky, Department of Biophysics, Albert Einstein College of

Medicine, Yeshiva University, Bronx, New York 10461 Klaas Krab, Department of Medical Chemistry, University of Helsinki, Sil-

tavuorenpenger 10, SF 00170 Helsinki 17, Finland Mark Kurzmack, Laboratory of Physiology and Biophysics, University of the

Pacific, San Francisco, California 94115 Dietrich Kuschmitz, Max-Planck Institut für Ernährungsphysiologie, Rhein­

landdamm 201, D-4600 Dortmund 1, West Germany Henry Lardy, Institute for Enzyme Research, University of Wisconsin, Madi-

son, Wisconsin 53706 Frank A . Lattanzio, Department of Pharmacology, University of Miami School

of Medicine, P.O. Box 520875, Miami, Florida 33152

.v.vv/77 Contributors of Volumes I and II

Pui-Wah Lau, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Paul C. Lauterbur, Department of Chemistry, State University of New York at Stonybrook, Stonybrook, New York 11794

C. P. Lee, Biochemistry Department, Wayne State University, School of Medicine, Detroit, Michigan 48201

K. S. Lee, Department of Physiology and Biophysics, University of Texas Med­ical Branch, Galveston, Texas 77550

Alison Lees, Institute of Science & Technology, Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109

Albert L . Lehninger, Department of Physiological Chemistry, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205

J. S. Leigh, Jr., Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadel­phia, Pennsylvania 19104

Aaron Lewis, Clark Hall, Cornell University, School of Applied and Engineer­ing Physics, Ithaca, New York 14853

David E. Lewis, Laboratory of Physiology and Biophysics, University of the Pacific, San Francisco, California 94115

R. A . Lieberman, Biophysics Research Division, Institute of Science and Technology, University of Michigan, Ann Arbor, Michigan 48109

David Y . Lo , Department of Genetics, Stanford University, School of Medicine, Stanford, California 94305

Paul A . Loach, Department of Biochemistry and Molecular Biology, North­western University, Evanston, Illinois 60301

Gilda Loew, Department of Genetics, Stanford University, School of Medicine, Stanford, California 94305

W. Scott Long, Department of Physiology, Yale University School of Medicine, New Häven, Connecticut 06510

M . Lorusso, Instituto di Chimica Biologica, Facolta di Medicina e Chirurgia, Universita di Bari, 70126 Bari, Italy

Bernd Ludwig, Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

Martha L . Ludwig, Biophysics Research Division and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Joel Lunardi, Laboratoire de Biochimie, Departement de Recherche Fondamen­tale, C E N - 8 5 X , 38041 Grenoble, Cedex, France

Elizabeth McCandlish, Department of Chemistry, Rutgers University, Busch Campus, New Brunswick, New Jersey 08903

Contributors of Volumes I and II xv/.v

Richard E. McCarty, Section of Biochemistry, Molecular, and Cell Biology, Cornell University, Ithaca, New York 14853

George G . McDonald, Department of Biochemistry and Biophysics, University of Pennsylvania Medical School, Johnson Research Foundation, Philadel­phia, Pennsylvania 19104

Allison McGeer, Max-Volmer Institut, Technische Universität Berlin, Strasse des 17 juni 135. 1 Berlin 12, Federal Republic of Germany

A . C . McLaughlin, Biology Department, Brookhaven National Laboratory, Up-ton, New York 11973

Stuart McLaughlin, Department of Physiology and Biophysics, Health Sciences Center, State University of New York at Stonybrook, Stonybrook, New York 11794

David H . MacLennan, C. H . Best Institute, University of Toronto, Toronto, Ontario, Canada M5G 1L6

Ronald P. Magnusson, Section of Biochemistry, Molecular, and Cell Biology, Cornell University, Ithaca, New York 14853

Henry R. Mahler, Department of Chemistry, Indiana University, Bloomington, Indiana 47401

T. Maida, Department of Biochemistry and Biophysics, University of Pennsyl­vania, School of Medicine, Johnson Research Foundation, Philadelphia, Pennsylvania 19104

Bruce L . Maloff, Department of Biological Sciences, State University of New York at Albany, Albany, New York 12222

Rimona Margalit, Department of Physiology, School of Medicine, Center for the Health Sciences, Los Angeles, California 90024

E. Margoliash, Department of Biochemistry and Molecular Biology, North­western University, Evanston, Illinois 60201

Takakatsu Matsumura, First Department of Internal Medicine, Osaka Univer­sity Medical School, Osaka, Japan

Ichiro Matsuoka, Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan

Avraham Mayevsky, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

J. Maylie, Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Werner Meesman, Pathologisches Institut, Universität Essen, Essen, West Germany

V. Miconi, Instituto di Patologia Generale, Via Loredan, Padova, Italy E. V . Mielczarek, Laboratory of Technical Development, National Heart,

A'.V.V Contributors o f Volu/nes I and II

Lung, and Blood Institute, National Institutes of Health, Bethesda, Mary­land 20014

A . S. Mildvan, Institute of Cancer Research, Fox Chase, Philadelphia, Pennsylvania 19111

Donald H . Miller, Department of Chemistry, Indiana University, Bloomington, Indiana 47401

Peter Mitchell, Glynn Research Laboratories, Bodmin, Cornwall PL30 4 A U , England

Roy Mitchell, Glynn Research Laboratories, Bodmin, Cornwall PL30 4 A U , England

M . Montal, Physics Department, University of California, San Diego, La Jolla, California 92037

C. Montecucco, Istituto di Patologia Generale, Universita di Padova, Padova, Italy

Anthony L . Moore, Department of Plant Sciences, King's College, London, England

M . Morad. Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Randall H . Morse, California Institute of Technology, Division of Chemistry and Chemical Engineering, Pasadena, California 91125

Jennifer Moyle, Glynn Research Laboratories, Bodmin, Cornwall PL30 4 A U , England

S. Nagaoka, Department of Biochemistry, State University of New York at A l ­bany, Albany, New York 12222

Takao Nakamura, Department of Biology, Faculty of Science, Osaka Univer­sity, Toyonaka, Osaka 560, Japan

Y . Nakase, Department of Biochemistry and Biophysics, University of Pennsyl­vania, Johnson Foundation, School of Medicine, Philadelphia, Pennsyl­vania 19104

Walter Neupert, Physiologisch-Chemisches Institut der Georg-August-Universität Göttingen, 34 Göttingen, Humboldtallee 7, West Germany

David Nicholls, University of Dundee, Department of Psychiatry, Ninewells Hospital, Dundee DD1 9SY, Scotland

Peter Nicholls, Department of Biological Sciences, Brock University, Glenridge Campus, St. Catharines, Ontario L2S 3A1, Canada

J. Wylie Nichols, Department of Zoology, University of California, Davis, California 95616

Mitsuo Nishimura, Department of Biology, Kyushu University, Fukuoka, 812 Japan

Contributors of Volumes I and II

Birgitta Norling, Department of Biochemistry, Arrhenius Laboratory, Univer­sity of Stockholm, Stockholm, Sweden

J. R. Norris, Chemistry Division D-200, Argonne National Laboratories, Ar­gonne, Illinois 60439

Barry E. North, Biology Department, Brookhaven National Laboratory, Upton, New York 11973

Svante Nyberg, Department of Chemistry, Section of Physiological Chemistry, University of Umea, S-901 87 Umea, Sweden

S. Ogawa, Bell Laboratories, Murray Hi l l , New Jersey 07974 Gabriel B . Ogunmola, Department of Chemistry, University of Ibadan, Ibadan,

Nigeria Per-Ingvar Ohlsson, Department of Chemistry, Section of Physiological

Chemistry, University of Umea, S-901 87 Umea, Sweden Tomoko Ohnishi, Department of Biochemistry and Biophysics, Johnson Re­

search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Yutaka Orii , Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka, Japan

Donald R. Ort, Department of Biochemistry, University of Washington, Seattle, Washington 98195

Neil Osheroff, Department of Biochemistry and Molecular Biology, Northwest­ern University, Evanston, Illinois 60201

Charles S. Owen, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

James Pachence, Departments of Chemistry and Biochemistry/Biophysics, Uni­versity of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104

N . K. Packham, Department of Biochemistry, University of Birmingham, Bir­mingham B15 2TT, United Kingdom

Ferdinando Palmieri, Department of Biochemistry, University of Bari, Via Amendola 165, Bari, Italy

Angela Pansini, Instituto di Chimica Biologica, Facolta di Medicina e Chirur-gia, Universita di Bari, 70126 Bari, Italy

S. Papa, Instituto di Chimica Biologica, Facolta di Medicina e Chirurgia, Uni­versita di Bari, 70126 Bari, Italy

William W. Parson, Department of Biochemistry, J405 Health Sciences Build­ing, SJ-70, University of Washington, Seattle, Washington 98195

Donald F. Parsons, Electron Optics Laboratory, State of New York Depart­ment of Health, Tower Building, Empire State Plaza, Albany, New York 12201

xxx ii Contributors o f Volumes / and II

Lehka Patel, Laboratory of Membrane Biochemistry, Roche Institute of Molecular Biology, Nutley, New Jersey 07110

Katherine A . Pattridge, Biophysics Research Division and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Karl Gustav Paul, Department of Medical Chemistry, University of Umea, S-901 87 Umea, Sweden

R. Paul, Laboratory of Technical Development, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014

Freddy Paulussen, Max-Planck Institut für Biophysikalische Chemie, D-3400 Göttingen-Nikolausberg, West Germany

Laraine Peiffer, Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Jack Peisach, Department of Molecular Pharmacology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461

R. A . Perreault, Department of Chemistry, University of Southern California, University Park, Los Angeles, California 90007

Gregory A . Petsko, Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201

K . M . Petty, Department of Biochemistry, University of Birmingham, Birming­ham B15 2TT, United Kingdom

R. M . Polichar, Science Applications, Inc., La Jolla, California Carol A . Popp, National Biomedical ESR Center, Department of Radiology,

Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Richard Pougeois, Laboratoire de Biochimie, Departement de Recherche Fon­

damentale, C E N - 85X, 38041 Grenoble, Cedex, France Linda Powers, Bell Telephone Laboratories, Murray H i l l , New Jersey 07974 T. Pozzan, Instituto di Patologia Generale, Via Loredan, Padova, Italy Berton C. Pressman, Department of Pharmacology, University of Miami School

of Medicine, P.O. Box 520875, Miami, Florida 33152 Roger C. Prince, Department of Biochemistry and Biophysics, Johnson Re­

search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

L . J. Prochaska, Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Alexandre T. Quintanilha, Membrane Bioenergetics Group, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720

Bjgrrn Quistorff, Department of Biochemistry A , Panum Institute, University of Copenhagen, DK-2200 Copenhagen N , Denmark

Contributors o f Volumes I and II x x x i i i

Edd Rabon, Laboratory of Membrane Biology, University of Alabama at Bir­mingham, Birmingham, Alabama 35294

Efraim Racker, Section of Biochemistry, Molecular, and Cell Biology, Cornell University, Ithaca, New York 14853

George K . Radda, Department of Biochemistry, University of Oxford, Oxford O X l 3 Q U , England

Hassan Rastegar, Harrison Department of Surgical Research, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

P. M . Rentzepis, Bell Laboratories, Murray Hi l l , New Jersey 07974 Baltazar Reynafarje, Department of Physiological Chemistry, Johns Hopkins

University, School of Medicine, Baltimore, Maryland 21205 S. G . Rhee, Laboratory of Biochemistry, National Heart, Lung, and Blood In­

stitute, National Institutes of Health, Bethesda, Maryland 20014 Peter R. Rieh, Department of Biochemistry, University of Cambridge, Cam­

bridge, England Lajos Rimai, Research Staff, Ford Motor Company, Dearborn, Michigan

48121 Dan E . Robertson, Laboratory of Membrane Biochemistry, Roche Institute of

Molecular Biology, Nutley, New Jersey 07110 David Rose, Department of Biochemistry, Wayne State University, School of

Medicine, Detroit, Michigan 48201 W. N . Ross, Department of Neurobiology, Harvard University, Boston, Mas­

sachusetts Hagai Rottenberg, Bell Laboratories, Murray Hi l l , New Jersey 07974 Andrew M . Rudin, Northport Veterans Administration Hospital, Northport,

New York Heinz Rupp, Department of Plant Sciences, King's College, London, England Frank J. Ruzicka, Enzyme Institute, University of Wisconsin, Madison, Wis­

consin 53706 Gaetano Saccomani, Laboratory of Membrane Biology, University of Alabama

at Birmingham, Birmingham, Alabama 35294 Roland Sacher, R W T H Aachen, Abt. Physiologische Chemie, Melatener

Strasse 211, D-5100 Aachen, West Germany George Sachs, Laboratory of Membrane Biology, University of Alabama at

Birmingham, Birmingham, Alabama 35294 A. Saito, Department of Molecular Biology, Vanderbilt University, Nashville,

Tennessee 37235 F. R. Salemme, Department of Chemistry, University of Arizona, Tucson,

Arizona 85721

xxxiv Contributors o f Volumes I and II

John C. Salerno, Department of Biochemistry, Duke University, School of Medicine, Durham, North Carolina

L . Salganicoff, Department of Pharmacology and Specialized Center for Thrombosis Research, Temple University, Philadelphia, Pennsylvania

Irving Salmeen, Ford Motor Company, Dearborn, Michigan 48121 B. M . Salzberg, Department of Physiology and Pharmacology, School of Dental

Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 R. H . Sands, University of Michigan, Institute of Science and Technology,

Biophysics Research Division, Ann Arbor, Michigan N - E Leo Saris, Department of Medical Chemistry, University of Helsinki, Si l-

tavuorenpenger 10, SF 00170 Helsinki 17, Finland Nobuhiro Sato, First Department of Internal Medicine, Osaka University Medi­

cal School, Osaka, Japan Michael Satre, Laboratoire de Biochimie, Departement de Recherche Fon­

damentale, Dentre d'Etudes Nucleaires, 85X, 38041 Grenoble Cedex, France

Antonio Scarpa, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Günter Schäfer, Med. Hochschule Hanover, Postfach 610 180, 3 Hanover 61, West Germany

Gottfried Schatz, Biocenter, University of Basel, CH-4056 Basel, Switzerland W. Robert Scheidt, Department of Chemistry, University of Notre Dame, Notre

Dame, Indiana 46556 C. C. Schenck, Department of Biochemistry, J405 Health Sciences Building,

SJ-70, University of Washington, Seattle, Washington 98195 Benno P. Schoenborn, Brookhaven National Laboratory, Upton, New York

11973 Charles P. Scholes, Department of Physics, State University of New York at

Albany, Albany, New York 12222 S. Schreier, Department of Bioquimica, Instituto de Quimica, Universidade de

Säo Paulo, Säo Paulo, Brazil Dana M . Scott, Department of Physiology, University of Pennsylvania, School

of Medicine, Philadelphia, Pennsylvania 19104 P. John Seeley, Department of Biochemistry, University of Oxford, Oxford

O X l 3QU, England C. H . A . Seiter, Department of Chemistry, University of Southern California,

University Park, Los Angeles, California 90007 Adil E. Shamoo, Department of Radiation Biology and Biophysics, University

of Rochester, Rochester, New York 14642

Contributors o f Volutnes I and II xxxv

C. V . Shank, Bell Telephone Laboratories, Holmdel, New Jersey Motoaki Shichiri, First Department of Internal Medicine, Osaka University

Medical School, Osaka, Japan R. G . Shulman, Bell Laboratories, Murray, H i l l , New Jersey 07974 H . Shuman, Departments of Physiology and Pathology, University of Pennsyl­

vania, School of Medicine, Philadelphia, Pennsylvania 19104 Vladimir. A . Shuvalov, Charles F. Kettering Research Laboratory, Yellow

Springs, Ohio 45387 Helmut Sies, Institut für Physiologische Chemie, Physikalische Biochemie, and

Zellbiologie, Universität München 2, Munich, West Germany Binah R. Silberstein, Department of Biochemistry and Biophysics, Johnson Re­

search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Ian A . Silver, Department of Pathology, University of Bristol Medical School, Bristol BS8 1TD, England

Michael B . Simson, Department of Cardiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Carolyn W. Slayman, Department of Human Genetics, Yale University, New Häven, Connecticut 06510

Clifford L . Slayman, Department of Physiology, Yale University School of Medicine, New Häven, Connecticut 06510

Jerry Smith, Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadel­phia, Pennsylvania 19104

Ward W. Smith, Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California

A. P. Somlyo, Pennsylvania Muscle Institute, Philadelphia, Pennsylvania 19104

A. V . Somlyo, Pennsylvania Muscle Institute, Philadelphia, Pennsylvania 19104

Nobuhito Sone, Jichi Medical School, Tochigi-ken, Japan 329-04 Annick Soumarmon, Section of Biochemistry, Molecular, and Cell Biology,

Cornell University, Ithaca, New York 14853 Thomas G . Spiro, Department of Chemistry, Princeton University, Princeton,

New Jersey 08540 John Springall, School of Chemical Sciences, University of East Anglia, Nor-

wich NR4 7TJ, England B. R. Sreenathan, Department of Biophysics, University of Western Ontario,

Health Sciences Center, London N6A 5C1, Canada

xxxvi Contributors of Volumes I and II

E. R. Stadtman, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014

James Stamatoff, Bell Telephone Laboratories, Murray H i l l , New Jersey 07974 Charles Steenbergen, Department of Biochemistry and Biophysics, Johnson Re­

search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Gerd J. Steffens, R W T H Aachen, Abt. Physiologische Chemie, Melatener Strasse 211, D-5100 Aachen, West Germany

Guy C. M . Steffens, R W T H Aachen, Abt. Physiologische Chemie, Melatener Strasse 211, D-5100 Aachen, West Germany

P. Stein, Institute for Cancer Research, Fox Chase, Philadelphia, Pennsylvania 19111

Paul Stein, Department of Chemistry, Princeton University, Princeton, New Jersey 08540

David L . Stetson, Department of Physiology, Yale University School of Medicine, New Häven, Connecticut 06510

Tom H . Stevens, California Institute of Technology, Division of Chemistry and Chemical Engineering, Pasadena, California 91125

Italo Stipani, Department of Biochemistry, University of Bari, Via Amendola 165, Bari, Italy

John D. Stong, Department of Chemistry, Princeton University, Princeton, New Jersey 08540

Bayard T. Storey, Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

R. Strasser, Departments of Physics and Biology, University of California at San Diego, La Jolla, California 92037

Peter Styles, Department of Biochemistry, University of Oxford, Oxford O X l 3QU, England

Jeanne Sweetland, Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

Ken-Ichiro Takamiya, Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Tsunehiro Takano, Department of Chemical Engineering, California Institute of Technology, Pasadena, California 91125

Hiroshi Takeda, Department of Surgery, Kyoto University, Medical School, Kyoto, Japan

C. P. S. Taylor, Department of Biophysics, University of Western Ontario, Health Sciences Center, London N6A 5C1, Canada

Contributors o f Volumes I and II xxxv i i

Henry Tedeschi, Department of Biological Sciences, State University of New York at Albany, Albany, New York 12222

A . Telfer, Department of Botany, Imperial College, London, United King­dom

Andrew Thomson, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, England

S. W. Thorne, CSIRO, Division of Plant Industry, Canberra, A . C . T . , Aus-tralia

M . C. Thurnauer, Chemistry Division, D-200, Argonne National Laboratories, Argonne, Illinois 60439

D. M . Tiede, Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, School of Medicine, Philadel­phia, Pennsylvania 19104

J. T. Tiffert, Department of Physiology, University of Maryland, School of Medicine, Baltimore, Maryland 21201

William F. Tivol, Department of Radiation Biology and Biophysics, University of Rochester, Rochester, New York 14642

D. R. Trentham, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

A. D. Trifunac, Chemistry Division D-200, Argonne National Laboratories, Argonne, Illinois 60439

Bernard L . Trumpower, Dartmouth Medical School, Hanover, New Hampshire 03755

Demetrius Tsernoglou, Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201

K. Ugurbil, Bell Laboratories, Murray Hi l l , New Jersey 07974 D. W. Urry, Laboratory of Molecular Biophysics, University of Alabama Med­

ical Center, University Station, Birmingham, Alabama 35294 Joan S. Valentine, Department of Chemistry, Rutgers University, Busch Cam­

pus, New Brunswick, New Jersey 08903 H. L . Van Camp, Biophysics Research Laboratory, University of Michigan,

Ann Arbor, Michigan 48104 K. van Dam, Universiteit van Amsterdam, Lab. voor Biochemie, B . C . P . Jarn­

sen Institut, Plantage Muidergracht 12, Amsterdam-C, Netherlands Willem H . van den Berg, Department of Biochemistry and Biophysics, Johnson

Research Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Jane Vanderkooi, Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

x x x v i i i Contributors o f Volumes I and II

R. van der Meer, Universiteit van Amsterdam, Lab. voor Biochemie, B . C . P . Jarnsen Institut, Plantage Muidergracht 12, Amsterdam-C, Netherlands

Sergio Verjovski-Almeida, Laboratory of Physiology and Biophysics, Univer­sity of the Pacific, San Francisco, California 94115

Paulette M . Vignais, Maitre de Recherche au C N R S , DRF/Biochemie C E N - G 85X, 38041 Grenoble, Cedex, France

Pierre V. Vignais, Laboratoire de Biochimie, Departement de Recherche Fon­damentale, C E N - 85X, 38041 Grenoble, Cedex, France

Steven Vik , Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

John L . Walker, Department of Physiology, University of Utah College of Medicine, Salt Lake City, Utah 84108

Terrence Walsh, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, England

C. T. Wang, Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235

A . Warshel, Department of Chemistry, University of Southern California, Uni­versity Park, Los Angeles, California 90007

Michael R. Wasielewski, Chemistry Division, Argonne National Laboratory, Argonne, Illinois 60439

Takahide Watanabe, Department of Biology, Faculty of Science, Osaka Univer­sity, Toyonaka, Osaka 560, Japan

Watt W. Webb, School of Applied and Engineering Physics, Cornell Univer­sity, Ithaca, New York 14853

Gregorio Weber, Roger Adams Laboratory, University of Illinois, Urbana, Il­linois 61801

Y . H . Wei, Laboratory of Bioenergetics, State University of New York at A l ­bany, Albany, New York 12222

R. B. Weisman, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104

J. Weiss, Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

R. Weiss, Department of Chemistry, University of Southern California, Univer­sity Park, Los Angeles, California 90007

Jürgen Werringloer, Department of Biochemistry, Southwestern Medical School, University of Texas, Dallas, Texas 75235

H . V . Westerhoff, Universiteit van Amsterdam, Lab. voor Biochemie, B . C . P . Jarnsen Institut, Plantage Muidergracht 12, Amsterdam-C, Netherlands

Contributors o f Volumes I and II x x x i x

W. R. Widger, Department of Chemistry and Laboratory of Bioenergetics, State University of New York at Albany, Albany, New York 12222

Volker Wiegand, Pathologisches Institut, Universität Essen, Essen, West Ger­many

Märten Wikström, Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10, SF 00170 Helsinki 17, Finland

John R. Williamson, Department of Biochemistry and Biophysics, Johnson Re­search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

M . T. Wilson, Department of Chemistry, University of Essex, Colchester, United Kingdom

M . W. Windsor, Department of Chemistry, Washington State University, Pullman, Washington

Raymond Wong, Section of Biochemistry, Molecular, and Cell Biology, Cor­nell University, Ithaca, New York 14853

Colin A . Wraight, Department of Botany, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Kurt Wüthrich, Institute für Molekularbiologie und Biophysik, E T H -Hönggerberg, CH-8093 Zürich, Switzerland

S. Yasui, Department of Physiology and Biophysics, University of Texas Medi­cal Branch, Galveston, Texas 77550

Takashi Yonetani, Department of Biochemistry and Biophysics, Johnson Re­search Foundation, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Masasuke Yoshida, Jichi Medical School, Tochigi-ken, Japan 329-04 Tatsuro Yoshida, Institute of Science and Technology, Biophysics Research Di­

vision, University of Michigan, Ann Arbor, Michigan 48109 C. A . Y u , Department of Biochemistry, State University of New York at A l ­

bany, Albany, New York 12222 L. Y u , Department of Chemistry and Laboratory of Bioenergetics, State Univer­

sity of New York at Albany, Albany, New York 12222 Richard Zimmermann, Physiologisch-Chemisches Institut der Georg-August-

Universität Göttingen, 34 Göttingen, Humboldtallee 7, West Germany

F R O N T I E R S O F BIOLOGICAL. E N E R G E T I C S , V O L U M E I

BIOSYNTHESIS AND INTRACELLULAR TRANSLOCATION OF MITOCHONDRIAL PROTEINS: CYTOCHROME C AND THE

CARBOXYATRACTYLOSIDE BINDING PROTEIN

Richard Zimmermann, Harald Korb and Walter Neupert

I n s t i t u t für Physiologische Chemie Universität Göttingen

Göttingen, GFR

I. INTRODUCTION

The vast majority of mitochondrial proteins i s t r a n s l a t e d on cytoplasmic ribosomes and must eventually be transported to t h e i r s i t e of function i n the mitochondrion. In previous stud­i e s we have presented evidence f or a tran s f e r mechanism which involves extramitochondrial precursors (Hallermayer et a l . , 1977; Harmey et a l . , 1977; Zimmermann et a l . , 1977) . These studies strongly argued against the proposal that as a general r u l e proteins are tra n s f e r r e d to mitochondria by d i r e c t In­s e r t i o n of nascent Polypeptide chains, c a r r i e d out by a sp e c i a l c l a s s of cytoplasmic. ribosomes bound to the outer mitochondrial membrane (Kellems and Butow, 1972; Kellems et a l . , 1 9 7 5 ) .

Attempts to further elucidate the mechanisms of tr a n s f e r have to take i n t o consideration that mitochondrial proteins d i f f e r widely with respect to t h e i r structure, physico-chemical properties and submitochondrial l o c a t i o n . Proteins of the outer membrane, the intermembrane Space, the inner membrane (peripheral and in t e g r a l ) and of the matrix space have to be distin g u i s h e d . I t may be expected that the mechanisms of tran s f e r are d i f f e r e n t f o r these various groups of protein s . Accordingly, the tra n s f e r processes have to be studied f o r i n -d i v i d u a l proteins of each group. The primary t r a n s l a t i o n products have to be analysed, extramitochondrial precursors must be i s o l a t e d , t h e i r s u b c e l l u l a r l o c a t i o n determined and th e i r s t r u c t u r a l r e l a t i o n s h i p to the fun c t i o n a l proteins

Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISBN 0-12-225401-5

P a r t I E/cctrochcmical Intcractions 147

investigated. F i n a l l y , the mechanism of transfer of extra­mitochondrial precursors must be studied i n reconstituted Systems, involving the precursor proteins and i s o l a t e d mitochondria.

Here we report on the synthesis and tra n s f e r of two mito­chondrial membrane proteins, cytochrome c and the carboxyatr-a c t y l o s i d e binding p r o t e i n , the ATP/ADP c a r r i e r of the inner mitochondrial membrane.

I I . RESULTS AND DISCUSSION

A. Cytochrome c

C e l l free homogenates from Neurospora crassa were employed to study biosynthesis and i n t r a c e l l u l a r t r a n s l o c a t i o n of cytochrome c. Antibodies against apocytochrome c and holo-cytochrome c were used to i s o l a t e these proteins by simple im-munoprecipitation. Preexistent proteins were discriminated from in. v i t r o synthesized proteins by dual l a b e l l i n g . For t h i s purpose, Neurospora c e l l s were grown i n 3 5-S-sulfate containing medium and l a b e l l i n g i n the c e l l free homogenate was performed with 3-H-leucine.

Apocytochrome c as well as holocytochrome c were found to be synthesized i n the c e l l free homogenate a f t e r incubation f o r 10 min under appropriate conditions. When pr o t e i n synthesis was then blocked with cycloheximide, the amount of iri v i t r o synthesized apocytochrome c decreased during a further 30 min incubation period. Simultaneously, the amount of newly syn­thesized holocytochrome c increased (Table I ) . The i d e n t i t i e s of the immunoprecipitated apo- and holocytochrome c were v e r i -f i e d by analysis of the cyanogen bromide fragments.

A c e l l free homogenate l a b e l l e d as described i n Table I was separated by d i f f e r e n t i a l c e n t r i f u g a t i o n into the mito­chondrial f r a c t i o n , the microsomal f r a c t i o n and the post-ribosomal supernatant. Immunoprecipitation with antibodies against apocytochrome c and holocytochrome c was c a r r i e d out with a l l f r a c t i o n s .

F i g . 1A and B show that iri v i t r o synthesized apocyto­chrome c i s present i n the postribosomal supernatant and that i t s amount decreases a f t e r blocking p r o t e i n synthesis. No pre­e x i s t e n t 3 5-S-labelled holocytochrome c i s p r e c i p i t a t e d by the antibody against apocytochrome c. From the mitochondrial f r a c ­t i o n anti-apocytochrome c p r e c i p i t a t e d neither 3-H-labelled apocytochrome c nor 3-H- or 3 5-S-labelled holocytochrome c.

Antibodies against holocytochrome c p r e c i p i t a t e d from the postribosomal f r a c t i o n 3 5-S-labelled holocytochrome c, which represents preexistent cytochrome c leaked out from the mito­chondria during preparation of the c e l l free homogenate. Also

148 R i c h a r d Z i m m e r m a n n et al.

TABLE I. Immunoprecipitation of Apocytochrome c and of Holocytochrome c from a C e l l Free Homogenate

A c e l l free homogenate was prepared from c e l l s grown i n the presence of 3 5-S-sulfate and incubated at 25 C for 10 min i n the presence of 3-H-leucine. Then cycloheximide (CHI) (100 yg/ ml) was added. One h a l f of the homogenate was withdrawn and kept at 0 C. The other h a l f was further incubated f o r 30 min at 25 C. Then from both portions apocytochrome c and #holocyto­chrome c were immunoprecipitated. The immunoprecipitates were analysed by SDS Polyacrylamide g e l e l e c t r o p h o r e s i s . 3-H- and 3 5 - S - r a d i o a c t i v i t i e s i n the peak f r a c t i o n were determined.

Antibody 10 min 10 min

+ 30 min CHI (counts x min Ra d i o a c t i v i t y

10 min + 30 min CHI (counts x min

A n t i - 3-H 1826 836 apocytochrome c 35-S 18 24

A n t i - 3-H 260 944 holocytochrome c

35-S 4420 4320

a small amount of in_ v i t r o synthesized holocytochrome c was p r e c i p i t a t e d which increased during incubation i n the presence of cycloheximide (from about 5 to lO% of t o t a l 3-H-holocyto-chrome c i n the homogenate). From the mitochondrial f r a c t i o n the antibody against holocytochrome c brought down preexistent 35-S-holocytochrome c. Also, 3-H-labelled holocytochrome c was found i n the p r e c i p i t a t e . I t s amount increased during i n ­cubation i n the absence of pro t e i n synthesis (Fig. IC and D).

From the microsomal f r a c t i o n no apocytochrome c could be immunoprecipitated, however a small amount of 3 5-S-holocyto­chrome c (3-4% of t o t a l ) . Also, 3-H-labelled holocytochrome c was detected i n t h i s f r a c t i o n (10-15% of to t a l ) which did not show a s i g n i f i c a n t increase or decrease during incubation i n the presence of cycloheximide.

These data suggest that apocytochrome c i n the post­ribosomal supernatant acts as a precursor of holocytochrome c i n the mitochondria.

Transfer of cytochrome c was further i n v e s t i g a t e d i n a re-constituted System, i n which a postribosomal supernatant con-t a i n i n g in_ v i t r o synthesized apocytochrome c was incubated with i s o l a t e d mitochondria from 3 5-S-labelled c e l l s . Table II shows that holocytochrome c appears i n the mitochondria which were r e i s o l a t e d a f t e r incubation. When unl a b e l l e d holo-

P a r t I Elcctrochemical Interactions 149

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FIGURE 1. Immunoprecipitation with antibodies against apo­cytochrome c from the postribosomal f r a c t i o n and with a n t i ­bodies against holocytochrome c from the mitochondrial f r a c t i o n . The immunoprecipitates were analysed by SDS gel el e c t r o p h o r e s i s . A,B: Ip with anti-apocytochrome c from the postribosomal super­natant; C,D: Ip with anti-holocytochrome c from the mito­chondria; A,C: 10 min incubation; B,D: 10 min incubation plus 30 min i n the presence of cycloheximide.

150 R i c h a r d Z i m m e r m a n n et al.

TABLE I I . Transfer of In V i t r o Synthesized Apocytochrome c i n t o Mitochondria and Conversion to Holo­cytochrome c

A postmitochondrial homogenate was incubated f o r 10 min with 3-H-leucine and a postribosomal supernatant was prepared. Mitochondria were i s o l a t e d from c e l l s grown i n the presence of 3 5-S-sulfate and resuspended with t h i s postribosomal supernat­ant. The Suspension was div i d e d i n t o three equal p o r t i o n s . One served as a c o n t r o l , to the second p o r t i o n holocytochrome c (1 nmol/ml) was added, to the t h i r d p o r t i o n apocytochrome c (1 nmol/ml). One h a l f of each p o r t i o n was immediately with-drawn a f t e r resuspension and kept at 0 C, the other h a l f was incubated f o r 30 min at 25 C. Then mitochondria were c o l l e c t e d from each sample by c e n t r i f u g a t i o n and washed with sucrose/ EDTA medium. The mitochondrial p e l l e t s were d i s s o l v e d with T r i t o n X -100 containing buffer and immunoprecipitation with anti-holocytochrome c was c a r r i e d out. The immunoprecipitates were subjected to SDS gel e l e c t r o p h o r e s i s . The 3-H- and 35-S-r a d i o a c t i v i t i e s i n the cytochrome c peaks were determined a f t e r s l i c i n g the gel s .

0 min 30 min Ra d i o a c t i v i t y (counts x min )

Control 3-H 26 440

35-S 3836 4224

+ Holocytochrome c 3-H 72 380

35-S 4016 4112

+ Apocytochrome c 3-H 20 78

35-S 4720 4048

cytochrome c was added to the incubation mixture, no s i g n i f i c a n t change of the amount of holocytochrome c appearing i n the mitochondria was observed. This should be expected since the postribosomal supernatant already contains appreciable amounts of holocytochrome c which leaked out from the mito­chondria during c e l l breakage (up to 40% of t o t a l cytochrome c ) . In contrast, a d d i t i o n of apocytochrome c leads to a marked reduction of the amount of holocytochrome c appearing i n the mitochondria.

On the basis of these r e s u l t s the fol l o w i n g mechanism of synthesis and assembly of cytochrome c i s proposed. Apocyto­chrome c i s synthesized on cytoplasmic ribosomes and released

P a r t 1 E l e c t r o c h e m i c a l I n t e r a c t i o n s 151

i n t o the c y t o s o l . I t seems reasonable to assume that apocyto­chrome c found i n the postribosomal supernatant i s a c t u a l l y present _in vivo i n the c y t o s o l i n free form. I t can however not d e f i n i t e l y be excluded that i t i s derived from some unknown very f r a g i l e structures which are destroyed during c e l l f r a c -t i o n a t i o n . I t i s f u r t h e r proposed that apocytochrome c d i f f ­uses to the mitochondrial membrane where the heme group i s c o v a l e n t l y linked to the apoprotein. According to our experi-ments, k i n e t i c data argue against a p o s s i b l e r o l e of microso-mal apo- or holocytochrome c as precursors of mitochondrial holocytochrome c. The conversion of apocytochrome c to holo­cytochrome c involves a d r a s t i c change i n the conformation of the molecule (Fisher et a l . , 1 9 7 3 ) . I t i s proposed that t h i s conformational change leads to the trapping of newly formed holocytochrome c i n the mitochondrial membrane.

C l e a r l y , our hypothesis r a i s e s a number of questions. They concern the permeability of the outer mitochondrial membrane fo r apocytochrome c, the mechanism by which the heme group i s cov a l e n t l y linked to the apoprotein and the problem, why newly synthesized cytochrome c i s bound to the membrane i n preference to the preexistent cytochrome c which leaked out from the mitochondria.

I t should be pointed out that i n a number of studies on the biogenesis i n r a t l i v e r i t was claimed that holocytochrome c i s synthesized as a whole by the microsomes and eventually t r a n s f e r r e d i n t o mitochondria (for reviews see Sherman and Stewart, 1971; Gonzalez-Cadavid, 1 9 7 4 ) . I t was however not p o s s i b l e i n these studies to discriminate between newly syn­thesized and preexistent cytochrome c? furthermore, apocyto­chrome c could not be separately determined. Moreover, the p o s s i b l e precursor r o l e of microsomal cytochrome c was recent-l y r u l e d out by Robbi e t a l . , (1978) (see a l s o Kadenbach, 1 9 7 0 ) .

B. Carboxyatractyloside Binding Protein

The carboxyatractyloside binding p r o t e i n (CAT-protein) or ATP/ADP c a r r i e r represents an i n t e g r a l p r o t e i n of the inner mitochondrial membrane which i s characterized by i t s hydro-phobic properties and an apparent molecular weight of about 32 ,000 (Klingenberg et a l . , 1 9 7 5 ) . I t s synthesis and transfer were studied i n e s s e n t i a l l y the same experimental Systems as described for cytochrome c.

A c e l l free homogenate derived from Neurospora c e l l s grown i n the presence of 3 5-S-sulfate was incubated with 3-H-leucine for 10 min; mitochondria were i s o l a t e d and the CAT-protein was immunoprecipitated. _In v i t r o synthesized CAT-protein was found i n the mitochondria (Fig. 2 A ). In order to detect possible

152 R i c h a r d Z i m m e r m a n n et al.

extramitochondrial precursors, the various s u b c e l l u l a r f r a c t i -ons were subjected to immunoprecipitation, adding mitochondria from unlabelled c e l l s as a source of c a r r i e r p r o t e i n or by double immunoprecipitation. With the ribosomal and microsomal f r a c t i o n s no c l e a r i n d i c a t i o n for the presence of In v i t r o synthesized CAT-protein was found. However, from the p o s t r i b o ­somal supernatant, 3-H-radioactivity was immunoprecipitated which upon SDS g e l electrophoresis y i e l d e d a peak with ident-i c a l e l e c t r o p h o r e t i c m o b i l i t y as the f u n c t i o n a l CAT-protein. No 35 - S - r a d i o a c t i v i t y was present i n t h i s peak, demonstrating absence of contaminating mitochondrial membrane fragments.

In order to v e r i f y the identy of the 3-H-labelled puta­t i v e CAT-protein from the postribosomal supernatant, an im-munoprecipitate obtained from t h i s f r a c t i o n was mixed with an immunoprecipitate obtained from mitochondria which were de­r i v e d from c e l l s l a b e l l e d in_ vivo with 14-C-leucine. The mixture was subjected to cyanogen bromide cleavage and the

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FIGURE 2. Immunoprecipitation of CAT-protein from mito­chondria and from a postribosomal p e l l e t . A: Mitochondria; immunoprecipitate obtained d i r e c t l y with antibodies against CAT-protein. B: P e l l e t obtained by 2 h u l t r a c e n t r i f u g a t i o n of a postribosomal supernatant; immunoprecipitate obtained by double p r e c i p i t a t i o n with antibodies against CAT-protein and antibodies against r a b b i t Immunoglobulins.

The immunoprecipitates were analysed by SDS g e l e l e c t r o ­phoresis .

P a r t I Electrochcmical Interactions 153

r a d i o a c t i v e cleavage products were analysed on phenol/formic a c i d Polyacrylamide gels. The 3-H and 14-C patterns showed s a t i s f a c t o r y coincidence.

Double immunoprecipitation employing anti-rabbit-immuno-globulin-antibodies from sheep lead also to the p r e c i p i t a t i o n of 3-H-labelled CAT-protein from the postribosomal supernatant, however only when T r i t o n X-100 was added. This indicated that the in_ v i t r o synthesized component does not e x i s t i n a free form i n the supernatant, but rather that i t s antigenic s i t e s are not a v a i l a b l e to the antibody. When the postribosomal supernatant was centrifuged f o r 2 h at 165,000 x g, a l l the 3-H-labelled CAT-protein was found i n the p e l l e t (Fig. 2B) .

The question a r i s e s whether the in_ v i t r o synthesized CAT-pr o t e i n i n t h i s f r a c t i o n i s a precursor of the mitochondrial CAT-protein. A c t u a l l y i t was found that the amount of 3-H-l a b e l l e d CAT-protein i n t h i s f r a c t i o n decreased when afte r the 10 min l a b e l l i n g period t r a n s l a t i o n was blocked with c y c l o ­heximide and the c e l l free homogenate incubated further. On the other hand, 3-H-labelled CAT-protein i n the mitochondrial f r a c t i o n increases under these conditions (Harmey et al.,1977). I t was however not possible to q u a n t i t a t i v e l y c o r r e l a t e these two processes. The main d i f f i c u l t i e s i n these experiments are the exceptional l a b i l i t y of the CAT-protein and the i n a b i l i t y to demonstrate tra n s f e r into f u n c t i o n a l p o s i t i o n s . The newly synthesized p r o t e i n appears to be e a s i l y subject to proteo-l y t i c degradation.

FIGURE 3. Tr a n s l a t i o n of Neurospora CAT-pro­t e i n i n a ra b b i t r e t i c u -cyte c e l l free System. Coelectrophoresis of immunoprecipitates from the heterologous System (35-S) and from mito­chondria l a b e l l e d in_ vivo (3-H).

20 40 Fraction no.

154 R i c h a r d Z i m m e r m a n n et al.

I t i s of i n t e r e s t i n t h i s context whether CAT-protein i s translated as a precursor with i d e n t i c a l or higher molecular weight as compared to the fu n c t i o n a l p r o t e i n i n the mitochond­r i a . Poly-A-containing RNA was i s o l a t e d from Neurospora and translat e d i n a r a b b i t r e t i c u l o c y t e l ysate i n the presence of 35-S-methionine. Double immunoprecipitation was c a r r i e d out a f t e r l y s i s with T r i t o n and the r e s u l t i n g p r e e i p i t a t e was mixed with an immunoprecipitate obtained from mitochondria i s o l a t e d from c e l l s l a b e l l e d in_ vivo with 3-H-leucine. The mixture was analysed by SDS gel ele c t r o p h o r e s i s . F i g . 3 shows that the 35-S peak and the 3-H peak with apparent molecular weights of about 32 ,000 coincide. This indicated that CAT-protein i s not translated as a large r molecule, at l e a s t not as a precursor d i f f e r i n g i n i t s apparent molecular weight by more than about 500 from the authentic p r o t e i n .

Further experiments w i l l have to c l a r i f y the precursor r o l e of a postribosomal CAT-protein. As a working hypothesis i t i s proposed that CAT-protein i s not tra n s l o c a t e d as a f r e e l y soluble precursor but rather that binding to l i p i d containing struetures i s involved.

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