Methods in Enzymology

Volume L V BIOMEMBRANES

Part F

METHODS IN E N Z Y M O L O G Y EDITORS- IN-CHIEF

Sidney P. Colowick Nathan O. Kaplan

Methods in Enzymology

Volume LV

Biomembranes Part F: Bioenergetics-Oxidative Phosphorylation

E D I T E D B Y

Sidney Fleischer D E P A R T M E N T O F M O L E C U L A R BIOLOGY

V A N D E R B I L T U N I V E R S I T Y , N A S H V I L L E , TENNESSEE

Lester Packer M E M B R A N E BIOENERGETICS GROUP

D E P A R T M E N T O F P H Y S I O L O G Y - A N A T O M Y

UN IVERS ITY OF C A L I F O R N I A , BERKELEY , C A L I F O R N I A

Editorial Advisory Board Lars Ernster Youssef Hatefi Ronald W. Estabrook Martin Klingenberg Frank Gibson David F. Wilson

A C A D E M I C PRESS N e w Y o r k San Franc isco L o n d o n 1979

A Subsidiary o f Harcourt Brace Jovanovich, Publishers

C O P Y R I G H T © 1979, B Y A C A D E M I C PRESS, I N C . A L L RIGHTS RESERVED. N O PART O F T H I S P U B L I C A T I O N M A Y B E REPRODUCED OR T R A N S M I T T E D I N A N Y F O R M OR B Y A N Y M E A N S , ELECTRONI C OR M E C H A N I C A L , I N C L U D I N G PHOTOCOPY, RECORDING, OR A N Y I N F O R M A T I O N STORAGE A N D RETRIEVAL SYSTEM, W I T H O U T PERMISS ION I N W R I T I N G F R O M T H E PUBLISHER.

A C A D E M I C P R E S S , I N C . I l l Fifth Avenue, New York, New York 10003

United Kingdom Edition published by A C A D E M I C P R E S S , I N C . ( L O N D O N ) L T D . 24/28 Oval Road, London NW1 7DX

Library of Congress Cataloging in Publication Data Main entry under t i t le :

Biomembranes.

(Methods in enzymology, v. 31 - 32, ) Part F has special t i t le : Bioenergetics, oxidative

phosphorylation. Includes bibliographical references. 1. Cell membranes. 2. Cell fractionation. 3. Cell

organelles. I . Fleischer, Sidney, ed. I I . Packer, Lester, ed. I I I . Series: Methods in enzymology; 31 [etc.] [ D N L M : 1. Cell membrane. W l ME9615K v. 31 /QH601 B6193] QP601.M49 vol. 3 1 - 3 2 [QH601] 574.1 ,925'08s [574.8'75] ISBN 0 - 1 2 - 1 8 1 9 5 5 - 8 (vol .55) 74 -11352

PR INTED I N T H E U N I T E D STATES OF AMERICA

79 80 81 82 9 8 7 6 5 4 3 2 1

To the memory of Dr. John W. Greenawalt, a devoted scholar of the mitochondria

T a b l e of C o n t e n t s

CONTRIBUTORS TO V O L U M E L V xv

PREFACE xx i

V O L U M E S I N SERIES xx i i i

Sec t i on I . Organe l l es a n d M e m b r a n e s

1. Overview—Preparation and Properties of Mito- J A N N E D E R G A A R D A N D chondria from Different Sources B A R B A R A C A N N O N 3

2. Long-Term Storage of Mitochondria to Preserve Energy-Linked Functions S I D N E Y F L E I S C H E R 28

3. Large-Scale Preparation of Rat L iver Mitochon- S I D N E Y F L E I S C H E R , J . dria O L I V E R M C I N T Y R E , A N D

J U A N C V I D A L 32

4. Isolation of Mitochondria with Emphasis on Heart L E E N A M E L A A N D S T E V E N Mitochondria from Small Amounts of Tissue S E I T Z 39

5. Preparation of Bovine Heart Mitochondria in High G I O V A N N I F E L I C E A Z Z O N E , Yield R A F F A E L E C O L O N N A , A N D

B R U N O Z I C H E 46

6. Preparation of Synaptic and Nonsynaptic Mito- J . C. K . L A I A N D J . B. chondria from Mammalian Brain C L A R K 51

7. Smooth Muscle Mitochondria A. SCARPA, J . V A L L I E R E S , B . S L O A N E , A N D A. P. S O M L Y O 60

8. Mitochondria from Brown Adipose Tissue: Iso- B A R B A R A C A N N O N A N D lation and Properties O L O V L I N D B E R G 65

9. Isolation of Mitochondria from Morris Hepatomas R. M . K A S C H N I T Z , Y O U S S E F H A T E F I , PETER L .

PEDERSEN, A N D H A R O L D P.

M O R R I S 79

10. Survey and Update of Outer and Inner Mitochon-drial Membrane Separation J O H N W . G R E E N A W A L T 88

11. Preparation o f Outer Membrane from Pig Heart J A N E C O M T E A N D D A N I E L E Mitochondria C. G A U T H E R O N 98

12. Tightly Coupled Beef Heart Submitochondrial Particles C H U A N - P U L E E 105

13. Separation o f Right-side-out and Inside-out Sub-mitochondrial Particles by Aff inity Chromatog- C A T H E R I N E G O D I N O T A N D raphy on Sepharose-Cytochrome c D A N I E L E C. G A U T H E R O N 112

14. Stabilization of Mitochondrial Functions wi th Dig- E R N E S T K U N , E V A K I R S T E N , itonin A N D W A L T E R N . P IPER 115

vi i

v i i i TABLE OF CONTENTS

15. The Kei l in-Hartree Heart Muscle Preparation A . D . V I N O G R A D O V A N D Tsoo E . K I N G 1 1 8

16. The Use o f a Viscosity Barrier in Subcellular A L E X A N D E R S. S U N A N D Fractionation B R I A N POOLE 127

17. General Methodology for Isolation and Characterization o f Mitochondria from Microorganisms D A V I D L L O Y D 135

18. Preparation of Ν eurospora crassa Mitochondria W A L T E R S E B A L D , W A L T E R N E U P E R T , A N D H A N N S W E I S S 144

19. Preparation of Yeast Mitochondria (Saccharo-myces cerevisiae) wi th Good P/O and Respira- B E R N A R D G U E R I N , P IERRE tory Control Ratios L A B B E , A N D M A R I E S O M L O 149

2 0 . Large- and Small-Scale Preparations o f Yeast Mi - Μ. Κ A YE T R E M B A T H A N D tochondria A L E X A N D E R T Z A G O L O F F 160

2 1 . Techniques for Measurement of Oxidative Phosphorylation in Intact Bacteria and in Membrane W A L T E R P. H E M P F L I N G A N D Preparations of Escherichia coli E L L I O T L . H E R T Z B E R G 164

2 2 . Properties o f Energy-Transducing Systems in Different Types of Membrane Preparations from A R N O L D F. B R O D I E , V I J A Y Mycobacterium phlei—Preparation, Resolu- K. K A L R A , S O O N - H O L E E , t ion, and Reconstruction A N D N A T A L I E S. C O H E N 175

2 3 . Assay of Citric Acid Cycle Intermediates and Related Compounds—Update wi th Tissue Metab- J O H N R. W I L L I A M S O N A N D olite Levels and Intracellular Distribution B A R B A R A E. C O R K E Y 2 0 0

Sec t i on I I . A T P Synthes is a n d R e g u l a t i o n

A . Phosphory la t i on Reactions and Regulat ion

2 4 . Respiratory Control and Oxidative Phosphoryla- K . V A N D A M A N D A. H . C. tion Measurements in Mitochondria A. W I E C H M A N N 2 2 5

2 5 . The Adenylate Energy Change in the Study of D A N I E L E. A T K I N S O N A N D Enzymes in Vitro A S T R I D G. C H A P M A N 2 2 9

2 6 . Measurement and Importance of Phosphorylation Potentials: Calculation of Free Energy of Hydrolysis in Cells E. C. S L A T E R 2 3 5

2 7 . Rapid Nucleotide Labeling and 1 8 0 Exchange Probes of Intermediate States in Electron- P A U L D . B O Y E R A N D K E R S T I N Transport-Coupled Phosphorylation S T E M P E L 2 4 5

TABLE OF CONTENTS i x

2 8 . Assay of Nicotinamide Nucleotide Transhydro-genases in Mammalian, Bacterial, and Recon-stituted Systems J . R Y D S T R Ö M 2 6 1

2 9 . Purification o f Mitochondrial Nicotinamide Nu- Bo HÖJEBERG A N D J A N cleotide Transhydrogenase from Beef Heart R Y D S T R Ö M 2 7 5

3 0 . The Use of Thin-Layer Chromatography on Poly(ethyleneimine)cellulose to Facilitate As-says of ATP -ADP Exchange, ATP-Pj Ex-change, Adenylate Kinase, and Nucleoside D i - PETER L . PEDERSEN A N D Phosphokinase Act iv i ty W I L L I A M A. C A T T E R A L L 2 8 3

3 1 . Pasteur and Crabtree Effects—Assay in Cells C H A R L E S E. W E N N E R 2 8 9

B. ATPase Complexes

3 2 . ATPase Associated wi th Electron Transport H A R V E Y S. PENEFSKY 2 9 7

3 3 . Preparation of Beef Heart Mitochondrial ATPase H A R V E Y S. PENEFSKY 3 0 4

3 4 . Preparation and Properties of Complex V D. L . S T I G G A L L , Υ. M . G A L A N T E , A N D Y . H A T E F I 3 0 8

3 5 . Preparation of Reconstitutively Active Rutamy-cin-Sensitive ATPase from Bovine Heart M i tochondria E F R A I M R A C K E R 3 1 5

36. An Improved Method for the Isolation o f Mitochondrial ATPase(F,) from Beef Heart G Ü N T E R V O G E L 3 1 7

3 7 . Purification of Fl ATPase from Rat Liver Mito- W I L L I A M A. C A T T E R A L L , chondria PETER L . PEDERSEN, D A V I D

O. L A M B E T H , A N D H E N R Y A.

L A R D Y 3 2 0

3 8 . Isolation o f an Oligomycin-Sensitive ATPase J O H N W . SOPER A N D PETER Complex from Rat Liver Mitochondria L . PEDERSEN 3 2 8

3 9 . Crystallization of F1 ATPase from Rat Liver M i - L . M A R I O A M Z E L A N D PETER tochondria L . PEDERSEN 3 3 3

4 0 . Preparation of the Soluble ATPase from Mito- P. E. L I N N E T T , A. D. chondria, Chlorophasts, and Bacteria by the M I T C H E L L , M . D. PART IS , Chloroform Technique A N D R. B . B E E C H E Y 3 3 7

4 1 . Mitochondrial ATPase Complex from Neurospora W A L T E R S E B A L D A N D G E R D crassa W I L D 3 4 4

4 2 . Oligomycin-Sensitive ATPase of Saccharomyces cerevisiae A L E X A N D E R T Z A G O L O F F 3 5 1

4 3 . ATP-Pj Exchange Preparation from Escherichia N . N E L S O N , R. C H I B O V S K Y , coli A N D D. L . G U T N I C K 3 5 8

χ TABLE OF CONTENTS

44. DCCD-Sensitive ATPase (TF 0-Fj) from a Thermophilic Bacterium: Purification, Dissociation into Functional Subunits, and Reconstruction into Vesicles Capable o f Energy Transforma- Y A S U O K A G A W A A N D t ion N O B U H I T O S O N E 364

45. Crystallization of ATPase (TFj) from a Thermophilic Bacterium Y A S U O K A G A W A 372

46. Preparation of Nucleotide-Depleted Ft and Binding o f Adenine Nucleotides and Analogs to the Depleted Enzyme H A R V E Y S. PENEFSKY 377

C. Coup l ing Factors

47. Coupling Factors—An Overview D. R A O S A N A D I A N D SAROJ J O S H I 380

48. Purification o f Coupling Factor Β SAROJ J O S H I A N D R. R A O

S A N A D I 384

49. Oligomycin-Sensitivity-Conferring Protein A. E. S E N I O R 391

50. Preparation of Coupling Factor 6 (F 6 ) E F R A I M R A C K E R 398

51. Mitochondrial ATPase Inhibitor: Properties and L A R S ERNSTER , C H R I S T I N E Applications C A R L S S O N , T O R I L L H U N D A L ,

A N D K E R S T I N N O R D E N B R A N D 399

52. Purification o f an ATPase Inhibitor Peptide Frac- N I T Z A M . C I N T R O N A N D tion f rom Rat Liver Mitochondria P E T E R L . PEDERSEN 408

53. Purification and Assay of a Proteolipid Ionophore T E T S U Y A K O N I S H I , L E S T E R from Yeast Mitochondrial ATP Synthetase PACKER, A N D R I C H A R D

G R I D D L E 414

54. ATPase Inhibitor from Candida utilis Mitochon- M I C H E L S A T R E , G E R A R D dria K L E I N , A N D P IERRE V.

V I G N A I S 421

55. Isolation o f Carbodimide-Binding Proteins from R. B . B E E C H E Y , P. E. Mitochondria and Escherichia coli L I N N E T T , A N D R. H .

F I L L I N G A M E 426

D. Special ized Reagents

56. Ionophores P E T E R W . R E E D 435

57. Mitochondrial Electron-Transport Inhibitors T H O M A S P. S I N G E R 454

58. Uncouplers o f Oxidative Phosphorylation PETER G . H E Y T L E R 462

59. Inhibitors o f the ATP Synthetase System P. E. L I N N E T T A N D R. B. B E E C H E Y 472

TABLE OF CONTENTS x i

60. 3 H - or 3 5S-Labeled Atractyloside and Carboxy-atractyloside, Atractyloside Derivatives Used for Af f inity Chromatography, Photoaffinity La- P A U L E T T E M . V I G N A I S , G. beling, and Spin Labeling, and 3 H - or 1 4 C-La - B R A N D O L I N , G. J . M . beled Bongkrekic Ac id L A U Q U I N , A N D J . C H A B E R T 518

61. Purification and Use of Colicins as Probes to Study Energy Transduction in Escherichia coli

and Derived Membrane Vesicles C H A R L E S A. P L A T E 532

E. M o d e l Systems

62. Model Reactions for Coupling Oxidation to Phos-phorylation J u i H . W A N G 536

63. A Model System for Transmembrane Redox Re-actions PETER C. H I N K L E 541

S e c t i o n I I I . M e m b r a n e P o t e n t i a l a n d I n t r a m i t o c h o n d r i a l p H

64. The Measurement of Membrane Potential and p H in CeJls, Organelles, and Vesicles H A G A I R O T T E N B E R G 547

65. The Use of Optical Probes to Monitor Membrane C. L . B A S H F O R D A N D J . C. Potential S M I T H 569

66. Membrane Potential and Reconstitution V. P. S K U L A C H E V 586

67. Lipid-Impregnated Filter Membranes: A Stable Planar Membrane System for Assay of Electri-cal Properties across Reconstituted Liposomes P A U L K. S H I E H , JANOS and Natural Membrane Vesicles L A N Y I , A N D L E S T E R PACKER 604

68. Electrometric Measurement of Rapid Proton- S. PAPA , F. G U E R R I E R I , A N D Transfer Reactions in Respiratory Chains L . R O S S I - B E R N A R D I 614

69. Measurement of - »H + /0 in Mitochondria and PETER M I T C H E L L , J E N N I F E R Submitochondrial Vesicles M O Y L E , A N D R O Y M I T C H E L L 627

70. Determination of the H +/Site and Ca 2 +/Site Ratios B A L T A Z A R R E Y N A F A R J E , of Mitochondrial Electron Transport M A R T I N D. B R A N D , A D O L F O

A L E X A N D R E , A N D A L B E R T L .

L E H N I N G E R 640

71 . Whole Cells for the Study of Transport Linked to C L I F F O R D L . S L A Y M A N A N D Membrane Potential: Neurospora crassa C A R O L Y N W. S L A Y M A N 656

72. The Use of K + Concentration Gradients for the R O N A L D S. C O C K R E L L A N D Synthesis of ATP by Mitochondria B E R T O N C. PRESSMAN 666

x i i TABLE OF CONTENTS

7 3 . The Use of K + Diffusion Gradients to Support Transport by Escherichia coli Membrane Vesi-cles H A J I M E H I R A T A 6 7 6

7 4 . The Use of Flow Dialysis for Determinations o f S O F I A R A M O S , S H I M O N p H and Active Transport S C H U L D I N E R , A N D H .

R O N A L D K A B A C K 6 8 0

7 5 . The Use of Cyanine Dyes for the Determination o f Membrane Potentials in Cells, Organelles, and Vesicles A L A N S. W A G G O N E R 6 8 9

Sec t i on I V . R e c o n s t i t u t i o n

76 . Reconstitution of Membrane Processes E F R A I M R A C K E R 6 9 9

7 7 . Reconstitution o f ATP Synthetase from Heart Mitochondria Y A S U O K A G A W A 7 1 1

7 8 . Reconstitution of Energy Conservation C. I A N R A G A N 7 1 5

7 9 . Resolution and Reconstitution o f ATP Synthesis and ATP-Dependent Functions o f Liver Mito- PETER L . PEDERSEN A N D chondria J O A N N E H U L L I H E N 7 3 6

8 0 . Reconstitution o f ATP Synthetase on a Collagen B R U N O B L A N C H Y , Membrane That Can Synthesize ATP Using a C A T H E R I N E G O D I N O T , A N D p H Gradient D A N I E L E C. G A U T H E R O N 7 4 2

8 1 . Proton Translocation by Cytochrome c Oxidase Incorporated into Phospholipid Vesicles PETER C. H I N K L E 7 4 8

8 2 . Methods for Reconstitution of the Electrogenic Function o f Membrane Proteins V. P. S K U L A C H E V 7 5 1

83 . Incorporation o f Purple Membrane into Vesicles Capable o f Light-Stimulated ATP Synthesis Y A S U O K A G A W A 7 7 7

8 4 . Soluble A T P a s e ^ ) from a Thermophilic Bacter-ium: Purification, Dissociation into Subunits, Y A S U O K A G A W A A N D and Reconstitution from Individual Subunits M A S A S U K E Y O S H I D A 7 8 1

85 . Reconstitution o f an Energy-Linked Reaction (Reduced Pyridine Nucleotide Transhydrogen-ation) in Fractionated Escherichia coli Mem-branes wi th Purified ATPase P. D. B R A G G 7 8 7

8 6 . Dissociation and Reconstitution of a Functional ATPase from Escherichia coli G Ü N T E R V O G E L 8 0 0

8 7 . Reconstitution o f Mitochondrial Nicotinamide Nucleotide Transhydrogenase from Beef Heart J A N R Y D S T R Ö M A N D S I D N E Y wi th Synthetic Phospholipids F L E I S C H E R 8 1 1

TABLE OF CONTENTS x i i i

A d d e n d a

Addendum to Art icle [34] Υ . Μ . G A L A N T E A N D Υ .

H A T E F I 8 1 9

Addendum to Art icle [47] Y. K A G A W A 821

A U T H O R I N D E X 823

SUBJECT I N D E X 851

Cont r i bu to r s to V o l u m e L V

Article numbers are in parentheses following the names of contributors. Affiliations listed are current.

A D O L F O A L E X A N D R E (70 ) , Department of

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

L . M A R I O A M Z E L ( 39 ) , Department of Bio

physics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

D A N I E L E . A T K I N S O N (25 ) , Biochemistry

Division, Department of Chemistry, University of California, Los Angeles, California 90024

G I O V A N N I F E L I C E A Z Z O N E ( 5 ) , (2 ) CNR.

Unit for the Study of Physiology of Mitochondria, Institute of General Pathology, University of Padova, Padova, Italy

C . L . B A S H F O R D ( 65 ) , Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

R . B . B E E C H E Y ( 4 0 , 5 5 , 5 9 ) , Shell Research Limited, Shell Biosciences Laboratory, Sittingbourne Research Centre, Sitting-bourne, Kent ME9 8AG, England

B R U N O B L A N C H Υ ( 8 0 ) , Laboratoire de Biol

ogie et Technologie, des Membranes (LBTM)-CNRS, Universite Claude Bernard de Lyon, 69621 Villeurbanne, France

P A U L D . B O Y E R (27 ) , Molecular Biology In

stitute, Department of Chemistry, University of California, Los Angeles, California

P. D . B R A G G (85 ) , Department of Biochemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1W5

M A R T I N D . B R A N D (70 ) , Department of Bio

chemistry, University of Cambridge, Cambridge CB2 1QW, England

G . B R A N D O L I N (60 ) , Laboratoire de Bioch-

imie, Department de Recherche Fonda-mentale, Centre d'Etudes Nucleaires, 85X 38041 Grenoble-cedex, France

A R N O L D F . B R O D I E ( 22 ) , Department of

Biochemistry, University of Southern California School of Medicine, Los Angeles, California 90033

B A R B A R A C A N N O N ( 1 , 8 ) , The Wenner-Gren

Institute, University of Stockholm, S-113 45 Stockholm, Sweden

C H R I S T I N E CARLSSON ( 51 ) , Department of

Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden

W I L L I A M A . C A T T E R A L L ( 3 0 , 3 7 ) , Depart

ment of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98105

J . C H A B E R T ( 6 0 ) , Laboratoire de Biochimie, Departement de Recherche Fondamentale, Centre d'Etudes Nucleaires, 85X 38041 Grenoble-cedex, France

A S T R I D G . C H A P M A N ( 25 ) , Biochemistry

Division, Department of Chemistry, University of California, Los Angeles, California 90024

R . C H I B O V S K Y ( 4 3 ) , Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

N I T Z A Μ . C I N T R O N ( 5 2 ) , Laboratory of

Molecular and Cellular Bioenergetics, Department of Physiological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

J . B . C L A R K ( 6 ) , Department of Biochemistry, St. Bartholomew's Hospital Medical- College, University of London, London ECIM 6BQ, England

xv

x v i CONTRIBUTORS TO VOLUME LV

R O N A L D S. C O C K R E L L ( 72 ) , Edward A.

Doisy Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104

N A T A L I E S. C O H E N ( 22 ) , Department of

Biochemistry, University of Southern California School of Medicine, Los Angeles, California 90033

R A F F A E L E C O L O N N A ( 5 ) , ( 2 ) C.N.R. Unit for the Study of Physiology of Mitochondria, Institute of General Pathology, University of Padova, Padova, Italy

J A N E C O M T E ( 1 1 ) , Laboratoire de Biologie et Technologie des Membranes LBTM— CNRS, Universite Claude Bernard de Lyon, 69621 Villeurbanne, France

B A R B A R A E . C O R K E Y ( 2 3 ) , Department of

Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

R I C H A R D C R I D D L E ( 53 ) , Niigata College of Pharmacy, 5829 Kamishin-ei, Niigata 950-21, Japan

L A R S ERNSTER ( 51 ) , Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden

R. H . F I L L I N G A M E ( 55 ) , Department of Physiological Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706

S I D N E Y FLE ISCHER ( 2 , 3 , 8 7 ) , Department

of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235

Υ. Μ . G A L A N T E ( 34 ) , Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037

D A N I E L E C . G A U T H E R O N ( 1 1 , 13, 8 0 ) , La

boratoire de Biologie et Technologie, des Membranes (LBTM) -CNRS, Universite Claude Bernard de Lyon, 69621 Villeurbanne, France

C A T H E R I N E G O D I N O T ( 1 3 , 8 0 ) , Laboratoire

de Biologie et Technologie, des Membranes (LBTM)-CNRS, Universite Claude Bernard de Lyon, 69621 Villeurbanne, France

J O H N W . G R E E N A W A L T ( 1 0 ) , Department of

Biochemistry, University of Tennessee, Knoxville, Tennessee 37916

B E R N A R D G U E R I N ( 1 9 ) , Laboratoire de

Biochemie, Universite de Bordeaux II, 33405 Talence, France

F . G U E R R I E R I ( 6 8 ) , Universita di Bari, In-stituto di Chimica Biologie a, Facolta di Medici na e Chirurgia, 70126 Bari, Italy

D . L . G U T N I C K ( 4 3 ) , Department of Microbiology, George Wise Center for Life Sciences, Tel Aviv University, Ramat Aviv, Israel

YOUSSEF H A T E F I ( 9 , 3 4 ) , Department of

Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037

W A L T E R P. H E M P F L I N G ( 2 1 ) , Department

of Biology, University of Rochester, Rochester, New York 14627

E L L I O T L . H E R T Z B E R G ( 2 1 ) , Department of

Cell Biology, The Rockefeller University, New York, New York 10021

PETER G . H E Y T L E R ( 5 8 ) , Central Research and Development Department, Experimental Station, Ε. I. Du Pont de Nemours & Company, Wilmington, Delaware 19898

PETER C . H I N K L E ( 6 3 , 8 1 ) , Section of Biochemistry, Molecular, and Cell Biology, Cornell University, Ithaca, New York 14853

H A J I M E H I R A T A ( 7 3 ) , Department of Biochemistry, Jichi Medical School, Tochigi, Japan 329-04

Bo HÖJEBERG ( 29 ) , Department of Bio-chemistry, Arrhenius Laboratory, Uni-versity of Stockholm, S-106 91 Stockholm, Sweden

CONTRIBUTORS TO VOLUME LV XVÜ

J O A N N E H U L L I H E N ( 7 9 ) , Laboratory for

Molecular and Cellular Bioenergetics, Department of Physiological Chemistry, Johns Hopkins University School of Med-icine, Baltimore, Maryland 21205

T O R I L L H U N D A L ( 5 1 ) , Department of Bio-chemistry, Arrhenius Laboratory, Uni-versity of Stockholm, S-106 91 Stockholm, Sweden

SAROJ J O S H I ( 4 7 , 4 8 ) , Department of Cell Physiology, Boston Biomedical Research Institute, Boston, Massachusetts 02114

H . R O N A L D K A B A C K ( 7 4 ) , The Roche Insti-tute of Molecular Biology, Nutley, New Jersey 07110

Y A S U O K A G A W A ( 4 4 , 4 5 , 7 7 , 8 3 , 84 ) , De-

partment of Biochemistry, Jichi Medical School, Japan 329-04

V I J A Y K . K A L R A ( 2 2 ) , Department of Bio-chemistry, University of Southern Cali-fornia School of Medicine, Los Angeles, California 90033

R. M . K A S C H N I T Z ( 9 ) , Institute for Molec-ular Biology, Austrian Academy of Sci-ences, A-5020 Salzburg, Austria

Tsoo E . K I N G ( 1 5 ) , Laboratory of Bioener-getics and Department of Chemistry, The State University of New York Albany, New York 12222

E V A K I R S T E N ( 1 4 ) , Departments of Phar-macology, Biochemistry, and Biophysics, The Cardiovascular Research Institute, University of California School of Medi-cine, San Francisco, California 94143

G E R A R D K L E I N ( 5 4 ) , Laboratoire de Bioch-imie, Department de Recherche Fonda-mentale, Centre d'Etudes Nucleaires, 38041 Grenoble-cedex, France

T E T S U Y A K O N I S H I ( 5 3 ) , Membrane Bio-energetics Group, Department of Physi-ology-Anatomy. University of California, Berkeley, California 95720

E R N E S T K U N ( 1 4 ) , Department of Pharma-

cology, Biochemistry, and Biophysics, The Cardiovascular Research Institute, University of California School of Medi-cine, San Francisco, California 94143

PIERRE L A B B E ( 1 9 ) , Laboratoire de Bioch-imie des Porphyrines, Universite de Paris VII, Tour 43, 75005 Paris, France

J . C . K . L A I ( 6 ) , Miriam Marks Department of Neurochemistry, Institute of Neurol-ogy (Queen's Square), University of Lon-don, London WCIM 3BQ, England

D A V I D O . L A M B E T H ( 3 7 ) , Department of

Biochemistry, University of North Da-kota, Grand Forks, North Dakota 58202

JANOS L A N Y I ( 6 7 ) , NASA Ames Research Center, Moffett Field, California 94035

H E N R Y A . L A R D Y ( 3 7 ) , The Institute of En-

zyme Research, The University of Wis-consin, Madison, Wisconsin 53706

G . J . M . L A U Q U I N ( 6 0 ) , Laboratoire de Biochimie, Department de Recherche Fondamentale, Centre d'Etudes Nu-cleaires, 38041 Grenoble-cedex, France

S O O N - H O L E E ( 22 ) , Department of Bio-chemistry, University of Southern Cali-fornia School of Medicine, Los Angeles, California 90033

C H U A N - P U L E E ( 12 ) , Department of Bio-chemistry, Wayne State University School of Medicine, Detroit, Michigan 48201

A L B E R T L . L E H N I N G E R ( 70 ) , Department of

Physiological Chemistry, Johns Hopkins University School of Medicine, Balti-more, Maryland 21205

O L O V L I N D B E R G ( 8 ) , The Wenner-Gren In-

stitute, University of Stockholm, S-113 45 Stockholm, Sweden

P. E . L I N N E T T ( 4 0 , 5 5 , 5 9 ) , Shell Research Limited, Shell Biosciences, Laboratory, Sittingbourne Research Centre, Sitting-bourne, Kent ME9 8AG, England

D A V I D L L O Y D ( 1 7 ) , Department of Micro-

x v i i i CONTRIBUTORS TO VOLUME LV

biology, University College, Cardiff CF2 I TA, Wales

J . O L I V E R M C I N T Y R E (3 ) , Department of

Molecular Biology, Vanderbilt Univer-sity, Nashville, Tennessee 37235

L E E N A M E L A ( 4 ) , Departments of Surgery and Biochemistry and Biophysics, Uni-versity of Pennsylvania School of Medi-cine, Philadelphia, Pennsylvania 19104

A . D . M I T C H E L L ( 40 ) , Shell Research Lim-ited, Shell Biosciences Laboratory, Sit-tingbourne Research Centre, Sitting-bourne, Kent ME9 8AG, England

P E T E R M I T C H E L L ( 6 9 ) , Glynn Research Laboratories, Bodmin, Cornwall PL30 4AU, England

R O Y M I T C H E L L ( 69 ) , Glynn Research Lab-oratories, Bodmin, Cornwall PL30 4AU, England

H A R O L D P. M O R R I S ( 9 ) , Department of Bio-chemistry, Howard University School of Medicine, Washington, D.C. 20059

J E N N I F E R M O Y L E ( 6 9 ) , Glynn Research Laboratories, Bodmin, Cornwall PL30 4AU, England

J A N N E D E R G A A R D ( 1 ) , The Wenner-Gren Institute, University of Stockholm, S-113 45 Stockholm, Sweden

N . N E L S O N ( 4 3 ) , Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

W A L T E R N E U P E R T ( 18 ) , Institut für Physil-ogische Chemie, Physikalische Biochemie und Zellbiologie der Universität München, 8 München 2, Federal Republic of Ger-many

K E R S T I N N O R D E N B R A N D ( 51 ) , Department

of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stock-holm, Sweden

L E S T E R PACKER ( 53 , 6 7 ) , Membrane Bio-energetics Group, Department of Physi-

ology-Anatomy, University of California at Berkeley, Berkeley, California 95720

S. P A P A (68 ) , Universitä di Bari, Istituto di Chimica Biologica, Facolta di Medicini e Chirurgia, 70126 Bari, Italy

M . D . PART IS ( 40 ) , Department of Molecu-lar Sciences, University of Warwick, Cov-entry CV4 7Ah, United Kingdom

PETER L . PEDERSEN ( 9 , 3 0 , 3 7 , 3 8 , 3 9 , 5 2 ,

7 9 ) , Laboratory for Molecular and Cel-lular Bioenergetics, Department of Phy-siological Chemistry, Johns Hopkins University School of Medicine, Balti-more, Maryland 21205

H A R V E Y S. PENEFSKY ( 3 2 , 3 3 , 4 6 ) , The Pub-

lic Health Research Institute, The City of New York, Inc., New York, New York 10016

W A L T E R N . P IPER ( 1 4 ) , Department of

Pharmacology, University of California School of Medicine, San Francisco, Cal-ifornia 94143

C H A R L E S A . P L A T E ( 61 ) , Department of Bi-

ology, Massachusetts Institute of Tech-nology, Cambridge, Massachusetts 02139

B R I A N POOLE (16 ) , Department of Pathol-ogy, Mt. Sinai School of Medicine, The Rockefeller University, New York, New York 10021

B E R T O N C . PRESSMAN ( 72 ) , Department of

Pharmacology, School of Medicine, Uni-versity of Miami, Miami, Florida 33152

E F R A I M R A C K E R (35 , 5 0 , 7 6 ) , Department

of Biochemistry, Molecular and Cell Bi-ology, Cornell University, Ithaca, New York 14853

C. I A N R A G A N ( 78 ) , Department of Bio-chemistry, School of Biochemical and Physiological Sciences, The University of Southampton, Southampton, S09 3TU, England

S O F I A R A M O S (74 ) , National Institute of

CONTRIBUTORS TO VOLUME LV x i x

Arthritis and Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014

PETER W . R E E D (56 ) , Department of Phar-macology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232

B A L T A Z A R R E Y N A F A R J E ( 70 ) , Department

of Physiological Chemistry, Johns Hop-kins University School of Medicine, Bal-timore, Maryland 21205

L . ROSSI -BERNARD] ( 6 8 ) , Cattedra di Chim-ica Biologic a, Universita di Milano, Milan, Italy

H A G A I ROTTENBERG ( 6 4 ) , Bell Laborato-ries, Murray Hill, New Jersey 07974

J A N RYDSTRÖM (28 , 2 9 , 87 ) , Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stock-holm, Sweden

D . R A O S A N A D I (47 ) , Department of Biolog-ical Chemistry, Harvard Medical School, Boston, Massachusetts 02115

R. R A O S A N A D I (48 ) , Boston Biomedical Research Institute, Boston, Massachu-setts 02115

M I C H E L SATRE (54 ) , Laboratoire de Bioch-imie, Department de Recherche Fonda-men tale, Centre d'Eludes Nucleaires, 85X 38041 Grenoble-cedex, France

A . SCARPA (7 ) , Department of Biochemistry and Biophysics, The Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, Penn-sylvania 19104

S H I M O N S C H U L D I N E R ( 7 4 ) , Department of

Molecular Biology, The Hebrew Univer-sity, Hadassah Medical School, Jerusa-lem, Israel

W A L T E R S E B A L D (18 , 4 1 ) , Gesellschaft für

Biotechnologische Forschung mbH. Abt lg. Stoffwechselregulation, 3300 Braun-schweig-Stockheim, Federal Republic of Germany

S T E V E N S E I T Z ( 4 ) , Departments of Surgery and of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

A . E . S E N I O R ( 4 9 ) , Department of Biochem-istry, University of Rochester Medical Center, Rochester, New York 14642

P A U L K . S H I E H ( 6 7 ) , Membrane Bioener-getics Group, University of California, Berkeley, California 94720

T H O M A S P. S I N G E R ( 5 7 ) , Molecular Biology Division, Veterans Administration Hos-pital, San Francisco, California 94121

V . P. S K U L A C H E V ( 6 6 , 8 2 ) , Department of Bioenergetics, A. N. Belozersky Labora-tory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow, USSR

E . C . S L A T E R ( 2 6 ) , Laboratory of Biochem-istry, B. C. P. Jansen Institute, University of Amsterdam, Amsterdam-C'., The Netherlands

C L I F F O R D L . S L A Y M A N ( 71 ) , Departments

of Physiology and Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

C A R O L Y N W . S L A Y M A N ( 7 1 ) , Departments

of Physiology and Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

B . S L O A N E ( 7 ) , Department of Biochemistry and Biophysics, The Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, Penn-sylvania 19104

J . C . S M I T H ( 6 5 ) , Johnson Research Foun-dation, University of Pennsylvania, Phil-adelphia, Pennsylvania 19104

M A R I E S O M L O ( 1 9 ) , Centre de Genetique Moleculaire, Gif 91190, France

A . P . S O M L Y O ( 7 ) , Departments of Physi-ology and Pathology, The Pennsylvania

XX CONTRIBUTORS TO VOLUME LV

Muscle Institute, Presbyterian-University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

N O B U H I T O S O N E ( 4 4 ) , Department of Biochemistry, Jichi Medical School, Tochigi, Japan 329-04

J O H N W . SOPER ( 3 8 ) , Department of Physiological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

K E R S T I N S T E M P E L ( 2 7 ) , Molecular Biology Institute, Department of Chemistry* University of California, Los Angeles, California 90024

D . L . S T I G G A L L ( 34 ) , Chemistry Department, San Diego State University, San Diego, California 92182

A L E X A N D E R S. S U N ( 1 6 ) , Department of Pathology, Mt. Sinai School of Medicine, The Rockefeller University, New York, New York 10021

M . K A Y E T R E M B A T H ( 20 ) , Department of

Biochemistry, Monash University, Clayton, Australia

A L E X A N D E R T Z A G O L O F F ( 2 0 , 4 2 ) , Public

Health Research Institute, The City of New York, Inc., New York, New York 10016

J . V A L L I E R E S ( 7 ) , Department of Physiology, School of Medicine, Laval University, Quebec, Canada

K . V A N D A M ( 2 4 ) , Universiteit van Amsterdam, Laboratorium voor Biochemie, Β. C. P. Jansen Institut, Amsterdam-C, The Netherlands

J U A N C . V I D A L ( 3 ) , Facultad de Medicina, Institut ο de Bioquimica, Buenos Aires, Argentina

P A U L E T T E M . V I G N A I S ( 6 0 ) , Laboratoire de

Biochimiedepartment de RechercheFon-damentale et ERA-CNRS, F-38041 Grenoble-cedex, France

PIERRE V . V I G N A I S ( 5 4 ) , Laboratoire de

Biochimie, Departement de Recherche Fondamentale, Centre d'Etudes Nucleaires, F-38041 Grenoble-cedex, France

A. D . V I N O G R A D O V ( 1 5 ) , Department of Biology, Moscow State University, Moscow, USSR

G Ü N T E R V O G E L ( 3 6 , 8 6 ) , Max-Planck-Insti-

tut für Biologie, D74 Tübingen, Federal Republic of Germany

A L A N S . W A G G O N E R ( 7 5 ) , Department of

Chemistry, Amherst College, Amherst, Massachusetts 01002

Jul H . W A N G ( 62 ) , Bioenergetics Labora-tory, State University of New York at Buf-falo, Buffalo, New York 14214

H A N N S WEISS ( 18 ) , European Molecular Biology Laboratory, 6900 Heidelberg, Federal Republic of Germany

C H A R L E S E . W E N N E R ( 3 1 ) , Department of

Experimental Biology, Roswell Park Memorial Institute, Buffalo, New York 14263

A. H . C . A . W I E C H M A N N ( 2 4 ) , Universiteit van Amsterdam, Laboratorium voor Biochemie, Β. C. P. Jansen Instit, Amsterdam-C, The Netherlands

G E R D W I L D ( 41 ) , Institut für Physiologische Chemie und Physikalische Biochemie der Universität, 8000 München 2, Federal Republic of Germany

J O H N R . W I L L I A M S O N ( 2 3 ) , Department of

Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Phil-adelphia, Pennsylvania 19104

M A S A S U K E Y O S H I D A ( 8 4 ) , Department of

Biochemistry, Jichi Medical School, To-chigi, Japan 329-04

B R U N O Z I C H E ( 5 ) , CN.R. Unit for the

Study of Physiology, and Mitochondria, Institute of General Pathology, Univer-sity of Padova, Padova, Italy

P r e f a c e

A great deal o f progress has taken place in b io log ica l ox idat ions and bioenerget ics since Oxidation and Phosphorylation edi ted by Rona ld W . Es tab rook and M a y n a r d E . Pu l lman (Vo lume X o f Methods in Enzymology) became avai lable i n 1967. T o update the mater ia l o r i g i -nal ly t reated in this vo lume , f ive vo lumes on b iomembranes (Vo lumes L I I - L V I , Parts C - G , respect ive ly ) have been prepared , three deal ing w i t h b io log i ca l ox idat ions and t w o w i t h bioenerget ics.

I n this v o l u m e , Part F o f " B i o m e m b r a n e s , ^ subt i t l ed k k Bioene rge t i c s -Ox ida t i v e P h o s p h o r y l a t i o n , " we br ing together new methodo logy on the prepara t i on o f organelles, membranes , A T P synthetase complexes , and pur i f i ed components re levant to the subject ; the measurement o f A T P synthesis and regulat ion; the measurement o f t ransmembrane po-tent ia l and in t rami tochondr i a l p H ; and the d issoc ia t ion and reconst i -t u t i o n o f energy-transducing membranes .

We are pleased to acknowledge the good counsel o f the members o f o u r A d v i s o r y Board for these five vo lumes . Special thanks are also due D r s . E. Cara fo l i , G. Palmer, H . Penefsky, and A . Scarpa f o r the i r he lp fu l comments on our out l ines fo r these vo lumes . Va luab le counse l f o r this v o l u m e was also p rov ided by Drs . D . E . Green , R H a r o l d , P. H i n k l e , R. K a b a c k , P. Pedersen, E . Racker , D . R. Sanadi , C. Schnai t -m a n , T . P. Singer, W. Stoeckenius, A . Tzagolo f f , and J . R. W i l l i a m s o n . W e were ve ry grat i f i ed by the enthusiasm and coopera t ion o f the par-t i c ipants i n the f i e ld o f b io log ica l ox idat ions and bioenerget ics whose adv ice , c omments , and cont r ibut i ons have enr iched and made possible these vo lumes . The f r i end ly cooperat ion o f the staf f o f Academic Press is grate fu l ly acknowledged .

S I D N E Y F L E I S C H E R

L E S T E R P A C K E R

xx i

METHODS IN E N Z Y M O L O G Y EDITED BY

Sidney P. Colowick and Nathan O. Kaplan V A N D E R B I L T UN IVERS ITY D E P A R T M E N T O F CHEMISTRY

SCHOOL O F M E D I C I N E UN IVERS ITY O F C A L I F O R N I A

N A S H V I L L E , TENNESSEE A T SAN D IEGO

L A J O L L A , C A L I F O R N I A

I . P reparat ion and Assay o f Enzymes I I . Preparat ion and Assay o f Enzymes

I I I . Preparat ion and Assay o f Substrates I V . Special Techniques fo r the Enzymo log i s t V . Preparat ion and Assay o f Enzymes

V I . Preparat ion and Assay o f Enzymes (Continued) Preparat ion o f Assay o f Substrates Special Techniques

V I I . Cumula t i v e Subject Index

xx i i i

METHODS I N E N Z Y M O L O G Y EDITORS- IN-CHIEF

Sidney P. Colo wick Nathan O. Kaplan

V O L U M E V I I I . Complex Carbohydrates Edited by E L I Z A B E T H F. N E U F E L D A N D V I C T O R G I N S B U R G

V O L U M E I X . Carbohydrate Me tabo l i sm Edited by W I L L I S A . W O O D

V O L U M E X . Ox ida t i on and Phosphory la t i on Edited by R O N A L D W . E S T A B R O O K A N D M A Y N A R D Ε . P U L L M A N

V O L U M E X I . Enzyme Structure Edited by C. H . W . H I R S

V O L U M E X I I . Nuc le ic Ac ids (Parts A and B ) Edited by L A W R E N C E G R O S S M A N A N D K I V I E M O L D A V E

V O L U M E X I I I . C i t r i c A c i d Cyc le Edited by J . M . L O W E N S T E I N

V O L U M E X I V . L ip ids

Edited ^ J . M . L O W E N S T E I N

V O L U M E X V . Steroids and Terpeno ids Edited by R A Y M O N D B . C L A Y T O N

V O L U M E X V I . Fast Reactions Edited by K E N N E T H K U S T I N

V O L U M E X V I I . Metabo l i sm o f A m i n o Ac ids and Amines (Parts A and B ) Edited by H E R B E R T T A B O R A N D C E L I A W H I T E T A B O R

V O L U M E X V I I I . V i tamins and Coenzymes (Parts A , B , and C) Edited by D O N A L D B . M C C O R M I C K A N D L E M U E L D . W R I G H T

V O L U M E X I X . Proteo lyt ic Enzymes Edited by G E R T R U D E E . P E R L M A N N A N D L A S Z L O L O R A N D

XXV

x x v i METHODS IN ENZYMOLOGY

V O L U M E X X . Nuc le i c Ac ids and Prote in Synthes is (Part C) Edited by K I V I E M O L D A V E A N D L A W R E N C E G R O S S M A N

V O L U M E X X I . Nuc le i c Ac ids (Part D) Edited by L A W R E N C E G R O S S M A N A N D K I V I E M O L D A V E

V O L U M E X X I I . Enzyme Pur i f i ca t ion and Related Techniques Edited by W I L L I A M B . J A K O B γ

V O L U M E X X I I I . Photosynthesis (Part A ) Edited by A N T H O N Y S A N P I E T R O

V O L U M E X X I V . Photosynthesis and N i t r ogen F i x a t i o n (Part B) Edited by A N T H O N Y S A N P I E T R O

V O L U M E X X V . Enzyme Structure (Part B) Edited by C. H . W . H I R S A N D S E R G E N . T I M A S H E F F

V O L U M E X X V I . Enzyme Structure (Part C) Edited by C. H . W . H I R S A N D S E R G E N . T I M A S H E F F

V O L U M E X X V I I . Enzyme Structure (Part D) Edited by C. H . W . H I R S A N D S E R G E N . T I M A S H E F F

V O L U M E X X V I I I . Complex Carbohydrates (Part B ) Edited by V I C T O R G I N S B U R G

V O L U M E X X I X . Nuc le i c Ac ids and Prote in Synthesis (Part E) Edited by L A W R E N C E G R O S S M A N A N D K I V I E M O L D A V E

V O L U M E X X X . Nuc l e i c Ac ids and Prote in Synthesis (Part F) Edited by K I V I E M O L D A V E A N D L A W R E N C E G R O S S M A N

V O L U M E X X X I . B iomembranes (Part A ) Edited by S I D N E Y F L E I S C H E R A N D L E S T E R P A C K E R

V O L U M E X X X I I . B iomembranes (Part B) Edited by S I D N E Y F L E I S C H E R A N D L E S T E R P A C K E R

V O L U M E X X X I I I . Cumula t i v e Subject Index Vo lumes I - X X X Edited by M A R T H A G . D E N N I S A N D E D W A R D A . D E N N I S

METHODS IN ENZYMOLOGY XXVÜ

V O L U M E X X X I V . A f f i n i t y Techniques (Enzyme Pur i f i ca t i on : Part B ) Edited by W I L L I A M B . J A K O B Y A N D M E I R W I L C H E K

V O L U M E X X X V . L ip ids (Part B ) Edited by J O H N M . L O W E N S T E I N

V O L U M E X X X V I . H o r m o n e A c t i o n (Part A : Stero id Hormones ) Edited by B E R T W . O ' M A L L E Y A N D J O E L G . H A R D M A N

V O L U M E X X X V I I . H o r m o n e A c t i o n (Part B : Peptide Hormones ) Edited by B E R T W . O ' M A L L E Y A N D J O E L G . H A R D M A N

V O L U M E X X X V I I I . H o r m o n e A c t i o n (Part C: Cyc l i c Nucleot ides ) Edited by J O E L G . H A R D M A N A N D B E R T W . O ' M A L L E Y

V O L U M E X X X I X . Ho rmone A c t i o n (Part D : Iso lated Cel ls, T issues, and Organ Systems) Edited by J O E L G . H A R D M A N A N D B E R T W . O ' M A L L E Y

V O L U M E X L . Hormone A c t i o n (Part E: Nuc lear St ructure and Func t i on ) Edited by B E R T W . O ' M A L L E Y A N D J O E L G . H A R D M A N

V O L U M E X L I . Carbohydrate Metabo l i sm (Part B ) Edited by W . A . W O O D

V O L U M E X L I I . Carbohydrate Me tabo l i sm (Part C) Edited by W . A . W O O D

V O L U M E X L I I I . An t ib i o t i c s Edited by J O H N H . H A S H

V O L U M E X L I V . Immob i l i z ed Enzymes Edited by K L A U S M O S B A C H

V O L U M E X L V . Proteolyt ic Enzymes (Part B ) Edited by L A S Z L O L O R A N D

V O L U M E X L V I . A f f i n i t y Labe l i ng Edited by W I L L I A M B . J A K O B Y A N D M E I R W I L C H E K

V O L U M E X L V I I . Enzyme S t ruc ture (Part E) Edited by C. H . W . H I R S A N D S E R G E N . T I M A S H E F F

x x v i i i METHODS IN ENZYMOLOGY

V O L U M E X L V I I I . E n z y m e Structure (Part F ) Edited by C. H . W . H I R S A N D S E R G E N . T I M A S H E F F

V O L U M E X L I X . Enzyme Structure (Part G ) Edited by C. H . W . H I R S A N D S E R G E N . T I M A S H E F F

V O L U M E L . Complex Carbohydrates (Part C) Edited by V I C T O R G I N S B U R G

V O L U M E L I . Purine and Pyr imid ine Nuc leo t ide Metabo l i sm Edited by P A T R I C I A A . H O F F E E A N D M A R Y E L L E N J O N E S

V O L U M E L I I . B iomembranes (Part C: Bio log ica l Oxidat ions) Edited by S I D N E Y F L E I S C H E R A N D L E S T E R P A C K E R

V O L U M E L I I I . B iomembranes (Part D : Bio log ica l Oxidat ions) Edited by S I D N E Y F L E I S C H E R A N D L E S T E R P A C K E R

V O L U M E L I V . B iomembranes (Part E : Bio log ica l Oxidat ions) Edited by S I D N E Y F L E I S C H E R A N D L E S T E R P A C K E R

V O L U M E L V . B iomembranes (Part F : Bioenergetics) Edited by S I D N E Y F L E I S C H E R A N D L E S T E R P A C K E R

V O L U M E L V I . B iomembranes (Part G : Bioenergetics) Edited by S I D N E Y F L E I S C H E R A N D L E S T E R P A C K E R

V O L U M E L V I I . B io luminescence and Chemiluminescence ( in preparat i on ) Edited by M A R L E N E A . D E L U C A

V O L U M E L V I I I . Cel l Cu l ture ( in preparat ion) Edited by W I L L I A M B . J A K O B Y A N D I R A H . P A S T A N

V O L U M E L I X . Nuc l e i c Ac ids and Prote in Synthesis, Part G ( in preparat ion )

Edited by K I V I E M O L D A V E A N D L A W R E N C E G R O S S M A N

V O L U M E L X . Nuc le i c Ac ids and Prote in Synthesis, Part Η ( in preparat i on )

Edited by K I V I E M O L D A V E A N D L A W R E N C E G R O S S M A N

METHODS IN ENZYMOLOGY x x i x

V O L U M E 6 1 . Enzyme S t ruc ture , Part Η ( in preparat ion) Edited by C. H . W . H I R S A N D S E R G E N . T I M A S H E F F

V O L U M E 62. V i tamins and Coenzymes, Part D ( in preparat ion) Edited by D O N A L D B . M C C O R M I C K A N D L E M U E L D . W R I G H T

V O L U M E 63. Enzyme K ine t i cs and Mechan ism ( in preparat ion ) Edited by D A N I E L L . P U R I C H

144 ORGANELLES AND MEMBRANES [18]

[18] P r e p a r a t i o n o f Neurospora crassa M i t o c h o n d r i a

By W A L T E R S E B A L D , W A L T E R N E U P E R T , and H A N N S W E I S S

The fungus Neurospora crassa represents a eukaryo t i c cel l w i t h high b iosynthe t i c act iv i t i es . Cel l mass doubles in 2-4 hr du r ing exponent ia l g r o w t h , even in simple salt media w i t h sucrose as the sole carbon source. T h e mic roorgan ism forms a myce l i um o f long hyphae dur ing vegetative g r o w t h . The m i t ochondr i a can be isolated under re la t ive ly gentle condi t ions since a few breaks in the thread l ike hyphae are suf f ic ient to cause the ou t f l ow o f the organelles. Th is art ic le describes t w o methods for the phys i ca l d i s rupt i on o f the hyphae: (1) The cells are opened in a g r ind mi l l be tween t w o ro ta t ing c o r u n d u m disks . Th is is a cont inuous and fast procedure and al lows large- and small-scale preparat ions o f m i t ochondr i a . (2) Hyphae are g round w i t h sand in a mor ta r and pestle. Th i s procedure can be appl ied to microscale preparat ions o f m i t o chondr i a s tar t ing w i t h m inu t e amounts o f cel ls. Other procedures for the i so la t ion o f Neurospora m i t ochondr i a after the phys ica l d i s rup t i on or the enzymat ic degrada t i on o f the cel l wa l l have been descr ibed e lsewhere . 1

C u l t i v a t i o n o f Ce l ls

Neurospora crassa w i l d type is g r o w n at 25° in VogeFs m in ima l m e d i u m plus 2% sucrose. 2 - 3 The i n o c u l u m is 1-2 χ 10 6 con id ia per m i l l i l i t e r .

Cul tures o f 50 ml are shaken in 200-ml Er l enmeye r flasks on a ro ta to ry shaker at about 150 r p m under steri le cond i t i ons . Larger cu l tures , 0.5-8 l i t e r s , are aerated in bott les w i t h a centra l in let tube under steri le con-

1 J . W. Greenawalt, D. O. Ha l l , and O. C. Wallis, this series, Vol . 10 [27]. 2 H . J . Vogel, Microb. Genet. Bull. 13, 42 (1956). 3 R. H . Davis and F. J. de Serres, this series, Vo l . I7A [4].

M E T H O D S I N E N Z Y M O L O G Y . V O L . L V

C o p y r i g h t © 1979 b y A c a d e m i c P r e s s . I n c . A l l r i g h t s o f r e p r o d u c t i o n in a n y f o r m r e s e r v e d .

I S B N 0 - 1 2 - 1 8 1 9 5 5 - 8

[18] P R E P A R A T I O N O F Ν. CMSSa M I T O C H O N D R I A 145

di t ions. F o r large-scale g r o w t h o f cel ls, 100 l i ters o f cu l ture m e d i u m are inoculated in a plastic t r ough w i t h 8 l i ters o f a cu l ture that was g r o w n f o r about 20 hr . The cells are aerated through a centra l in let tube . I t is not necessary to steri l ize the t r o u g h , the m e d i u m , and the air .

The g r o w t h per iod is 14-24 hr for al l three types o f cu l tures . H y p h a e g row exponent ia l ly up to 20-24 hr . The cells are harvested by filtration on filter paper or l inen . Large amounts o f cu l ture med ium are centr i fuged in a laundry- type spin dr i e r th rough a bag o f l i nen . The cells are washed w i t h d is t i l l ed water . The y ie ld o f cells is 5-20 g wet weight per l i t e r cu l ture med ium. One g ram wet weight corresponds to about 100 mg d r y weight o r 40-50 mg p ro t e in . Fo r special purposes the cells can be f rozen and stored at -20°

D i s r u p t i o n o f the C e l l Wa l l s

A l l operat ions are per fo rmed at 0°-4°. The iso lat ion med ium consists o f 0.25 Μ sucrose, 1 mM E D T A , and 10 mM Tr is-acetate, p H 7.2.

Grind Mill (Large- and Small-scale Preparation).4 The filtered cells are resuspended in 5-10 vo lumes (w/v) o f iso lat ion med ium by a 60-sec homogenizat ion in a k i t chen blender. The suspended cells are d i s rupted in a g r ind m i l l (F ig . 1) between t w o ro ta t ing c o r u n d u m disks. The ce l l suspension is poured in to a reservo i r and is d r i ven by centr i fugal force through the gr ind ing wheels. The homogenate is co l lected in the case and f lows through the out le t tube.

F igure 1 shows the sect ion o f a g r ind m i l l const ructed for large-scale preparat ions . W i t h this mode l a f l ow rate o f 10-12 liters/hr is obta ined at a g r ind ing pressure o f 0.1-0.15 k i l opound/cm 2 . F o r small-scale preparat ions a m i l l can be bu i l t i n w h i c h al l parts are reduced in size by a fac tor o f about 3.

Sand and Mortar (Microscale Preparation). The filtered ce l ls , 0. Ι Ο.5 g wet we ight , are m i x ed w i t h an equal we ight o f quartz sand (washed and ca lc inated , reagent grade) i n a mor tar ( inner d iameter 4-5 cm ) . One v o l u m e (v/w) o f i so lat ion m e d i u m is added, and the m i x tu r e is g r ound w i t h a pestle un t i l a smooth paste is obta ined (about 2 m in ) . A n o t h e r v o lume o f med ium is added, and the g r ind ing is cont inued for a fu r the r 2 m i n . F ina l l y , the homogenized s lurry is transferred w i t h 2 vo lumes o f i so la t i on med ium into microcentr i fuge tubes o f 1.4 m l (Fa. Eppendor f f , H a m b u r g , Germany ) .

4 H . Weiss, G. von Jagow, M. Klingenberg, and T. Bücher, Eur. J . Biochem. 14, 75 (1970).

146 ORGANELLES AND MEMBRANES [18]

F I G . I . Section of the grind mi l l . The upper (D) and lower (E) grinding wheels consist of corundum (Edelkorund 89 A60 M5 V, Fa. Tyrol i t , Schwaz, Austria). The upper wheel (D) is glued into the round plastic disk (C) made from hard polyvinyl chloride. The reservoir (A) is a tube of Plexiglas 30 cm long and is screwed into the disk (C). The lead rings (B) have a weight of 2 kg each. The holding device for the lower wheel (E) is the screw (F), which fits exactly into the central hole. The screw (F) has on the bottom side a square bore, which fits to the end of the movable shaft (J). On its upper side the screw carries a stirrer. The shaft (J) rotates in the ball bearings ( I , K) and is protected from moisture by the covering (G) and the gasket (H). The case (L) is made from hard polyvinyl chloride. The shaft (J) is connected by means of the flexible coupling (M) to the electric motor (N) not shown in the figure (1300 rpm; at least 150 W). The diameter of the case is 180 mm. The lower grinding wheel is rotated by the electric motor. Its central hole is closed by a holding device. The upper grinding wheel does not rotate and can be li fted. Its central hole is open and connected with the reservoir cylinder. The upper grinding wheel is pressed against the lower wheel by the lead weights. The grinding area is reduced by a conical cut of the lower surface of the upper wheel.

[18 ] PREPARATION OF Ν. crassa MITOCHONDRIA 147

The ef f ic iency o f the d i s rup t i on procedures can be checked by i n spect ion in a phase-contrast microscope . The empty cells appear da rk , whereas the broken cells are br ight and show part icu late cel l con ten ts . 4

I s o l a t i o n o f M i t o c h o n d r i a

The m i t ochondr i a are isolated f r o m cells d is rupted by e i ther o f the t w o methods described above b y di f ferent ia l centr i fugat ion between 1200 g (10 min) and 8000 g (30 m in ) . The low-speed sediment is usual ly comple te l y colorless. The high-speed centr i fugat ion may also be perf o rmed at 15,000 g for 15 m i n . W h e n " m i t o c h o n d r i a " are isolated f r o m f rozen cells the t ime for high-speed centr i fugat ion is doub led . The crude m i t ochondr i a l f rac t ion is resuspended in iso la t ion med ium (10% o f the s tar t ing vo lume ) , and the low- and high-speed centr i fugat ions are repeated. Fo r the microscale preparat ion o f m i t o chondr i a the microtubes can be used in a preparat ive refr igerated centr i fuge w i t h appropr iate adaptors .

The y ie ld o f m i t ochondr i a is 2-3 mg/g wet weight o f cel ls. A b o u t 25 % o f the tota l m i t ochondr i a are isolated, w h i c h corresponds to 4 - 6 % o f to ta l ce l lu lar p r o t e i n . 4

Prope r t i e s o f I s o l a t ed N e u r o s p o r a M i t o c h o n d r i a

Respiratory Chain and Oxidative Phosphorylation. The mi tochondr ia isolated by means o f the gr ind m i l l show a high rate o f resp irat ion w i t h py ruva te plus malate, succinate, N A D H , and N A D P H , wh i ch is fa i r ly we l l coupled to ox idat i ve phosphory la t i on . Three phosphory la t i on steps are i nvo l v ed in the ox ida t i on o f pyruva te , and t w o are invo l ved in the ox i da t i on o f succinate, N A D H , and N A D P H . 4 Rotenone, a n t i m y c i n , and K C N inh ib i t the e lectron f l ow at the k n o w n steps. O l i g omyc in , v en tur i -c i d i n , and d i c y c l ohexy l ca rbod i im ide (DCCD) inh ib i t m i t ochondr ia l A T P ase ha l f -max imal l y at 0.2, 0.2, and 1 μglmg m i t o chondr i a l p ro t e in , respect ive ly .The molar rat io o f cy tochromes aa%> c, c i , b-^2-> and b-^% is 1.0:2.9:1.0:0.9:0.9. The content o f c y tochrome aa3 is 0.24-0.34 jutmol/ mg m i t ochondr i a l p r o t e i n . 5

In Vitro Protein Synthesis. The m i tochondr i a synthesize prote ins at a rate o f 5-10 p m o l leucine incorporated per minute and mi l l i g ram m i t ochondr ia l p r o t e i n . 6 The leucine inco rpora t i on is in the same range as that observed w i t h m i t ochondr i a isolated after enzymat ic degradat ion o f

5 G. von Jagow, H. Weiss, and M. Klingenberg, Eur. J . Biochem. 33, 140 (1973). 6 A. von Ruecker, S. Werner, and W. Neupert, FEBS Lett. 4 7 , 290 (1974).

148 O R G A N E L L E S A N D M E M B R A N E S [18]

the cell w a l l , 7 but it does not exceed 4 -8% o f the inco rpora t i on rate observed in vivo in intact cel ls . The in vitro synthesis o f c y tochrome oxidase subunits cou ld be demons t ra t ed . 6

Isolation of Mitochondrial Components. The m i t ochondr i a have been used as s tar t ing mater ia l f o r the iso lat ion o f c y tochrome ox idase , 8 ' 9 cytochrome b,10,11 ATPase comp lex and Fx ATPase , 1 2 ' 1 3 ca rboxya t rac ty l o -s ide-binding p r o t e i n , 1 4 and r i bosomes 1 5 and for the separat ion o f outer and inner m e m b r a n e . 1 6 The prote ins can be prepared f r om mi tochondr i a labeled homogeneously w i t h rad ioact ive amino ac ids . 1 7 This offers the advantage that pro te in is de termined by rad ioac t i v i t y measurements.

The cy tochromes and F t ATPase can be prepared f r o m cells f rozen for several weeks at -20° . The complete subunit pat tern o f the F ! ATPase is obta ined on ly when m i t o chondr i a have been isolated in the presence o f the protease i nh ib i t o r pheny lme thy l su l f ony l f luor ide ( PMSF ) . The presence o f proteinase in the m i t o chondr i a l preparat ion is largely due to the co iso lat ion o f prote inase-conta in ing ves ic les . 1 8 M i t o chondr i a can be freed o f the proteinase vesicles by sucrose density gradient centr i fugat i on . ^ M i t o c h o n d r i a " isolated f r om frozen cells are largely free o f this proteinase, since the proteinase vesicles are d isrupted by freeze-thaw-i n g . 1 H

Immunoprecipitation. For biogenetic s tud ies , 1 7 membrane-bound enzyme complexes can be isolated by immuno log i ca l techniques. Neurospora m i t ochondr i a are a lmost comple te ly dissolved by T r i t o n X-100. F r o m the lysate, c y t ochrome ox idase , 1 9 a free subuni t o f c y t ochrome ox idase , 1 9 and the ATPase c o m p l e x 1 2 , 1 3 have been immunoprec ip i t a t ed by means o f specific ant isera. I n these studies the microscale preparat ion o f m i t o chondr i a has been appl ied to smal l amounts o f cells h ighly labeled by in vivo i nco rpora t i on o f radioact ive amino ac ids . 1 7

7 W. Sebald, Τ. Bücher, Β. Olbrich, and F. Kaudewitz, FEBS Leu. 1, 235 (1968). 8 H . Weiss, W. Sebald, and T. Bücher, Eur. J . Biochem. 2 2 , 19 (1971). 9 H. Weiss and W. Sebald, this series, Vol . 53 [ I I ] .

1 0 Η. Weiss and Β. Ziganke, Eur. J . Biochem. 41 , 63 (1974). 1 1 H . Weiss and B. Ziganke, this series, Vol . 53 [23]. 1 2 G. Jackl and W. Sebald, Eur. J . Biochem. 54, 97 (1975). 1 3 W. Sebald and G. Wi ld , this volume, Article [41]. 1 4 M. Klingenberg, Η. Aquila, P. Ricchio, Β. B. Buchanan, W. Eiermann, and H . Hack

enberg, in "E lec t ron Transfer Chains and Oxidative Phophorylation" (E. Quagliariello et al., eds.), p. 431. North-Hol land Publ., Amsterdam, 1975.

1 5 W. Neupert, G. Hallermeyer, and R. Michel, this series, Vo l . 56 [8]. 1 6 W. Neupert and G. D. Ludwig , Eur. J . Biochem. 19, 523 (1971). 1 7 W. Sebald, S. Werner, and H . Weiss, this series, Vol . 56 [5]. 1 8 R. Michel, A. L iebl , A. Hartmann, and W. Neupert, Hoppe-Seylers Z. Physiol. Chem.

357,415 (1976). 1 9 S. Werner, Eur. J . Biochem. 43, 39 (1974).

A U T H O R I N D E X 823

A u t h o r I n d e x

Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although his name is not cited in the text.

A

Aas, Μ, 9, 14(10), 75, 76(31) Aberdeen, Ε., 40 Abram, Α., 805 Abrams, Α., 298, 337, 397, 434, 497, 499(99),

515, 802 Ackre i l , Β. A. C , 460, 461 Addanki, S., 31 , 562 Addink, A. D. F., 89 Adelson, 176 Adler, L. W., 498, 502(113) Adolfsen, R., 301, 483, 485(33a), 517(33a),

5l8(33a) Adr ian. R. H . , 656 Afzelius, Β. Α., 77, 78(44) Agin, D. P., 604 Agnila, H . , 303 Ahlers, J . , 796 Ahonen, P., 60 Aithal , Η. N . , 176, 183, 186(5), 187(5) Aka i , Α., 434 Akerboom, Τ. P. M . , 20, 21(128), 243, 244 Akerman, Κ. E., 571(31), 572, 573(31) Akerman, Κ. E. O., 559 Alberty, R. Α., 237 Aldridge, W. N. , 507, 508, 509, 518 Alexandre, Α., 641, 642(16), 643(16), 645 A lkony i , I . , 221 Al len, J . R., 76 Al lred, J . B. , 210, 211 A l m , B., 406 Almazan, A. M . , 405 Altendorf, K., 427, 429(11), 431(11), 497,

498(104), 499(104), 502(104), 555, 676, 677(5), 678(4, 6), 679(4, 5), 800

Alvares, A. P., 282 Amar-Costesec, Α., 130 Ames, Β. N . , 718 Ammoumi , Α. Α. , 17

Amzel , L . M. , 336 Anan, F. K., 18 Anderson, D., 209 Anderson, L. E., 252 Andreo, C. S., 516 Andreol i , Τ. E., 382 Angielsky, S., 11, 12 Angliker, E., 460 Angl in, Μ., 31 Anraku, Υ., 516, 794, 795, 796, 805 Apelmans, F., 102 Aquila, Η , 148 Arai , Τ., 513, 514 Arie l , Ν., 504 Ariga, Τ., 371 Aringer, L. , 20, 21(126) Ar ion, W. J . , 730 Arndt , U . W., 334 Arntzen, C. J . , 493 Arrhenius, E., 13 Arselin-Dechateaubodeau, G., 157 Asai, J . , 397 Asami, K., 383, 401, 402(o, q), 403, 406,

407(16, 25), 408, 413, 414(2, 17), 426 Asano, Α., 176, 177, 180(8), 181(8), 182(8),

183(8), 185, 186(4, 5, 7), 187(5) Asghar, S. S., 497(108), 498, 499(108),

502(108) Ashkenazi, I . , 571(22), 572, 573(22) Atkinson, D. E., 231, 233 Auslander, W., 565, 566(26) Avi-dor, Y., 504 Avran, P., 709 Avron, M . , 229, 407, 483, 503(29), 550, 551,

552(8), 555, 557(24), 559, 563, 564, 565, 566(28), 571(37), 572, 573(37), 666

Azoulay, E., 805 Azz i , Α., 427, 485, 497, 499(90), 500, 501

(122, 123, 124), 559, 571(27), 572, 573 (27), 667, 694

Azzone, G. F., 12, 46, 666, 667, 673(8)

824 A U T H O R I N D E X

Β

Baak, J . Μ., 164 Baarda, J . R., 434, 497(106), 498, 499(99,

106), 515 Baccarini-Melandri, Α., 563 Bader, P., 456 Bäuerlein, Ε., 536(6, 8, 9) Baginski, Ε. S., 311 Baginsky, Μ. L. , 700 Bahr, D. P., 559, 571(24), 572, 573(24),

579(24), 586(24), 667 Bahr, J. Τ., 25 Baker, P. F., 571(18), 572, 573(18) Baker, R., 177, 180(8), 181(8), 182(8), 183(8) Bakkenist, A. R. J . , 402(k), 403, 404, 405,

426 Bakker, E. P., 229, 466, 541, 551, 555 Balcavage, W. X. , 27, 28(186), 140(4, 5, 6),

141, 149, 151, 152(1, 2), 423 Baldassare, J . , 454 Baldwin, C. L. , 477(6), 478, 481(6) Bal l , E. G., 75 Ballantine, J . , 185 Baltcheffsky, M. , 559 Baltscheffsky, H . , 503 Baltscheffsky, M. , 503, 577, 585(48), 694 Bandlow, W., 89 Banerjee, R., 700, 706(8), 710 Bangham, J. Α., 510 Bäräny, Μ., 566 Barbehenn, Ε. Κ., 209 Barber, J . , 449, 453(50) Barbour, R. L . , 402(g), 403, 404, 405, 412,

413(12), 426 Bardawill , C. J . , 81 , 309, 312(4) Barden, R. E., 70 Barlow, C., 217 Barnard, T., 16, 17(90), 66, 67, 76(15), 77

(15), 78 Barnes, Ε. M . , 497, 499(101) Barnes, Jr., Ε. M . , 551, 680, 685, 687(11) Barnes, R., 137 Baron, C., 397, 434, 497, 499(99), 515, 802 Barrett, R. H . , 340, 341(8), 342(8), 516 Barsky, E. L. , 756, 761, 761(16), 762(16),

763(16), 764, 768(9, 16), 770 Bartoov, B. , 11 Bashford, C. L . , 576, 579, 584(46) Basselt, D., 19, 20(123)

Bates, R. G., 228, 680 Batra, S., 14, 15(76) Batra, S. C., 60 Bauchop, T. , 164 Baudhuin, P., 39 Bauer, P., 89 Baum, Η., 478, 479(7), 482(7), 487 Baylor, S. M . , 695 Beattie, D. S., 13 Beaufay, H . , 39, 130, 132 Bechara, E. J . H . , 536 Beck, J. C., 27 Bednärovä, Η., 27, 140(3), 141 Beechey, R. B. , 105, 106, 303, 317, 338,

339(6). 342(6, 17), 343, 344, 380, 382, 383, 427, 428, 434(18), 478, 479(7), 481, 482, 483(23), 485(4), 487, 497, 498(14, 23), 499(23. 79, 80, 85), 500(79, 80), 502(23), 504, 505, 506, 512(154), 515, 782

Beem, E. P., 568, 641 Begg, R. W., 465 Beigel, M. , 516 Beinert, H . , 10, 455, 456, 457, 460 Bell , J . J . , 10 Bellum, A. S., 10 Bensch, K., 19, 20(124) Ben-Shaul, Y., 497, 499(93) Bentzel, C. J . , 551 Berden, J . Α., 407, 461, 462(40) Berends, W., 528, 529(16) Berg, D. H . , 435, 439(8), 440(8), 443(8),

445(8) Berger, J . , 445(43), 447 Berger, Μ. Α., 558, 559, 560(19) Berger, R., 59 Berger, R. L. , 619, 621, 622(10) Berger, S. J . , 207, 217 Bergmeyer, H . -U . , 200, 205, 239, 244(7),

294 Bergstrand, Α., 102 Bering, C. L. , 517 Berl , S., 51 Bernson, V. S. M . , 16, 76 Berry, Ν. M . , 203 Bertina, R. M . , 482, 483(21), 485(21) Bertol i , E., 427, 434(8), 497, 499(87) Beyer, R. E., 105, 249, 257, 498, 500(109,

120), 502(109) Bezanilla, F., 695

A U T H O R I N D E X 825

Bhalia, R. C , 60 Bhatnagar, P., 492, 493(65) Bhattacharyya, P., 497, 499(101), 536 Biemann, K., 530 Bienvenüe, Α., 519, 524(4), 527(4) Bigon, E., 400, 402(f). 403, 407(7) Birdsall, N . J . M . , 701 Birkett, D. J . , 489 Bittman, R., 438, 439, 440(29), 441, 448(24,

29) Björkhem, I . , 10 Blackman, G. E., 465 Blackmore, P. F., 302 Blair, J . E., 340, 341(9), 342(9), 343(9) Biair. P., 305 Blair, P. V. , 308, 309 Blanchaer, M . C , 13, 14, 91 Blanchy. B . , 114 Blanco, Α., 12 Blaustein, Μ. P., 571(23), 572, 573(23) Blouin, Α., 39 Böhm, Μ., 341, 342(12), 343(12). 344 B0hmer, Τ., 211 Boer, P., 89 Bogin, Ε., 177, 183, 187(9), 188(9), 189,

190(28), 192(28), 194(28), 516 Boguslavsky, L. I . , 773, 775, 776 Boheim, G., 571(40), 572, 573(40) Bolender, R. P., 39 Bonner, W. D., 25 Bonner, Jr., W. D., 139 Bookelman, H . , 244 Booth, R. F. G., 59 Boothroyd, B., 505 Borowitz, G., 438, 439(24), 448(24) Borowitz, I . J . , 438, 439 Borst, P., 9, 25(12), 26(12), 28(12) Bortignon, C , 491, 492(59) Bosmann, Η. B. , 18 Botham, Κ. M. , 12 Boyd, G. S., 10 Boyer, P. D. , 245, 246, 248, 252, 253, 254

(16), 255(16), 257(16), 258(11, 16), 259 (11, 16), 261, 286, 287(11), 463

Brabcova, J . , 89 Bracey, Α., 465, 467(16) Bradbeer, J . W., 516 Braden, B. , 444 Bradford, Μ. M . , 277 Bradley, L . B. , 277, 320, 409, 412(6)

Brady, B. L . , 494 Brag, P. D., 298, 299(7) Bragadin, M . , 427, 500, 501(122, 123) Bragg, P. D., 382, 497, 498, 499(96), 500(96),

502(115), 515, 532, 788, 790(6), 791, 792, 793, 794(11, 12, 13), 795, 796(6, 13), 797(6, 7, 10, 13), 798, 799(6, 10, 12), 800, 802(1), 805

Bramhatl, J . S., 571(16, 17), 572, 573(16, 17) Brand, J . S., 209 Brand, K., 291 Brand, L. , 576 Brand, M . , 226 Brand, M . D., 17, 636, 640(9), 641, 642(12,

13), 643(12, 13), 645(12, 13), 646(13), 649(13)

Brandolin, G., 519, 522(2), 524(2, 3), 526(3), 527(2), 531(3), 532(2, 3)

Brandt, K. G.. 272 Brawand, F., 204 Brdiczka, D.. 89 Bremer. J . , 212 Brewer, G., 571(39). 572, 573(39) Brewer, G. J . , 532 Bridges, B. J . , 15, 16(78) Brierley, G. P., 667, 680 Brinigar, W. S., 536, 537 Brink, T . W., 498, 500(120) Briquet, M . , 27, 149, 515 Britten, A. Z., 571(16, 17), 572, 573(16, 17) Brocks, D. G., 208, 214 Brodie, A. F., 164, 176, 177, 178, 179, 180,

181(8), 182(6, 8), 183, 184, 185, 186(4, 5, 7, 24), 187(5, 9), 188, 189, 190(28), 192(25,27, 28), 193(11, 12), 194(11,28), 195, 196(12, 32), 197, 263, 272, 497, 498(98), 499(98, 98a), 516

Bronk, J . R., 301 Brooker, R. Μ., 477(6), 478, 481(6) Brooks, J . C., 298, 317, 343, 383, 400,

402(c), 403, 404, 426, 743 Brosnan, J . R., 200 Broughall, J . M. , 434,497, 499(85), 504, 505,

506(151) Brown, J . G., 205, 206(14), 209 Browning, Ε. T. , 213 Brufani, M . , 502, 504, 505(130) Bruni , Α. , 400, 402(f), 403, 407(7, 8), 427,

428(14), 491, 492(59), 497, 498, 499(83), 704

826 A U T H O R I N D E X

Bruns, F. Η., 294 Brunt, R. V., 140(7), 141, 149 Bryan, D. M . , 252, 258(11), 259(11) Bryant, C. P., 512 Bryla, J . , 14, 203, 208, 211 Buchanan, Β. B., 148, 303 Buchanan, P. J . , 138 Bucher, T. , 139, 291 Buchwald, Η. Ε., 585 Budd, Κ., 664 Büchel, Κ. Η., 468 Bücher, Τ., 26, 145, 147(4), 148 Buetow, D. Ε., 138 Bukowiecki , L. , 76, 77(41) Bullock, G. R., 13, 79(10), 80 Bulos, B. , 762 Bulos, Β. Α., 23, 24 Bülten, E. J . , 518 Bulychev, Α., 78 Burchiel, K., 415 Burckhardt, G., 571(15), 572, 573(15),

580(15), 585(15) Burgos, J . , 455 Burk, D., 294 Burns, D. D., 497, 499(94), 516(94) Burseil, E., 4, 23(1) Burstein, C., 113 Burt, C. T. , 566 Burton, W. C., 221 Bustamante, E., 330, 409, 737 Bustamente, E., 515 Butler, K. W., 78 But l in , J. D., 794, 797(15) Butow, R. Α., 19, 138 Buttery, P. J . , 209 Bygrave, F., 283 Bygrave, F. L. , 22, 649 Byington, Κ. H. , 302

c Cahill, F. D., 31, 562 Cahn, T. L . , 408 Cain, K., 510, 511 Cammer, W., 10, 11(18) Campbell, L . C , 25 Cannon, B., 16, 17, 66, 69(9, 10), 70(9, 10,

16), 71 , 72(16, 25), 74, 75, 76, 77(15, 25, 42), 78,318

Cantley, L . C , 300, 301, 489, 490, 491, 492(55, 56)

Capaldi, R. Α., 351 Capano, M . , 14 Caplan, S. R., 226, 551, 555 Carafoli, E., 7, 8, 12, 14, 23, 24(151), 27,

439, 440(37), 441(37), 443(37), 448, 667 Cardini, C. E., 174 Carlson, N . , 555 Carlsson, C , 301, 302(36), 401, 407, 517 Carmeli, C , 301, 407 Camay, L. , 571, 572, 573(10), 578(8) Carofoli, E., 650 Caroni, P., 439, 440(37), 441, 443(37), 448

(37) Carpenedo, F., 491, 492(59) Carrol l , R., 699 Carrol l , R. C , 728 Carter, Ε. E., 13, 79(10), 80 Carter, J. Α., 207, 217 Carver, Μ. Α., 343 Casida, J . E., 455, 456, 457(5, 7) Cassadio, 563 Cassuto, Y., 76 Casteels, R., 60 Caswell, A. H . , 668, 672 Cattell, K. J . , 105, 303, 380, 382, 383, 427,

428(13), 497, 515 Catterall, W. Α., 88, 284, 298, 299(12), 320,

323, 324, 329, 333, 354, 382, 481, 482, 737, 738

Cederblad, G., 211 Cellai, L . , 504 Cerna, H . , 79 Cerrini , S., 504 Chaffee, R. R. J . , 76, 559, 571(24), 572,

573(24), 579(24), 586(24), 667 Chaikoff, I . L. , 135 Chambon, H . , 152, 153(16), 155 Champness, J. N. , 334 Chan, S. H . P., 402(g), 403, 404, 405, 407,

412, 413(12), 426 Chan, T . L . , 87, 88(20), 331, 737 Chance, B., 14, 40, 57, 59, 64, 65, 81, 89,

107, 136(2), 137, 138, 153, 175,217, 225, 291, 467, 559, 565, 574, 577, 578, 585 (48), 614, 619, 622(8), 671, 694

Chandler, D. E., 449 Chandler, W. K., 695 Chang, Τ. M . , 301, 478, 481, 482(13), 485(8,

13) Chappell, J . B. , 12, 13, 14, 17, 20, 23, 24

(146), 27, 61, 143, 287, 435, 438(5),

AUTHOR INDEX 827

439(5), 440(5), 442(5), 443(5), 444(5), 446(5), 453, 644, 674

Charnock, J . S., 579 Chase, F. Α., 211 Chavin, W., 11 Cheah, K. S., 12, 13(49) Cheema-Dhadli, S., 14 Chen, C.-H., 641 Chen, R. F., 107 Chen, Τ. Τ. T. , 9, 10, 11(13, 19) Cheng, W., 694 Cheng, W. K., 577, 585(48) Chien, T . F., 316, 703, 704(22), 730, 732(29),

733(29), 734(29) Chiesara, E., 21 Chmieliauskaite, V., 555 Chrambach, Α., 192 Christensen, Ε. L. , 25 Christensen, R. Ζ., 212 Christiansen, Ε. Ν., 16, 66, 69(11), 70(11,

14), 71(14), 77 Christiansen, R. Ο., 113 Chu, L . V., 229 Chude, O., 111(19), 112 Chung, A. E., 275 Cierkosz, Β. I . T., 111(18), 112 Cilento, G., 536 Cintron, N. , 300 Ciott i , Μ. M. , 263 Clark, D. J . , 497, 499(95) Clark, J . B., 17, 18, 21(101), 51, 52(1), 53,

59 Clark, L . C , 569 Clark, W. ML, 536 Clarke, D. D., 51 Clegg, R. Α., 457 Cleland, K. W., 119, 121(2) Cleland, W. W., 70 Cl intron, Ν. M . , 412 Cobley, J . G., 456, 457 Cockburn, Α., 634 Cockrel l , R. S., 452, 666, 668, 672, 673(2),

674(23), 675(2) Coffey, J . W., 39 Cohen, G. N. , 802 Cohen, J . , 423 Cohen, L . , 294 Cohen, L . B. , 570, 571, 572, 573(12), 574,

575, 576(6, 7), 578(6), 580, 581, 689, 693, 694, 695(9)

Cohen, N . S., 176, 177, 178, 180(8), 181(8),

182(8), 183(8), 184, 186(5), 187(5), 188(11), 193(11, 12), 194(11), 195, 196(12, 32), 197(12), 497, 499(98a)

Cohen, P. T . , 271 Cohn, D., 707 Cohn, Μ., 252, 463 Cohn, W. Ε., 538 Coleman, J . , 401, 402(q), 403, 406(16),

407(16) Coleman, J . D., 25 Coleman, J . O. D., 508 Coles, C. J . , 456, 460 Coll , K., 215 Colleran, Ε. M . , 137 Colonna, R., 46 Colowick, S. P., 681 Colson, A . -M . , 515 Comes, P., 19 Comte, J . , 89, 99, 104(3, 4), 114 Connelly, J . L. , 482, 483(22) Conover, Τ. Ε., i06, 736, 737(3) Contessa, A. R., 427, 428(14), 497, 498,

499(83) Conti , F., 571(1 la, 1 lb ) , 572, 573(1 la, l i b ) ,

580(1 la) , 581 Cooper, C , 463 Cooper, S., 551 Cooper, Τ. Α., 536(4), 537 Cooperman, B. S., 559, 574 Cordes, A. W., 492 Corkey, Β. E., 200, 201, 202, 203, 205(2),

206, 207(2), 208(2), 209(2), 211(2) Coruzzi., G., 161 Coty, W. Α., 298, 299(12), 324, 333, 354 Coulet, P. R., 745 Cowan, M . C., 657 Cox, G. B. , 788, 794, 797 Cox, H . C , 528, 529(16) Crabtree, H . G., 296 Cramer, W. Α., 532, 535 Crane, F. L . , 113, 313, 385, 456, 517, 794,

796, 797(15) Crankshaw, D. L . , 498, 500(109, 120),

502(109) Cremona, T . , 272 Criddle, R. S., 414(1), 415, 416, 420 Crofts, A. R., 404, 442(40), 444, 541, 544(4),

558, 569, 571(32), 572, 573(32), 585(3, 32), 586, 667, 674

Crompton, M . , 12, 14, 25 Crook, Κ. E., 501, 503(126), 512

828 A U T H O R I N D E X

Cross, R. J . , 462 Crosse, R., 505 Cuatrecasas, P., 113, 524 Cubero Robles, E., 812 Cunarro, J . , 466, 471(32) Curthoys, N. P., 12

D

Dabbeni-Sala, F., 400, 407(8) Daikiw, V. , 438, 439(24), 448(24) Daliner, G., 296 Damon, S., 211 Dancshazy, F., 760, 761(16), 762(16), 763

(16). 764(16), 768(16), 770(16) Dancshazy. Z., 772 Daniel, Ε. E., 60 Danielson, L. , 412, 413 Danks, S. M. , 23 Danon, Α., 497, 499,(102) Dashti, N . , 88 Datta, Α., 192, 297, 301, 304, 307(1), 313,

316, 317, 320, 328(4), 408, 413(5), 736. 737(2), 746

David, Μ. M. , 81, 309, 312(4) Davies, D. R., 142 Davies, P. L. , 298, 299(7), 788, 792, 795,

797(10), 799(10), 800(10), 805 Davila, Η. V. , 570, 571, 572, 573(12), 574(6,

7), 575(6, 7), 576(6, 7), 578(6), 580(6). 581(6, 7), 693, 695(9)

Davis, B . D., 677 Davis, E. J . , 14, 241 Davis, R. C , 502(136), 503 Davis, R. H . , 144 Dawson, A. P., 509, 510(164), 518, 584 Day, D. Α., 25 Deak, S. B., 501, 503(126) de Alvare, L. R., 11 Deamer, D. W., 541, 544(4) Dean Nelson, B., 313 de Boer, B. L . , 400, 402(p), 403, 406(9), 413,

414(18), 426 Debras, G., 60 Decker, G., 330 Decker, G. L. , 89, 90(16), 98(16) de Deken, R. H . , 465, 470(21) de Duve, C , 39, 102, 103

Defaye, G., 519. 520, 521(5), 522, 524(2), 527(2), 531(5, 6, 8), 532(2, 5, 6, 8)

de Goede, G. W., 26 de Guzman, Ν. T., 438, 440(23), 443(23),

446(23), 447(23) de Jerphanion, M.-B.. 402(h), 403, 404, 405,

413, 422, 425(1), 426(1) Dei Campo, F. F., 532 DeLceuw, G. Α., 203, 217 DeirAntone, P., 46 Delorme, P., 17 DeLuca, H . F.. 11, 12 Demetriou, J. M. , 515 Dennis, S. C , 17, 21(101), 51. 53. 59, 60 Denton, R. M . , 15, 16 de Pierre, J . , 77, 78(44) de Serres, F. J . , 144 Deters, D., 142, 155 Deters, D. W., 298, 299(10), 489, 490(43),

491(43) Deutch, E. W., 466, 469(28) Devaux, P. F., 519, 524(4), 527(4) Devine, C. E., 65 Devlin, Τ. E., 79 Devlin, Τ. M. , 22, 463 Dilley, R. Α.. 517 Dim, Κ. Η., 466, 469(29) Di Pietro, Α., 743, 746(7) Dolecki, G., 209 Domenech, C. E., 12 Domissiere, J . , 303 Douce, R., 25 Douraghi-Zadeh, D., 496, 498(75) Dow, D. S., 13, 28, 29, 30(2), 31(2, 3), 32(2) Downie, J. Α., 641 Drachev, L. Α., 587, 588, 590(9), 592, 594,

595, 597(9), 599(9), 601, 603, 604, 609(1, 2), 610(1), 715, 752, 755, 756, 757, 760, 761(16), 762( 16), 763, 764, 765, 768, 769, 770, 771, 772, 773, 774, 776

Drahota, Z., 66, 69(8), 70(8), 74, 75, 76, 77, 78, 89, 317

Drysdale, G. R., 463 Duee, E. D., 531 Duell , Ε. Α., 139, 140(1), 141, 149, 157(6) Duffy, Τ. Ε., 218(e), 219 Dumont, J . Ε., 18 Duplaa, Α. Μ., 530, 532(18) Durbin, R. D., 492, 493(65) Duszynski, J . , 77 Duvert, M . , 89

AUTHOR INDEX 829

Ε

Ebel, R. Ε., 301, 328, 48 ! , 482(18) Ebner, Ε.. 402(i), 403, 404, 405, 413, 426 Edmondson, D. E., 455, 456(8) Edwards, D. L . , 25, 26 Edwards, Ο. E., 512 Edwards, Ρ. Α., 341, 342(14) Edwards, S. W., 136(3), 137 Eiermann, W., 148 Eisenbach, M . , 551 Eisenberg, M . , 435. 438(10) Eisenhardt, R. H . , 175 Eklund, H . , 334 Ellerman, J . , 221 Ell iott , Β., 505, 518 El lory. J. C , 579 Elsden, S., 164 Elvehjem, C. Α.. 462 Eizinga, M. , 389, 390(9) Emanuel, C. F., 135 Eneroth, P., 20, 21(126) Engelhardt, V. Α., 291 Enns, R., 415, 416 Epel, D., 59 Ephrussi, B. , 155 Ergle, Jr., D. H . , 252 Eriksson, L . E. G., 78 Ernster, L. , 10, 13, 21, 22(138), 102, 105,

106, 107, I I I , 112, 124, 141, 167, 225, 261, 262(1), 263, 265, 266(1), 267(1), 269(1), 270, 271(1), 273(12), 274(1), 275, 277, 296, 301, 302(36), 313, 383, 393, 401, 402(o, q), 403, 406, 407, 408, 412, 413, 414(2, 17), 426, 482, 501, 517, 726, 732, 787(1), 788

Erwin , V. G., 81 , 91 Eschenbrenner, E., 207 Eska, T. , 88, 333 Essig, Α., 226 Estabrook, R. W., 10, 11(18), 64, 65, 81 ,

143, 463, 668, 749 Estrada-O, S., 435, 439(4), 440, 441(4, 38),

442(38), 444(4), 450(4), 453(4) Evans, D. Α., 138 Evans, Jr., D. J . , 805 Evans, R. H . , 445(43), 447 Evans, R. M . , 19, 20(120) Eytan, E., 415, 701, 704, 705(29), 706(29) Eytan, G. D., 707, 708, 709(35), 728

F

Fabbri, E., 483, 503(31) Fagan, J . , 518 Falcone, Α. Β., 463 Fantes, Κ. Η., 505 Farmer, I . S., 164 Farr, A. L . , 102, 249, 257(9), 320, 330, 345,

346(5), 365, 409 Farrant, J . , 32 Farron, F., 298 Fedeli, W., 504 Feingold, D. S., 532, 533(5) Ferguson, J . . 299 Ferguson. S. J . , 401, 406(22), 407, 483, 489,

490. 503(33), 507(33), 517, 518 Ferreira, Ν. P., 480 Fertel, R., 221 Fessenden, J . M . , 105, 106. 385, 392, 398 Fessenden-Raden, J. M. , 303, 380, 383, 390,

398 Fields, K. L. , 532, 533(2) Filiingame, R. H . . 415, 427, 428, 429(9),

430(9), 431, 432(20), 433(20), 434(20), 497

Finkelstein, Α., 466 Fiolet, J. W. T. , 229, 801 Fioranvanti, R., 571(1 lb ) , 572, 573(1 lb ) ,

581(1 lb) Fishbein, P., 40 Fisher, A. B. , 19, 20(123) Fisher, L . W., 701, 705(15), 706(15) Fisher, R. F., 514 Fisher, R. J . , 396, 498, 502(116), 788 Fisher, R. R., 263, 271 Fiske, C. H . , 191, 192, 422 Fitzpatrick, D. F., 60 Flatman, P., 447 Fiatmark, T. , 5, 16, 65, 69, 72(21), 75, 76(4),

78(19, 33) Flavell, R. Α., 9, 25(12), 26(12), 28(12) Fleet, G. W. J . , 526 Fleischer, B. , 39, 102, 311 Fleischer, S., 7, 20(6), 28, 29, 31 , 32(2, 10),

33, 35(2), 36(2), 37(2), 39, 42. 82, 102, 272, 311, 700, 718

Fluharty, Α., 471 Foa, P. P., 311 Folch, J . , 430, 432, 762 Folch-Pi, J . , 415, 417(9)

830 A U T H O R I N D E X

Folkers, K., 456 Font, B. , 99, 114, 743 Ford, C , 216 Ford, G. D., 60 Foster, D. W., 211, 212 Foury, F., 515 Fowler, S., 39 Franke, W. W., 19 Frankel, Ε. P., 221 Franks, Α., 335 Frazier, H . S., 656 Freedman, J . C., 580, 584(52), 691, 694 Frehn, J . L. , 66 Fridovich, I . , 170 Friedberg, I . , 497, 499(93) Friedl, P., 799, 801, 810(6) Friedrichs, D., 215 Friend, D. S., 203 Friend, W. H . , 139 Frigeri, L. , 400, 402(f), 403 Fritz, I . B., 76 Frolov, V. N . , 587, 594, 595(10), 604, 609(2),

752, 755(2), 756(2, 4), 760(4), 763(2, 4), 765(4), 768(2, 4), 769(2), 770(2, 4), 771(4), 772(2), 775

Frumkin, A. N. , 773, 775(28), 776 Fujimoto, K., 468 Fukushima, K., 513 Furlan, R., 400, 407(8) Futai, M. , 168, 382, 794, 795(18), 796, 800,

805(2), 806(2), 808 Futar, M. , 489, 491(52)

G

Gadkari, D., 164 Gaensslen, R. E., 563 Gaffney, B. J . , 527 Gains, N . , 584 Galante, Y., 302, 497, 499(89), 516(89), 743 Galante, Υ. M . , 308, 314(1), 315(1) Galmiche, J . ML, 300 Gambino, A. M . , 10 Gamble, J . G., 21 Gamble, J . L . , 463 Gamble, R. L . , 650 Gamow, R. I . , 657 Gams, W., 494

Garbus, J . , 12, 13(46) Garland, P. B., 21 , 27, 141, 209, 457, 638,

641 Garret, Ν. E., 736, 737(4) Garrett, Ν. E., 300, 377, 378(2, 3, 4), 379(2),

380(2, 4) Garton, G,, 465 Garty, H . , 551 Gassman, D. G., 510, 511(177) Gautheron, D., 114 Gautheron, D. C , 89, 99, 104(3, 4), 114,

743, 745, 746(7) Gavo, G. Α., 462 Gavrikova, Ε. V., 127 Gayet, J . , 17 Gazzotti, P., 66, 69(8), 70(8), 439, 440(37),

441(37), 443(37), 448(37) Gear, A. R. L. , 516 Gennis, R. B., 532 Gensch, Κ. H . , 536(7) George, P., 237 Gergely. J . , 402(m), 403, 405 Ghazarian, J. G., 11, 12 Gheradina, P., 667 Gherardini, P., 559, 571(27), 572, 573(27) Ghosh, P. B. ,489 Gianetto, R., 102 Gibson, F., 788, 794, 797 Gibson, Q. H . , 175 Gilchrist, M. J. R., 532, 533(4), 536 Gimpel, J. Α., 203 Gingras, G., 760 Giordano, G., 805 Girault, G., 300 Gitler, C., 415 Glass, R., 445(43), 447 Glenn, J . L . , 385 Glinski , R. P., 512 Glonek, T. , 566 Glynn, I , M. , 287 Gmay, P., 11 Godfrey, S. S., 12 Godinot, C., 99, 114, 743, 746(7) Goffeau, Α., 27, 141, 149, 283, 402(j), 403,

413, 426, 515 Goldberg, N . D., 205, 206(15), 207(18) Golder, R. H . , 297 Goldin, S. M . , 701, 702 Goldman, D. E., 582 Goldring, J. M . , 571(23), 572, 573(23)

AUTHOR INDEX 831

Goldsby, R. Α., 465, 470(23) Goldstein, Α., 473 Goldstein, L. , 12 Goloveshkina, V. G., 127 Gomez-Fernandez, J . C., 400, 402(s), 403 Gomez-Lojero, C , 435, 439(15), 440, 441

(38), 442(38), 450(15), 452, 453(15), 454(15)

Gomez-Puyou, Α., 317, 401, 402(r), 403, 435, 439(15), 440(15), 450(15), 452, 453(15), 454(15), 516

Gomperts, B. D., 447 Gonze, J . , 18, 19, 40 Goodwin, C. W., 40 Gornau, A. G., 81 , 309, 312(4) Gosalvez, M . , 292 Goto, T., 486 Gottardo, G., 10 Gould, J. M . , 509 Gould, R. F., 466, 469(27) Graber, P., 569 Gradmann, D., 657, 662 Graf, T . , 350, 351(9) Graham, N. , 578 Grav, H . J . , 16 Grav, H . J . , 6 6 , 68, 69(11, 17), 70(1 i , 14, 16,

17), 71 , 72(16), 75(24), 77(11) Graven, S. N. , 435, 439(4), 440(4), 441(4),

444(4), 450(4), 453(4) Gray, C. T . , 183 Graziott i , P., 14, 62, 485 Greaser, ML L., 402(m), 403, 405 Grebe. K.. 303 Green, Α. Α., 327 Green, D. E., 89, 385, 462, 466, 719, 720 Greenawalt, Η. N . , 115, 116 Greenawalt, J . W., 25, 79, 81 , 84, 87, 88,

89, 90(1, 16), 91 , 93, 98(16), 139, 144, 321, 330, 331, 408, 737

Greenlee, W. J . , 510, 511(177) Greenwald, R., 446 Greiff, D., 31 Greksäk, M . , 27, 140(3), 141 Grif f i ths, D. E., 343, 427, 434(8), 495, 497,

499(85, 86, 87), 502, 503(128), 504, 506, 512(128), 518, 716

Grinius, L . , 555 Grinius, L . L . , 262, 273, 597, 601(13), 602,

811 Griniuviene, B., 555

Grinvald, Α., 571, 574(7), 575(7), 576(7), 580, 581(7), 689, 693, 694(1), 695(9)

Groen, A. K., 244 Gromet-Elhanan, Z., 452, 555, 563, 571(38),

572, 573(38) Groot, G. S. P., 149, 157(8) Groot, P. Η. E., 12, 13(50) Grossman, S., 456, 457 Grotyohan, L . W., 203, 212(7) Groves, P. M . , 657 Grunwald, T. , 550, 551, 552(8), 563, 564,

565(23) Grynne, B., 75, 76(31) Guarnieri, M . , 140(6), 141, 149, 151(2),

152(2) Gudz, Τ. I . , 515 Günther, Τ., 796 Guerieri, F., 551 Guerin, Β., 89, 157 Guerin, Μ., 157 Guerra, F. C., 20 Guerrieri, F., 618, 623(12), 624, 625(12),

626(12), 627 Gugeshashvili, Μ. I . , 773, 775(28) Guil lory, R. J . , 263, 514 Gunter, Τ. F., 438 Gustafsson, J . Α., 10 Gutfreund, Η., 245, 246 Gutman, Μ., 455, 456, 457(5), 459 Gutnick, D., 176, 801 Gutnick, D. L. , 272, 360, 363(5), 489, 490

(51), 498, 502(111), 503(111), 794, 795 (17), 796(17)

Gutnik, D. L . , 805 Gutowski, G. E., 512 Guy, D. G., 210, 211 Guynn, R. W., 207, 212, 237, 244

Η

Haarhoff, Κ. N. , 674 Haas, D. W., 124 Haavik, A. G., 79(9), 80, 716 Hackenberg, H . , 148 Hackenbrock, C. R., 89, 411, 412(9), 413(9),

737 Hackney, D. D. , 252, 253(12), 261 Hadler, Η. I . , 515 Hagihara, B. , 14, 27, 40, 141, 149, 151(7),

157(7), 171, 508

832 A U T H O R I N D E X

Hahn, P., 66, 69(8), 70(8), 74, 75(30c), 76 Haksar, Α., 9, 10(15) Hal l , C , 456 Hal l , D. O., 25, 139, 144 Hal l , J. D., 113 Hal l , Τ. Α., 65 Hallermeyer, G., 148 Hallett, M. , 571(11), 572, 573(11), 580(11) Halperin, M . L . , 14 Hattrop, M. , 26 Hamer, F. M . , 574 Hamil l , R. L . , 435, 439(6), 440(6), 443(6),

493, 494(69), 495(69), 513(69). 518 Hamilton, J. Α., 444 Hammes, G. G., 300, 301, 489, 490, 491,

492(55, 56) Hammill , R. H . , 435, 439(8), 440(8), 443(8),

445(8) Hammon, Κ. M. , 411, 412(9), 413(9), 737 Hanes, C. S., 284, 287 Hanning, K., 113 Hansch, C , 466, 469(28. 29) Hansford, R. G., 14, 23, 24, 27, 210, 641 Hanson, R. L. , 382, 805 Hanson. R. W., 15 Hanstein, W. G., 302, 303, 497, 499(89),

516(89), 716, 743 Harding, B. W., 10 Hare, J. F., 427, 434(10), 497, 499(97) Harmon, H . J . , 113 Harold, F. M. , 434, 450, 452(53, 54), 453

(53), 497, 498, 499(99, 104, 106, 107, 108), 502(104, 108), 515, 555, 676. 677(5), 678, 679(4, 5), 800

Harper, R. D., 15, 16,(82) Harris, D. Α., 300, 301(20), 377, 378(1), 400,

401, 402(s), 403, 404, 406(22), 407(22) Harris, E. J . , 228, 239, 297, 435, 439(1),

430(1), 450(1), 453, 510, 666, 673(2), 674, 675(2)

Harris, R. D., 439, 440(31), 441(31) Harrison, R., 24 Harrison, W. Α., 461 Harrison, W. H . , 463 Hartman, Η., 571(22), 572, 573(22) Hartmann, Α., 148 Hartree, Ε. F., 118, 119, 121, 123, 124, 125,

126 Hartshorne, D. I . , 402(L), 403, 405 Hasebe, H . , 405

Haslam, J . M . , 143 Hatefi , Y., 31 , 79, 80, 82(12), 83(12), 84(12),

85(12), 302, 303, 308, 309, 314(1), 315(1), 387, 388, 391, 497, 499(89), 516(89), 700, 716, 743

Hato, M . , 657 Hauska, G., 452 Hawkins, R. Α., 218(g), 219 Haworth, R., 335, 343 Hawthorne, D. C , 27 Hayashi, H . , 102 Haynes, B. , 149 Haynes, D. H . , 438. 439, 440(22, 35). 443

(35), 447(35), 451(35), 584 Heaton, G. M . , 73, 75, 453, 667 Hecht, Η. H . , 656 Hecht, P., 214, 222, 551 Hedman, R., 13 Heeb, M . J . , 439, 440(34). 443(34), 446(34) Heidrich, H . G.. 113 Heidt, H . W., 71. 72(27). 241. 263 Hel inski , D. R., 533 Hemon, P., 78 Hempfling. W. P.. 164, 165(4), 227 Hems, D. Α., 220 Hems, R., 204 Hemsworth, Β. Α., 18 Henderson, P. J. F., 435, 439(5, 17), 440(5),

442(5), 443(5), 444(5), 446(5), 450(17), 451(17), 452(17), 674

Henry, H . L . , 12 Heppel, L . Α., 382, 400, 489, 491(52), 794,

795, 800, 805(2), 806(2), 808(2), 809 Hernandez-Asensio, M . , 532 Hertzberg, Ε., 167, 168 Hertzberg, Ε. L. , 806, 808 Heslot, Η., 141 Hess, Β., 291, 341, 342(12), 343(12), 344 Hess, Μ. L . , 60 Hewitt , Ε. J . , 520 Heytler, P. G., 465, 469, 470(23) Higashi, Τ., 177, 183, 187(9), 188(9), 189,

190(28), 192(28), 194(28), 516 Higashiyama, T. , 303 Higgins, C. E., 502, 503(129) Higgins, E. S., 139 Higuchi , T. , 536(7), 537 Hilden, S., 701, 703(14) H i l l , Α. E., 657 H i l l , B. S., 657

A U T H O R I N D E X 833

Hind, G., 618 Hinds, T. R., 176, 186(5), 187(5) Hinkle, P. C , 167, 168, 197, 225, 245, 452,

541, 542, 543, 544(2), 637, 638, 641, 706, 707. 710, 716, 742, 748, 749(2), 751(2), 806, 808

Hiraga, M. , 18 Hiraoka, M . , 656 Hirata, H . , 176, 185(7), 186(5, 7), 187(5),

297, 298(3). 299(3), 302(3). 335, 364, 365(1), 366, 368, 370(3). 371, 372, 373. 415, 452, 497, 498, 499(100), 502(117), 503(100), 509(100), 516, 555, 571(41), 572. 573(41), 676. 677, 678(4, 6), 679(4, 5), 680, 702, 704(20), 711, 742, 777, 778(8), 779, 781, 782(1), 784(6), 786, 787(3)

Hirata, Y., 486 Hirose, S., 401. 402(t. u), 403, 405 Hittelman, K. J . , 16, 66, 69(9). 70(9), 74(9),

75(9) Hladky, S. B. , 571(21), 572, 573(21), 585

(21). 689, 691(3), 692, 693 Hoberman, H. D., 275 Hochman, Y., 301 Hodgkin, A. L . , 582 Hoeck, J. B. , 787(1), 788 Hoehn, Μ. M . , 502, 503(129) Höjeberg, Β., 270, 276, 283, 811, 815(3) Hoek, J. B. , 261, 262(1), 266(1), 267(1),

269(1), 270(1), 271(1), 274(1) Hofer, M. P., 453 Hoffman, J . F., 571(19, 20), 572, 573(19, 20),

574(20), 576(20), 578(20), 580, 582(19), 583(19), 584(52), 689, 690(2), 691, 692, 693(2), 694

Hoffman, J . H . , 559 Hogben, C. A . M. , 656 Hohorst, H . J . , 16, 66, 69(12), 70(12), 71(12) Hok in , L . E., 701, 703(14) Hol loway, C. T . , 106, 427, 428, 434(18), 481,

482, 483(23), 497, 498(23), 499(23, 79, 80), 500(79, 80), 502(23)

Holmes, N . G., 586 Holtzman, D. , 17 Homberger, Η., 142, 155 Hong, J. S., 676 Honn, Κ. V. , 11 Honovä, E. , 66, 69(8), 70(8), 74, 75(30c) Hooper, I . R., 501, 503(126), 512

Hope, A