Proteolytic Enzymes A Practical Approach

Name: Proteolytic Enzymes A Practical Approach
Availability: OutOfStock
ISBN: 9780199630585

ISBN-10: 0199630585

ISBN-13: 9780199630585

Edition: 1989

Authors: Rob J. Beynon, Judith S. Bond

List price: $95.00

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This book offers comprehensive, unified coverage of proteolytic enzymes, which are useful in a multitude of important physiological processes. The emphasis here is on the practical aspects of the handling, characterization, inhibition, and use of these enzymes. The book provides general advice as well as specific examples. Written in a clear, readable style, the volume is an excellent resource for scientists who study proteases, or those who use them as probes for experimental and therapeutic purposes.

Book details

List price: $95.00
Copyright year: 1989
Publisher: Oxford University Press, Incorporated
Publication date: 10/26/1989
Binding: Hardcover
Pages: 278
Size: 7.00" wide x 9.75" long x 0.75" tall
Weight: 1.386
Language: English

Preface	p. v
List of protocols	p. xv
Abbreviations	p. xvii
Proteolytic enzymes: nomenclature and classification	p. 1
Introduction	p. 1
Terminology and nomenclature	p. 1
Peptidase and related terms	p. 1
Specificity-subsite terminology	p. 3
Catalytic type	p. 3
Homology	p. 3
The EC classification of peptidases	p. 4
What is the EC system?	p. 4
What information does the EC list contain?	p. 6
When and how can a newly-discovered peptidase be added to the EC list?	p. 7
The MEROPS system for the classification of peptidases	p. 12
Families	p. 13
Clans	p. 17
Individual peptidases	p. 17
Uses of the MEROPS system	p. 18
Steps one might take on discovering a new peptidase	p. 19
Acknowledgement	p. 20
References	p. 20
Purification of proteolytic enzymes	p. 23
Introduction	p. 23
Prelude to purification	p. 24
Assay	p. 24
Initial considerations	p. 26
Source	p. 26
Buffer composition	p. 26
Membrane-bound or soluble?	p. 26
Membrane-bound enzymes	p. 27
Endogenous inhibitors and activators	p. 29
General scheme for purification of proteolytic enzymes	p. 30
Initial steps	p. 30
Intermediate and final steps	p. 32
Specialized techniques for proteolytic enzymes	p. 33
Peptidyl aldehyde affinity chromatography	p. 33
Affinity columns for cysteine proteinases	p. 34
Affinity columns for trypsin-like enzymes	p. 34
Affinity columns for metalloproteinases	p. 35
Other affinity columns	p. 35
Optimization of the purification protocol	p. 37
Determination of homogeneity	p. 38
Purification of the proteasome; EC 3.4.99.46	p. 38
Conclusions: many roads lead to Rome	p. 42
References	p. 42
Protease assay methods	p. 45
Introduction	p. 45
Assays with natural substrates	p. 45
Endopeptidase assays	p. 45
Exopeptidase assays	p. 55
Assays with synthetic substrates	p. 55
Endopeptidase and aminopeptidase substrates	p. 55
Spectrophotometric assays	p. 56
Fluorimetric assays	p. 59
Miscellaneous fluorimetric methods	p. 62
Carboxypeptidase substrates	p. 64
Radiometric assays	p. 64
HPLC assays for peptidases	p. 65
Capillary electrophoretic analyses of proteases	p. 67
Solid-phase protease assays	p. 69
Gel electrophoretic methods	p. 70
Plate assays	p. 73
Miscellaneous solid-phase assays	p. 73
Assays for histochemical studies	p. 74
Acknowledgements	p. 75
References	p. 75
Determination of protease mechanism	p. 77
Introduction--the importance of mechanistic classification	p. 77
The serine peptidases	p. 78
The cysteine peptidases	p. 80
The aspartic peptidases	p. 81
The metallopeptidases	p. 81
Methods for determining the mechanistic class	p. 83
Classification based on 'standard' inhibitors	p. 83
Chemical modification/identification	p. 86
Site-directed mutagenesis	p. 87
Mechanistic distinctions--intermediates	p. 88
Kinetic studies to probe the mechanism in more detail	p. 91
Notes on 'ideal' assays	p. 91
Kinetic determination of K[subscript m] and k[subscript cat]	p. 92
pH dependence of the kinetic parameters	p. 95
Solvent deuterium isotope effects	p. 96
Transition state analogues and substrate alteration	p. 97
Determination of primary specificity of a protease	p. 97
Degradation of standard proteins and peptides	p. 98
Cleavage of homologous synthetic peptides	p. 99
References	p. 102
Inhibition of proteolytic enzymes	p. 105
Introduction: which inhibitors?	p. 105
Principles for using irreversible and reversible inhibitors	p. 106
General structure of synthetic inhibitors	p. 106
How to live with crossreactivity	p. 106
Practical use of inhibition constants	p. 107
Irreversible inhibitors	p. 107
Reversible inhibitors	p. 108
Non-specific inhibitors	p. 109
[alpha]-Macroglobulins	p. 109
Peptide aldehydes	p. 110
Peptide chloromethyl ketones	p. 110
Metal chelators	p. 111
Class-specific inhibitors	p. 112
Serine proteases	p. 112
Cysteine proteases	p. 116
Proteasome	p. 119
Metalloproteases	p. 120
Aspartic proteases	p. 122
Inhibitors as active-site titrants	p. 122
Cysteine proteases	p. 122
Serine proteases	p. 124
Protease inhibitors in cell culture	p. 125
Suppression of proteolysis	p. 126
Therapeutic value of protease inhibitors	p. 127
References	p. 128
Finding, purification and characterization of natural protease inhibitors	p. 131
Introduction	p. 131
The meaning of inhibition	p. 131
Finding protease inhibitors	p. 132
Screening of inhibitors from natural sources	p. 132
Finding inhibitors by reverse zymography	p. 133
Finding inhibitors from DNA sequences	p. 135
Combinatorial protease inhibitors	p. 137
Purification of natural protease inhibitors	p. 137
Use of the target enzyme as an affinity ligand	p. 137
Conventional purification	p. 137
Reverse zymography	p. 138
Characterization of inhibitors: inhibition kinetics	p. 138
The importance of kinetics	p. 138
IC[subscript 50] and percentage inhibition	p. 138
Practical inhibitor kinetics	p. 139
Reversible inhibitors	p. 140
Irreversible inhibitors	p. 143
Practical applications of protein inhibitors	p. 146
Acknowledgements	p. 146
References	p. 146
Mass spectrometry of proteolysis-derived peptides for protein identification	p. 149
Introduction	p. 149
Mass spectrometry	p. 150
Methods of ionization	p. 150
Mass analysis	p. 152
Tandem mass spectrometry	p. 153
Interrogation of sequence databases using mass spectrometry data	p. 155
Choice of protease	p. 155
Peptide mass mapping	p. 157
Database identification via tandem mass spectrometry	p. 159
Analytical strategy for the identification or cloning of proteins using mass spectrometry	p. 161
Low-level protein preparation for characterization by mass spectrometry	p. 161
Protein visualization	p. 163
Proteolytic cleavage of gel separated proteins	p. 165
Protein identification by MALDI peptide mass mapping	p. 167
Peptide sequencing by nanoelectrospray tandem mass spectrometry	p. 172
Towards cloning of proteins using mass spectrometry data	p. 177
Post-translationally modified proteins	p. 181
Concluding remarks	p. 183
Acknowledgements	p. 183
References	p. 183
Using proteinases for Edman sequence analysis and peptide marking	p. 187
Introduction	p. 187
The need for digesting proteins to peptides	p. 187
Substrate preparation	p. 188
Purification techniques	p. 188
Sample requirements	p. 189
Reduction and alkylation of proteins	p. 191
Digestion	p. 193
Choice of proteinase	p. 193
Conditions for proteolytic digestions	p. 196
Digestion of proteins isolated on polyacrylamide gels	p. 199
Monitoring a reaction	p. 200
Analysis of the proteolytic digestion	p. 200
Mass spectrometry	p. 200
Electrophoresis	p. 200
Ion exchange	p. 200
Reverse-phase chromatography	p. 201
Data interpretation	p. 209
Acknowledgments	p. 209
References	p. 209
Prevention of unwanted proteolysis	p. 211
Introduction	p. 211
Proteolytic susceptibility of native proteins	p. 213
Intrinsic factors determining the susceptibility of proteins to proteolysis	p. 213
The influence of other molecules on susceptibility to proteolysis	p. 213
Properties of endogenous proteinases	p. 214
Identification of proteolysis as a problem	p. 214
Changes in protein properties	p. 214
Mimicking an effect with added proteinases	p. 216
Checking samples for proteinase activity	p. 216
Inhibition of proteinases	p. 217
Outline of approaches for reducing proteinase activity	p. 217
Suppression of endogenous proteinase activity	p. 217
Preventing proteolysis by denaturation	p. 217
Use of proteinase inhibitors	p. 220
Removal of proteinases	p. 227
Choice of starting material	p. 227
Cell disruption and fractionation	p. 230
Selective removal of proteinases during purification	p. 231
Acknowledgements	p. 231
References	p. 231
Proteolysis of native proteins as a structural probe	p. 233
Introduction	p. 233
Factors influencing susceptibility	p. 235
Molecular recognition and limited proteolysis	p. 235
Prediction of nicksites	p. 236
A tool to aid in prediction of sites of limited proteolysis	p. 239
Experimental considerations	p. 240
Choice of proteinase	p. 242
Ratio of proteinase to substrate	p. 245
Solution conditions	p. 245
Determination of site of proteolysis	p. 246
Strategies for limited proteolysis experiments	p. 247
Analysis of limited proteolysis data and simulations	p. 249
Obtaining quantitative data	p. 250
Analysing the data by non-linear curve fitting	p. 254
Example reaction schemes	p. 257
Simulations and modelling	p. 258
References	p. 264
Proteases in peptide synthesis	p. 265
Introduction	p. 265
Enzyme properties influencing the product yield and steric purity in protease catalysed peptide synthesis	p. 267
Kinetically controlled synthesis	p. 267
Equilibrium-controlled peptide synthesis	p. 270
Selecting the optimal protease	p. 271
Purity of the protease	p. 271
P[subscript 1] and P'[subscript 1] specificity	p. 272
Factors controlling the yield and steric purity in the synthesis of a peptide bond with a given enzyme	p. 278
Protection of the P'[subscript 1] and activation of the P[subscript 1]-carboxyl group	p. 279
pH	p. 279
Temperature	p. 281
Ionic strength	p. 282
Solvent composition	p. 283
Peptide synthesis in suspensions with solid product or substrate	p. 284
Planning a protease-catalysed synthesis of a peptide bond	p. 286
What enzyme?	p. 286
Equilibrium-controlled or kinetically controlled synthesis?	p. 287
Free or immobilized enzyme?	p. 287
Experimental methods for protease-catalysed peptide synthesis	p. 288
Enzyme purity and purification	p. 288
Enzyme immobilization	p. 288
Substrates and buffers	p. 288
Monitoring the synthesis; purification of products	p. 289
Optimizing the yield	p. 289
Proteases in peptide synthesis: limitations and perspectives	p. 289
References	p. 291
The Schechter and Berger nomenclature for proteinase subsites	p. 293
Some commercially available proteases	p. 295
Commercially available proteinase inhibitors	p. 317
List of suppliers	p. 331
Index	p. 337
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