Proteolytic enzymes: a practical approach
Saved in:
| Format: | Book |
|---|---|
| Language: | English |
| Published: |
Oxford
Oxford University Press
2001
|
| Edition: | 2. ed., 1. publ. |
| Series: | The practical approach series
247 |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XVIII, 340 S. Ill., graph. Darst. |
| ISBN: | 019963663X 0199636621 |
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MARC
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| 245 | 1 | 0 | |a Proteolytic enzymes |b a practical approach |c ed. by Robert Beynon ... |
| 250 | |a 2. ed., 1. publ. | ||
| 264 | 1 | |a Oxford |b Oxford University Press |c 2001 | |
| 300 | |a XVIII, 340 S. |b Ill., graph. Darst. | ||
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| 490 | 1 | |a The practical approach series |v 247 | |
| 650 | 7 | |a Peptidases |2 ram | |
| 650 | 4 | |a Peptide Hydrolases | |
| 650 | 4 | |a Proteolytic enzymes | |
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Record in the Search Index
| _version_ | 1804128670450712576 |
|---|---|
| adam_text | Proteolytic Enzymes
Second Edition
^ [pFaeftfeal Approaelhi
Edited by
Robert Beynon
Department of Veterinary Preclinical Sciences,
University of Liverpool, U K
and
Judith S Bond
Department of Biochemistry and
Molecular Biology,
The Pennsylvania State University, U S A
OXPORD
UNIVERSITY PRESS
Contents
Preface page v
List of protocols page xv
Abbreviations xvii
1 Proteolytic enzymes: nomenclature and classification 1
Alan J Barrett
1 Introduction 1
2 Terminology and nomenclature l
Peptidase and related terms 1
Specificity-subsite terminology 3
Catalytic type 3
Homology 3
3 The EC classification of peptidases 4
What is the EC system? 4
What information does the EC list contain? 6
When and how can a newly-discovered peptidase be added to the EC list?
4 The MEROPS system for the classification of peptidases 12
Families 13
Clans 17
Individual peptidases 17
— Uses of the MEROPS system 18
5 Steps one might take on discovering a new peptidase 19
Acknowledgement 20
References 20
2 Purification of proteolytic enzymes 23
Sherwin Wilk
1 Introduction 23
2 Prelude to purification 24
Assay 24
3 Initial considerations 26
Source 26
CONTENTS
Buffer composition 26
Membrane-bound or soluble? 26
Membrane-bound enzymes 27
Endogenous inhibitors and activators 29
4 General scheme for purification of proteolytic enzymes 30
Initial steps 30
Intermediate and final steps 32
5 Specialized techniques for proteolytic enzymes 33
Peptidyl aldehyde affinity chromatography 33
Affinity columns for cysteine proteinases 34
Affinity columns for trypsin-like enzymes 34
Affinity columns for metalloproteinases 35
Other affinity columns 35
6 Optimization of the purification protocol 37
7 Determination of homogeneity 38
8 Purification of the proteasome; EC 3 4 99 46 38
9 Conclusions: many roads lead to Rome 42
References 42
3 Protease assay methods 45
Gautam Sarath, Michael G Zeece and Alan R Periheiter
1 Introduction 45
2 Assays with natural substrates 45
Endopeptidase assays 45
Exopeptidase assays 55
3 Assays with synthetic substrates 55
Endopeptidase and aminopeptidase substrates 55
Spectrophotometric assays 56
Fluorimetric assays 59
Miscellaneous fluorimetric methods 62
Carboxypeptidase substrates 64
Radiometric assays 64
HPLC assays for peptidases 65
Capillary electrophoretic analyses of proteases 67
4 Solid-phase proteaseassays 69
Gel electrophoretic methods 70
Plate assays 73
Miscellaneous solid-phase assays 73
Assays for histochemical studies 74
Acknowledgements 75
References 75
4 Determination of protease mechanism 77
Ben M Dunn
1 Introduction—the importance of mechanistic classification
The serine peptidases 78
The cysteine peptidases 80
The aspartic peptidases 81
The metallopeptidases 81
2 Methods for determining the mechanistic class 83
Classification based on standard inhibitors 83
Chemical modification/identification 86
Site-directed mutagenesis 87
Mechanistic distinctions—intermediates 88
3 Kinetic studies to probe the mechanism in more detail
Notes on ideal assays 91
Kinetic determination of fCm and kac 92
pH dependence of the kinetic parameters 95
Solvent deuterium isotope effects 96
Transition state analogues and substrate alteration 97
4 Determination of primary specificity of a protease 97
Degradation of standard proteins and peptides 98
Cleavage of homologous synthetic peptides 99
References 102
5 Inhibition of proteolytic enzymes 105
Guy S Salvesen and Hideaki Nagase
1 Introduction: which inhibitors? 105
2 Principles for using irreversible and reversible inhibitors
General structure of synthetic inhibitors 106
How to live with crossreactivity 106
3 Practical use of inhibition constants l07
Irreversible inhibitors 107
Reversible inhibitors 108
4 Non-specific inhibitors 109
a-Macroglobulins 109
Peptide aldehydes 110
Peptide chloromethyl ketones 110
~~Metal chelators 111
5 Class-specific inhibitors 112
Serine proteases 112
Cysteine proteases 116
Proteasome 119
Metalloproteases 120
Aspartic proteases 122
6 Inhibitors as active-site titrants 122
Cysteine proteases 122
Serine proteases 124
7 Protease inhibitors in cell culture 125
8 Suppression of proteolysis 126
9 Therapeutic value of protease inhibitors 127
References 128
CONTENTS
6 Finding, purification and characterization of natural protease
inhibitors 131
Hideaki Nagase and Guy S Salvesen
1 Introduction 131
The meaning of inhibition 131
2 Finding protease inhibitors 132
Screening of inhibitors from natural sources 132
Finding inhibitors by reverse zymography 133
Finding inhibitors from DNA sequences 135
Combinatorial protease inhibitors 137
3 Purification of natural protease inhibitors 137
Use of the target enzyme as an affinity ligand 137
Conventional purification 137
Reverse zymography 138
4 Characterization of inhibitors: inhibition kinetics 138
The importance of kinetics 138
ICJQ and percentage inhibition 138
Practical inhibitor kinetics 139
Reversible inhibitors 140
Irreversible inhibitors 143
5 Practical applications of protein inhibitors 146
Acknowledgements 146
References 146
7 Mass spectrometry of proteolysis-derived peptides for protein
identification 149
Berrihard Kiister, Andrej Shevchenko and Matthias Mann
1 Introduction 149
2 Mass spectrometry 150
Methods of ionization 150
Mass analysis 152
- Tandem mass spectrometry 153
3 Interrogation of sequence databases using mass spectrometry data 155
Choice of protease 155
Peptide mass mapping 157
Database identification via tandem mass spectromeny 159
4 Analytical strategy for the identification or cloning of proteins using
mass spectrometry 161
Low-level protein preparation for characterization by mass spectrometry 161
Protein visualization 163
Proteolytic cleavage of gel separated proteins 165
Protein identification by MALDI peptide mass mapping 167
Peptide sequencing by nanoelectrospray tandem mass spectrometry 172
Towards cloning of proteins using mass spectrometry data 177
5 Post-translationally modified proteins 181
6 Concluding remarks 183
x
CONTENTS
Acknowledgements 183
References 183
8 Using proteinases for Edman sequence analysis and peptide
marking 187
John Shannon
1 Introduction 187
The need for digesting proteins to peptides 187
2 Substrate preparation 188
Purification techniques 188
Sample requirements 189
Reduction and alkylation of proteins 191
3 Digestion 193
Choice of proteinase 193
Conditions for proteolytic digestions 196
Digestion of proteins isolated on polyacrylamide gels 199
Monitoring a reaction 200
4 Analysis of the proteolytic digestion 200
Mass spectrometry 200
Electrophoresis 200
Ion exchange 200
Reverse-phase chromatography 201
Data interpretation 209
Acknowledgments 209
References 209
9 Prevention of unwanted proteolysis 211
Michael J North and Robert J Beynon
1 Introduction 211
2 Proteolytic susceptibility of native proteins 213
Intrinsic factors determining the susceptibility of proteins to proteolysis 213
The influence of other molecules on susceptibility to proteolysis 213
Properties of endogenous proteinases 214
3 Identification of proteolysis as a problem 214
Changes in protein properties 214
Mimicking an effect with added proteinases 216
Checking samples for proteinase activity 216
4 Inhibition of proteinases 217
Outline of approaches for reducing proteinase activity 217
Suppression of endogenous proteinase activity 217
Preventing proteolysis by denaturation 217
Use of proteinase inhibitors 220
5 Removal of proteinases 227
Choice of starting material 227
Cell disruption and fractionation 230
Selective removal of proteinases during purification 231
Acknowledgements 231
References 231
xi
CONTENTS
10 Proteolysis of native proteins as a structural probe 233
Simon Hubbard and Robert J Beynon
1 Introduction 233
2 Factors influencing susceptibility 235
Molecular recognition and limited proteolysis 235
Prediction of nicksites 236
A tool to aid in prediction of sites of limited proteolysis 239
3 Experimental considerations 240
Choice of proteinase 242
Ratio of proteinase to substrate 245
Solution conditions 245
Determination of site of proteolysis 246
Strategies for limited proteolysis experiments 247
4 Analysis of limited proteolysis data and simulations 249
Obtaining quantitative data 250
Analysing the data by non-linear curve fitting 254
Example reaction schemes 257
Simulations and modelling 258
References 264
11 Proteases in peptide synthesis 265
Volker Kasche
1 Introduction 265
2 Enzyme properties influencing the product yield and steric purity
protease catalysed peptide synthesis 267
Kinetically controlled synthesis 267
Equilibrium-controlled peptide synthesis 270
3 Selecting the optimal protease 271
Purity of the protease 271
P, and P, specificity 272 ,
4 Factors controlling the yield and steric purity in the synthesis of a
peptide bond with a given enzyme 278
Protection of the P, and activation of the P,-carboxyl group 279
pH 279
Temperature 281
Ionic strength 282
Solvent composition 283
Peptide synthesis in suspensions with solid product or substrate 284
5 Planning a protease-catalysed synthesis of a peptide bond 286
What enzyme? 286
Equilibrium-controlled or kinetically controlled synthesis? 287
Free or immobilized enzyme? 287
6 Experimental methods for protease-catalysed peptide synthesis 2
Enzyme purity and purification 288
Enzyme immobilization 288
Substrates and buffers 288
Monitoring the synthesis; purification of products 289
Optimizing the yield 289
xii
CONTENTS
7 Proteases in peptide synthesis: limitations and perspectives 289
References 291
A1 The Schechter and Berger nomenclature for proteinase subsltes 293
A2 Some commercially available proteases 295
A3 Commercially available proteinase inhibitors 317
A4 List of suppliers 33i
Index 337
xiii
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| spelling | Proteolytic enzymes a practical approach ed. by Robert Beynon ... 2. ed., 1. publ. Oxford Oxford University Press 2001 XVIII, 340 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series 247 Peptidases ram Peptide Hydrolases Proteolytic enzymes Proteasen (DE-588)4047521-9 gnd rswk-swf Proteasen (DE-588)4047521-9 s DE-604 Beynon, Robert J. Sonstige oth The practical approach series 247 (DE-604)BV007921321 247 HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009464948&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Proteolytic enzymes a practical approach The practical approach series Peptidases ram Peptide Hydrolases Proteolytic enzymes Proteasen (DE-588)4047521-9 gnd |
| subject_GND | (DE-588)4047521-9 |
| title | Proteolytic enzymes a practical approach |
| title_auth | Proteolytic enzymes a practical approach |
| title_exact_search | Proteolytic enzymes a practical approach |
| title_full | Proteolytic enzymes a practical approach ed. by Robert Beynon ... |
| title_fullStr | Proteolytic enzymes a practical approach ed. by Robert Beynon ... |
| title_full_unstemmed | Proteolytic enzymes a practical approach ed. by Robert Beynon ... |
| title_short | Proteolytic enzymes |
| title_sort | proteolytic enzymes a practical approach |
| title_sub | a practical approach |
| topic | Peptidases ram Peptide Hydrolases Proteolytic enzymes Proteasen (DE-588)4047521-9 gnd |
| topic_facet | Peptidases Peptide Hydrolases Proteolytic enzymes Proteasen |
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