quinones and quinone enzymes, part b

Nucleic Acids and Protein Synthesis Part C Edited by Kivie Moldave and Lawrence Grossman Volume XXI.. Nucleic Acids and Protein Synthesis Part E Edited by Lawrence Grossman and Kivie Mol

Trang 1

EDITORS-IN-CHIEFJohn N Abelson Melvin I Simon

DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY

PASADENA, CALIFORNIA

FOUNDING EDITORSSidney P Colowick and Nathan O Kaplan

Trang 2

Article numbers are in parentheses and following the names of contributors.

Affiliations listed are current.

L Mario Amzel (9), Interim Director,

Department of Biophysics and

Bio-physical Chemistry, The Johns

Hop-kins University School of Medicine,

Baltimore, Maryland 21205

Z ´ ilvinas Anusevicˇius (15), Institute of

Biochemistry, LT-2600 Vilnius, Lithuania

Gad Asher (16), Department of

Molecu-lar Genetics, Weizmann Institute of

Science, Rehovot 76100, Israel

Michel Aubier (4), Laboratoire de

Bio-physique and IFR 02, Faculte´ X Bichat,

75018 Paris, France

Alessandra Baracca (1), Dipartimento

Di Biochimica, Universita Di Bologna,

40126 Bologna, Italy

Raymond P Baumann (12), Department

of Pharmacology and Developmental

Therapeutics Program, Cancer Center,

Yale University School of Medicine,

New Haven, Connecticut 06520

M Flint Beal (26), Department of

Neu-rology and Neuroscience, Weill Medical

College of Cornell University, New

York, New York 10021

Asher Begleiter (18), Manitoba Institute

of Cell Biology, CancerCare Manitoba,

Department of Internal Medicine;

De-partment of Pharmacology and

Thera-peutics, University of Manitoba,

Manitoba R3E 0V9, Canada

Rosario I Bello (13), Departamento de

Biologı´a Celular, Fisiologı´a, e

Immunol-gı´a, Campus Rabanales, Universidad de

Co´rdoba, 14014 Co´rdoba, Spain

Mario A Bianchet (9), Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School

of Medicine, Baltimore, Maryland 21205 Jorge Boczkowski (4), Laboratoire de Biophysique and IFR 02, Faculte´ X Bichat, 75018 Paris, France

Micahel K Bowman (2), William R ley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland Washington 99352 John Butler (10), Department of Biologi- cal Sciences, The University of Salford, Salford M5 4WT, United Kingdom Jonathan Cape (2), Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340 Maria Cecilia Carreras (4), Laboratory

Wi-of Oxygen Metabolism, University pital, University of Buenos Aires, 1120 Buenos Aires, Argentina

Hos-Ercole Cavalieri (17), Eppley Institute for Research in Cancer and Allied Dis- eases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805 Narimantas Cˇe ` nas (15), Institute of Bio- chemistry, LT-2600 Vilnius, Lithuania Dhrubajyoti Chakravarti (17), Eppley Institute for Research in Cancer and Al- lied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805

Tom S Chan (6), Department of ceutical Sciences, University of Toronto, Toronto, Ontario M5S 2S2, Canada

Pharma-xi

Trang 3

Shiuan Chen (11), Department of

Sur-gery, Beckman Research Institute of

the City of Hope, Duarte, California

91010-0269

Ching Kuang Chow (7), Graduate Center

for Nutritional Sciences and Kentucky

Agricultural Experiment Station,

Univer-sity of Kentucky, Lexington, Kentucky

40506

Angela DiFrancesco (10), Division of

Pediatric Oncology, Policlinico Gemelli,

00168 Rome, Italy

Albena T Dinkova-Kostova (14, 19, 23),

Department of Pharmacology and

Mo-lecular Science, Lewis B and Dorothy

Cullman Cancer Center

Chemoprotec-tion Center, Johns Hopkins University

School of Medicine; Center for Human

Nutrition, Johns Hopkins University

Bloomberg School of Public Health,

Baltimore, Maryland 21205-2185

Manuchair Ebadi (27), School of

Medi-cine and Health Sciences, University

of North Dakota, Grand Forks, North

Dakota 58203

Joshua Eken (27), School of Medicine and

Health Sciences, University of North

Da-kota, Grand Forks, North Dakota 58203

Hesham El Rafaey (27), School of

Med-icine and Health Sciences, University

of North Dakota, Grand Forks, North

Dakota 58203

Margarita Faig (9), Department of

Bio-physics and Biophysical Chemistry, The

Johns Hopkins University School of

Medicine, Baltimore, Maryland 21205

Jed W Fahey (14, 23), Department of

Phar-macology and Molecular Science, Johns

Hopkins University School of Medicine,

Baltimore, Maryland 21205-2185

Romana Fato (1), Dipartimento Di

Bio-chimica, Universita Di Bologna, 40126

Bologna, Italy

Jeanne Fourie (18), Manitoba Institute

of Cell Biology, CancerCare Manitoba, Department of Pharmacology and Thera- peutics, University of Manitoba, Manitoba R3E 0V9, Canada

Maria Luisa Genova (1), Dipartimento

Di Biochimica, Universita Di Bologna,

40126 Bologna, Italy Consuelo Go´mez-Dıáz (13), Departa- mento de Biologıá Celular, Fisiologıá, e Immunolgıá, Campus Rabanales, Uni- versidad de Co´rdoba, 14014 Co´rdoba, Spain

Cindy Hagen (27), School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58203

Andrew M James (3), Medical Research Council, Dun Human Nutrition Unit, Cambridge CB2 2XY, United Kingdom Elizabeth H Jeffery (25), Department of Food Science and Human Nutrition, University of Illinois, Urbana, Illinois 61801

Young-Hwa Kang (21), College of macy, University of Illinois at Chicago, Chicago, Illinois 60612

Phar-Thomas W Kensler (22), Department of Environmental Health Sciences, School

of Public Health, Johns Hopkins versity Bloomberg, Baltimore, Maryland 21205

Uni-Geoffrey F Kelso (3), Department of Chemistry, University of Otago, Dunedin, New Zealand

Mohsen Kheradphezhou (27), School of Medicine and Health Sciences, Univer- sity of North Dakota, Grand Forks, North Dakota 58203

Richard J Knox (11), Enact Pharma PLC, Salisbury SP4 0JQ, United Kingdom

Trang 4

David M Kramer (2), Institute of

Biologi-cal Chemistry, Washington State

Univer-sity, Pullman, Washington 99164-6340

Mi-Young Kwak (22), Department of

En-vironmental Health Sciences, School of

Public Health, Johns Hopkins

Univer-sity Bloomberg, Baltimore, Maryland

21205

Giorgio Lenaz (1), Dipartimento Di

Bio-chimica, Universita Di Bologna, 40126

Bologna, Italy

Constanza L Lisdero (4), Laboratory of

Oxygen Metabolism, University

Hospi-tal, University of Buenos Aires, 1120

Buenos Aires, Argentina

Joseph Lotem (16), Department of

Mole-cular Genetics, Weizmann Institute of

Mariana Melani (4), Laboratory of

Oxy-gen Metabolism, University Hospital,

University of Buenos Aires, 1120 Buenos

Aires, Argentina

Alain Meulemans (4), Laboratoire de

Biophysique and IFR 02, Faculte´ X.

Bichat, 75018 Paris, France

Lina Misevicˇiene` (15), Institute of

Bio-chemistry, LT-2600 Vilnius, Lithuania

Arnold Munnich (5), Department of

Ge-netics, Hoˆpital Necker-Enfants Malades,

75014 Paris, France

Florian Muller (2), Institute of

Biologi-cal Chemistry, Washington State

Univer-sity, Pullman, Washington 99164-6340

Micahel P Murphy (3), Medical

Re-search Council, Dun Human Nutrition

Unit, Cambridge CB2 2XY, United

Kingdom

Christine M Munday (24), AgResearch,

Ruakura Agricultural Research Centre,

Hamilton, New Zealand

Rex Munday (20, 24), AgResearch, kura Agricultural Research Centre, Ha- milton, New Zealand

Rua-Pla´cido Navas (13), Centro Andaluz de Biologı´a del Desarrollo, Universidad Pablo de Olavide, 41013 Sevilla, Spain Henrikas Nivinskas (15), Institute of Bio- chemistry, LT-2600 Vilnius, Lithuania Peter J O’brien (6), Department of Pharma- ceutical Sciences, University of Toronto, Toronto, Ontario M5S 2S2, Canada Philip J Penketh (12), Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520 John M Pezzuto (21), Schools of Phar- macy, Nursing, and Health Sciences, Pur- due University, West Lafayette, Indiana 47907-7880

Juan Jose´ Poderoso (4), Laboratory of Oxygen Metabolism, University Hospi- tal, University of Buenos Aires, 1120 Buenos Aires, Argentina

Minerva Ramos-Gomez (22), Department

of Environmental Health Sciences, School

of Public Health, Johns Hopkins sity Bloomberg, Baltimore, Maryland 21205

Univer-Arthur G Roberts (2), Institute of gical Chemistry, Washington State Uni- versity, Pullman, Washington 99164-6340 Eleanor Rogan (17), Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805

Biolo-David Ross (8), Department of maceutical Sciences, School of Phar- macy, University of Colorado, Denver, Colorado 80262

Trang 5

Phar-Agne`s Ro¨tig (5), Department of Genetics,

Hoˆpital Necker-Enfants Malades, 75014

Paris, France

Pierre Rustin (5), Department of

Genet-ics, Hoˆpital Necker-Enfants Malades,

75014 Paris, France

Leo Sachs (16), Department of Molecular

Genetics, Weizmann Institute of Science,

Rehovot 76100, Israel

Jonas Sˇarlauskas (15), Institute of

Bio-chemistry, LT-2600 Vilnius, Lithuania

Alan C Sartorelli (12), Department of

Pharmacology and Developmental

Ther-apeutics Program, Cancer Center, Yale

University School of Medicine, New

Haven, Connecticut 06520

Helen A Seow (12), Department of

Phar-macology and Developmental

Thera-peutics Program, Cancer Center, Yale

University School of Medicine, New

Haven, Connecticut 06520

Yosef Shaul (16), Department of

Molecu-lar Genetics, Weizmann Institute of

Sushil Sharma (27), School of Medicine and

Shaik Shavali (27), School of Medicine and

David Siegel (8), Department of

Pharma-ceutical Sciences, School of Pharmacy,

University of Colorado, Denver, Colorado

80262

Robin A.J Smith (3), Department of

Chemistry, University of Otago,

Dune-din, New Zealand

Katherine K Stephenson (14), ment of Pharmacology and Molecular Science, Lewis B and Dorothy Cullman Cancer Center Chemoprotection Center, Johns Hopkins University School of Med- icine, Baltimore, Maryland 21205-2185 Kristin E Stewart (25), Division of Nu- tritional Sciences, University of Illinois, Urbana, Illinois 61801

Depart-Paul Talalay (14, 19, 23), Department of Pharmacology and Molecular Science, Lewis B and Dorothy Cullman Cancer Center Chemoprotection Center, Johns Hopkins University School of Medicine; Center for Human Nutrition, Johns Hopkins University Bloomberg School

of Public Health, Baltimore, Maryland 21205-2185

Jose´ M Villalba (13), Departamento de Biologıá Celular, Fisiologıá, e Immunol- gıá, Campus Rabanales, Universidad de Co´rdoba, 14014 Co´rdoba, Spain Nobunao Wakabayashi (22), Department

of Environmental Health Sciences, School

of Public Health, Johns Hopkins sity Bloomberg, Baltimore, Maryland 21205

Univer-Timothy H Ward (10), Drug ment Unit, Paterson Institute, Christie Hospital, Manchester M20 9BX, United Kingdom

Develop-John X Wilson (6), Department of siology, University of Western Ontario, London, Ontario N6A 5C1, Canada

Trang 6

Phy-Developments in genomics and proteomics rapidly generated focus on new-omics, particularly metabolomics and phenomics Quinones, hydroquinones,semiquinones and their metabolites are naturally occurring compounds thatserve as wonderful examples for this new paradigm of interdigitating ,-omics Inaddition to a role as substrates and products in metabolism, quinone com-pounds are intermediates in many pathways of gene regulation, enzyme proteininduction, feedback control, and waste product elimination Quinones play apivotal role in energy metabolism (Peter Mitchell’s proton-motive, Q cycle’),many other key processes, and even in chemotherapy where redox cyclingdrugs are utilized.

The present volume of Methods in Enzymology on quinones and quinoneenzymes serves to bring together current methods and concepts on this topic Itfocuses on the role in the so-called Phase II of drug metabolism (xenobiotics),but include aspects on Phase I (CYP, cytochromes P-450) and Phase III(transport systems) as well This volume of Methods in Enzymology, Part Baddresses mitochondrial ubiquinone and reductases, anticancer quinones, andthe role of quinone reductases in chemoprevention and nutrition, as well as therole of quinones in age-related diseases, whereas (Part A) focused on quinonesand quinone enzymes in terms of coenzyme Q (detection and quinone reduc-tases), plasma membrane quinone reductases, and the role of quinones

in cellular signaling and modulation of gene expression Phase II Enzymes,Part C, will be focusing on glutathione, glutathione S-transferases, and otherconjugation enzymes

The enzyme, NAD(P)H:quinone oxidoreductase, is the subject of a majorsection in this volume This enzyme, discovered in 1958 in Stockholm by LarsErnster, and named DT-Diaphorase by him, has multiple roles, some of whichwere only recently discovered

Human polymorphisms exist in these enzymes that relate to variations incancer risk, and enzymes targeted by quinones are being investigated.Modern methods in assaying quinone reactions and, indeed, various quinonesthemselves, are also included in this volume

Following its discovery in 1957, ubiquinone (coenzyme Q10) as a majornaturally occurring quinone became a highlight of scientific interest and anestablished role in mitochondrial electron transport by Frederick Crane.Fundamental contributions were made by Karl Folkers on its supplemental

xv

Trang 7

use for health benefits in disease prevention and by Andre´s O.M Stoppani, apioneer of Argentinian biochemistry, in utilizing quinones for the treatment ofChagas disease.

We thank the Advisory Committee (Enrique Cadenas, Los Angeles; GustavDallner, Stockholm; Tom Kensler, Baltimore; Lars-Oliver Klotz, Du¨sseldorf;David Ross, Denver) for their valuable suggestions and wisdom in selecting thecontributions for this volume

Helmut Sies and Lester Packer

Trang 8

METHODS IN ENZYMOLOGY

Volume I Preparation and Assay of Enzymes

Edited by Sidney P Colowick and Nathan O Kaplan

Volume II Preparation and Assay of Enzymes

Volume III Preparation and Assay of Substrates

Volume IV Special Techniques for the Enzymologist

Volume V Preparation and Assay of Enzymes

Volume VI Preparation and Assay of Enzymes (Continued)

Preparation and Assay of Substrates

Special Techniques

Volume VII Cumulative Subject Index

Volume VIII Complex Carbohydrates

Edited by Elizabeth F Neufeld and Victor Ginsburg

Volume IX Carbohydrate Metabolism

Edited by Willis A Wood

Volume X Oxidation and Phosphorylation

Edited by Ronald W Estabrook and Maynard E Pullman

Volume XI Enzyme Structure

Edited by C H W Hirs

Volume XII Nucleic Acids (Parts A and B)

Edited by Lawrence Grossman and Kivie Moldave

Volume XIII Citric Acid Cycle

Edited by J M Lowenstein

Volume XIV Lipids

Edited by J M Lowenstein

Volume XV Steroids and Terpenoids

Edited by Raymond B Clayton

xvii

Trang 9

Volume XVI Fast Reactions

Edited by Kenneth Kustin

Volume XVII Metabolism of Amino Acids and Amines (Parts A and B)Edited by Herbert Tabor and Celia White Tabor

Volume XVIII Vitamins and Coenzymes (Parts A, B, and C)

Edited by Donald B McCormick and Lemuel D Wright

Volume XIX Proteolytic Enzymes

Edited by Gertrude E Perlmann and Laszlo Lorand

Volume XX Nucleic Acids and Protein Synthesis (Part C)

Edited by Kivie Moldave and Lawrence Grossman

Volume XXI Nucleic Acids (Part D)

Volume XXII Enzyme Purification and Related Techniques

Edited by William B Jakoby

Volume XXIII Photosynthesis (Part A)

Edited by Anthony San Pietro

Volume XXIV Photosynthesis and Nitrogen Fixation (Part B)

Volume XXV Enzyme Structure (Part B)

Edited by C H W Hirs and Serge N Timasheff

Volume XXVI Enzyme Structure (Part C)

Volume XXVII Enzyme Structure (Part D)

Volume XXVIII Complex Carbohydrates (Part B)

Edited by Victor Ginsburg

Volume XXIX Nucleic Acids and Protein Synthesis (Part E)

Volume XXX Nucleic Acids and Protein Synthesis (Part F)

Volume XXXI Biomembranes (Part A)

Edited by Sidney Fleischer and Lester Packer

Volume XXXII Biomembranes (Part B)

Volume XXXIII Cumulative Subject Index Volumes I-XXX

Edited by Martha G Dennis and Edward A Dennis

Volume XXXIV Affinity Techniques (Enzyme Purification: Part B)Edited by William B Jakoby and Meir Wilchek

Trang 10

Volume XXXV Lipids (Part B)

Edited by John M Lowenstein

Volume XXXVI Hormone Action (Part A: Steroid Hormones)

Edited by Bert W O’Malley and Joel G Hardman

Volume XXXVII Hormone Action (Part B: Peptide Hormones)

Edited by Bert W O’Malley and Joel G Hardman

Volume XXXVIII Hormone Action (Part C: Cyclic Nucleotides)

Edited by Joel G Hardman and Bert W O’Malley

Volume XXXIX Hormone Action (Part D: Isolated Cells, Tissues, and OrganSystems)

Edited by Joel G Hardman and Bert W O’Malley

Volume XL Hormone Action (Part E: Nuclear Structure and Function)Edited by Bert W O’Malley and Joel G Hardman

Volume XLI Carbohydrate Metabolism (Part B)

Edited by W A Wood

Volume XLII Carbohydrate Metabolism (Part C)

Edited by W A Wood

Volume XLIII Antibiotics

Edited by John H Hash

Volume XLIV Immobilized Enzymes

Edited by Klaus Mosbach

Volume XLV Proteolytic Enzymes (Part B)

Edited by Laszlo Lorand

Volume XLVI Affinity Labeling

Edited by William B Jakoby and Meir Wilchek

Volume XLVII Enzyme Structure (Part E)

Volume XLVIII Enzyme Structure (Part F)

Volume XLIX Enzyme Structure (Part G)

Volume L Complex Carbohydrates (Part C)

Volume LI Purine and Pyrimidine Nucleotide Metabolism

Edited by Patricia A Hoffee and Mary Ellen Jones

Volume LII Biomembranes (Part C: Biological Oxidations)

Volume LIII Biomembranes (Part D: Biological Oxidations)

Trang 11

Volume LIV Biomembranes (Part E: Biological Oxidations)

Volume LV Biomembranes (Part F: Bioenergetics)

Volume LVI Biomembranes (Part G: Bioenergetics)

Volume LVII Bioluminescence and Chemiluminescence

Edited by Marlene A DeLuca

Volume LVIII Cell Culture

Edited by William B Jakoby and Ira Pastan

Volume LIX Nucleic Acids and Protein Synthesis (Part G)

Volume LX Nucleic Acids and Protein Synthesis (Part H)

Volume 61 Enzyme Structure (Part H)

Volume 62 Vitamins and Coenzymes (Part D)

Volume 63 Enzyme Kinetics and Mechanism (Part A: Initial Rate andInhibitor Methods)

Edited by Daniel L Purich

Volume 64 Enzyme Kinetics and Mechanism (Part B: Isotopic Probes andComplex Enzyme Systems)

Volume 65 Nucleic Acids (Part I)

Volume 66 Vitamins and Coenzymes (Part E)

Volume 67 Vitamins and Coenzymes (Part F)

Volume 68 Recombinant DNA

Edited by Ray Wu

Volume 69 Photosynthesis and Nitrogen Fixation (Part C)

Volume 70 Immunochemical Techniques (Part A)

Edited by Helen Van Vunakis and John J Langone

Volume 71 Lipids (Part C)

Trang 12

Volume 72 Lipids (Part D)

Volume 73 Immunochemical Techniques (Part B)

Edited by John J Langone and Helen Van Vunakis

Volume 74 Immunochemical Techniques (Part C)

Volume 75 Cumulative Subject Index Volumes XXXI, XXXII, XXXIV–LXEdited by Edward A Dennis and Martha G Dennis

Volume 76 Hemoglobins

Edited by Eraldo Antonini, Luigi Rossi-Bernardi, and Emilia ChianconeVolume 77 Detoxication and Drug Metabolism

Volume 78 Interferons (Part A)

Edited by Sidney Pestka

Volume 79 Interferons (Part B)

Volume 80 Proteolytic Enzymes (Part C)

Edited by Laszlo Lorand

Volume 81 Biomembranes (Part H: Visual Pigments and Purple Membranes, I)Edited by Lester Packer

Volume 82 Structural and Contractile Proteins (Part A: Extracellular Matrix)Edited by Leon W Cunningham and Dixie W Frederiksen

Volume 83 Complex Carbohydrates (Part D)

Volume 84 Immunochemical Techniques (Part D: Selected Immunoassays)Edited by John J Langone and Helen Van Vunakis

Volume 85 Structural and Contractile Proteins (Part B: The ContractileApparatus and the Cytoskeleton)

Edited by Dixie W Frederiksen and Leon W Cunningham

Volume 86 Prostaglandins and Arachidonate Metabolites

Edited by William E M Lands and William L Smith

Volume 87 Enzyme Kinetics and Mechanism (Part C: Intermediates,

Stereo-chemistry, and Rate Studies)

Volume 88 Biomembranes (Part I: Visual Pigments and Purple Membranes, II)Edited by Lester Packer

Volume 89 Carbohydrate Metabolism (Part D)

Trang 13

Volume 90 Carbohydrate Metabolism (Part E)

Volume 91 Enzyme Structure (Part I)

Volume 92 Immunochemical Techniques (Part E: Monoclonal Antibodies andGeneral Immunoassay Methods)

Volume 93 Immunochemical Techniques (Part F: Conventional Antibodies,

Fc Receptors, and Cytotoxicity)

Volume 94 Polyamines

Edited by Herbert Tabor and Celia White Tabor

Volume 95 Cumulative Subject Index Volumes 61–74, 76–80

Edited by Edward A Dennis and Martha G Dennis

Volume 96 Biomembranes [Part J: Membrane Biogenesis: Assembly andTargeting (General Methods; Eukaryotes)]

Edited by Sidney Fleischer and Becca Fleischer

Volume 97 Biomembranes [Part K: Membrane Biogenesis: Assembly andTargeting (Prokaryotes, Mitochondria, and Chloroplasts)]

Volume 98 Biomembranes (Part L: Membrane Biogenesis: Processing andRecycling)

Volume 99 Hormone Action (Part F: Protein Kinases)

Edited by Jackie D Corbin and Joel G Hardman

Volume 100 Recombinant DNA (Part B)

Edited by Ray Wu, Lawrence Grossman, and Kivie Moldave

Volume 101 Recombinant DNA (Part C)

Edited by Ray Wu, Lawrence Grossman, and Kivie Moldave

Volume 102 Hormone Action (Part G: Calmodulin and Calcium-BindingProteins)

Edited by Anthony R Means and Bert W O’Malley

Volume 103 Hormone Action (Part H: Neuroendocrine Peptides)

Edited by P Michael Conn

Volume 104 Enzyme Purification and Related Techniques (Part C)

Volume 105 Oxygen Radicals in Biological Systems

Edited by Lester Packer

Volume 106 Posttranslational Modifications (Part A)

Edited by Finn Wold and Kivie Moldave

Trang 14

Volume 107 Posttranslational Modifications (Part B)

Edited by Finn Wold and Kivie Moldave

Volume 108 Immunochemical Techniques (Part G: Separation and

Characterization of Lymphoid Cells)

Edited by Giovanni Di Sabato, John J Langone, and Helen Van VunakisVolume 109 Hormone Action (Part I: Peptide Hormones)

Edited by Lutz Birnbaumer and Bert W O’Malley

Volume 110 Steroids and Isoprenoids (Part A)

Edited by John H Law and Hans C Rilling

Volume 111 Steroids and Isoprenoids (Part B)

Edited by John H Law and Hans C Rilling

Volume 112 Drug and Enzyme Targeting (Part A)

Edited by Kenneth J Widder and Ralph Green

Volume 113 Glutamate, Glutamine, Glutathione, and Related CompoundsEdited by Alton Meister

Volume 114 Diffraction Methods for Biological Macromolecules (Part A)Edited by Harold W Wyckoff, C H W Hirs, and Serge N TimasheffVolume 115 Diffraction Methods for Biological Macromolecules (Part B)Edited by Harold W Wyckoff, C H W Hirs, and Serge N TimasheffVolume 116 Immunochemical Techniques (Part H: Effectors and Mediators ofLymphoid Cell Functions)

Edited by Giovanni Di Sabato, John J Langone, and Helen Van VunakisVolume 117 Enzyme Structure (Part J)

Volume 118 Plant Molecular Biology

Edited by Arthur Weissbach and Herbert Weissbach

Volume 119 Interferons (Part C)

Volume 121 Immunochemical Techniques (Part I: Hybridoma Technologyand Monoclonal Antibodies)

Volume 122 Vitamins and Coenzymes (Part G)

Edited by Frank Chytil and Donald B McCormick

Volume 123 Vitamins and Coenzymes (Part H)

Edited by Frank Chytil and Donald B McCormick

Volume 124 Hormone Action (Part J: Neuroendocrine Peptides)

Trang 15

Volume 125 Biomembranes (Part M: Transport in Bacteria, Mitochondria,and Chloroplasts: General Approaches and Transport Systems)

Volume 126 Biomembranes (Part N: Transport in Bacteria, Mitochondria, andChloroplasts: Protonmotive Force)

Volume 127 Biomembranes (Part O: Protons and Water: Structure andTranslocation)

Volume 128 Plasma Lipoproteins (Part A: Preparation, Structure, andMolecular Biology)

Edited by Jere P Segrest and John J Albers

Volume 129 Plasma Lipoproteins (Part B: Characterization, Cell Biology, andMetabolism)

Edited by John J Albers and Jere P Segrest

Volume 130 Enzyme Structure (Part K)

Volume 131 Enzyme Structure (Part L)

Volume 132 Immunochemical Techniques (Part J: Phagocytosis and

Cell-Mediated Cytotoxicity)

Edited by Giovanni Di Sabato and Johannes Everse

Volume 133 Bioluminescence and Chemiluminescence (Part B)

Edited by Marlene DeLuca and William D McElroy

Volume 134 Structural and Contractile Proteins (Part C: The ContractileApparatus and the Cytoskeleton)

Edited by Richard B Vallee

Volume 135 Immobilized Enzymes and Cells (Part B)

Volume 136 Immobilized Enzymes and Cells (Part C)

Volume 137 Immobilized Enzymes and Cells (Part D)

Volume 138 Complex Carbohydrates (Part E)

Volume 139 Cellular Regulators (Part A: Calcium- and Calmodulin-BindingProteins)

Edited by Anthony R Means and P Michael Conn

Trang 16

Volume 141 Cellular Regulators (Part B: Calcium and Lipids)

Edited by P Michael Conn and Anthony R Means

Volume 142 Metabolism of Aromatic Amino Acids and Amines

Edited by Seymour Kaufman

Volume 143 Sulfur and Sulfur Amino Acids

Edited by William B Jakoby and Owen Griffith

Volume 144 Structural and Contractile Proteins (Part D: Extracellular Matrix)Edited by Leon W Cunningham

Volume 145 Structural and Contractile Proteins (Part E: Extracellular Matrix)Edited by Leon W Cunningham

Volume 146 Peptide Growth Factors (Part A)

Edited by David Barnes and David A Sirbasku

Volume 147 Peptide Growth Factors (Part B)

Edited by David Barnes and David A Sirbasku

Volume 148 Plant Cell Membranes

Edited by Lester Packer and Roland Douce

Volume 149 Drug and Enzyme Targeting (Part B)

Edited by Ralph Green and Kenneth J Widder

Volume 150 Immunochemical Techniques (Part K: In Vitro Models of B and TCell Functions and Lymphoid Cell Receptors)

Edited by Giovanni Di Sabato

Volume 151 Molecular Genetics of Mammalian Cells

Edited by Michael M Gottesman

Volume 152 Guide to Molecular Cloning Techniques

Edited by Shelby L Berger and Alan R Kimmel

Volume 153 Recombinant DNA (Part D)

Edited by Ray Wu and Lawrence Grossman

Volume 154 Recombinant DNA (Part E)

Edited by Ray Wu and Lawrence Grossman

Volume 155 Recombinant DNA (Part F)

Edited by Ray Wu

Volume 156 Biomembranes (Part P: ATP-Driven Pumps and RelatedTransport: The Na, K-Pump)

Volume 157 Biomembranes (Part Q: ATP-Driven Pumps and RelatedTransport: Calcium, Proton, and Potassium Pumps)

Volume 158 Metalloproteins (Part A)

Edited by James F Riordan and Bert L Vallee

Trang 17

Volume 159 Initiation and Termination of Cyclic Nucleotide ActionEdited by Jackie D Corbin and Roger A Johnson

Volume 160 Biomass (Part A: Cellulose and Hemicellulose)

Edited by Willis A Wood and Scott T Kellogg

Volume 161 Biomass (Part B: Lignin, Pectin, and Chitin)

Edited by Willis A Wood and Scott T Kellogg

Volume 162 Immunochemical Techniques (Part L: Chemotaxis

and Inflammation)

Volume 163 Immunochemical Techniques (Part M: Chemotaxis

and Inflammation)

Volume 164 Ribosomes

Edited by Harry F Noller, Jr., and Kivie Moldave

Volume 165 Microbial Toxins: Tools for Enzymology

Edited by Sidney Harshman

Volume 166 Branched-Chain Amino Acids

Edited by Robert Harris and John R Sokatch

Volume 167 Cyanobacteria

Edited by Lester Packer and Alexander N Glazer

Volume 168 Hormone Action (Part K: Neuroendocrine Peptides)Edited by P Michael Conn

Volume 169 Platelets: Receptors, Adhesion, Secretion (Part A)

Edited by Jacek Hawiger

Volume 170 Nucleosomes

Edited by Paul M Wassarman and Roger D Kornberg

Volume 171 Biomembranes (Part R: Transport Theory: Cells and ModelMembranes)

Volume 172 Biomembranes (Part S: Transport: Membrane Isolation andCharacterization)

Volume 173 Biomembranes [Part T: Cellular and Subcellular Transport:Eukaryotic (Nonepithelial) Cells]

Volume 174 Biomembranes [Part U: Cellular and Subcellular Transport:Eukaryotic (Nonepithelial) Cells]

Trang 18

Volume 176 Nuclear Magnetic Resonance (Part A: Spectral Techniques andDynamics)

Edited by Norman J Oppenheimer and Thomas L James

Volume 177 Nuclear Magnetic Resonance (Part B: Structure and Mechanism)Edited by Norman J Oppenheimer and Thomas L James

Volume 178 Antibodies, Antigens, and Molecular Mimicry

Edited by John J Langone

Volume 179 Complex Carbohydrates (Part F)

Volume 180 RNA Processing (Part A: General Methods)

Edited by James E Dahlberg and John N Abelson

Volume 181 RNA Processing (Part B: Specific Methods)

Edited by James E Dahlberg and John N Abelson

Volume 182 Guide to Protein Purification

Edited by Murray P Deutscher

Volume 183 Molecular Evolution: Computer Analysis of Protein and NucleicAcid Sequences

Edited by Russell F Doolittle

Volume 184 Avidin-Biotin Technology

Edited by Meir Wilchek and Edward A Bayer

Volume 185 Gene Expression Technology

Edited by David V Goeddel

Volume 186 Oxygen Radicals in Biological Systems (Part B: Oxygen Radicalsand Antioxidants)

Edited by Lester Packer and Alexander N Glazer

Volume 187 Arachidonate Related Lipid Mediators

Edited by Robert C Murphy and Frank A Fitzpatrick

Volume 188 Hydrocarbons and Methylotrophy

Edited by Mary E Lidstrom

Volume 189 Retinoids (Part A: Molecular and Metabolic Aspects)

Volume 190 Retinoids (Part B: Cell Differentiation and Clinical Applications)Edited by Lester Packer

Volume 191 Biomembranes (Part V: Cellular and Subcellular Transport:Epithelial Cells)

Volume 192 Biomembranes (Part W: Cellular and Subcellular Transport:Epithelial Cells)

Trang 19

Volume 193 Mass Spectrometry

Edited by James A McCloskey

Volume 194 Guide to Yeast Genetics and Molecular Biology

Edited by Christine Guthrie and Gerald R Fink

Volume 195 Adenylyl Cyclase, G Proteins, and Guanylyl CyclaseEdited by Roger A Johnson and Jackie D Corbin

Volume 196 Molecular Motors and the Cytoskeleton

Volume 197 Phospholipases

Edited by Edward A Dennis

Volume 198 Peptide Growth Factors (Part C)

Edited by David Barnes, J P Mather, and Gordon H Sato

Volume 199 Cumulative Subject Index Volumes 168–174, 176–194Volume 200 Protein Phosphorylation (Part A: Protein Kinases: Assays,Purification, Antibodies, Functional Analysis, Cloning, and Expression)Edited by Tony Hunter and Bartholomew M Sefton

Volume 201 Protein Phosphorylation (Part B: Analysis of ProteinPhosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases)Edited by Tony Hunter and Bartholomew M Sefton

Volume 202 Molecular Design and Modeling: Concepts and Applications(Part A: Proteins, Peptides, and Enzymes)

Volume 203 Molecular Design and Modeling: Concepts and Applications(Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides,and Drugs)

Volume 204 Bacterial Genetic Systems

Edited by Jeffrey H Miller

Volume 205 Metallobiochemistry (Part B: Metallothionein and RelatedMolecules)

Volume 206 Cytochrome P450

Edited by Michael R Waterman and Eric F Johnson

Volume 207 Ion Channels

Edited by Bernardo Rudy and Linda E Iverson

Volume 208 Protein–DNA Interactions

Edited by Robert T Sauer

Volume 209 Phospholipid Biosynthesis

Edited by Edward A Dennis and Dennis E Vance

Trang 20

Volume 210 Numerical Computer Methods

Edited by Ludwig Brand and Michael L Johnson

Volume 211 DNA Structures (Part A: Synthesis and Physical Analysis ofDNA)

Edited by David M J Lilley and James E Dahlberg

Volume 212 DNA Structures (Part B: Chemical and Electrophoretic Analysis

of DNA)

Edited by David M J Lilley and James E Dahlberg

Volume 213 Carotenoids (Part A: Chemistry, Separation, Quantitation, andAntioxidation)

Volume 214 Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis)Edited by Lester Packer

Volume 215 Platelets: Receptors, Adhesion, Secretion (Part B)

Edited by Jacek J Hawiger

Volume 216 Recombinant DNA (Part G)

Volume 219 Reconstitution of Intracellular Transport

Edited by James E Rothman

Volume 220 Membrane Fusion Techniques (Part A)

Edited by Nejat Du¨zgu¨nes,

Volume 221 Membrane Fusion Techniques (Part B)

Volume 222 Proteolytic Enzymes in Coagulation, Fibrinolysis, and

Complement Activation (Part A: Mammalian Blood Coagulation Factors andInhibitors)

Edited by Laszlo Lorand and Kenneth G Mann

Volume 223 Proteolytic Enzymes in Coagulation, Fibrinolysis, and

Complement Activation (Part B: Complement Activation, Fibrinolysis, andNonmammalian Blood Coagulation Factors)

Edited by Laszlo Lorand and Kenneth G Mann

Volume 224 Molecular Evolution: Producing the Biochemical Data

Edited by Elizabeth Anne Zimmer, Thomas J White, Rebecca L Cann, andAllan C Wilson

Volume 225 Guide to Techniques in Mouse Development

Edited by Paul M Wassarman and Melvin L DePamphilis

Trang 21

Volume 226 Metallobiochemistry (Part C: Spectroscopic and PhysicalMethods for Probing Metal Ion Environments in Metalloenzymes andMetalloproteins)

Volume 227 Metallobiochemistry (Part D: Physical and SpectroscopicMethods for Probing Metal Ion Environments in Metalloproteins)

Volume 228 Aqueous Two-Phase Systems

Edited by Harry Walter and Go¨te Johansson

Volume 230 Guide to Techniques in Glycobiology

Edited by William J Lennarz and Gerald W Hart

Volume 231 Hemoglobins (Part B: Biochemical and Analytical Methods)Edited by Johannes Everse, Kim D Vandegriff, and Robert M WinslowVolume 232 Hemoglobins (Part C: Biophysical Methods)

Edited by Johannes Everse, Kim D Vandegriff, and Robert M WinslowVolume 233 Oxygen Radicals in Biological Systems (Part C)

Volume 234 Oxygen Radicals in Biological Systems (Part D)

Volume 235 Bacterial Pathogenesis (Part A: Identification and Regulation ofVirulence Factors)

Edited by Virginia L Clark and Patrik M Bavoil

Volume 236 Bacterial Pathogenesis (Part B: Integration of PathogenicBacteria with Host Cells)

Volume 237 Heterotrimeric G Proteins

Edited by Ravi Iyengar

Volume 238 Heterotrimeric G-Protein Effectors

Edited by Ravi Iyengar

Volume 239 Nuclear Magnetic Resonance (Part C)

Edited by Thomas L James and Norman J Oppenheimer

Volume 240 Numerical Computer Methods (Part B)

Edited by Michael L Johnson and Ludwig Brand

Volume 241 Retroviral Proteases

Edited by Lawrence C Kuo and Jules A Shafer

Volume 242 Neoglycoconjugates (Part A)

Edited by Y C Lee and Reiko T Lee

Volume 243 Inorganic Microbial Sulfur Metabolism

Edited by Harry D Peck, Jr., and Jean LeGall

Trang 22

Volume 244 Proteolytic Enzymes: Serine and Cysteine Peptidases

Edited by Alan J Barrett

Volume 245 Extracellular Matrix Components

Edited by E Ruoslahti and E Engvall

Volume 246 Biochemical Spectroscopy

Edited by Kenneth Sauer

Volume 247 Neoglycoconjugates (Part B: Biomedical Applications)

Edited by Y C Lee and Reiko T Lee

Volume 248 Proteolytic Enzymes: Aspartic and Metallo Peptidases

Edited by Alan J Barrett

Volume 249 Enzyme Kinetics and Mechanism (Part D: Developments inEnzyme Dynamics)

Volume 250 Lipid Modifications of Proteins

Edited by Patrick J Casey and Janice E Buss

Volume 251 Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, andThiyl Radicals)

Volume 252 Biothiols (Part B: Glutathione and Thioredoxin; Thiols in SignalTransduction and Gene Regulation)

Volume 253 Adhesion of Microbial Pathogens

Edited by Ron J Doyle and Itzhak Ofek

Volume 254 Oncogene Techniques

Edited by Peter K Vogt and Inder M Verma

Volume 255 Small GTPases and Their Regulators (Part A: Ras Family)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 256 Small GTPases and Their Regulators (Part B: Rho Family)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 257 Small GTPases and Their Regulators (Part C: Proteins Involved

in Transport)

Edited by W E Balch, Channing J Der, and Alan Hall

Volume 258 Redox-Active Amino Acids in Biology

Edited by Judith P Klinman

Volume 259 Energetics of Biological Macromolecules

Edited by Michael L Johnson and Gary K Ackers

Volume 260 Mitochondrial Biogenesis and Genetics (Part A)

Edited by Giuseppe M Attardi and Anne Chomyn

Volume 261 Nuclear Magnetic Resonance and Nucleic Acids

Edited by Thomas L James

Trang 23

Volume 262 DNA Replication

Edited by Judith L Campbell

Volume 263 Plasma Lipoproteins (Part C: Quantitation)

Edited by William A Bradley, Sandra H Gianturco, and Jere P SegrestVolume 264 Mitochondrial Biogenesis and Genetics (Part B)

Edited by Giuseppe M Attardi and Anne Chomyn

Volume 265 Cumulative Subject Index Volumes 228, 230–262

Volume 266 Computer Methods for Macromolecular Sequence AnalysisEdited by Russell F Doolittle

Volume 267 Combinatorial Chemistry

Edited by John N Abelson

Volume 268 Nitric Oxide (Part A: Sources and Detection of NO; NOSynthase)

Volume 269 Nitric Oxide (Part B: Physiological and Pathological Processes)Edited by Lester Packer

Volume 270 High Resolution Separation and Analysis of Biological

Macromolecules (Part A: Fundamentals)

Edited by Barry L Karger and William S Hancock

Volume 271 High Resolution Separation and Analysis of Biological

Macromolecules (Part B: Applications)

Edited by Barry L Karger and William S Hancock

Volume 272 Cytochrome P450 (Part B)

Edited by Eric F Johnson and Michael R Waterman

Volume 273 RNA Polymerase and Associated Factors (Part A)

Edited by Sankar Adhya

Volume 274 RNA Polymerase and Associated Factors (Part B)

Edited by Sankar Adhya

Volume 275 Viral Polymerases and Related Proteins

Edited by Lawrence C Kuo, David B Olsen, and Steven S CarrollVolume 276 Macromolecular Crystallography (Part A)

Edited by Charles W Carter, Jr., and Robert M Sweet

Volume 277 Macromolecular Crystallography (Part B)

Volume 278 Fluorescence Spectroscopy

Volume 279 Vitamins and Coenzymes (Part I)

Edited by Donald B McCormick, John W Suttie, and Conrad Wagner

Trang 24

Volume 280 Vitamins and Coenzymes (Part J)

Edited by Donald B McCormick, John W Suttie, and Conrad WagnerVolume 281 Vitamins and Coenzymes (Part K)

Edited by Donald B McCormick, John W Suttie, and Conrad WagnerVolume 282 Vitamins and Coenzymes (Part L)

Edited by Donald B McCormick, John W Suttie, and Conrad WagnerVolume 283 Cell Cycle Control

Edited by William G Dunphy

Volume 284 Lipases (Part A: Biotechnology)

Edited by Byron Rubin and Edward A Dennis

Volume 285 Cumulative Subject Index Volumes 263, 264, 266–284, 286–289Volume 286 Lipases (Part B: Enzyme Characterization and Utilization)Edited by Byron Rubin and Edward A Dennis

Volume 287 Chemokines

Edited by Richard Horuk

Volume 288 Chemokine Receptors

Edited by Richard Horuk

Volume 289 Solid Phase Peptide Synthesis

Edited by Gregg B Fields

Volume 290 Molecular Chaperones

Edited by George H Lorimer and Thomas Baldwin

Volume 291 Caged Compounds

Edited by Gerard Marriott

Volume 292 ABC Transporters: Biochemical, Cellular, and Molecular AspectsEdited by Suresh V Ambudkar and Michael M Gottesman

Volume 293 Ion Channels (Part B)

Volume 294 Ion Channels (Part C)

Volume 295 Energetics of Biological Macromolecules (Part B)

Edited by Gary K Ackers and Michael L Johnson

Volume 296 Neurotransmitter Transporters

Edited by Susan G Amara

Volume 297 Photosynthesis: Molecular Biology of Energy Capture

Edited by Lee McIntosh

Volume 298 Molecular Motors and the Cytoskeleton (Part B)

Trang 25

Volume 299 Oxidants and Antioxidants (Part A)

Volume 300 Oxidants and Antioxidants (Part B)

Volume 301 Nitric Oxide: Biological and Antioxidant Activities (Part C)Edited by Lester Packer

Volume 302 Green Fluorescent Protein

Volume 303 cDNA Preparation and Display

Edited by Sherman M Weissman

Volume 304 Chromatin

Edited by Paul M Wassarman and Alan P Wolffe

Volume 305 Bioluminescence and Chemiluminescence (Part C)

Edited by Thomas O Baldwin and Miriam M Ziegler

Volume 306 Expression of Recombinant Genes in Eukaryotic SystemsEdited by Joseph C Glorioso and Martin C Schmidt

Volume 307 Confocal Microscopy

Volume 308 Enzyme Kinetics and Mechanism (Part E: Energetics of EnzymeCatalysis)

Edited by Daniel L Purich and Vern L Schramm

Volume 309 Amyloid, Prions, and Other Protein Aggregates

Edited by Ronald Wetzel

Volume 310 Biofilms

Edited by Ron J Doyle

Volume 311 Sphingolipid Metabolism and Cell Signaling (Part A)

Edited by Alfred H Merrill, Jr., and Yusuf A Hannun

Volume 312 Sphingolipid Metabolism and Cell Signaling (Part B)

Edited by Alfred H Merrill, Jr., and Yusuf A Hannun

Volume 313 Antisense Technology (Part A: General Methods, Methods ofDelivery, and RNA Studies)

Edited by M Ian Phillips

Volume 314 Antisense Technology (Part B: Applications)

Volume 315 Vertebrate Phototransduction and the Visual Cycle (Part A)Edited by Krzysztof Palczewski

Volume 316 Vertebrate Phototransduction and the Visual Cycle (Part B)Edited by Krzysztof Palczewski

Trang 26

Volume 317 RNA–Ligand Interactions (Part A: Structural Biology Methods)Edited by Daniel W Celander and John N Abelson

Volume 318 RNA–Ligand Interactions (Part B: Molecular Biology Methods)Edited by Daniel W Celander and John N Abelson

Volume 319 Singlet Oxygen, UV-A, and Ozone

Edited by Lester Packer and Helmut Sies

Volume 320 Cumulative Subject Index Volumes 290–319

Volume 321 Numerical Computer Methods (Part C)

Edited by Michael L Johnson and Ludwig Brand

Volume 322 Apoptosis

Edited by John C Reed

Volume 323 Energetics of Biological Macromolecules (Part C)

Edited by Michael L Johnson and Gary K Ackers

Volume 324 Branched-Chain Amino Acids (Part B)

Edited by Robert A Harris and John R Sokatch

Volume 325 Regulators and Effectors of Small GTPases (Part D: Rho Family)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 326 Applications of Chimeric Genes and Hybrid Proteins (Part A:Gene Expression and Protein Purification)

Edited by Jeremy Thorner, Scott D Emr, and John N Abelson

Volume 327 Applications of Chimeric Genes and Hybrid Proteins (Part B:Cell Biology and Physiology)

Volume 328 Applications of Chimeric Genes and Hybrid Proteins (Part C:Protein–Protein Interactions and Genomics)

Volume 329 Regulators and Effectors of Small GTPases (Part E: GTPasesInvolved in Vesicular Traffic)

Edited by W E Balch, Channing J Der, and Alan Hall

Volume 330 Hyperthermophilic Enzymes (Part A)

Edited by Michael W W Adams and Robert M Kelly

Volume 331 Hyperthermophilic Enzymes (Part B)

Volume 332 Regulators and Effectors of Small GTPases (Part F: Ras Family I)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 333 Regulators and Effectors of Small GTPases (Part G: Ras Family II)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 334 Hyperthermophilic Enzymes (Part C)

Trang 27

Volume 335 Flavonoids and Other Polyphenols

Volume 336 Microbial Growth in Biofilms (Part A: Developmental andMolecular Biological Aspects)

Volume 337 Microbial Growth in Biofilms (Part B: Special Environments andPhysicochemical Aspects)

Volume 338 Nuclear Magnetic Resonance of Biological Macromolecules(Part A)

Edited by Thomas L James, Volker Do¨tsch, and Uli Schmitz

Volume 339 Nuclear Magnetic Resonance of Biological Macromolecules(Part B)

Edited by Thomas L James, Volker Do¨tsch, and Uli Schmitz

Volume 340 Drug–Nucleic Acid Interactions

Edited by Jonathan B Chaires and Michael J Waring

Volume 341 Ribonucleases (Part A)

Edited by Allen W Nicholson

Volume 342 Ribonucleases (Part B)

Edited by Allen W Nicholson

Volume 343 G Protein Pathways (Part A: Receptors)

Edited by Ravi Iyengar and John D Hildebrandt

Volume 344 G Protein Pathways (Part B: G Proteins and Their Regulators)Edited by Ravi Iyengar and John D Hildebrandt

Volume 345 G Protein Pathways (Part C: Effector Mechanisms)

Edited by Ravi Iyengar and John D Hildebrandt

Volume 346 Gene Therapy Methods

Volume 347 Protein Sensors and Reactive Oxygen Species (Part A:

Selenoproteins and Thioredoxin)

Edited by Helmut Sies and Lester Packer

Volume 348 Protein Sensors and Reactive Oxygen Species (Part B: ThiolEnzymes and Proteins)

Volume 349 Superoxide Dismutase

Volume 350 Guide to Yeast Genetics and Molecular and Cell Biology (Part B)Edited by Christine Guthrie and Gerald R Fink

Volume 351 Guide to Yeast Genetics and Molecular and Cell Biology (Part C)Edited by Christine Guthrie and Gerald R Fink

Trang 28

Volume 352 Redox Cell Biology and Genetics (Part A)

Edited by Chandan K Sen and Lester Packer

Volume 353 Redox Cell Biology and Genetics (Part B)

Edited by Chandan K Sen and Lester Packer

Volume 354 Enzyme Kinetics and Mechanisms (Part F: Detection andCharacterization of Enzyme Reaction Intermediates)

Volume 355 Cumulative Subject Index Volumes 321–354

Volume 356 Laser Capture Microscopy and Microdissection

Volume 357 Cytochrome P450, Part C

Edited by Eric F Johnson and Michael R Waterman

Volume 358 Bacterial Pathogenesis (Part C: Identification, Regulation, andFunction of Virulence Factors)

Volume 359 Nitric Oxide (Part D)

Edited by Enrique Cadenas and Lester Packer

Volume 360 Biophotonics (Part A)

Edited by Gerard Marriott and Ian Parker

Volume 361 Biophotonics (Part B)

Edited by Gerard Marriott and Ian Parker

Volume 362 Recognition of Carbohydrates in Biological Systems (Part A)Edited by Yuan C Lee and Reiko T Lee

Volume 363 Recognition of Carbohydrates in Biological Systems (Part B)Edited by Yuan C Lee and Reiko T Lee

Volume 364 Nuclear Receptors

Edited by David W Russell and David J Mangelsdorf

Volume 365 Differentiation of Embryonic Stem Cells

Edited by Paul M Wassauman and Gordon M Keller

Volume 366 Protein Phosphatases

Edited by Susanne Klumpp and Josef Krieglstein

Volume 367 Liposomes (Part A)

Volume 368 Macromolecular Crystallography (Part C)

Volume 369 Combinational Chemistry (Part B)

Edited by Guillermo A Morales and Barry A Bunin

Volume 370 RNA Polymerases and Associated Factors (Part C)

Edited by Sankar L Adhya and Susan Garges

Trang 29

Volume 371 RNA Polymerases and Associated Factors (Part D)Edited by Sankar L Adhya and Susan Garges

Volume 372 Liposomes (Part B)

Edited by Negat Du¨zgu¨nes,

Volume 373 Liposomes (Part C)

Edited by Negat Du¨zgu¨nes,

Volume 374 Macromolecular Crystallography (Part D)

Edited by Charles W Carter, Jr., and Robert W Sweet

Volume 375 Chromatin and Chromatin Remodeling Enzymes (Part A)Edited by C David Allis and Carl Wu

Volume 376 Chromatin and Chromatin Remodeling Enzymes (Part B)Edited by C David Allis and Carl Wu

Volume 377 Chromatin and Chromatin Remodeling Enzymes (Part C)Edited by C David Allis and Carl Wu

Volume 378 Quinones and Quinone Enzymes (Part A)

Volume 379 Energetics of Biological Macromolecules (Part D)Edited by Jo M Holt, Michael L Johnson, and Gary K AckersVolume 380 Energetics of Biological Macromolecules (Part E)

Edited by Jo M Holt, Michael L Johnson, and Gary K AckersVolume 381 Oxygen Sensing (in preparation)

Edited by Chandan K Sen and Gregg L Semenza

Volume 382 Quinones and Quinone Enzymes (Part B)

Volume 383 Numerical Computer Methods (Part D)

Volume 384 Numerical Computer Methods (Part E)

Volume 385 Imaging in Biological Research (Part A) (in preparation)Edited by P Michael Conn

Volume 386 Imaging in Biological Research (Part B) (in preparation)Edited by P Michael Conn

Trang 30

[1] Mitochondrial Quinone Reductases: Complex I

By Giorgio Lenaz, Romana Fato,Alessandra Baracca, and Maria Luisa Genova

Introduction

The NADH:quinone (Coenzyme Q, CoQ, ubiquinone) oxidoreductase(Complex I) is the most complicated enzyme of the respiratory chain in mi-tochondria and aerobic bacteria.1–3Mitochondrial Complex I is a multisub-unit enzyme that uses the energy associated to NADH oxidation by CoQ topump hydrogen ions across the inner membrane, thus significantly contrib-uting to the formation of an electrochemical proton gradient (Hþ) andconsequently to the efficiency of the oxidative phosphorylation process.The number of protons pumped by Complex I is estimated to be 2 to 5(probably 4) Hþ/2e.4–8

In spite of recent improvements of our knowledge on both structural andfunctional properties of Complex I,9–11the atomic structure and the detailedreaction mechanism of the enzyme are still unknown: it is the only enzyme

of the membrane-bound respiratory chain to remain a ‘‘L shaped blackbox.’’12The reason for this lack of information is principally due to the com-plexity of this enzyme: in fact, the bovine enzyme consists of 46 differentpolypeptides13,14with different prosthetic groups: an FMN, 8 to 9 Fe-S clus-ters and one or more molecules of quinones The need to clarify the molecu-lar mechanism of the enzyme complex is strongly supported by different

1 T Friedrich, K Stainmuller, and H Weiss, FEBS Lett 367, 107 (1995).

2 J E Walker, J M Skehel, and S K Buchanan, this series, Vol 260, p 14.

3 K L Soole and R I Menz, J Bioenerget Biomembr 27, 397 (1995).

4 F Di Virgilio and G F Azzone, J Biol Chem 257, 4106 (1982).

5 M K F Wikstrom, FEBS Lett 169, 300 (1984).

6 G C Brown and M D Brand, Biochem J 252, 473 (1988).

7 H Weiss and T Friedrich, J Bioenerg Biomembr 23, 743 (1991).

8 T Yano, Mol Aspects Med 23, 345 (2002).

9 N Grigorieff, Curr Opin Struct Biol 9, 476 (1999).

10 A D Vinogradov, Biochim Biophys Acta 1364, 169 (1998).

11 M Degli Esposti, A Ngo, G L McMullen, A Ghelli, F Sparla, B Benelli, M Ratta, and

A W Linnane, Biochem J 313, 327 (1996).

12 A Matsuno-Yagi and T Yagi, J Bioenerg Biomembr 33, 155 (2001).

13 I M Fearnley, J Carroll, R J Shannon, M J Runswick, J E Walker, and J Hirst, J Biol Chem 276, 38345 (2001).

14 J Hirst, J Cazzoll, I M Fearnley, R J Shannon, and J E Walker, Biochim Biophys Acta

1604, 35 (2003).

Trang 31

research fields Besides representing a major target of bioenergetics, sincemany neurodegenerative disorders as well as the aging process have beenassociated with Complex I deficiency,15–17knowledge of the enzyme struc-ture-function relationship is essential for better understanding of thesephysiological-pathological dysfunctions.

Therefore the availability of reliable methods allowing the study of theComplex I activity is very important to investigate the mechanism of elec-tron transfer and proton translocation by the enzyme and to assess cellbioenergetic damage occurring in mitochondrial diseases and aging.Assay of Redox Activities of Complex I

NADH-CoQ Reductase

Investigation of electron transfer in the complex along its redox groups

is very difficult to perform Moreover, isolation of Complex I in an activeform is not an easy task,18so the best way to assay its activity is to studythe enzyme in situ, in mitochondrial membranes or in submitochondrialparticles From the functional point of view, Complex I activity can be iso-lated from the other respiratory complexes by the action of specific inhibi-tors such as antimycin A and mucidin for Complex III (acting respectively

on center ‘‘i’’ and ‘‘o’’) and cyanide for Complex IV Myxothiazol should

be avoided, since it also inhibits Complex I.19Difficulties in assaying plex I activity may arise from a limited permeability of its substrates; inparticular, beef heart mitochondria, which are the choice material for Com-plex I studies, must be permabilized to NADH, and this may be achievedthrough freezing and thawing cycles (from 1 to 3 cycles) On the otherhand, the complex uses Coenzyme Q10as physiological electron acceptor;however, this molecule is too hydrophobic and cannot be used as exoge-nous substrate For this reason Complex I activity is normally assayed byusing short-chain analogs of CoQ10: the most widely used are CoQ1(withonly one isoprenoid unit in the side chain) and decylubiquinone (DB) with

Com-a 10-cCom-arbon-Com-atom lineCom-ar sCom-aturCom-ated side chCom-ain.20,21 This lack of a suitable

15 S Rhaman, R B Blok, H H Dahl, D M Danks, D M Kirby, C W Chow,

J Christodoulou, and D R Thorburn, Ann Neurol 39, 343 (1996).

16 J L Loeffen, J A Smeitink, J M Trijbels, A J Janssen, R H Triepels, R C Sengers, and

L P van den Heuvel, Hum Mutat 15, 123 (2000).

17 A H Schapira, Biochim Biophys Acta 1364, 261 (1998).

18 C I Ragan, Curr Top Bioenerg 15, 1 (1987).

19 M Degli Esposti, A Ghelli, M Crimi, E Estornell, R Fato, and G Lenaz, Biochem Biophys Res Commun 190, 1090 (1993).

20 G Lenaz, Biochim Biophys Acta 1364, 207 (1998).

21 E Estornell, R Fato, F Pallotti, and G Lenaz, FEBS Lett 332, 127 (1993).

Trang 32

assay method with endogenous substrates makes Complex I activitymeasurements considerably hampered It has to be borne in mind thatthe activity of Complex I with these exogenous acceptors may be stronglyunderestimated.20,22,23

In Bovine Heart Mitochondria and Submitochondrial Particles

Beef heart mitochondria (BHM) are obtained by a large-scale dure24and submitochondrial particles (SMP) by sonic irradiation of frozenand thawed mitochondria.25SMP obtained by this method are essentiallybroken membrane fragments26; alternatively, coupled closed particles (elec-tron transfer particles or ETPH) are prepared by the method of Hansen andSmith27or EDTA-particles by the method of Lee and Ernster.28

proce-All preparations are kept frozen at 80 at a stock concentrationranging between 40 and 60 mg/ml of protein detected by the biuret method

by Gornall et al.29BHM are frozen and thawed two or three times beforeuse, while SMP are used after thawing once: under these conditions thepermeability barrier for NADH is completely lost, as demonstrated bythe lack of further stimulation by detergents

NADH-CoQ reductase is assayed essentially as described by Yagi30and modified by Degli Esposti et al.19and Estornell et al.21

Reagents

Buffer, 50 mM KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4

NADH, 30 mM freshly prepared solution in water

23 M L Genova, C Castelluccio, R Fato, G Parenti Castelli, M Merlo Pich, G Formiggini,

C Bovina, M Marchetti, and G Lenaz, Biochem J 311, 105 (1995).

24 A L Smith, Methods Enzymol 10, 81 (1967).

25 R E Beyer, Methods Enzymol 10, 519 (1967).

26 R Fato, M Cavazzoni, C Castelluccio, G Parenti Castelli, G Palmer, and G Lenaz, Biochem J 290, 225 (1993).

27 M Hansen and A L Smith, Biochim Biophys Acta 81, 214 (1964).

28 C P Lee and L Ernster, Eur J Biochem 3, 391 (1968).

29 A G Gornall, C J Bardawill, and M M David, J Biol Chem 177, 752 (1949).

30 T Yagi, Arch Biochem Biophys 281, 305 (1990).

Trang 33

Procedure KCN and Antimycin A are added directly to the buffer to

a final concentration of 2 mM and 2 M, respectively; because of thealkaline hydrolysis of CN, it is necessary to adjust the pH to 7.4 to 7.5 withHCl KCN and Antimycin A are added to avoid the electron flow throughthe cytochrome system (complexes III and IV)

The reaction is started by the addition of 10 to 20 g/ml of mitochondrialprotein (either BHM or SMP) and different amounts of the quinone analogsused as external electron acceptor and is followed spectrophotometrically

by the decrease of absorbance at 340 minus 380 nm of a saturating amount

of NADH (75 M) in a double wavelength spectrophotometer (Jasco V-550equipped with a double wavelength accessory and a rapid mixing apparatus)with an extinction coefficient of 3.5 mM1cm1 This extinction coefficientwas directly calculated, taking in account that, in the double wavelengthspectrophotometer used, the light transfer is due to an optical fiberdevice; the extinction coefficient may vary with other spectrophotometers

We have used this method to study the kinetic parameters of differentshort chain quinone homologs such as CoQ0, CoQ1, CoQ2, CoQ3and analogshaving straight saturated chain such as 6-pentyl and 6-decylubiquinones(respectively PB and DB).21,31The results obtained are listed inTable I

As shown in the table, different quinones elicited different CoQ reductase activity, and this is principally due to the capability

NADH-of quinone analogs to interact with the physiological active site NADH-ofComplex I The widely used method to test this capability is to measurethe effect of a specific Complex I inhibitor such as rotenone: NADH-CoQ1, -CoQ2, -CoQ3, -DB, -PB are at least 90% sensitive to rotenone(2 M), suggesting that all quinones are able to interact with the physio-logical site in Complex I Nevertheless, only CoQ1, PB and DB can elicithigh activity; this observation could be explained taking in account thatCoQ2is also a Complex I inhibitor,31–33whereas CoQ3and higher homo-logs are too hydrophobic to be used as external electron acceptors Onthe other hand, CoQ0 as well as the tetramethyl benzoquinone analogduroquinone (DQ) are too hydrophilic and can accept electrons alsofrom a non-physiological site in the complex upstream with respect to therotenone inhibition site

31 R Fato, E Estornell, S Di Bernardo, F Pallotti, G Parenti Castelli, and G Lenaz, Biochemistry 35, 2705 (1996).

32 G Lenaz, P Pasquali, E Bertoli, and G Parenti Castelli, Arch Biochem Biophys 169,

217 (1975).

33 L Landi, P Pasquali, L Cabrini, A M Sechi, and G Lenaz, J Bioenerg Biomembr 16, 153 (1984).

Trang 34

In Mitochondria From Different Rat Tissues

Reagents

Reagents used are same as above

Procedure Liver, heart and muscle mitochondria from rats are pared by the procedure of Kun et al.,34and the enzyme activity is assayed

pre-at 30 after one cycle of freezing and thawing to remove the permeabilitybarrier to NADH without damaging the enzyme The assay medium isthe same used for BHM or SMP The only difference is a higher residualactivity after rotenone inhibition in rat liver mitochondria (RLM) because

of a NADH-dehydrogenase activity of the outer mitochondrial membrane.The rotenone-insensitive component is more evident when using CoQ1assubstrate, so that DB seems to be a more situable acceptor InTable IIarereported some results when using a quasi-saturating concentration of DB(20 to 40 M) and NADH (75 M)

In Mitochondria From Human Platelets

Reagents

Reagents used are same as above

Procedure Platelets are obtained from venous blood samples and fied as described by Degli Esposti et al.35and mitochondrial membranes areprepared as described by Merlo Pich et al.36

puri-34 E Kun, E Kirsten, and W N Piper, Methods Enzymol 55, 115 (1979).

TABLE I Kinetic Constants for NADH-CoQ Reductase in Bovine Heart SMP by

Using Various Acceptorsa

Quinone homologs or analogs Vmaxb (mol min1mg1) Kmb(M)

Trang 35

The activity is measured at 32by utilizing a quasi saturating tion of DB (about 20 to 40 M) as external electron acceptor and NADH(75 M) with an activity value of about 4.6 1.6 nmol min1mg1.

concentra-In Cultured Cells

The major problem in this case is the preparation of the mitochondrialfraction from cells: we have prepared mitochondria from the human osteo-sarcoma 143B cell line by using the method described by Trounce et al.,37starting from about 1 to 2 108 cells (6 to 12 semiconfluent 150-mmdishes) The mitochondrial fraction is kept frozen at80 Complex I activ-ity is assayed after only one thawing process in a buffer containing 250 mMsucrose, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4), 75 M NADH, and

2 mM KCN The reaction is started with 50 M DB and followed photometrically at 340 minus 380 nm for 3 minutes at 30in a Jasco V-550,

spectro-a double-wspectro-avelength spectrophotometer, with the extinction coefficient

of 3.5 mM1 cm1; in another sample, the activity is measured after theaddition of 2 M rotenone; the activity in the presence of rotenone is sub-tracted from the previous one, giving the specific Complex I activity (40 to

50 nmol min1mg1)

35 M Degli Esposti, V Carelli, A Ghelli, M Ratta, M Crimi, S Sangiorgi, P Montagna,

G Lenaz, E Lugaresi, and P Cortelli, FEBS Lett 352, 375 (1994).

36 M Merlo Pich, C Bovina, G Formiggini, G G Cometti, A Ghelli, G Parenti Castelli,

M L Genova, M Marchetti, S Semeraro, and G Lenaz, FEBS Lett 380, 176 (1996).

37 I A Trounce, Y L Kim, A S Jun, and D C Wallace, Methods Enzymol 264, 484 (1996).

TABLE II NADH-DB Reductase Activity in Mitochondria From

Different Rat Tissues

Mitochondria

source

NADH-DB (nmol min1mg1)

G Lenaz, FEBS Lett 410, 467 (1997).

Trang 36

Determination of NADH Dehydrogenase by Using Water-Soluble

Electron Acceptors

Complex I may transfer electrons to water soluble acceptors such as ricyanide and 2,6-dichlorophenolindophenol (DCIP), but the reduction ofthese compounds is not coupled to energy transduction.38Among these ac-ceptors, ferricyanide accepts electrons before the physiological reductionsite, as shown by the lack of inhibition by 2 M rotenone of NADH-ferricyanide reductase activity39and by retention of ferricyanide reductionactivity by the solubilized type I NADH dehydrogenases40 that lack thehydrophobic sector of the enzyme

fer-NADH-Ferricyanide Reductase Activity

Reagents

Antimycin A, 2 mM in ethanol

KCN, 2 mM in buffer solution

Rotenone, 2 mM in ethanol

Variable amounts of potassium ferricyanide

Procedure The reaction is started by adding 150 M NADH to the action mixture containing 10 to 20 g/ml of mitochondrial protein (eitherBHM or SMP), 2 M of antimycin A and 2 mM of KCN and followingspectrophotometrically the decrease of ferricyanide absorbance at 420minus 500 nm in a Jasco V-550 spectrophotometer equipped with a doublewavelength apparatus with an extinction coefficient of 1 mM1cm1at 30and under continuous mixing

re-The rate of ferricyanide reduction is several times that of Coenzyme Q1,the most effective quinone acceptor either in BHM or in SMP, with valuesranging between 20 to 25 mol min1 mg1 in BHM and 40 to 50 molmin1 mg1 in SMP The method has been successfully used also inmitochondrial membranes from human platelets.41

The specific activity of ferricyanide reductase with 2 mM potassiumferricyanide was used to estimate the content of active Complex I in themembrane by considering half of maximum turnover (8 105 min1 as

38 A B Kotlyar and M Gutman, Biochim Biophys Acta 1140, 169 (1992).

39 Y Hatefi, A G Haavik, and D E Griffiths, J Biol Chem 237, 1676 (1962).

40 R L Ringler, S Minakami, and T P Singer, J Biol Chem 238, 352 (1963).

41 V Carelli, A Ghelli, L Bucchi, P Montagna, A De Negri, V Leuzzi, C Carducci,

G Lenaz, E Lugaresi, and M Degli Esposti, Ann Neurol 45, 320 (1999).

Trang 37

in Cremona and Kearney42), since this concentration of ferricyanide isapproximately equal to the Km.43,44

Nevertheless, the amount of Complex I determined by this method may

be overestimated so that it is better to estimate the FMN content by thehigh-performance liquid chromatography (HPLC) method of Light et al.45after water extraction at 80 according to Yagi,46 with a Nova-Pack C18column, 3.9 150 mm (Waters) and 20% methanol in water containing

5 mM ammonium acetate (pH 6) as mobile phase, at a flow rate of0.8 ml/min, in a Waters Millennium 2010 Chromatography Managerequipped with a Waters 996 Photodiode Array Detector

NADH-2,6-Dichlorophenolindophenol (DCIP) Reductase ActivityReagents

Metabolic Flux Control

It is known that Complex I may undergo functional alterations affectingmitochondrial bioenergetics, particularly in aging and in mitochondrialdiseases.47–49The decrease in an individual enzyme activity in a metabolic

42 T Cremona and E B Kearney, J Biol Chem 239, 2328 (1964).

43 S Smith, I R Cottingham, and C I Ragan, FEBS Lett 110, 279 (1980).

44 M Degli Esposti, A Ghelli, M Ratta, D Cortes, and E Estornell, Biochem J 301, 161 (1994).

45 D R Light, C Walsh, and A Merletta, Anal Biochem 109, 87 (1980).

46 K Yagi, Methods Enzymol 18B, 290 (1971).

47 G Lenaz, C Bovina, C Castelluccio, R Fato, G Formiggini, M L Genova, M Marchetti,

M Merlo Pich, F Pallotti, G Parenti Castelli, and G Biagini, Mol Cell Biochem 174, 329 (1997).

48 G Lenaz, M D’Aurelio, M Merlo Pich, M L Genova, B Ventura, C Bovina, G Formiggini, and G Parenti Castelli, Biochim Biophys Acta 1459, 397 (2000).

Trang 38

pathway is meaningful only if it is able to affect the rate of the wholepathway, and this will depend on the degree of flux control exerted

by the individual step itself50; in other words, it depends on how muchthe step is rate controlling on the whole pathway Analysis of the literatureshows that mitochondrial Complex I activity is among the major rate-controlling steps in electron transfer chain,51but the control of respiration

is shared between different biochemical steps and its distribution changesaccording to the metabolic rate performed by mitochondria.52,53

Quantitative Analysis of Metabolic Flux Control

The metabolic control exerted by a single enzyme in a metabolicpathway can be expressed by a coefficient (Cvicalculated from the experi-mentally measured percent changes in the enzyme activities [global activityand individual step]) on addition of small concentrations of a specificinhibitor Thus,

that is, the ratio of the initial slope of the inhibition curve of the globalactivity (J) to the initial slope of the inhibition curve of the individual step(vi) The titration curves can be obtained by a nonlinear regression-fittingprocedure, performed on the experimental data with a commercial app-lication program.54 The initial slope of each curve is calculated as thelimit of the derivative of the function for inhibitor concentration tending

to zero

The profile, particularly the initial slope, of the global flux curve isindicative of the control exerted by the examined enzyme: the steeperthe curve, the greater the similarity to the hyperbolic titration curve

of the single enzyme step and the higher (closer to 1) the flux control ficient If a metabolic pathway is composed of distinct enzymes, theextent to which each enzyme is rate-controlling may be different and the

coef-49 G Lenaz, A Baracca, C Bovina, M Cavazzoni, M D’Aurelio, S Di Bernardo, R Fato,

G Formiggini, M L Genova, A Ghelli, M Merlo Pich, F Pallotti, G Parenti Castelli, and

B Ventura, in ‘‘Recent Research Developments in Bioenergetics’’ (S G Pandalai, ed.), Vol 1, p 63 Transworld Research Network, Trivandrum (India), 2000.

50 A Kacser and J A Burns, Biochem Soc Trans 7, 1149 (1979).

51 B Ventura, M L Genova, C Bovina, G Formiggini, and G Lenaz, Biochim Biophys Acta 1553, 249 (2002).

52 T Letellier, M Malgat, and J-P Mazat, Biochim Biophys Acta 1141, 58 (1993).

53 G P Davey and J B Clark, J Neurochem 66, 1617 (1996).

54 Jandel SigmaPlotÕ Scientific Graphing Software, Jandel Corporation.

Trang 39

sum of all the flux control coefficients for the different enzymes should beequal to unity.55

Metabolic Control of Complex I Over the Respiratory Chain ActivityThis requires the preliminary determination of experimental inhibitioncurves of the NADH oxidase (global activity) and the NADH-ubiquinoneoxidoreductase activity (individual step) The two activities can be deter-mined in succession51 by using a dual-wavelength spectrophotometerequipped with a temperature-controlled compartment and a rapid mixingdevice

Reagents

Antimycin A, 2 mM in ethanol

KCN, 0.2 M freshly prepared in buffer solution

DB, 30 mM solution in ethanol

Rotenone, 12 M, solution in ethanol

Procedure Frozen and thawed mitochondria (2 mg protein/ml) arepulse sonicated five times at 10-sec periods (150 W) with 50-sec intervals

in an ice water bath under nitrogen gas An aliquot of the mitochondrialsuspension (i.e., 80 g protein/ml, final concentration for rat livermitochondria) is diluted in buffer solution The reaction is started with

75 M NADH, and the oxidation of the substrate at 30 is followed atthe wavelength couple of 340 minus 380 nm (e ¼ 3.5 mM1 cm1), first

in the absence and then in the presence of antimycin A (2 M) plus cyanide (1 mM) as downstream inhibitors and decylubiquinone (DB,

K-60 M) as electron acceptor: the former assay condition indicates theNADH oxidase activity of the whole mitochondrial respiratory chain,the latter represents the specific activity of Complex I Inhibition curvesfor both activities are determined experimentally by employing rotenone-titrated mitochondrial suspensions, preincubated for 5 min with increas-ing amounts (0 to 100 pmoles/mg protein) of a concentrated solution

of the specific inhibitor for Complex I The activity rates are expressed aspercentage of the control that has no inhibitor present and plotted againstthe rotenone concentration

55 A K Groen, R J A Wanders, H V Westerhoff, R van der Meer, and J M Tager, J Biol Chem 257, 2754 (1982).

Trang 40

Metabolic Control of Complex I Over Coupled Mitochondrial

Respiration

To determine flux control exerted by Complex I over coupled tion in intact mitochondria, experiments are performed of stepwise inhibi-tion of oxygen consumption in the presence of NAD-linked substrates(global activity), in comparison with stepwise inhibition of NADH-ubiqui-none oxidoreductase activity (individual step) The latter is necessarilydetermined in mitochondrial membrane fragments in the presence of Com-plex I substrates (NADH and DB) and downstream inhibitors (antimycin

respira-A and KCN), essentially as described in the previous section, while theformer is assayed in freshly prepared mitochondria by using a thermostat-ically controlled oxygraph apparatus equipped with a Clark’s electrode and

a rapid mixing device.51

Reagents

Buffer, 100 mM KCl, 75 mM mannitol, 25 mM sucrose, 10 mM

KH2PO4, 2 mM MgCl 10 mM Tris-HCl and 50 M EDTA, pH 7.4Rotenone, 40 M, solution in ethanol

Na-Glutamate, 0.8 M, Na-Malate 0.4 M solution in water

ADP, 0.5 M

Procedure In a typical experiment, mitochondria (1 to 2 mg tein) are incubated for 5 min at 30 in a small volume (about 800 l) ofrespiration buffer in the absence or in the presence of increasing amounts

pro-of rotenone (0 to 100 pmoles/mg protein), and then diluted 1:1 with thesame buffer solution State 4 respiration is started by addition of 10 mMglutamate plus 5 mM malate and monitored for approximately 2 min, thenstate 3 is induced by the addition of 0.5 mM ADP.56Activity rates are ex-pressed as percentage of the control where no inhibitor is present and plot-ted against the rotenone concentration for determination of the fluxcontrol coefficient

Other Activities of Complex I

Proton Translocation

The proton pumping activity of Complex I can be studied by a trofluorometric assay in submitochondrial particles (EDTA particles)prepared from mitochondria of different sources as beef heart and

spec-56 B Chance and G R Williams, Adv Enzymol 17, 65 (1956).

Tiêu đề	Quinones and Quinone Enzymes, Part B
Tác giả	John N. Abelson, Melvin I. Simon
Người hướng dẫn	L. Mario Amzel, Mario A. Bianchet, Jorge Boczkowski, Źilvinas Anusevičius, Michael K. Bowman, Gad Asher, Michel Aubier, John Butler, Alessandra Baracca, Jonathan Cape, Raymond P. Baumann, Maria Cecilia Carreras, Ercole Cavalieri, M. Flint Beal, Narimantas Čènas, Asher Begleiter, Dhrubajyoti Chakravarti, Rosario I. Bello, Tom S. Chan, Shiuan Chen, Jeanne Fourie, Ching Kuang Chow, Maria Luisa Genova, Angela DiFrancesco, Consuelo Gómez-Dı́az, Albena T. Dinkova-Kostova
Trường học	California Institute of Technology
Chuyên ngành	Biology
Thể loại	Methods in Enzymology
Thành phố	Pasadena

Định dạng
Số trang	602
Dung lượng	6,44 MB