Proteolytic Enzymes A Practical Approach

Note: Supplemental materials are not guaranteed with Rental or Used book purchases.

ISBN: 9780199636624 | 0199636621
Cover: Paperback
Copyright: 3/22/2001

Rent

(Recommended)

$71.52

Term

Due

Price

Semester

Aug 2

$71.52

Quarter

Jul 19

$67.95

Short Term

Jun 21

$64.38

*This item is part of an exclusive publisher rental program and requires an additional convenience fee. This fee will be reflected in the shopping cart.
Buy New

Usually Ships in 3-5 Business Days

$97.71
eBook

Available Instantly

Online: 180 Days

Downloadable: 180 Days

$68.01

Like the popular first edition, this new edition of Proteolytic Enzymes emphasizes practical aspects of the handling, characterization, inhibition, and use of proteolytic enzymes giving general advice and specific examples. The text and protocols have been thoroughly updated to take account ofthe advances made in the last 10 years in both the increased understanding of the role of peptidases in many critical cellular processes e.g. apoptosis and new technological developments e.g. in recombinant protein expression, protein sequencing, and structural studies. The topics covered are:nomenclature and classification; purification; assay methods; determination of mechanism; inhibition and prevention of unwanted proteolytic activity; characterizing natural inhibitors; proteolytic enzymes in peptide mapping and primary structure elucidation by mass spectrometry and Edman sequencing;limited proteolysis as a structural probe; synthetic function. This book will be as invaluable as the first edition in providing ideas and protocols for scientists either studying proteases or using proteases as a research tool.

Preface

List of protocols

Abbreviations

xvii

Proteolytic enzymes: nomenclature and classification

(22)

Alan J. Barrett

Introduction

(1)

Terminology and nomenclature

(3)

Peptidase and related terms

(2)

Specificity-subsite terminology

(1)

Catalytic type

(1)

Homology

(1)

The EC classification of peptidases

(8)

What is the EC system?

(2)

What information does the EC list contain?

(1)

When and how can a newly-discovered peptidase be added to the EC list?

(5)

The MEROPS system for the classification of peptidases

(7)

Families

(4)

Clans

(1)

Individual peptidases

(1)

Uses of the MEROPS system

(1)

Steps one might take on discovering a new peptidase

(4)

Acknowledgement

(1)

References

(3)

Purification of proteolytic enzymes

(22)

Sherwin Wilk

Introduction

(1)

Prelude to purification

(2)

Assay

(2)

Initial considerations

(4)

Source

(1)

Buffer composition

(1)

Membrane-bound or soluble?

(1)

Membrane-bound enzymes

(2)

Endogenous inhibitors and activators

(1)

General scheme for purification of proteolytic enzymes

(3)

Initial steps

(2)

Intermediate and final steps

(1)

Specialized techniques for proteolytic enzymes

(4)

Peptidyl aldehyde affinity chromatography

(1)

Affinity columns for cysteine proteinases

(1)

Affinity columns for trypsin-like enzymes

(1)

Affinity columns for metalloproteinases

(1)

Other affinity columns

(2)

Optimization of the purification protocol

(1)

Determination of homogeneity

(1)

Purification of the proteasome; EC 3.4.99.46

(4)

Conclusions: many roads lead to Rome

(3)

References

(3)

Protease assay methods

(32)

Gautam Sarath

Michael G. Zeece

Alan R. Penheiter

Introduction

(1)

Assays with natural substrates

(10)

Endopeptidase assays

(10)

Exopeptidase assays

(1)

Assays with synthetic substrates

(14)

Endopeptidase and aminopeptidase substrates

(1)

Spectrophotometric assays

(3)

Fluorimetric assays

(3)

Miscellaneous fluorimetric methods

(2)

Carboxypeptidase substrates

(1)

Radiometric assays

(1)

HPLC assays for peptidases

(2)

Capillary electrophoretic analyses of proteases

(2)

Solid-phase protease assays

(8)

Gel electrophoretic methods

(3)

Plate assays

(1)

Miscellaneous solid-phase assays

(1)

Assays for histochemical studies

(1)

Acknowledgements

(1)

References

(2)

Determination of protease mechanism

(28)

Ben M. Dunn

Introduction---the importance of mechanistic classification

(6)

The serine peptidases

(2)

The cysteine peptidases

(1)

The aspartic peptidases

(1)

The metallopeptidases

(2)

Methods for determining the mechanistic class

(8)

Classification based on `standard' inhibitors

(3)

Chemical modification/identification

(1)

Site-directed mutagenesis

(1)

Mechanistic distinctions---intermediates

(3)

Kinetic studies to probe the mechanism in more detail

(6)

Notes on `ideal' assays

(1)

Kinetic determination of Km and kcat

(3)

pH dependence of the kinetic parameters

(1)

Solvent deuterium isotope effects

(1)

Transition state analogues and substrate alteration

(1)

Determination of primary specificity of a protease

(8)

Degradation of standard proteins and peptides

(1)

Cleavage of homologous synthetic peptides

(3)

References

102

(3)

Inhibition of proteolytic enzymes

105

(26)

Guy S. Salvesen

Hideaki Nagase

Introduction: which inhibitors?

105

(1)

Principles for using irreversible and reversible inhibitors

106

(1)

General structure of synthetic inhibitors

106

(1)

How to live with crossreactivity

106

(1)

Practical use of inhibition constants

107

(2)

Irreversible inhibitors

107

(1)

Reversible inhibitors

108

(1)

Non-specific inhibitors

109

(3)

α-Macroglobulins

109

(1)

Peptide aldehydes

110

(1)

Peptide chloromethyl ketones

110

(1)

Metal chelators

111

(1)

Class-specific inhibitors

112

(10)

Serine proteases

112

(4)

Cysteine proteases

116

(3)

Proteasome

119

(1)

Metalloproteases

120

(2)

Aspartic proteases

122

(1)

Inhibitors as active-site titrants

122

(3)

Cysteine proteases

122

(2)

Serine proteases

124

(1)

Protease inhibitors in cell culture

125

(1)

Suppression of proteolysis

126

(1)

Therapeutic value of protease inhibitors

127

(4)

References

128

(3)

Finding, purification and characterization of natural protease inhibitors

131

(18)

Hideaki Nagase

Guy S. Salvesen

Introduction

131

(1)

The meaning of inhibition

131

(1)

Finding protease inhibitors

132

(5)

Screening of inhibitors from natural sources

132

(1)

Finding inhibitors by reverse zymography

133

(2)

Finding inhibitors from DNA sequences

135

(2)

Combinatorial protease inhibitors

137

(1)

Purification of natural protease inhibitors

137

(1)

Use of the target enzyme as an affinity ligand

137

(1)

Conventional purification

137

(1)

Reverse zymography

138

(1)

Characterization of inhibitors: inhibition kinetics

138

(8)

The importance of kinetics

138

(1)

IC50 and percentage inhibition

138

(1)

Practical inhibitor kinetics

139

(1)

Reversible inhibitors

140

(3)

Irreversible inhibitors

143

(3)

Practical applications of protein inhibitors

146

(3)

Acknowledgements

146

(1)

References

146

(3)

Mass spectrometry of proteolysis-derived peptides for protein identification

149

(38)

Bernhard Kuster

Andrej Shevchenko

Matthias Mann

Introduction

149

(1)

Mass spectrometry

150

(5)

Methods of ionization

150

(2)

Mass analysis

152

(1)

Tandem mass spectrometry

153

(2)

Interrogation of sequence databases using mass spectrometry data

155

(6)

Choice of protease

155

(2)

Peptide mass mapping

157

(2)

Database identification via tandem mass spectrometry

159

(2)

Analytical strategy for the identification or cloning of proteins using mass spectrometry

161

(20)

Low-level protein preparation for characterization by mass spectrometry

161

(2)

Protein visualization

163

(2)

Proteolytic cleavage of gel separated proteins

165

(2)

Protein identification by MALDI peptide mass mapping

167

(5)

Peptide sequencing by nanoelectrospray tandem mass spectrometry

172

(5)

Towards cloning of proteins using mass spectrometry data

177

(4)

Post-translationally modified proteins

181

(2)

Concluding remarks

183

(4)

Acknowledgements

183

(1)

References

183

(4)

Using proteinases for Edman sequence analysis and peptide marking

187

(24)

John Shannon

Introduction

187

(1)

The need for digesting proteins to peptides

187

(1)

Substrate preparation

188

(5)

Purification techniques

188

(1)

Sample requirements

189

(2)

Reduction and alkylation of proteins

191

(2)

Digestion

193

(7)

Choice of proteinase

193

(3)

Conditions for proteolytic digestions

196

(3)

Digestion of proteins isolated on polyacrylamide gels

199

(1)

Monitoring a reaction

200

(1)

Analysis of the proteolytic digestion

200

(11)

Mass spectrometry

200

(1)

Electrophoresis

200

(1)

Ion exchange

200

(1)

Reverse-phase chromatography

201

(8)

Data interpretation

209

(1)

Acknowledgments

209

(1)

References

209

(2)

Prevention of unwanted proteolysis

211

(22)

Michael J. North

Robert J. Beynon

Introduction

211

(2)

Proteolytic susceptibility of native proteins

213

(1)

Intrinsic factors determining the susceptibility of proteins to proteolysis

213

(1)

The influence of other molecules on susceptibility to proteolysis

213

(1)

Properties of endogenous proteinases

214

(1)

Identification of proteolysis as a problem

214

(3)

Changes in protein properties

214

(2)

Mimicking an effect with added proteinases

216

(1)

Checking samples for proteinase activity

216

(1)

Inhibition of proteinases

217

(10)

Outline of approaches for reducing proteinase activity

217

(1)

Suppression of endogenous proteinase activity

217

(1)

Preventing proteolysis by denaturation

217

(3)

Use of proteinase inhibitors

220

(7)

Removal of proteinases

227

(6)

Choice of starting material

227

(3)

Cell disruption and fractionation

230

(1)

Selective removal of proteinases during purification

231

(1)

Acknowledgements

231

(1)

References

231

(2)

Proteolysis of native proteins as a structural probe

233

(32)

Simon Hubbard

Robert J. Beynon

Introduction

233

(2)

Factors influencing susceptibility

235

(5)

Molecular recognition and limited proteolysis

235

(1)

Prediction of nicksites

236

(3)

A tool to aid in prediction of sites of limited proteolysis

239

(1)

Experimental considerations

240

(9)

Choice of proteinase

242

(3)

Ratio of proteinase to substrate

245

(1)

Solution conditions

245

(1)

Determination of site of proteolysis

246

(1)

Strategies for limited proteolysis experiments

247

(2)

Analysis of limited proteolysis data and simulations

249

(16)

Obtaining quantitative data

250

(4)

Analysing the data by non-linear curve fitting

254

(3)

Example reaction schemes

257

(1)

Simulations and modelling

258

(6)

References

264

(1)

Proteases in peptide synthesis

265

(28)

Volker Kasche

Introduction

265

(2)

Enzyme properties influencing the product yield and steric purity in protease catalysed peptide synthesis

267

(4)

Kinetically controlled synthesis

267

(3)

Equilibrium-controlled peptide synthesis

270

(1)

Selecting the optimal protease

271

(7)

Purity of the protease

271

(1)

P1 and P1' specificity

272

(6)

Factors controlling the yield and steric purity in the synthesis of a peptide bond with a given enzyme

278

(8)

Protection of the P1' and activation of the P1-carboxyl group

279

(1)

279

(2)

Temperature

281

(1)

Ionic strength

282

(1)

Solvent composition

283

(1)

Peptide synthesis in suspensions with solid product or substrate

284

(2)

Planning a protease-catalysed synthesis of a peptide bond

286

(2)

What enzyme?

286

(1)

Equilibrium-controlled or kinetically controlled synthesis?

287

(1)

Free or immobilized enzyme?

287

(1)

Experimental methods for protease-catalysed peptide synthesis

288

(1)

Enzyme purity and purification

288

(1)

Enzyme immobilization

288

(1)

Substrates and buffers

288

(1)

Monitoring the synthesis; purification of products

289

(1)

Optimizing the yield

289

(1)

Proteases in peptide synthesis: limitations and perspectives

289

(4)

References

291

(2)

A1 The Schechter and Berger nomenclature for proteinase subsites

293

(2)

A2 Some commercially available proteases

295

(22)

A3 Commercially available proteinase inhibitors

317

(14)

A4 List of suppliers

331

(6)

Index

337

Please wait while the item is added to your bag...

FREE SHIPPING

Amazon no longer offers textbook rentals. We do!

Proteolytic Enzymes A Practical Approach

Summary

Table of Contents

Supplemental Materials