cell cycle control

BALASUBRAMANIAN 37, Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 UDO BARON ]2, Zentrum fiir M

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P r e f a c e Since the mid-1980s, there has been an explosion in our understanding

of the mechanisms underlying cell cycle transitions At the core of this progress is the realization that a family of cyclin-dependent kinases (Cdks) catalyzes the events of the cell cycle by phosphorylating numerous target proteins, thereby triggering the replication and segregation of the chromo- somes Despite this key insight, much remains to be learned about the regulation and action of the Cdks The elucidation of the regulatory net- works impinging on the Cdks is an ongoing challenge Moreover, the cata- loging of the substrates of Cdks and an understanding of how phosphorylation of these targets results in the execution of cell cycle events remain unfulfilled goals in most experimental systems Apart from the unraveling

of the mechanisms of cell cycle transitions, the description of the many biological contexts in which cell cycle control is a key determinant is an everblossoming area of research It is well established (or highly likely) that cell cycle regulators play critical roles in the commitment to terminal differentiation, progression to malignant transformation, triggering of apoptosis, and attainment of senescence The study of cell cycle molecules

in these contexts will be of great intellectual and practical importance

A critical feature of cell cycle research is that complementary studies

of diverse organisms and experimental systems have resulted in a synergistic rate of progress in our understanding of key regulatory paradigms The conserved nature of cell cycle control mechanisms in vertebrates, marine invertebrates, and yeast has facilitated a rapid acquisition of universally applicable principles In recognition of this process of discovery, this volume

is largely subdivided according to the major experimental systems that are widely used by current cell researchers Specifically, sections of the volume are devoted to mammalian cells, various yeasts, and several "embryonic" systems In addition, there is one section on techniques that generally can

be applied regardless of the experimental organism

We thank the various authors who took the time to describe their experimental methods in a clear and accessible manner It is hoped that these articles will serve as a resource for future progress in this rapidly burgeoning area of science

WILLIAM G D U N P H Y

XV

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C o n t r i b u t o r s to V o l u m e 2 8 3 Article numbers are in parentheses following the names o f contributors

Affiliations listed are current

PETER D A D A M S ( 5 ) , Dana-Farber Cancer

Institute, Boston, Massachusetts 02115

NATALIE G AnN (3), Howard Hughes Medi-

cal Institute, Department of Chemistry

and Biochemistry, University of Colorado,

Boulder, Colorado 80309

SOREN S L ANDERSEN (2), EMBL, Cell

Biology Programme, D 69117 Heidelberg,

Germany

CHANG BAI (11), Verna and Marrs McLean

Department of Biochemistry, Howard

Hughes Medical Institute, Baylor College of

Medicine, Houston, Texas 77030

MOHAN K BALASUBRAMANIAN (37), Howard

Hughes Medical Institute and Department

of Cell Biology, Vanderbilt University

School of Medicine, Nashville, Tennessee

37232

UDO BARON (]2), Zentrum fiir Molekulare

Biologie, 69120 Heidelberg, Germany

DOUGLAS E BASSETr, JR (10), Department of

Molecular Biology and Genetics, The Johns

Hopkins University School of Medicine,

Baltimore, Maryland 21205-2185, and The

National Center for Biotechnology Infor-

mation, National Library of Medicine,

National Institutes of Health, Bethesda,

Maryland 20894

J JULIAN BLOW (41), Imperial Cancer Re-

search Fund, Clare Hall Laboratories,

South Mimms, Hefts EN6 3LD, England

MARK BOGUSKI (10), The National Center for

Biotechnology Information, National Li-

brary of Medicine, National Institutes of

Health, Bethesda, Maryland 20894

ANGELIKA L BON1N (12), BASF Bioresearch

Corporation, Worcester, Massachusetts

01605-4314

LINDA L BREEDEN (25), Fred Hutchinson

Cancer Research Center, Division of Basic

Sciences, Seattle, Washington 98109

HERMANN BUJARD (12), Zentrum far Molek- ulare Biologie, 69120 Heidelberg, Germany

JUDITH L CAMPBELL (30), Braun Labora- tories, California Institute of Technology, Pasadena, California 91125

ANTONY M CARR (36), MRC Cell Mutation Unit, Sussex University, Brighton BN1 9RR, United Kingdom

REY-HuEI CHEN (43), Department of Physiol- ogy, University of California, San Fran- cisco, San Francisco, California 94143-0444

YONG CH1 (28), California Institute of Tech- nology, Pasadena, California 91125

JAMES P J CHONG (41), Imperial Cancer Re- search Fund, Clare Hall Laboratories, South Mimms, Hefts EN6 3LD, England

DAWN COVERLEY (40), Wellcome/CRC Institute, Cambridge CB2 1QR, United Kingdom

VINCENT L CRYNS (7), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

JAMES DEGREGORI (15), Department of Ge- netics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

RAYMOND J DESHAIES (28), California Insti- tute of Technology, Pasadena, California

91125

JOHN F X DIFFLEY (29), Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hefts EN6 3LD, United Kingdom

WENDY J DIXON (30), Braun Laboratories, California Institute of Technology, Pasa- dena, California 91125

WILLIAM G DUNPHY (42), Division of Biol- ogy, Howard Hughes Medical Institute, Cal- ifornia Institute of Technology, Pasadena, California 91125

ix

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X CONTRIBUTORS TO VOLUME 283

B D DYNLACHT (17), Department of Molecu-

lar and Cellular Biology, Harvard Medical

School, Cambridge, Massachusetts 02138

RHIAN J EDWARDS (36), MRC Cell Mutation

Unit, Sussex University, Brighton BN1 9RR,

United Kingdom

S J ELLEDGE (11, 17, 31), Verna and Marts

McLean Department of Biochemistry, De-

partment of Molecular and Human Genet-

ics, Baylor College of Medicine, Howard

Hughes Medical Institute, Houston, Texas

77030

PETER A FANTES (34), Institute of Cell and

Molecular Biology, University of Edin-

burgh, Edinburgh EH9 3 J T , United

Kingdom

ANNE FERNANDEZ (6), Cell Biology Unit,

Centre de Recherche de Biochimie Macro°

moleculaire, Centre National de la Recher-

che Scientifique, 34033 Montpellier Cedex,

France

RUSSELL R PINCHER (39), The Weis Center

for Research, Geisinger Clinic, Danville,

Pennsylvania 17822

ROBERT P FISHER (19), Program in Cell Biol-

ogy and Genetics, Memorial Sloan-Ketter-

ing Cancer Center, New York, New York

10021

ELLA FREULICH (18), Department of Biologi-

cal Sciences, Columbia University, New

York, New York 10027

SABINE FREUNDLIEB (12), Zentrum far Mo-

lekulare Biologie, 69120 Heidelberg,

Germany

ANDREW M FRY (20), Department of Molecu-

lar Biology, University of Geneva, 1211 Ge-

neva 4, Switzerland

MARTIN FUNK (23), MediGene AG, D-82152

Martinsried/Munich, Germany

DAVID M GLOVER (47), Cancer Research

Campaign, Cell Cycle Genetics Group, De-

partment of Anatomy and Physiology, Med-

icaI Sciences Institute, University of Dundee,

Dundee DD1 4HN, Scotland

MANFRED GOSSEN (12), Division of Biochem-

istry and Molecular Biology, Department of

Molecular and Cell Biology, University of

California, Berkeley, California 94720

KATHLEEN L GOULD (37), Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

WOLFF GRAULICH (23), Institut far Moleku- larbiologie und Tumorforschung (IMT), Philipps- Universititt Marburg, D-35033 Marburg, Germany

STEVEN I HAASE (24), Department of Molec- ular Biology, The Scripps Research Insti- tute, La Jolla, California 92037

E HARLOW (17), Massachusetts GeneralHos- pital Cancer Center, Charlestown, Massa- chusetts 021292

J W HARPER (16, 17), Department of Bio- chemistry, Baylor College of Medicine, Houston, Texas 77030

AVRAM HERSHKO (46), Unit of Biochemistry, Rappaport Faculty of Medicine and Rappa- port Institute for Research in Medical Sci- ences, Technion-Israel Institute of Technol- ogy, Haifa 31096, Israel

PHILIP HIETER (10), Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185

M1NGXIA HUANG (31), Verna and Marts McLean Department of Biochemistry, De- partment of Molecular and Human Genet- ics, Baylor College of Medicine, Howard Hughes Medical Institute, Houston, Texas

77030

LASZLO JAKOI (15), Howard Hughes Medical Institute, Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710

LATA JAYARAMAN (18), Department of Bio- logical Sciences, Columbia University, New York, New York 10027

ANTHONY L JOHNSON (26), Division of Yeast Genetics, National Institute for Medical Research, London NW7 1AA, United Kingdom

LELAND H JOHNSTON (26), Division of Yeast Genetics, National Institute for Medical Research, London NW7 1AA, United Kingdom

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CONTRIBUTORS TO VOLUME 283 xi WILLIAM G KAELIN, JR (5), Dana-Farber

Cancer Institute, Boston, Massachusetts

02115

ERIC KARSENTI (2), EMB L, Cell Biology Pro-

gramme, D 69117 Heidelberg, Germany

BRUCE E KEMP (31), St Vincent' s Institute of

Medical Research, Victoria 3065, Australia

SUNG-Hou KIM (9), Melvin Calvin Labora-

tory, University of California, Berkeley,

Berkeley, California 94720

RANDALL W KING (7), Department of Cell

Biology, Harvard Medical School, Boston,

Massachusetts 02115

MARC W KIRSCHNER (7), Department of Cell

Biology, Harvard Medical School, Boston,

SALLY KORNBLUTH (45), Department of Mo-

lecular Cancer Biology, Duke University

Medical Center, Durham, North Carolina

27710

AKIKO KUMAGAI (42), Division of Biology,

California Institute of Technology, Pasa-

dena, California 91125

NED J C LAMB (6), Cell Biology Unit, Centre

de Recherche de Biochimie Macromolecu-

laire, Centre National de la Recherche Sci-

entifique, 34033 Montpellier Cedex, France

XIAOHONG LENG (10, Department of Bio-

chemistry, Baylor College of Medicine,

Houston, Texas 77030

GUSTAVO LEONE (15), Howard Hughes Medi-

cal Institute, Department of Genetics, Duke

University Medical Center, Durham, North

Carolina 27710

DANIEL J LEW (24), Department of Molecular

Cancer Biology, Duke University Medical

Center, Durham, North Carolina 27710

PETER LOPEZ (5), Cytomation, Inc., Fort Col-

lins, Colorado 80525

KEVIN D LUSTIG (7), Department of Cell Bi-

ology, Harvard Medical School, Boston,

DAVID LYDALL (32), Department of Molecu-

lar and Cellular Biology, University of Ari-

zona, Tucson, Arizona 85721

STUART A MACNEILL (34), Institute of Cell and Molecular Biology, University of Edin- burgh, Edinburgh EH9 3JR, United Kingdom

MARK A MADINE (40), Wellcome/CRC Institute, Cambridge CB2 1QR, United Kingdom

HIRO M H MAHBUBANI (41), Imperial Can- cer Research Fund, Clare Hall Labora- tories, South Mimms, Herts EN6 3LD, England

DANNELL McCOLLUM (37), Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

THOMAS J MCGARRY (7), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

PAUL E MEAD (7), Howard Hughes Medical Institute and Children's Hospital, Harvard Medical School, Boston, Massachusetts

02115

LAURENT MEIJER (9), Centre National de la Recherche Scientifique, Station Biologique

de Roscoff, 29682 Roscoff Cedex, France

SEROIO MORENO (4), Instituto de Microbi- ologia Bioquimica, CSIC/Universidad de Salamanca, Edificio Departmental, Campus Miguel de Unamuno, 37007 Salamanca, Spain

ROLE MOLLER (23), Institut far Molekular- biologie und Tumorforschung (1MT), Philipps- Universiti~t Marburg, D-35033 Marburg, Germany

DOMINIK MUMBERG (23), Department of Pathology, The University of Chicago, Chicago, Illinois 60637

MONICA S MURAKAMI (44), ABL-Basic Re-

search Program, NCI-Frederick Cancer Re- search and Development Center, Frederick, Maryland 21702

ANDREW W MURRAY (33, 42), Department

of Physiology, University of California at San Francisco, San Francisco, California 94143-0444

KENTARO NABESHIMA (35), Department of Biophysics, Faculty of Science, Kyoto Uni- versity, Kyoto 606-01, Japan

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xii CONTRIBUTORS TO VOLUME 283

JOSEPH R NEVINS (15), Howard Hughes Med-

ical Institute, Department of Genetics, Duke

University Medical Center, Durham, North

Carolina 27710

C NGWU (17), Massachusetts General Hospi-

tal Cancer Center, Charlestown, Massachu-

setts 021292

ERICH A NIGG (20), Department of Molecu-

lar Biology, University of Geneva, 1211 Ge-

neva 4, Switzerland

TAKEHARU NISHIMOTO (22), Department of

Molecular Biology, Graduate School of

Medical Science, Kyushu University, Fuku-

oka 812-82, Japan

CHRIS NORBURY (4), Molecular Oncology

Laboratory, Institute of Molecular Medi-

cine, John Radcliffe Hospital, Oxford OX3

9DU, United Kingdom

STEPHEN A OSMANI (39), The Weis Center

for Research, Geisinger Clinic, Danville,

Pennsylvania 17822

MATH-IIAS PETER (27), Swiss Institute for Ex-

perimental Cancer Research, (ISREC),

1066 Epalinges, Switzerland

DAWN E PHELPS (14), Lineberger Compre-

hensive Cancer Center, The University of

North Carolina at Chapel Hill, Chapel Hill,

North Carolina 27599-3280

JONATHON PINES (8), WeUcome/CRC Institute

and Department of Zoology, Cambridge

CB2 1QR, United Kingdom

RANDY Y C POON (21), Department of BiD-

chemistry, Hong Kong University of Science

and Technology, Clearwater Bay, Kowloon,

Hong Kong

CAROL PRIVES (18), Department of Biological

Sciences, Columbia University, New York,

New York 10027

KATHERYN A RESING (3), Department of

Chemistry and Biochemistry, University of

Colorado, Boulder, Colorado 80309

VOLKER R(bNICKE (23), Max-Planck-lnstitut

fiir klinische und physiologische Forschung,

Kerckhoff-Institut, D-61231 Bad Nau-

heim, Germany

ALISON ROWELS (41), Imperial Cancer Re-

search Fund, Clare Hall Laboratories,

South Mimms, Herts EN6 3LD, England

JOAN V RUDERMAN (46), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

PAUL RUSSELL (38), Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

ALICIA A RUSSO (1), Memorial Sloan-Ketter- ing Cancer Center, Cellular Biochemistry and Biophysics Program, New York, New York 10021

SHIGEAKI SAITOH (35), Department of Bio- physics, Faculty of Science, Kyoto Univer- sity, Kyoto 606-01, Japan

YOLANDA SANCHEZ (31), Verna and Marts McLean Department of Biochemistry, Bay- lot College of Medicine, Houston, Texas

77030

CORRADO SANTOCANALE (29), Imperial Can- cer Research Fund, Clare Hall Labora- tories, South Mimms, Hens EN6 3LD, United Kingdom

WILLIAM R SELLERS (5), Dana-Farber Can- cer Institute, Boston, Massachusetts 02115

ROBERT J SHEAFF (13), Division of Basic Sci- ences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104

KAZUHIRO SHIOZAKI (38), Department of Molecular Biology, The Scripps Research Institute, La JoUa, California 92037

LILIA STEPANOVA (16), Department of Bio- chemistry, Baylor College of Medicine, Houston, Texas 77030

AARON F STRAIGHT (33), Department of Physiology, University of California at San Francisco, San Francisco, California 94143-0444

P TODD STUKENBERG (7), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

VALERY SUDAKIN (46), Unit of Biochemistry, Rappaport Faculty of Medicine and Rappa- port Institute for Research in Medical Sci- ences, Technion-Israel Institute of Technol- ogy, Haifa 31096, Israel

E C SWINDELL (17), Verna and Marts McLean Department of Biochemistry, Bay- lot College of Medicine, Houston, Texas

77030

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CONTRIBUTORS TO VOLUME 283 xiii /~kLVARO TAVARES (47), Cancer Research

Campaign, Cell Cycle Genetics Group, De-

partment of Anatomy and Physiology, Med-

ical Sciences Institute, University of Dundee,

Dundee DD1 4HN, Scotland

PIA THt3MMES (41), Imperial Cancer Research

Fund, Clare Hall Laboratories, South

Mimms, Hefts EN6 3LD, England

NICOLE VALTZ (27), Programs in Genetics

and Cell Biology, Department of Biochem-

istry and Biophysics, University of Califor-

nia, San Francisco, San Francisco, Califor-

nia 94143-0448

GEORGE F VANDE WOUDE (44), ABL-Basic

Research Program, NCI-Frederick Cancer

Research and Development Center, Freder-

ick, Maryland 21702

RATI VERMA (28), California Institute of

Technology, Pasadena, California 91125

TED WEINERT (33), Department of Molecular

and Cellular Biology, University of Ari-

zona, Tucson, Arizona 85721

HEIKE WILHELM (2), EMBL, Cell Biology

Programme, D 69117Heidelberg, Germany

J WINSTON (17), Department of Biochemistry,

Baylor College of Medicine, Houston,

Texas 77030

YUE XIONG (14), Department of Biochemistry and Biophysics, Program in Molecular Bi- ology and Biotechnology, Lineberger Com- prehensive Cancer Center, The University

of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280

GANG XU (39), Department of Pathology and Laboratory Medicine, University of Penn- sylvania School of Medicine, Philadelphia, Pennsylvania 19104

MITSUH1RO YANAGIDA (35), Department of Biophysics, Faculty of Science, Kyoto Uni- versity, Kyoto 606-01, Japan

XIAN~ S YE (39), The Weis Center for Re- search, Geisinger Clinic, Danville, Pennsyl- vania 17822

JUNYING YUAN (7), Department of Cell Biol- ogy, Harvard Medical School, Boston, Mas- sachusetts 02115

ZHENG ZHOC (31), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

LEONARD I ZON (7), Howard Hughes Medi- cal Institute and Children's Hospital, Har- vard Medical School, Boston, Massachu- setts 02115

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M E T H O D S IN E N Z Y M O L O G Y

VOLUME I Preparation and Assay of Enzymes

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

Preparation and Assay of Substrates

Special Techniques

VOLUME VII Cumulative Subject Index

VOLUME VIII Complex Carbohydrates

VOLUME IX Carbohydrate Metabolism

Edited by WILLIS A WOOD

VOLUME X Oxidation and Phosphorylation

VOLUME XI Enzyme Structure

Edited by C H W HIRS

VOLUME XII Nucleic Acids (Parts A and B)

VOLUME XIII Citric Acid Cycle

Edited by J M LOWENSTEIN

VOLUME XIV Lipids

Edited by J M LOWENSTEIN

VOLUME XV Steroids and Terpenoids

VOLUME XVI Fast Reactions

xvii

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xviii M E T H O D S IN E N Z Y M O L O G Y

VOLUME XVII Metabolism of Amino Acids and Amines (Parts A and B)

Edited by HERBERT TABOR AND CELIA WHITE TABOR

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 MOLDAVZ AND LAWRENCE GROSSMAN

VOLUME XXI Nucleic Acids (Part D)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE

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)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVZ

Edited by Klvm MOLDAVE AND LAWRENCE GROSSMAN

VOLUME XXXI Biomembranes (Part A)

Edited by SIDNEY FLEISCHER AND LESTER PACKER

VOLUME XXXII Biomembranes (Part B)

VOLUME XXXIII Cumulative Subject Index Volumes I - X X X

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

VOLUME XXXW Lipids (Part B)

Edited by JOHN M LOWENSTEIN

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METHODS IN ENZYMOLOGY xix 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)

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 Organ Systems)

Edited by JOEL G HARDMAN AND BERT W O'MALLEY

VOLUME XL Hormone Action (Part E: Nuclear Structure and Function)

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

VOLUME XLVII Enzyme Structure (Part E)

Edited by C H W HIRS AND SERGE N TIMASHEFF

VOLUME XLVIII Enzyme Structure (Part F)

VOLUME XLIX Enzyme Structure (Part G)

VOLUME n Complex Carbohydrates (Part C)

Edited by VICTOR GINSBURG

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)

VOLUME LIV Biomembranes (Part E: Biological Oxidations)

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XX METHODSIN ENZYMOLOGY

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)

Edited by KIVlE MOLDAVE AND LAWRENCE GROSSMAN

VOLUME LX Nucleic Acids and Protein Synthesis (Part H)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN

VOLUME 61 Enzyme Structure (Part H)

VOLUME 62 Vitamins and Coenzymes (Part D)

Edited by DONALD B McCoRMICK AND LEMUEL D WRIGHT

VOLUME 63 Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods)

Edited by DANIEL L PURICH

VOLUME 64 Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Com- plex Enzyme Systems)

VOLUME 65 Nucleic Acids (Part I)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE

VOLUME 66 Vitamins and Coenzymes (Part E)

Edited by DONALD B McCORMICK AND LEMUEL D WRIGHT

VOLUME 67 Vitamins and Coenzymes (Part F)

Edited by DONALD B McCORMICK AND LEMUEL D WRIGHT

VOLUME 68 R e c o m b i n a n t D N A

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)

VOLUME 72 Lipids (Part D)

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METHODS IN ENZYMOLOGY xxi

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-LX

Edited by EDWARD A DENNIS AND MARTHA G DENNIS

VOLUME 76 Hemoglobins

Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONE

VOLUME 77 Detoxication and Drug Metabolism

VOLUME 78 Interferons (Part A)

VOLUME 79 Interferons (Part B)

VOLUME 80 Proteolytic Enzymes (Part C)

VOLUME 81 Biomembranes (Part H: Visual Pigments and Purple Mem-

branes, I)

VOLUME 82 Structural and Contractile Proteins (Part A: Extracellular Matrix)

VOLUME 83 Complex Carbohydrates (Part D)

VOLUME 84 Immunochemical Techniques (Part D: Selected Immunoassays)

VOLUME 85 Structural and Contractile Proteins (Part B: The Contractile Appara- tus and the Cytoskeleton)

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 Mem-

branes, II)

VOLUME 89 Carbohydrate Metabolism (Part D)

VOLUME 90 Carbohydrate Metabolism (Part E)

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xxii M E T H O D S IN E N Z Y M O L O G Y

VOLUME 91 Enzyme Structure (Part I)

VOLUME 92 Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)

VOLUME 93 Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity)

VOLUME 94 Polyamines

VOLUME 95 Cumulative Subject Index Volumes 61-74, 76-80

VOLUME 96 Biomembranes [Part J: Membrane Biogenesis: Assembly and Tar- geting (General Methods; Eukaryotes)]

VOLUME 97 Biomembranes [Part K: Membrane Biogenesis: Assembly and Tar- geting (Prokaryotes, Mitochondria, and Chloroplasts)]

VOLUME 98 Biomembranes (Part L: Membrane Biogenesis: Processing and Re- cycling)

VOLUME 99 Hormone Action (Part F: Protein Kinases)

VOLUME 100 Recombinant D N A (Part B)

VOLUME 101 Recombinant D N A (Part C)

VOLUME 102 Hormone Action (Part G: Calmodulin and Calcium-Binding Pro- teins)

VOLUME 103 Hormone Action (Part H: Neuroendocrine Peptides)

VOLUME 105 Oxygen Radicals in Biological Systems

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METHODS IN ENZYMOLOGY xxiii VOLUME 108 Immunochemical Techniques (Part G: Separation and Characteriza- tion of Lymphoid Cells)

HELEN VAN VUNAKIS

VOLUME 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)

VOLUME 113 Glutamate, Glutamine, Glutathione, and Related Compounds

Edited by ALTON MEISTER

VOLUME 114 Diffraction Methods for Biological Macromolecules (Part A)

VOLUME 115 Diffraction Methods for Biological Macromolecules (Part B)

VOLUME 116 Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions)

VUNAKIS

VOLUME 117 Enzyme Structure (Part J)

Edited by C H W HIRS AND SERGE N T1MASHEFF

VOLUME 118 Plant Molecular Biology

VOLUME 119 Interferons (Part C)

Edited by SIDNEY PESTKA

VOLUME 121 Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)

VOLUME 122 Vitamins and Coenzymes (Part G)

VOLUME 123 Vitamins and Coenzymes (Part H)

VOLUME 124 Hormone Action (Part J: Neuroendocrine Peptides)

Edited by P MICHAEL CONN

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

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xxiv METHODS IN ENZYMOLOGY

VOLUME 126 Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

VOLUME 127 Biomembranes (Part O: Protons and Water: Structure and Translo- cation)

Edited by LESTER PACKER

VOLUME 128, Plasma Lipoproteins (Part A: Preparation, Structure, and Molecu- lar Biology)

Edited by JERE P SEGREST AND JOHN J ALBERS

VOLUME 129 Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism)

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-Medi- ated Cytotoxicity)

Edited by GIOVANNI OI 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 Contractile Appa- ratus and the Cytoskeleton)

Edited by RICHARD B VALLEE

VOLUME 135 Immobilized Enzymes and Cells (Part B)

Edited by KLAUS MOSBACH

VOLUME 136 Immobilized Enzymes and Cells (Part C)

VOLUME 137 Immobilized Enzymes and Cells (Part D)

VOLUME 138 Complex Carbohydrates (Part E)

Edited by VICTOR G1NSBURG

VOLUME 139 Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins)

Edited by ANTHONY R MEANS AND P MICHAEL CONN

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

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VOLUME 143 Sulfur and Sulfur A m i n o Acids

VOLUME 144 Structural and Contractile Proteins (Part D: Extracellular Matrix)

VOLUME 145 Structural and Contractile Proteins (Part E: Extracellular Matrix)

VOLUME 146 Peptide Growth Factors (Part A )

VOLUME 147 Peptide Growth Factors (Part B)

VOLUME 148 Plant Cell Membranes

VOLUME 149 Drug and Enzyme Targeting (Part B)

Cell Functions and Lymphoid Cell Receptors)

VOLUME 151 Molecular Genetics of Mammalian Cells

VOLUME 152 Guide to Molecular Cloning Techniques

VOLUME 153 Recombinant D N A (Part D)

VOLUME 154 Recombinant D N A (Part E)

VOLUME 155 Recombinant D N A (Part F)

Edited by RAY W u

VOLUME 156 Biomembranes (Part P: ATP-Driven Pumps and Related Trans- port: The Na,K-Pump)

VOLUME 157 Biomembranes (Part Q: ATP-Driven Pumps and Related Trans- port: Calcium, Proton, and Potassium Pumps)

VOLUME 158, Metalloproteins (Part A )

VOLUME 159 Initiation and Termination of Cyclic Nucleotide Action

VOLUME 160 Biomass (Part A: Cellulose and Hemicellulose)

Edited by WILLIS A WOOD AND Scoqq" T, KELLOGG

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xxvi METHODS IN ENZYMOLOGY

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 Inflam- mation)

Edited by GIOVANNI D I SABATO

VOLUME 163 Immunochemical Techniques (Part M: Chemotaxis and Inflam- mation)

Edited by GIOVANNI DI SABATO

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 Model Mem- branes)

VOLUME 172 Biomembranes (Part S: Transport: Membrane Isolation and Char- acterization)

VOLUME 173 Biomembranes [Part T: Cellular and Subcellular Transport: Eukary- otic (Nonepithelial) Cells]

VOLUME 174 Biomembranes [Part U: Cellular and Subcellular Transport: Eukar- yotic (Nonepithelial) Cells]

VOLUME 176 Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dy- namics)

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

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M E T H O D S IN E N Z Y M O L O G Y xxvii

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)

VOLUME 181 RNA Processing (Part B: Specific Methods)

VOLUME 182 Guide to Protein Purification

VOLUME 183 Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences

VOLUME 184 Avidin-Biotin Technology

VOLUME 185 Gene Expression Technology

VOLUME 186 Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants)

VOLUME 187 Arachidonate Related Lipid Mediators

VOLUME 188 Hydrocarbons and Methylotrophy

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

VOLUME 190 Retinoids (Part B: Cell Differentiation and Clinical Applications)

VOLUME 191 Biomembranes (Part V: Cellular and Subcellular Transport: Epithe- lial Cells)

VOLUME 192 Biomembranes (Part W: Cellular and Subcellular Transport: Epi- thelial Cells)

VOLUME 193 Mass Spectrometry

VOLUME 194 Guide to Yeast Genetics and Molecular Biology

VOLUME 195 Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase

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°.°

VOLUME 196 Molecular Motors and the Cytoskeleton

Edited by RICHARD B VALLEE

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 200 Protein Phosphorylation (Part A: Protein Kinases: Assays, Purifica- tion, Antibodies, Functional Analysis, Cloning, and Expression)

Edited by TONY HUNTER AND BARTHOLOMEW M SEFFON

VOLUME 201 Protein Phosphorylation (Part B: Analysis of Protein Phosphoryla- tion, 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 Related Mole- cules)

Edited by JAMES F RIORDAN AND BERT L VALLEE

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 P r o t e i n - D N A I n t e r a c t i o n s

Edited by ROBERT T SAUER

VOLUME 209 Phospholipid Biosynthesis

Edited by EDWARD A DENNIS AND DENNIS E VANCE

VOLUME 210 Numerical Computer Methods

Edited by LUDWIG BRAND AND MICHAEL L JOHNSON

VOLUME 211 DNA Structures (Part A: Synthesis and Physical Analysis of DNA)

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

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M E T H O D S IN E N Z Y M O L O G Y xxix

VOLUME 213 Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation)

VOLUME 214 Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis)

VOLUME 219 Reconstitution of Intracellular Transport

VOLUME 222 Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors)

VOLUME 223 Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors)

VOLUME 224 Molecular Evolution: Producing the Biochemical Data

AND ALLAN C WILSON

VOLUME 225 Guide to Techniques in Mouse Development

VOLUME 226 Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins)

VOLUME 227 Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins)

VOLUME 228 Aqueous Two-Phase Systems

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XXX METHODS IN ENZYMOLOGY

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)

VOLUME 232 Hemoglobins (Part C: Biophysical Methods)

VOLUME 233 Oxygen Radicals in Biological Systems (Part C)

Edited by LESTER PACKER

VOLUME 234 Oxygen Radicals in Biological Systems (Part D)

VOLUME 235 Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors)

Edited by VIRGINIA L CLARK AND PATRIK M BAVOIL

VOLUME 236 Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells)

Edited by VIRGINIA L CLARK AND PATRIK M BAVOIL

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

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 SAVER

VOLUME 247 Neoglycoconjugates (Part B: Biomedical Applications)

Edited by Y C LEE AND REIKO T LEE

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METHODS IN ENZYMOLOGY x x x i VOLUME 248 Proteolytic Enzymes: Aspartic and Metallo Peptidases

Edited by ALAN J BARRETt

VOLUME 249 Enzyme Kinetics and Mechanism (Part D: Developments in En- zyme 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, and Thiyl Radicals)

VOLUME 252 Biothiols (Part B: Glutathione and Thioredoxin; Thiols in Signal Transduction and Gene Regulation)

VOLUME 253 Adhesion of Microbial Pathogens

Edited by R o y J DOYLE AND ITZHAK OFEK

VOLUME 254 Oncogene Techniques

VOLUME 255 Small GTPases and Their Regulators (Part A: Ras Family)

VOLUME 256 Small GTPases and Their Regulators (Part B: Rho Family)

VOLUME 257

Transport)

Edited by W

Small GTPases and Their Regulators (Part C: Proteins Involved in

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

VOLUME 261 Nuclear Magnetic Resonance and Nucleic Acids

Edited by THOMAS L JAMES

VOLUME 262 D N A Replication

VOLUME 263 Plasma Lipoproteins (Part C: Quantitation)

VOLUME 264 Mitochondrial Biogenesis and Genetics (Part B)

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xxxii METHODS IN ENZYMOLOGY

VOLUME 265 Cumulative Subject Index Volumes 228, 230-262

VOLUME 266 Computer Methods for Macromolecular Sequence Analysis

Edited by RUSSELL F DOOLIT!rLE

VOLUME 267 Combinatorial Chemistry

VOLUME 268 Nitric Oxide (Part A: Sources and Detection of NO; NO Synthase)

VOLUME 269 Nitric Oxide (Part B: Physiological and Pathological Processes)

VOLUME 270 High Resolution Separation and Analysis of Biological Macromole- cules (Part A: Fundamentals)

VOLUME 271 High Resolution Separation and Analysis of Biological Macromole- cules (Part B: Applications)

VOLUME 272 Cytochrome P450 (Part B)

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

VOLUME 276 Macromolecular Crystallography (Part A)

VOLUME 277 Macromolecular Crystallography (Part B)

VOLUME 278 Fluorescence Spectroscopy

VOLUME 279 Vitamins and Coenzymes, Part I

VOLUME 280 Vitamins and Coenzymes, Part J

VOLUME 281 Vitamins and Coenzymes, Part K

Edited by DONALD B McCoRMICK, JOHN W SUTrIE, AND CONRAD WAGNER

VOLUME 282 Vitamins and Coenzymes, Part L (in preparation)

Edited by DONALD B McCORMICK, JOHN W SUTTIE, AND CONRAD WAGNER

VOLUME 283 Cell Cycle Control

Edited by WILLIAM G DUNPHY

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METHODS IN ENZYMOLOGY xxxiii VOLUME 284 Lipases (Part A: Biotechnology) (in preparation)

Edited by BYRON RUBIN AND EDWARD A DENNIS

VOLUME 285 Cumulative Subject Index Volumes 263, 264, 266-289 (in preparation)

VOLUME 286 Lipases (Part B: Enzyme Characterization and Utilization) (in preparation)

Edited by BYRON RUBIN AND EDWARD A DENNIS

VOLUME 287 Chemokines (in preparation)

Edited by RICHARD HORUK

VOLUME 288 Chemokine Receptors (in preparation)

Edited by RICHARD HORUK

VOLUME 289 Solid-Phase Peptide Synthesis (in preparation)

Edited by GREGG B FIELDS

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[ 1] PURIFICATION OF CYCLIN-DEPENDENT KINASE 2 3

of CDKs is tightly regulated by multiple mechanisms that involve activating

or inhibitory p r o t e i n - p r o t e i n interactions as well as covalent modifications

by phosphorylation 2 T h e activating regulatory mechanisms involve binding

of the cyclin subunit and phosphorylation on the regulatory T loop of the

C D K (Thr-160 for C D K 2 ) by the CDK-activating kinase (CAK)3,4 whereas inhibitory mechanisms involve phosphorylation at a threonine/tyrosine pair

of residues (Thr-14 and Tyr-15 for CDK22), as well as the binding of the CDK-inhibitory proteins ( C K I s ) J T h e isolated C D K subunit is inactive as

a protein kinase and requires the binding of its cyclin subunit for partial activation 6 This is a key step responsible for much of the temporal regulation of C D K activity, as cyclin protein levels are tightly controlled in a cell cycle-dependent m a n n e r by ubiquitin-dependent degradation and by transcription 2 Cyclin binding also appears to facilitate the phosphorylation

of the C D K subunit by C A K , which is involved in the full activation of the CDK 3

T h e mechanisms of most of these regulatory steps have been elucidated

by the crystallographic analyses of C D K 2 in four distinct states (Fig I): the inactive CDK2 m o n o m e r ] the partially active cyclin A - C D K t complex, 8 the fully active cyclin A - C D K 2 complex phosphorylated on Thr-160

of CDK2, 9 and the phosphorylated cyclin A - C D K 2 complex bound to the

i j Pines, Cancer Biol 6, 63 (1995)

2 D O Morgan, Nature (London) 374, 131 (1995)

-~ R P Fisher and D O Morgan, Cell 78, 713 (1994)

4 T P Mtikel~, J P Tassan, E A Nigg, S Frutiger, G J Hughes, and R A Weinberg,

Nature (London) 3/1, 254 (1994)

s C J Sherr and J M Roberts, Genes Dev 9, 1149 (1995)

,1L Connell-Crowley, M J Solomon, N Wei, and J W Harper, Mol BioL Cell 4, 79 (1993)

7 H L De Bondt, J Rosenblatt, J Jancarik, H D Jones, D O Morgan, and S Kim, Nature (London) 363, 595 (1993)

P D Jeffrey, A A Russo, K Polyak, E Gibbs, J Hurwitz, J Massagu6, and N P Pavletich,

Nature (London) 376, 313 (1995)

9 A A Russo, P D Jeffrey, and N P Pavletich, Nature Strucr Biol 3, 696 (1996)

Trang 25

4 GENERAL METHODOLOGIES [ 1]

Inactive Inactive 1 O0/~/-1 see-1 30,000 ~-1 sec-1

Fxo 1 The serine/threonine protein kinase activity of CDK2 is regulated by multiple mechanisms involving protein-protein interactions as well as covalent modification by phosphorylation The kc~t/Km values were obtained from a Lineweaver-Burk plot (1/velocity vs

1/[substrate]), using a consensus substrate peptide Encircled P indicates the phosphate group

p27 Kip1 CKI, in a fully inhibited state 1° A comparison of the structure of inactive, free CDK2 with the structure of CDK2 in the partially active cyclin A - C D K 2 complex revealed that cyclin activates the CDK by re- aligning catalytic site residues (bringing them into correct register for cataly- sis) as well as by relieving the steric blockade of the catalytic cleft imposed

by the regulatory T loop in the free form of the kinase, s The crystal structure

of the fully active phosphorylated CDK2-cyclin A complex showed that phosphorylation induces additional conformational changes in the T loop

of CDK2 that result in additional cyclin A contacts, thus stabilizing the cyclin-CDK complex, as well as resulting in structural changes in the puta- tive substrate-binding site of CDK2 9 The crystal structure of the p27 Kipt inhibitory domain bound to the phosphorylated CDK2-cyclin A complex revealed the structural basis of inhibition by the Kip/Cip family of CKIs

In this complex, p27 binds as an extended structure interacting with both cyclin A and CDK2; p27 binding inhibits the CDK catalytic activity because its interactions with the CDK2 induce conformational changes that change the shape of the catalytic cleft, and because p27 inserts into and fills up the catalytic cleft, mimicking ATP 1°

The cyclin A - C D K 2 complex was used as the model system in these studies because of the availability of highly suitable baculovirus expression clones constructed in the laboratory of D O Morgan al and the apparently greater stability of the complex relative to that of other CDK-cyclin complexes Several laboratories have described methods to express and purify CDK2 H and cyclin A 6, as well as the CDK-activating kinase (CAK) 3 and

to A A Russo, P D Jeffrey, A K Patten, J Massagu6, and N P Pavletich, Nature (London)

382, 325 (1996)

11 j Rosenblatt, H De Bondt, J Jancarik, D O Morgan, and S Kim, J Mol Biol 230,

1317 (1993)

Trang 26

[ 1 ] PURIFICATION OF CYCLIN-DEPENDENT KINASE 2 5 the inhibitor molecule p27.12 These methods have been adapted and modi- fied for the large-scale production of these proteins necessary for X-ray crystallographic analysis

Methods that are described in this chapter include the following: the

purification of CDK2 from baculovirus-infected insect cells; the Escherichia coli expression, purification, and proteolytic truncation of cyclin A; forma-

tion and purification of a CDK2-cyclin A complex; the expression and purification of CAK (CDK7/cyclin H) from coinfected baculovirus insect

cells; the in vitro activation of the CDK2-cyclin A complex through phos-

phorylation by CAK and subsequent purification of the activated complex;

the E coli expression and purification of the inhibitory domain of the

p27 Kipl CKI and the formation and purification of the CDK2-cyclin A-p27 ternary complex Also described are assays for CDK2 catalytic activity

Methods

Overexpression and Purification of Cyclin-Dependent Kinase 2

The baculovirus construct expressing CDK2 has been generously pro- vided by D O Morgan l~ To produce large quantities of CDK2, 200 plates (150 c m 2) of High 5 insect cells (Invitrogen, San Diego, CA) at 80% conflu- ency (approximately 5 x 107 cells/plate), grown in Grace's medium (20 ml/plate; GIBCO, Grand Island, NY) with 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT), are infected with 0.5 ml of viral supernatant [1 × 108 plaque-forming units (pfu)/ml] The infection is carried out by removing the medium from the cells and gently adding 0.5 ml of viral supernatant to the plate, being careful not to disturb the cells Over the period of 1 hr, the plates are gently rocked every 15 min to ensure complete coverage of the cells with the viral supernatant After incubation with the viral supernatant, 15 ml of the Grace's medium containing 10% (v/v) FBS

is added back to the plates After a 48-hr incubation at 27 ° the cells are harvested, using sterile technique, by pipetting them off the plate with a 10-ml pipette, pooling them together in a sterile conical centrifuge tube, and centrifuging them at 650 g in a swinging bucket rotor (Sorvall, Newtown, CT) for 5 min at 4 ° After removal of the supernatant from the cell pellet, the cells are resuspended in 200 ml of hypotonic buffer containing 10.0

mM tris(hydroxymethyl)aminomethane hydrochloride (Tris), 25.0 mM NaC1, 1.0 mM ethylenediaminetetraacetic acid (EDTA), 1.0 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (1.0/xg/ml), aprotinin (2.0/zg/ L~_ K Polyak, M Lee, H Erdjument-Bromage, A Koff, J M Roberts, P Tempst, and J Massagu6, Cell 78, 59 (1994)

Trang 27

6 GENERAL METHODOLOGIES [ 1] ml), and 2.0 mM 1,4-dithiothreitol (DTI'), pH 7.4 and are incubated at 4 ° for 40 min to swell them The swollen cells are poured into a 40-ml tissue grinder (Dounce; Wheaton, Millville, N J), ground 10 times with the "tight"- fitting pestle, incubated on ice for 1 hr to allow for the release of the cell contents from the perforated cells into the buffer, and centrifuged at 30,000

g at 4 ° for 30 min in an SS34 (Sorvall) rotor to remove the nuclei and cell debris The supernatant is loaded at 10 ml/min onto a 75-ml Q-Sepharose Fast Flow column (Pharmacia, Piscataway, NJ) preequilibrated with 10.0

mM Tris, 25.0 mM NaC1, and 5.0 mM DTT, pH 7.5 The flow-through of the Q-Sepharose column containing the recombinant CDK2 is loaded at 5 ml/min onto a 25-ml ATP-agarose column (Sigma, St Louis, MO) 12a that has been preequilibrated with 10.0 mM 4-(2-hydroxyethyl)-l-piperazineeth- anesulfonic acid, sodium salt (HEPES), 25.0 mM NaCI, 10% (v/v) glycerol, and 5.0 mM DTT, pH 7.5 The column is washed with the equilibration buffer until the ultraviolet (UV) absorbance returns to zero, and the CDK2

is eluted with a 10-column volume gradient from 25.0 to 500.0 mM NaCI

in 10.0 mM HEPES (pH 7.4), 10% (v/v) glycerol, and 5.0 mM DTT A peak of CDK2 protein is obtained at approximately 225.0 mM NaC1 Ap- proximately 1 mg of CDK2 is obtained per plate of infected cells

Overexpression and Purification of Cyclin A

Human cyclin A is overexpressed from the T7 promoter in BL21DE3

E coli cells 8 Twenty-four liters of LB medium containing ampicillin (200 /zg/ml) is inoculated with an overnight culture of the cyclin A clone (0.2 ml/liter of medium), which is grown at 20 ° in a floor shaker to a n OD600

of 1.0, at which point 1.0 mM isopropyl-/3-D-thiogalactopyranoside (IPTG)

is added and the culture induced for 18 hr at 20 ° The cells are harvested

by centrifugation in a GS3 rotor (Sorvall) at 5000 g at 4 ° for 10 min, resuspended in 25.0 mM HEPES, 300.0 mM NaC1, 0.1 mM PMSF, 5.0

mM DTT (pH 7.5), and lysed in a cell disrupter (EmulsiFlex-C5; Avisten, Ottawa, Ontario, Canada) under a pressure of 10,000 psi The lysed cells are centrifuged at 30,000 g in an SS34 rotor to remove cell debris and insoluble proteins The supernatant is then diluted 1 : 2 (v/v) with 40.0 mM HEPES, 5.0 mM DTT (pH 7.0) and loaded onto a 40-ml S-Sepharose column (Pharmacia) at 4 ° preequilibrated with 40.0 mM HEPES, 150.0 mM NaCI, and 5.0 mM DTT, pH 7.0 The column is washed with 10 column volumes of equilibration buffer and eluted with 40.0 mM HEPES, 400.0

mM NaCI, 5.0 mM DTT, pH 7.0 Cyclin A is about 80% pure after this initial step A gradient from this column is not useful because the cyclin

12a Sigma adenosine 5'-triphosphate 4% beaded agarose, cyanogen bromide activated, attached

at ribose hydroxyls, with an eleven-atom spacer

Trang 28

[ 1 ! PURIFICATION OF CYCLIN-DEPENDENT KINASE 2 7

A elutes as a broad peak (100.0 to 300.0 mM NaC1) and no further purification is obtained

Characterization of Cyclin A-CDK2 Complex

The cyclin A - C D K 2 complex remains intact during gel-filtration chro- matography, indicating a long half-life and high-affinity interaction The cyclin A and CDK2 preparations are mixed at an approximate equimolar ratio, incubated on ice for 30 min, and fractionated on a Superdex 200 gel- filtration column (Pharmacia) at 4 ° The complex elutes at an apparent molecular mass of 55 kDa, with no detectable dissociation (monomeric CDK2 and monomeric cyclin A each elute at approximately 30 kDa) The ability of cyclin A to convey basal kinase activity to CDK2 is assayed by

a histone H1 kinase assay The reaction contains 25.0 mM HEPES, 50.0

mM NaC1, 5.0 mM MgC12, 0.1 mM adenosine 5'-triphosphate (ATP), 6.25

nM [y-32p]ATP (4000 Ci/mmol), 1.0/xg of histone H1 (Boehringer Mann- heim, Indianapolis, IN), and 100 ng of CDK2-cyclin A (pH 7.5) in a total volume of 20/zl and is carried out at room temperature for 30 min The reaction is separated on a 10% (w/v) sodium dodecyl sulfate (SDS)- polyacrylamide gel, and the histone H1 phosphorylation is quantitated by scanning an autoradiogram and determining the amount of radioactivity incorporated An approximate 40,000-fold stimulation is measured in the presence of cyclin A, consistent with previous reports 6

Characterization of Cyclin A by Proteolytic Digestion

To address the question of whether a structural domain of cyclin A is responsible for binding and activating CDK2, the cyclin A preparation is digested by the protease subtilisin (Boehringer Mannheim) Subtilisin is chosen for its low amino acid sequence preference, as this results in a digestion pattern more sensitive to the domain organization of the protein, instead of a pattern based on digestion at sequences sensitive to certain proteases Subtilisin is added to the cyclin A preparation (1-3 mg/ml) at

a final concentration of 10/xg/ml After a 30-min incubation at 4 °, a 20-/xl aliquot is quenched with 0.1 mM PMSF (final concentration) The rest of the digestion is frozen in liquid nitrogen to pause the digestion while the aliquot is being separated on a 15% (w/v) SDS-polyacrylamide gel for analysis of the progress of the digestion This procedure is repeated until 80-90% of the protein is a 31-kDa product, as determined by SDS- polyacrylamide gel electrophoresis (SDS-PAGE), at which point the entire digestion is stopped by the addition of 0.1 mM PMSF The reaction is then diluted to 75.0 mM NaC1 with 40.0 mM HEPES and 5.0 mM DTT (pH 7.0) and loaded onto a 10-ml Mono S column (Pharmacia) at 4 ° preequilibrated with 40.0 mM HEPES, 75.0 mM NaC1, and 5.0 mM DTT, pH 7.0

Trang 29

8 GENERAL METHODOLOGIES [ 1] The column is washed with 20 column volumes of the equilibration buffer and the protein is eluted with a 20-column volume gradient from 75.0 to 400.0 mM NaC1 The purified 31-kDa product is determined to contain amino acids 174-432 of cyclin A by N-terminal sequencing and mass spectroscopy 8 This proteolytic fragment has CDK2-binding and stimulatory properties essentially identical to those of full-length cyclin A, 8 consistent with previous studies showing a similar fragment having mitotic stimulatory activity in Xenopus oocytes, t3'14 This procedure yields approximately 75

mg of the 31-kDa proteolytic digestion product of cyclin A from 24 liters

of culture

Formation of CDK2-Cyclin A Fragment Binary Complex

To form the CDK2-cyclin A complex, 50 mg of the CDK2 preparation and 75 mg of the cyclin A fragment preparation are incubated at 4 ° for 30 min and concentrated by ultrafiltration through a 10-kDa cutoff membrane (Amicon, Danvers, MA) to a concentration of 30 mg/ml The complex is then loaded onto a 24-ml Superdex 200 gel-filtration column at 4 ° preequilibrated with 40.0 mM HEPES, 200.0 mM NaCI, and 5.0 mM D T r , pH 7.0 The peak of interest containing both proteins is collected and concentrated

to 20 mg/ml by ultrafiltration The ratio is determined to be 1 : 1 by SDS-

P A G E of the complex after gel filtration, and by UV absorbance at 280

nm The activity of the CDK2-cyclin A complex is verified by a histone H1 kinase assay 8

Overexpression and Purification of Cyclin-Dependent

48 hr at 27 ° and the cells are harvested and lysed as previously described for the CDK2 purification However, the viral supernatant is not collected

by sterile technique for future use, because a coinfection does not yield equal amounts of both viruses in the supernatant and subsequent passages may lose one of the viruses The protein supernatant from the final step

in the lysing protocol is brought to 40.0 mM Bis-Tris-propane (BTP), pH

t3 H Kobayashi, E Stewart, R Poon, J P Adamczewski, J Gannon, and T Hunt, MoL Biol Cell 3, 1279 (1992)

14 E M Lees and E Harlow, Mol Cell Biol 13, 1194 (1993)

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[ 1] PURIFICATION OF CYCLIN-DEPENDENT KINASE 2 9 8.5, and loaded at 5 ml/min onto a 10-ml Source Q column (Pharmacia) preequilibrated with 40.0 mM BTP, 25.0 mM NaC1, and 5.0 mM DTT, pH 8.5 The column is washed with 20 column volumes of the equilibration buffer and eluted with a 20-column volume gradient from 25.0 to 400.0

mM NaC1 Fractions (5 ml) are collected and assayed for their ability to phosphorylate CDK2 in the CDK2-cyclin A complex In a 20-tzl reaction containing 5 mg of CDK2-cyclin A complex, 25.0 mM HEPES, 100.0 mM NaC1, 1.0 mM MgCI2, and 0.5 mM ATP (pH 7.5), CAK activity is assayed

by adding 5 tzl of the CAK fractions from the anion-exchange column and incubating at room temperature for 1 hr The reactions are then separated

on a 15% (w/v) SDS-polyacrylamide gel and the phosphorylation of the CDK2 is visualized by Coomassie staining of the gel, thus verifying a change

in mobility of the CDK2 3 The fractions containing CAK activity are pooled and diluted to 25.0 mM NaC1 with 40.0 mM BTP (pH 8.5) and are further purified and concentrated on a 1-ml Source Q column at 4 °, eluted with a 20-column volume NaC1 gradient (0-500 mM) Fractions with the highest specific activity are pooled to yield 80-90% CDK7 and cyclin H proteins

in an approximately equimolar ratio

Large-Scale Phosphorylation and Purification of Cyclin-Dependent Kinase 2 Bound to Cyclin A Fragment

The phosphorylation reaction is scaled up proportionally from the conditions used to assay the CAK fractions In a volume of 200 ml, 50 mg of the CDK2-cyclin A complex is incubated with 1.0 ml (approximately 1 mg) of the CAK preparation at 16 ° under the conditions described above The phosphorylation of CDK2 is verified by the S D S - P A G E mobility shift,

as described above, and the reaction is purified when the phosphorylation

is near 100% complete usually after 3-5 hr The phosphorylation reaction

is concentrated to 10 mg/ml by ultrafiltration as previously described and loaded onto a 120-ml Superdex 200 column (Pharmacia) at 4 ° preequilibrated with 40.0 mM HEPES, 200.0 mM NaC1, and 5.0 mM DTT, pH 7.0 The CDK2-cyclin A binary complex is separated from what appears to be a CDK7-cyclin H-CDK2-cyclin A quaternary complex during gel filtration The binary complex is concentrated to 20 mg/ml by ultrafiltration

Purification of Inhibitory Domain of p27

The full-length p27 protein, overexpressed and purified according to published procedures, 12 is digested by subtilisin to investigate its structural organization The digestion reveals that p27 can be cleaved to produce two fragments: an N-terminal fragment (residues 28-96) containing C D K 2 - cyclin A inhibitory activity essentially identical to that of the full-length p27,1° and a C-terminal fragment (residues 123-198) that does not contain

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10 GENERAL METHODOLOGIES [ 11 any inhibitory activity The N-terminal fragment (residues 22-106) is cloned into the pET3d expression vector (Novagen, Madison, WI) To purify large quantities of this fragment, a l-liter culture of LB medium with ampicillin (200/zg/ml) is inoculated with a fresh colony and grown overnight at 25 ° The overnight culture is used to inoculate twenty-four l-liter cultures, which are grown at 37 ° to an OD600 of 0.5, induced with 1.0 mM IPTG (final concentration), and grown for an additional 3 hr at 37 ° The cells are harvested and lysed as previously described for the cyclin A purification

in a buffer containing 10 mM Tris, 100 mM NaC1, 10 mM DTT, leupeptin (1/zg/ml), aprotinin (1/zg/ml), and 0.1 mM PMSF, pH 7.5 The extract is loaded onto a 75-ml Q-Sepharose Fast Flow column at 4 ° that has been preequilibrated in 10.0 mM Tris, 100.0 mM NaC1, 10.0 mM DTT, pH 7.5 The p27 fragment flows through the Q-Sepharose and is dialyzed against 10.0 mM Tris, 25.0 mM NaC1, 10.0 mM DTT (pH 7.5) overnight at 4 ° The dialyzed flow-through is loaded onto a 10-ml Mono Q column (Pharmacia)

at 4 ° preequilibrated with the dialysis buffer The p27 fragment flows through the Mono Q column and is approximately 75% pure The flow- through is brought to 40.0 mM 2-(N-morpholino)ethanesulfonic acid (MES,

pH 6.0), loaded onto a 10-ml heparin sulfate column (Tosohaus, Montgom- eryville, PA) preequilibrated in 40.0 mM MES, 25.0 mM NaC1, 10.0 mM DTT (pH 6.0), and eluted with a 20-column volume gradient from 25

to 500 mM NaC1 The p27 fragment elutes at approximately 150 to 200

mM NaC1

Formation and Purification of Ternary Cyclin A-CDK2-p27 Complex

To form and purify the CDK2-cyclin A-p27 ternary complex, 10 mg

of the phosphorylated CDK2-cyclin A complex is incubated with 5 mg of the p27 N-terminal fragment (this is a threefold molar excess of the p27 fragment) on ice for 30 min The binding reaction is concentrated to 15 mg/ml by ultrafiltration with a 50-ml Amicon filtration device, using a 3-kDa molecular weight cutoff membrane The concentrated ternary complex is fractionated through a 24-ml Superdex 200 column preequilibrated with 40.0 mM HEPES, 200.0 mM NaC1, and 5.0 mM DTT, pH 7.0 The complex contains a 1 : 1 molar ratio of the binary CDK2-cyclin A to p27 as determined by UV absorption at 280 nm The ternary complex (containing CDK2, the cyclin A fragment, and the N-terminal p27 fragment) is concentrated to 20 mg/ml by ultrafiltration as previously described

Catalytic Activity of Four Forms of Cyclin-Dependent Kinase 2

Figure 2 shows the histone H1 kinase activity of monomeric CDK2, unphosphorylated CDK2-cyclin A binary complex, phosphorylated CDK2-cyclin A complex, and the CDK2-cyclin A-p27 ternary complex

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of phosphate incorporated per 25/xmol of the consensus peptide in the presence of 30 nM kinase for 30 min

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12 GENERAL METHODOLOGIES 121 Similar results are obtained using a consensus substrate peptide 15 For the assays involving a peptide substrate, the reactions contain 30 nM kinase,

500 nM consensus peptide, 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 50 mM NaCl, 10 mM MgC12, 1.0 mM ATP, 6.25 nM [y-azp]ATP, 2.0 mM DTT, and bovine serum albumin (100/xg/ml, pH 7.0) in a 50-/xl volume The reactions are incubated for 30 min at 30 °, spotted on P81 cation-exchange filter paper (Whatman, Clifton, N J), and washed with 30% (v/v) acetic acid to remove free radiolabeled ATP from the phosphorylated peptide, which remains bound to the filter paper The filters are briefly washed with 100% acetone and allowed to air dry The radiolabeled phosphate incorporated into the peptide during the phosphorylation reaction

is then measured by scintillation counting

15 Z Songyang, S Blechner, N Hoagland, M F Hoekstra, H Piwinica-Worms, and L C

Cantley, Curt Biol 4, 973 (1994)

1 L Brizuela, G Draetta, and D Beach, Proc Natl Acad Sci U.S.A 86, 4362 (1989)

2 j C L a b b r , A Picard, G Peaucellier, J C Cavadore, P Nurse, and M D o r r e , Cell 57,

253 (1989)

3 j C Labbr, J P Capony, D Caput, J C Cavadore, J Derancourt, M Kaghdad, J M

Lelias, A Picard, and M D o r r e , E M B O J 8, 3053 (1989)

4 M J Lohka, M K Hayes, and J L Mailer, Proc Natl Acad Sci U.S.A 85, 3009

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12 GENERAL METHODOLOGIES 121 Similar results are obtained using a consensus substrate peptide 15 For the assays involving a peptide substrate, the reactions contain 30 nM kinase,

500 nM consensus peptide, 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 50 mM NaCl, 10 mM MgC12, 1.0 mM ATP, 6.25 nM [y-azp]ATP, 2.0 mM DTT, and bovine serum albumin (100/xg/ml, pH 7.0) in a 50-/xl volume The reactions are incubated for 30 min at 30 °, spotted on P81 cation-exchange filter paper (Whatman, Clifton, N J), and washed with 30% (v/v) acetic acid to remove free radiolabeled ATP from the phosphorylated peptide, which remains bound to the filter paper The filters are briefly washed with 100% acetone and allowed to air dry The radiolabeled phosphate incorporated into the peptide during the phosphorylation reaction

is then measured by scintillation counting

15 Z Songyang, S Blechner, N Hoagland, M F Hoekstra, H Piwinica-Worms, and L C

Cantley, Curt Biol 4, 973 (1994)

1 L Brizuela, G Draetta, and D Beach, Proc Natl Acad Sci U.S.A 86, 4362 (1989)

2 j C L a b b r , A Picard, G Peaucellier, J C Cavadore, P Nurse, and M D o r r e , Cell 57,

253 (1989)

3 j C Labbr, J P Capony, D Caput, J C Cavadore, J Derancourt, M Kaghdad, J M

Lelias, A Picard, and M D o r r e , E M B O J 8, 3053 (1989)

4 M J Lohka, M K Hayes, and J L Mailer, Proc Natl Acad Sci U.S.A 85, 3009

Trang 35

[2] CYCLIN B 1 / c d c 2 KINASE PURIFICATION 13 rus, 5 yeast, 6 human cell systems, 7'8 or Xenopus 9 Here, we describe a procedure that makes use of glutathione S-transferase (GST)-tagged recombinant cyclin that complexes to and activates the native cdkl protein present in

X e n o p u s egg extracts devoid of mitotic cyclins This method was first described by Solomon et aL 9 Here we describe an improved version of this preparation as well as the characterization of the purified enzyme The principle of the method is diagrammed in Fig 1: the cyclin is added to the concentrated interphase extract that contains inactive free cdc2 protein

An inhibitor of type 2 A phosphatase (microcystin) is added to the extract

to keep cdc25 active l°'ll and produce a fully active cyclin B 1-cdc2 complex The cdc2-cyclin B1 complex is then retrieved on glutathione beads, eluted, concentrated, and further purified on a Mono S column (Fig 1)

Cyclin Purification

Materials and Solutions

EX: Phosphate-buffered saline (PBS: 137 mM NaC1, 2.7 mM KC1, 10

mM Na2HPO4, 1.5 mM KH2PO4) containing 1 mM EGTA, 1 mM EDTA, 0.1% (v/v) Tween 20, lysozyme (0.1 mg/ml), and protease inhibitor mix [1 mM phenylmethylsulfonyl fluoride (PMSF), 10 ~g/

ml each of aprotinin, pepstatin, and leupeptin]

WB: PBS with 300 mM KC1, 1 mM D3T, and protease inhibitor mix ELB: 50 mM Tris (pH 8.1), 300 mM KCI, protease inhibitor mix, and

5 mM reduced glutathione (Sigma, St Louis, MO)

FB: 50 mM HEPES (pH 7.6), 250 mM KC1, and 30% (v/v) glycerol The buffers may be stored without protease inhibitors and reduced glutathione at 4 °

GT-agarose: glutathione-agarose (Sigma)

Reduced glutathione (Sigma)

GST-cyclin B1 plasmid: A kind gift from D Kellogg [in B1 21 (DE3) pLys S strain]

Procedure

1 Grow 100 ml of preculture overnight in Luria-Bertani medium (LB) with ampicillin (0.1 mg/ml) at 37 °

5 D Desai, Y Gu, and D O Morgan, Mol Biol Cell 3, 571 (1992)

D Leroy, V Baldin, and B Ducommun, Yeast 10, 1631 (1994)

7 Z Q Pan, A Amin, and J Hurwitz, I Biol Chem 268, 20443 (1993)

Z Q Pan and J Hurwitz, J Biol Chem 268, 20433 (1993)

9 M J Solomon, T Lee, and M W Kirschner, Mol Biol Cell 3, 13 (1992)

t~ M A F61ix, P Cohen, and E Karsenti, E M B O J 9, 675 (1990)

11 I Hoffmann, P R Clarke, M J Marcote, E Karsenti, and G Draetta, EMBOJ 12, 53 (1993)

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GST Cyclin B1/cdc2

0 Other proteins of the extract

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[2] CYCLIN B1/cdc2 KINASE PURIFICATION 15

2 Dilute, the next day, 1 : 50 (v/v) in LB with ampicillin

3 G r o w until an OD60o of 0.6 is r e a c h e d and induce with 0.4 m M isopropyl-/3-D-thiogalactopyranoside ( I P T G ) overnight at 18 °

4 Pellet the cells and, after washing once in ice-cold PBS, freeze the pellet in liquid nitrogen and store at - 8 0 °

5 Lyse the cells by adding a 5 × pellet volume of E X to the frozen pellet and stir for 30 min on ice

6 Sonicate five to seven times, for 30 sec each, at setting 7 - 8 on a

B r a n s o n sonifier at 30-sec intervals

7 Bring the suspension to 300 m M KC1 and 15 m M dithiothreitol ( D T T )

8 Dialyze in a 20-fold suspension v o l u m e against W B without p r o t e a s e inhibitor mix C h a n g e the buffer three times (45 min each)

9 Clarify by ultracentrifugation for 1 hr at 100,000 g

10 Bind the s u p e r n a t a n t to G T - a g a r o s e equilibrated in WB (use 1 ml

of G T - a g a r o s e for every 1 liter of cells) R o t a t e for 1 hr at 4 °

11 W a s h the suspension once batchwise, and then p o u r it into a column (e.g., a Poly-Prep c h r o m a t o g r a p h y column; Bio-Rad, R i c h m o n d ,

C A ) and wash until no p r o t e i n can be detected in the flow-through

12 Elute with 10 column v o l u m e s of ELB

13 Dialyze p e a k fractions separately overnight in FB (200 times the fraction volume)

14 Freeze aliquots in liquid nitrogen and store at - 8 0 °

15 Analyze p r e p a r a t i o n b y sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) ,

we added microcystin, which inactivates the type 2A phosphatase that dephosphorylates cdc25 (b) Two-step purification of the active GST-cyclin B1, first on glutathione (GT) beads and then on a Mono S column

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FIG 2 (a) Purification of GST-cyclin B1 from bacterial extract Coomassie blue staining

of an SDS-polyacrylamide gel Lane 1, total extract after sonication; lane 2, 100,000 g supernatant; lane 3, flow-through of the GT-agarose column; lanes 4-7, eluted fractions 1-4; lane

8, molecular mass marker proteins (kDa) (b) cdc2 kinase activation by addition of GST-cyclin B1 to an interphase extract (ll) No microcystin; (©) 0.2/.~M microcystin added (final concentration)

c d c 2 K i n a s e A c t i v a t i o n in I n t e r p h a s e E x t r a c t

P r e g n a n t m a r e s e r u m g o n a d o t r o p i n (PMSG; Intergonan, V e r m i e Vet- erinar, T0nisvorst, G e r m a n y ) , 200 units/ml

H u m a n chorionic g o n a d o t r o p i n ( H C G , Sigma), 2000 units/ml

MMR" 100 m M NaCI, 2 m M KCI, 1 m M MgC12, 2 m M CaClz, 0.1 m M

E G T A , 5 m M H E P E S ( p H 7.8) (2 liters for a typical p r e p a r a t i o n ) Cysteine hydrochloride (2%, w/v; Sigma), adjusted to p H 7.8 with

N a O H (2 liters for a typical p r e p a r a t i o n )

Calcium i o n o p h o r e A23187 (Sigma), 20 m g / m l in dimethyl sulfoxide ( D M S O )

Cycloheximide (Sigma), 10 m g / m l in w a t e r

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[9.] CYCLIN B 1 / c d c 2 KINASE PURIFICATION 17 Cytochalasin D (Sigma), 10 mg/ml in DMSO

Microcystin LR (GIBCO-BRL, Gaithersburg, MD), 1 mM in DMSO

X B 1 2 : 1 0 0 mM KC1, 0.1 mM CaCI2, 1 mM MgC12, 10 mM potassium HEPES (pH 7.7), 50 mM sucrose, cytochalasin D diluted 1:1000 (v/v), and protease inhibitor mix (100 ml of XB for a typical preparation)

Energy mix (20×): 150 mM creatine phosphate, 20 mM ATP, creatine kinase (0.2 mg/ml), 20 mM MgC12

3 Collect the eggs and wash in MMR two or three times

4 Remove the jelly coat by washing the eggs in several changes of 2% (w/v) cysteine hydrochloride followed by three or four washes with MMR

5 Activate the eggs in MMR with calcium ionophore diluted 1 : 10,000 (v/v) for 2 min (first rinse once to change the buffer; total volume used is 200 ml)

6 Wash in MMR and incubate for 20 min in MMR with cycloheximide diluted 1 : 50 (v/v) (first rinse once to change the buffer; total volume used is 200 ml)

7 Wash again in MMR and finally in XB

8 Transfer into SW50.1 tubes (Beckman, Palo Alto, CA) containing

XB and pack them by spinning at 16 ° in a Heraeus Megafuge 1.0

R (Osterocle, Germany) for 60 sec at 180 g

9 Remove the excess buffer and place tubes (plus contents) in a 12-

ml polypropylene tube (Sarstedt, Ntimbrecht, Germany)

10 Crush the eggs at 4 ° in a Sorvall (Newton, CT) centrifuge, using the HB4 rotor (with rubber adaptor) for 15 min at 16,000 g

11 Collect, on ice, the cytoplasmic layer between the bottom yolk pellet and the top lipid layer; use a 2-ml syringe and 18-gauge needle to puncture the side of the tube

12 Add energy mix (1 : 20, v/v) and cytochalasin D (1 : 1000, v/v), then freeze in 200-/xl aliquots in liquid nitrogen

12 A Murray, "Cell Cycle Extracts," pp 581-605 Academic Press, New York, 1991

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3 Activate the cdc2 kinase in the extract by adding GST-cyclin B1 to

a final concentration of 200 nM in the presence of 0.2/,tM microcystin-

L R at 20 ° for 45 min Proceed to the next section

Result

Normally, the cyclin B1 activates cdc2 kinase in the interphase extracts after a lag phase that can vary from a few minutes to half an hour (Fig 2b) Sometimes, it does not activate at all We have noticed that this variabil- ity occurs even in aliquots of the same extract preparation It is not yet clear why this happens, but we believe this is a consequence of the mechanism

of activation of the kinase, which involves a nonlinear autoamplification loop 13-I5 To avoid irreproducibility, we routinely add microcystin to the extracts By inhibiting a type-2A phosphatase this keeps cdc25 active This procedure allows an immediate activation of cdc2 kinase by the GST-cyclin B1 in all extracts

Purification of Active Kinase

KDB: 80 mM fl-glycerophosphate (pH 7.3), 20 mM EGTA, 15 mM MgC12, 300 mM KCI, 1 mM DTT, and protease inhibitor mix GT-agarose

Reduced glutathione

KWB: KDB containing 0.5% (v/v) Nonidet P-40 (NP-40), bovine serum albumin (BSA, 10/zg/ml; Sigma) (approximately 99% pure) KEB: KWB with 50 mM reduced glutathione

Centricon-30 cells (Amicon, Beverly, MA)

MDB: 40 mM Tris (pH 6.8), 1 mM EGTA, 0.5% (v/v) NP-40, 5% (v/v)

13 B Novak and J J Tyson, J Cell Sci 106, 1153 (1993)

14 I Hoffmann and E Karsenti, J Cell Sci 18, 75 (1994)

15 I Hoffmann, G Draetta, and E Karsenti, E M B O J 13, 4302 (!994)

Tiêu đề	Cell Cycle Control
Tác giả	William G. Dunphy
Chuyên ngành	Cell Biology
Thể loại	review article

Định dạng
Số trang	725
Dung lượng	12,56 MB