Biomembrane Protocols 11. Architecture and Function

Methods in Molecular Biology John M, Walker, SERIES EDITOR

27. Biomembrane Protocols: II. Architecture and Function, edited by John M. Graham and Joan A. Higgins, 1994

26. Protocols for Oligonucleotide Conjugates, edited by Sxidhir Agrawal, 1994 25. Computer Analysis of Sequence Data: Part II, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 24. Computer Analysis of Sequence Data: Part I, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 23. DNA Sequencing Protocols, edited by flu^^ G. Griffin

and Annette M. Griffin, 1993 22. Optical Spectroscopy, Microscopy, and Macroscopic Techniques,

edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomtts, 1994

21. Protocols in Molecular Parasitology, edited by John E. Hyde, 1993 20. Protocols for Oligonucleotides and Analogs, edited by

SudhirAgrawal, 1993 19. Biomembrane Protocols: /. Isolation and Analysis, edited by

John M. Graham and Joan A. Higgins, 1993 18. Transgenesis Techniques, edited by David Murphy

and David A. Carter, 1993 17. Spectroscopic Methods and Analyses, edited by Christopher Jones,

Barbara Mulloy, and Adrian H. Thomas, 1993 16. Enzymes of Molecular Biology, edited by Michael M. Burrell, 1993 15. PCR Protocols, edited by Bruce A. White, 1993 14. Glycoprotein Analysis in Biomedicine, edited by

Elizabeth F. Hounsell, 1993 13. Protocols in Molecular Neurobiology, edited by Alan Longstaff

and Patricia Revest, 1992 12. Pulsed-Field Gel Electrophoresis, edited by Margit Burmeister

and Levy Ulanovsky, 1992 11. Practical Protein Chromatography, edited by Andrew Kenney

and Susan Fowell, 1992 10. Immunochemical Protocols, edited by Margaret M. Manson, 1992

9. Protocols in Human Molecular Genetics, edited by Christopher G. Mathew, 1991

8. Practical Molecular Virology, edited by Mary K. L. Collins, 1991 7. Gene Transfer and Expression Protocols, edited by

Edward J. Murray, 1991 6. Plant Cell and Tissue Culture, edited by Jeffrey W. Pollard

and John M. Walker, 1990 5. Animal Cell Culture, edited by Jeffrey W. Pollard and John M. Walker, 1990 4. New Nucleic Acid Techniques, edited by John M. Walker, 1988 3. New Protein Techniques, edited by John M. Walker, 1988 2. Nucleic Acids, edited by John M. Walker, 1984 1. Proteins, edited by John M. Walker, 1984

Methods in Molecular Biology • 27

Biomembrane Protocols

//. Architecture and Function

Edited by

John M. Graham Merseyside Innovation Centre,

Liverpool, UK

Joan A. Higgins Department of Molecular Biology and Biotechnology,

University of Sheffield, UK

H u m a n a P r e s s ^ j ^ Totowa, New Jersey

© 1994 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

All rights reserved.

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Library of Congress Cataloging in Pubhcation Data Main entry under title:

Methods in molecular biology.

Biomembrane protocols / edited by John M. Graham, Joan A. Higgins. p . cm. — (Methods in molecular biology ; 19, 27)

Includes indexes. Contents: 1. Isolation and analysis — 2. Architecture and function. 1. Membranes (Biology). 2. Membranes (Biology)—Research—Methodology.

I. Graham, John M. II. Higgins, Joan A. III. Series: Methods in molecular biology (Totowa, NJ) ; 19, 27

QH601.B5257 1993 574.87'5 93-13471 ISBN 0-89603-236-1 (v. 1) CIP ISBN 0-89603-250-7 (v. 2)

Preface

There have been many important advances in our understanding of biological membrane structure and function over the last decade. Much of this progress has been driven by the development of new techniques for studying membrane components and their interactions. Tradition­ally, the investigation of membranes has largely occurred within the domains of biochemistry, physical chemistry, and cell biology; but many of the most significant advances have resulted from an expansion into other disciplines, such as molecular biology, immunology, and the clinical sciences. In these two volumes we have attempted to bring together some of these techniques—to combine the old and the new. Biomembrane Protocols: II. Architecture and Function is concerned exclusively with the architecture and activities of membranes, whereas the companion volume, Biomembrane Protocols: I. Isolation and Analysis, deals with their isolation and compositional analysis.

The aim of each chapter is to provide detailed technical and meth­odological information that will allow the reader to perform the tech­nique successfully, without the need to consult other texts. Detailed reviews of membrane structure and function are not included, except where they are relevant to the choice or efficacy of a particular proce­dure. In addition to Materials and Methods sections, each chapter has a Notes section that explains the reasons for taking certain steps and pro­vides practical tips for applying the techniques to other systems. Of neces­sity, some chapters may diverge from this simple format; some deal with techniques that require adapting to suit a specific apphcation, whereas others include a combination of different techniques. In these cases, the text may be more discursive; nevertheless detailed examples are always given. Sometimes the Notes section is considerably expanded to pro­vide worked-out examples or to help in interpreting experimental results.

Biomembrane Protocols: II. Architecture and Function is concemed with the organization of membrane components and how this organiza­tion controls function. Methods for the investigation of the structure and topography of membrane proteins; the transverse distribution of mem-

vi Preface

brane lipids; and the interactions between lipids and proteins in intact and reconstituted membranes are described. There are also chapters on the dynamic aspects of membrane structure, including the translocation and intracellular movement of lipids and membrane fluidity. Methods concerned with the function of membranes are of necessity limited because of their diversity, but we have included chapters that cover top­ics central to a number of key areas in modern membrane research— areas such as membrane transport and the role of membrane-bound receptors in the regulation of cell growth and metabolism. Therefore, there are chapters on ligand binding and processing; on G-proteins, pro­tein kinases, phosphatidylinositol metabolism, and intracellular calcium; and on ion transport and membrane permeabilization.

The study of biological membranes covers an enormous range of topics and employs a great diversity of techniques. In Biomembrane Protocols: II. Architecture and Function, we have attempted to cover most of the techniques that are likely to be of substantial interest to bio­chemists and cell biologists. Inevitably, some important methodology will have escaped treatment; the editors will be grateful to receive com­ments and suggestions from our readers that might be incorporated in future editions.

John M. Graham Joan A. Higgins

Contents Preface v Contributors xi

CH. 1. Crystallization of Membrane Proteins for X-Ray Analysis, Brian J. Sutton and Maninder K. Sohi 1

CH. 2. Determination of Cell-Surface Polarity by Solid-Phase Lactoperoxidase lodination,

William A. Muller 19 CH. 3. Biochemical Methods to Determine Cell-Surface Topography,

William A. Muller Part A: Labeling of Oxidized Glycoproteins

with'H-Borohydride 31 Part B: Identification and Separation of Integral Membrane

Proteins Using Triton X-114 33 Part C: Surface-Selective Labeling of Polarized Proteins

on Tight Epithelia Using Sulfo-A^-Hydroxysuccinimido-Biotin (S-NHS-B) 38

CH. 4. Use of Antipeptide Antibodies for the Isolation and Study of Membrane Proteins,

Stephen A. Baldwin Part A: Preparation of Antibodies 43 Part B: Affinity-Purification of Antipeptide Antibodies 52 Part C: Purification of Membrane Proteins

by Immunoaffinity Chromatography 55 Part D: Competitive ELISA for Determining Membrane

Protein Topology 58 CH. 5. The Production of Monoclonal Antibodies to Membrane Proteins,

Lynda J. Partridge Part A: Production of Hybridoma Cell Lines 65 Part B: Screening for Monoclonal Antibody Production 78

CH. 6. Purification of a Membrane Protein (Ca^*/Mg^*-ATPase) and Its Reconstitution into Lipid Vesicles,

J. Malcolm East 87 CH. 7. Measurement of Protein-Protein Interactions in Reconstituted

Membrane Vesicles Using Fluorescence Spectroscopy, Anthony G. Lee 95

Vll

via Contents

CH. 8. Measurement of Lipid-Protein Interactions in Reconstituted Membrane Vesicles Using Fluorescence Spectroscopy,

Anthony G. Lee 101 CH. 9. Determination of the Transverse Topography of Membrane

Lipids Using Enzymes and Covalent Labels as Probes, Joan A. Higgins 709

CH. 10. Determination of the Transverse Topography of Membrane Phospholipids Using Phospholipid Transfer Proteins as Tools,

Joan A. Higgins 125 CH. 11. Prothrombinase Complex as a Tool to Assess Changes

in Membrane Phospholipid Asymmetry, Paul Comfurius, Edouard Bevers, and Robert F. A. Zwaal 131

CH. 12. Fluorescent Glycerolipid Probes: Synthesis and Use for Examining Intracellular Lipid Trafficking,

Richard G. Sleight Part A: Synthesis of Fluorescent Glycerolipid Probes 143 Part B: Delivery of Fluorescent Glycerophospholipid Probes

to Cultured Cells 154 CH. 13. Synthesis and Use of Spin-Labeled Lipids for Studies

of the Transmembrane Movement of Phospholipids, Pierre Fellmann, Alain Zachowski, and Philippe F. Devaux.... 767

CH. 14. Measurement of Membrane Fluidity and Membrane Fusion with Fluorescent Probes,

C Lindsay Bashford 777 CH. 15. Extraction and Assay of Cyclic Nucleotides,

Guy St. J. Whitley 189 CH. 16. Analysis of G-Proteins Regulating Signal Transduction Pathways,

Margaret M. Harnett Part A: Identification of G-Proteins 799 Part B: GTPase Studies 204 Part C: Reconstitution of Second Messenger Pathways in

Permeabilized Cell Preparations Using GTP Analogs and Receptor Ligands 205

CH. 17. Assay of Protein Kinases and Protein Phosphorylation, Margaret M. Harnett Part A: Protein Kinase-Mediated Phosphorylation Events 213 Part B: Measurement of Protein Kinase Expression 279 Part C: Measurement of Protein Kinase Activity

and Translocation 227

Contents ix

CH. 18. Analysis of Cellular Phosphoinositides and Phosphoinositols by Extraction and Simple Analytical Procedures,

Ian M. Bird Part A: Biosynthesis and Extraction of Phosphoinositides

and Phosphoinositols 227 Part B: Separation of Phosphoinositols by Anion-Exchange

Chromatography 234 Part C: Separation of Phosphoinositides

by Thin-Layer Chromatography 238 Part D: Deacylation of Phospholipids and Separation

of Products by Anion-Exchange Chromatography 241 CH. 19. Analysis of Cellular Phosphoinositides and Phosphoinositols

by High-Performance Liquid Chromatography, Ian M. Bird Part A: Sample and Standards Preparation 249 Part B: HPLC Analytical Methods 260

CH. 20. Cytosolic Free Calcium Measurements in Single Cells Using Calcium-Sensitive Fluorochromes,

Mone Zaidi, A. S. M. Towhidul Alam, Christopher Box, Vijay Shankar, Peter J. R. Bevis, Christopher L. H. Huang, Michael Pazianas, and Baljit S. Moonga 279

CH. 21. Membrane Permeabilization with Bacterial Toxins, C. Lindsay Bashford 295

CH. 22. Measurement of Ion Fluxes and pH Gradients Across Cell Membranes,

C. Lindsay Bashford 307 CH. 23. Ligand Binding and Processing: r/ie Pe//tti'eJLjver

as a Model System, Carol A. Renfrew, Livia A. Casciola-Rosen,

and Ann L. Hubbard 325 CH. 24. The Binding of Protein-Ligands to Cell-Surface Receptors,

David A. W. Grant 343

Appendix: 1. Density Gradient Media 351

Appendix: 2. Balanced Salt Solutions 352

Index 353

Contributors

A. S. M. TowHiDUL ALAM • Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, UK; and The Physiological Laboratory, University of Cambridge, UK

STEPHEN A. BALDWIN • Departments of Biochemistry and Chemistry, and Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine (University of London), London, UK

C. LINDSAY BASHFORD • Department of Cellular and Molecular Sciences, Division of Biochemistry, St. George's Hospital Medical School, London, UK

CHRISTOPHER BAX • Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, UK; and The Physiological Laboratory, University of Cambridge, UK

EDOUARD BEVERS • Department of Biochemistry, Cardiovascular Research Institute Maastricht, University ofLimburg, Maastricht, The Netherlands

PETER J. R. BEVIS • Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, UK; and The Physiological Laboratory, University of Cambridge, UK

IAN M . BIRD • University Department of Clinical Chemistry, The Royal Infirmary, Edinburgh, Scotland {Current address: Cecil H. and Ida Green Center, University of Texas Southwestern Medical Center, Dallas, TX)

LiviA A. CASCIOLA-ROSEN • Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD

PAUL COMFURIUS • Department of Biochemistry, Cardiovascular Research Institute Maastricht, University ofLimburg, Maastricht, The Netherlands

PHILIPPE F . DEVAUX • Institut de Biologie Physico-Chimique, Paris, France

J. MALCOLM EAST • Department of Biochemistry, University of Southampton, UK

xi

xii Contributors

PIERRE FELLMANN • Institut de Biologie Physico-Chimique, Paris, France

DAVID A. W. GRANT • Leukaemia Research Fund, London, UK MARGARET M . HARNETT • Department of Biochemistry, University

of Glasgow, Scotland JOAN A. HIGGINS • Department of Molecular Biology and Biotechnology,

University of Sheffield, UK CHRISTOPHER L . H . HUANG • Department of Cellular and Molecular

Sciences, St. George's Hospital Medical School, London, UK; and The Physiological Laboratory, University of Cambridge, UK

ANN L . HUBBARD • Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD

ANTHONY G . LEE • Department of Biochemistry, University of Southampton, UK

BALJIT S . MOONGA • Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, UK; and The Physiological Laboratory, University of Cambridge, UK

WILLIAM A. MULLER • Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York

LYNDA J. PARTRIDGE • Department of Molecular Biology and Biotechnology, University of Sheffield, UK

MICHAEL PAZIANAS • Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, UK;

and The Physiological Laboratory, University of Cambridge, UK CAROL A. RENFREW • Department of Cell Biology and Anatomy,

Johns Hopkins University School of Medicine, Baltimore, MD VIJAY SHANKAR • Department of Cellular and Molecular Sciences,

St. George's Hospital Medical School, London, UK; and The Physiological Laboratory, University of Cambridge, UK

RICHARD G . SLEIGHT • Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH

MANINDER K. SOHI • Biophysics Section, King's College, London, UK BRIAN J. SUTTON • Biophysics Section, King's College, London, UK GUY ST. J. WHITLEY • Department of Cellular and Molecular Sciences,

St. George's Hospital Medical School, London, UK ALAIN ZACHOWSKI • Institut de Biologie Physico-Chimique, Paris,

France

Contributors xiii

MoNE ZAIDI • Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, UK; and The Physiological Laboratory, University of Cambridge, UK

ROBERT F . A. ZWAAL • Department of Biochemistry, Cardiovascular Research Institute Maastricht, University ofLimburg, Maastricht, The Netherlands