General Description of the Volume:Small GTPases play a key role in many aspects of contemporary cell biology: control of cell growth and differentiation; regulation of cell adhesion and cell movement; the organization of the actin cytoskeleton; and the regulation of intracellular vesicular transport. This volume plus its companion Volumes 255 and 257 cover all biochemical and biological assays currently in use for analyzing the role of small GTPases in these aspects of cell biology at the molecular level. It is the first compendium of practical techniques for working with small GTPases of the Rho group.General Description of the Series:The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.
Author(s): John N. Abelson, Melvin I. Simon, W. E. Balch, Channing J. Der, Alan Hall
Series: Methods in Enzymology 256
Edition: 1st
Publisher: Academic Press
Year: 1995
Language: English
Pages: 414
Table of Contents......Page 6
Contributors to Volume 256......Page 10
Preface......Page 13
Section I Expression. Purification. and Posttranslational Modification......Page 14
[ 1] Purification of Recombinant Rho / Rac / G25K from Escherichia coli......Page 16
[2] Purification of Baculovirus-Expressed Cdc42Hs......Page 24
[3] Purification and Properties of Rac2 from Human Leukemia Cells......Page 28
[4] Purification of Rac2 from Human Neutrophils......Page 38
[5] Purification of Rac-GDP Dissociation Inhibitor Complex from Phagocyte Cytosol......Page 46
[6] Purification and Properties of Recombinant Rho-GDP Dissociation Inhibitor......Page 54
[7] Prenylcysteine-Directed Carboxyl Methyltransferase Activity in Human Neutrophil Membranes......Page 62
Section II Guanine Nucleotide Exchange and Hydrolysis......Page 78
[8] Measurement of Intrinsic Nucleotide Exchange and GTP Hydrolysis Rates......Page 80
[9] Guanine Nucleotide Exchange Catalyzed by dbl Oncogene Product......Page 90
[10] Stimulation of Nucleotide Exchange on Ras- andRho-Related Proteins by Small GTP-Binding Protein GDP Dissociation Stimulator......Page 98
[11] Interaction of Ect2 and Dbl with Rho-Related GTPases......Page 103
[13] Purification and GTPase-Activating Protein Activity of Baculovirus Expressed p 190......Page 118
[ 14] GTPase-Activating Protein Activityof n(al)-Chimaerin and Effect of Lipids......Page 127
[15] Characterization of Breakpoint Cluster Region Kinase and SH2-Binding Activities......Page 138
[16] Identification of GTPase-Activating Proteins by Nitrocellulose Overlay Assay......Page 143
[17] Identification of 3BP- 1 in cDNA Expression Libraryby SH3 Domain Screening......Page 153
Section III Cell Expression and in Vitro Analysis......Page 162
[18] Serum Induction of RhoG Expression......Page 164
[19] Microinjection of Epitope-Tagged Rho Family cDNAs and Analysis by Immunolabeling......Page 175
[20] Purification and Assay of Recombinant C3 Transferase......Page 187
[21] In Vitro ADP-Ribosylation of Rho by Bacterial ADP-Ribosyltransferases......Page 197
[22] Preparation of Native and Recombinant Clostridiumbotulinum C3 ADP-Ribosyltransferase and Identification of Rho Proteins by ADP-Ribosylation......Page 209
[23] In Vitro Binding Assay for Interactions of Rho and Rac with GTPase-Activating Proteins and Effectors......Page 220
[24] Purification and Assay of Kinases That Interact with Rac / Cdc42......Page 228
[25] Yeast Two-Hybrid System to Detect Protein-Protein Interactions with Rho GTPases......Page 241
[26] Assay for Rho-Dependent Phosphoinositide 3-Kinase Activity in Platelet Cytosol......Page 254
[27] Neutrophil Phospholipase D: Inhibition by Rho-GDP Dissociation Inhibitor and Stimulation by Small GTPase GDP Dissociation Stimulator......Page 259
[28] Measurement of Rac Translocation from Cytosol to Membranes in Activated Neutrophils......Page 269
[29] Reconstitution of Cell-Free NADPH Oxidase Activity by Purified Components......Page 281
Section IV. Biological Activity......Page 292
[30] Genetic and Biochemical Analysis of Cdc42p Function in Saccharomyces cerevisiae and Schizosaccharomyces pombe......Page 294
[31] Lymphocyte Aggregation Assay and Inhibition by Clostridium botulinum C3 ADP-Ribosyltransferase......Page 303
[32] Inhibition of p21 Rho in Intact Cells by C3 Diphtheria Toxin Chimera Proteins......Page 310
[33] Growth Factor-Induced Actin Reorganization in Swiss 3T3 Cells......Page 319
[34] Microinjection of Rho and Rac into Quiescent Swiss 3T3 Cells......Page 326
[35] Inhibition of Lymphocyte-Mediated Cytotoxicity by Clostridium botulinum C3 Transferase......Page 333
[36] Neutrophil Chemotaxis Assay and Inhibition by C3 ADP-Ribosyltransferase......Page 340
[37] Cell Motility Assay and Inhibition by Rho-GDP Dissociation Inhibitor......Page 349
[38] Cell Transformation by dbl Oncogene......Page 360
[39] Inhibition of Rac Function Using Antisense Oligonucleotides......Page 371
Author Index......Page 380
Subject Index......Page 398
