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Methods In Enzymology, Volume 475 : Thiol Redox Transitions in Cell Signaling, Part B - Lesley Packer

Methods In Enzymology, Volume 475

Thiol Redox Transitions in Cell Signaling, Part B

Hardcover

Published: 8th July 2010
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This volume, along with its companion (volume 475), presents methods and protocols dealing with thiol oxidation-reduction reactions and their implications as they relate to cell signaling. The critically acclaimed laboratory standard for 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. Over 450 volumes have been published to date, and much of the material is relevant even today--truly an essential publication for researchers in all fields of life sciences.
*Along with companion volume, provides a full overview of techniques necessary to the study of thiol redox in relation to cell signaling
* Gathers tried and tested techniques from global labs, offering both new and tried-and-true methods
* Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines

Contributorsp. xiii
Prefacep. xix
Volumes in Seriesp. xxi
Engineering of Fluorescent Reporters into Redox Domains to Monitor Electron Transfersp. 1
Introductionp. 2
The Problem: Low Sensitivity and Improperly Rate-Limited Assays for Redox Functions of Bacterial Peroxiredoxin Systemsp. 3
The Solution: Engineering of Fluorescent Redox Reporters into the N-Terminal Domain of AhpF and E. coli Grx1p. 4
Engineering of Disulfide-Containing Electron Acceptor Domains to Detect Electron Transfers via Fluorescence Changes; Linkage of Fluorescein to Bacterial AhpC via a Reducible Disulfide Bondp. 7
Materialsp. 8
Methodsp. 10
Summaryp. 19
Referencesp. 19
Blot-Based Detection of Dehydroalanine-Containing Glutathione Peroxidase with the Use of Biotin-Conjugated Cysteaminep. 23
Introductionp. 24
Oxidative Inactivation of Glutathione Peroxidase and the Conversion of Its Active Site Sec to DHAp. 25
Preparation of Biotin-Conjugated Cysteaminep. 27
Blot-Based Detection of DHA-GPx1 in RBCsp. 28
Effects of Oxidative Stress on the Formation of DHA-GPx1 in RBCsp. 29
Concluding Remarksp. 30
Acknowledgmentp. 32
Referencesp. 32
Analysis of the Redox Regulation of Protein Tyrosine Phosphatase Superfamily Members Utilizing a Cysteinyl-Labeling Assayp. 35
Introductionp. 36
Active-Site Structure, Catalysis, and Oxidationp. 37
Detection Methodsp. 39
General Principle of the Assayp. 40
Solutionsp. 42
Preparation of the Lysis Bufferp. 42
Preparation of the Hypoxic Glove Boxp. 43
Preparation of Cell Lysatesp. 43
Cysteinyl-Labeling Assayp. 44
Acute Stimulus-Induced Reversible Oxidation of PTPsp. 44
Perspectivesp. 45
Conclusionp. 48
Referencesp. 48
Measuring the Redox State of Cellular Peroxiredoxins by Immunoblottingp. 51
Introductionp. 52
Measurement of Prx Dimerizationp. 54
Measurement of Prx Hyperoxidationp. 59
Discussionp. 61
Acknowledgmentsp. 64
Referencesp. 64
Thiol Redox Transitions by Thioredoxin and Thioredoxin-Binding Protein-2 in Cell Signalingp. 67
Functional Regulation of Redox-Sensitive Proteins by Thiol Modificationp. 68
Thiol Reduction by the Thioredoxin Redox Systemp. 70
Thioredoxin Superfamilyp. 73
Reversible Redox and Signal Regulation by Thioredoxin and Thioredoxin-binding Protein-2 (TBP-2)p. 74
Conclusionp. 76
Referencesp. 76
Detection of Protein Thiols in Mitochondrial Oxidative Phosphorylation Complexes and Associated Proteinsp. 83
Introductionp. 84
Mitochondria Isolation and Protein Thiol Labelingp. 86
Application of Blue Native-PAGE for the Isolation of Oxidative Phosphorylation Protein Subunits and Other Proteins Associated with the Complexesp. 89
Detection of IBTP-Labeled Protein Thiols in Protein Complexesp. 94
Analysis and Mass Spectrometry Identification of Proteinp. 95
Other Considerationsp. 104
Conclusionp. 105
Acknowledgmentsp. 106
Referencesp. 106
Mitochondrial Thioredoxin Reductase: Purification, Inhibitor Studies, and Role in Cell Signalingp. 109
Introductionp. 110
Purification of Thioredoxin Reductase from Isolated Mitochondria, Cultured Cells, and Whole Organsp. 111
Estimation of Thioredoxin Reductase Activityp. 117
Inhibitor Studies of Thioredoxin Reductasep. 118
Role in Cell Signalingp. 118
Referencesp. 120
Measuring Mitochondrial Protein Thiol Redox Statep. 123
Introductionp. 124
Quantification of Mitochondrial Protein Thiolsp. 127
Quantification of Glutathionylation of Mitochondrial Proteinsp. 130
Assessment of S-Nitrosated Protein Thiolsp. 135
Measurement of the Thioredoxin and Peroxiredoxin Redox Statesp. 139
Conclusionsp. 143
Acknowledgmentsp. 144
Referencesp. 144
Measurement of Extracellular (Exofacial) Versus Intracellular Protein Thiolsp. 149
Measurement of Mitochondrial Thiol Statusp. 150
Measurement of Cytosolic Thiol Statusp. 156
Measurement of Exofacial Thiolsp. 157
Exofacial Thiol Status and Cancerp. 160
Referencesp. 162
Redox Clamp Model for Study of Extracellular Thiols and Disulfides in Redox Signalingp. 165
Introductionp. 166
Key Concepts for Usep. 166
Principles for Experimental Designp. 167
Summary of Available Redox Clamp Studiesp. 171
Perspectives and Conclusionp. 177
Acknowledgmentsp. 178
Referencesp. 178
Redox State of Human Serum Albumin in Terms of Cysteine-34 in Health and Diseasep. 181
Backgroundp. 182
HPLC Analysisp. 182
Albumin Thiol State and Exercisep. 187
Influence of Supplementationp. 188
Albumin Oxidation in Diseasep. 188
Albumin Thiol State During Agingp. 191
Summaryp. 193
Referencesp. 193
Methods for Studying Redox Cycling of Thioredoxin in Mediating Preconditioning-Induced Survival Genes and Proteinsp. 197
Hormetic Mechanism: Role of Redox Cycling of Thioredoxinp. 198
Redox Functioning of Thioredoxinp. 199
Implications of Preconditioning Protection from Preclinical and Clinical Studiesp. 200
Drugs Mimic Thioredoxin-Medicated Preconditioning-Induced Signaling and Protection in Cellsp. 202
Methods and Materialsp. 203
Referencesp. 209
Oxidative Stress, Thiol Redox Signaling Methods in Epigeneticsp. 213
Introductionp. 216
Histone Acetylation Assays Using [3H]-Acetate Incorporationp. 219
Histone Acetylation by Immunoblottingp. 221
HAT Activity Assayp. 222
HDAC Activity Assay Using [3H]-Labeled Histonesp. 223
HDAC Activity Assayp. 225
HDACs Levels by Immunoblottingp. 227
Posttranslational Modifications of HDACs and SIRTs (Sirtuins 1-7) by Immunoprecipitationp. 228
Preparation of Whole Cell Lysatep. 229
Preparation of Cytoplasmic and Nuclear Proteinsp. 229
Redox-Mediated Posttranslational Modification Assaysp. 230
Chromatin Immunoprecipitation (ChIP) Assayp. 233
Conclusionsp. 238
Acknowledgmentsp. 239
Referencesp. 239
Characterization of Protein Targets of Mammalian Thioredoxin Reductasesp. 245
Introductionp. 246
Preparation of TR-immobilized Affinity Resinsp. 247
Identification of Targets of Mammalian TRs in Cell Lysatesp. 250
Concluding Remarks and Future Perspectivesp. 252
Acknowledgmentsp. 253
Referencesp. 253
Alteration of Thioredoxin Reductase 1 Levels in Elucidating Cancer Etiologyp. 255
Introductionp. 256
Materials and Methodsp. 257
Results and Discussionp. 263
Conclusions and Future Perspectivesp. 271
Acknowledgmentsp. 272
Referencesp. 272
Regulation of Apoptosis Signal-Regulating Kinase 1 in Redox Signalingp. 277
Overviewp. 278
Materialsp. 281
Methodsp. 283
Commentp. 286
Referencesp. 287
Protocols for the Detection of S-Glutathionylated and S-Nitrosylated Proteins In Situp. 289
Introductionp. 290
Protein S-Glutathionylationp. 290
Protein S-Nitrosylationp. 293
Summaryp. 295
Referencesp. 295
Synthesis, Quantification, Characterization, and Signaling Properties of Glutathionyl Conjugates of Enalsp. 297
Introductionp. 298
Synthesis, Quantification, and Characterization of Reagent Glutathionyl Conjugates of HNEp. 300
Metabolism of HNEp. 305
Signaling Properties of Glutathionyl Conjugates of HNEp. 309
Conclusionsp. 310
Acknowledgmentsp. 311
Referencesp. 311
Thioredoxin and Redox Signaling in Vasculature-Studies Using Trx2 Endothelium-Specific Transgenic Micep. 315
Introductionp. 316
Methodsp. 318
Referencesp. 323
Author Indexp. 325
Subject Indexp. 333
Table of Contents provided by Ingram. All Rights Reserved.

ISBN: 9780123810038
ISBN-10: 0123810035
Series: Methods in Enzymology : Book 474
Audience: Professional
Format: Hardcover
Language: English
Number Of Pages: 392
Published: 8th July 2010
Publisher: Elsevier Science Publishing Co Inc
Country of Publication: US
Dimensions (cm): 22.9 x 15.2  x 2.29
Weight (kg): 0.75
Edition Number: 474