Receptor-effector coupling: a practical approach
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Oxford u.a.
IRL Press at Oxford Univ. Press
1990
|
| Schriftenreihe: | The practical approach series
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Literaturangaben |
| Beschreibung: | XVIII, 224 S. graph. Darst. |
| ISBN: | 019963095X 0199630941 |
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| 264 | 1 | |a Oxford u.a. |b IRL Press at Oxford Univ. Press |c 1990 | |
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| 650 | 4 | |a Receptors, Neurotransmitter | |
| 650 | 4 | |a Signal Transduction | |
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Datensatz im Suchindex
| _version_ | 1804122426436485120 |
|---|---|
| adam_text | Receptor-Effector
Coupling
A Practical Approach
Edited by
ECHULME
Division ofPhysicalBiochemistry
National Institute for Mcdical Research
MillHill,London,NW71AAUK
QliiA E^SiS • 1
at
OXFORD UNIVERSITY PRESS
Oxford New York Tokyo
Contents
List of contributors xv
Abbreviations xvii
1 Preparation of G-proteins and their subunits l
P C Sternweis and Iok-Hou Pang
1 Introduction 1
2 Procedures for purification of G-proteins 3
Materials 3
Preparation of membranes 5
Extraction and general purification of G-proceins from membranes
of bovine brain 9
Additional procedures for further purification of Gs and Gj
proteins 16
Preparation of Gt (transducin) 20
Resolution of a- and /fy-subunits 21
3 Detection techniques and assays 23
Binding of GTPyS 24
Gs activity 25
Use of bacterial toxins 26
Analysis of G-proteins by electrophoresis 28
Acknowledgements 29
References 29
2 Receptor-G-protein complexes in solution 31
D Poyner
1 Introduction 31
2 Preparation of R-G complexes 32
Introduction 32
Membrane preparation 32
Solubilization 33
Assay of solubilized complexes 34
Solubilization of muscarinic receptor-G-protein complexes from
the cortex 34
ix
Conten l s
3 Characterization of R—G complexes in solution 35
Introduction 35
Ligand-binding studies 35
GTPase activity of R-G complexes 36
Effects of agonists on guanine nucleotide binding 37
Gel filtration of R-G complexes 38
Sucrose density gradient centrifugation of R-G complexes 41
Calculation of hydrodynamic parameters 42
4 Purification of R—G complexes 47
Introduction 47
Separation of R from R-G 47
Separation of R-G complexes from G-proteins 49
5 G-protein identification 51
Introduction 51
Toxin labelling 52
Antibody detection 52
Labelling with GTP 55
Acknowledgements 55
References 56
Appendix 57
3 Reconstitution of hormone-sensitive adenylate
cyclase and tyrosine kinases 59
R A Cerione
1 Introduction 59
2 Reconstitution of a hormone-responsive adenylate
cyclase system 60
Insertion of the /^-adrenergic receptor into phospholipid vesicles 61
Functional reconstitution of receptor-G-protein interactions in
phospholipid vesicles 64
Reconstitution of a receptor/G-protein/adenylate cyclase signal
transduction system 70
3 Reconstitution of related receptor-coupled signal
transduction systems 74
Vertebrate phototransduction 74
Reconstitution of growth factor receptor/tyrosine kinases 77
4 Conclusions 80
References 81
x
Conten t s
4 Interactions of muscarinic acetylcholine
receptors (mAChRs) with G-proteins 83
T Haga and K Haga
1 Introduction 83
2 Preparation of mAChR, G-proteins, lipids, and
membranes 85
mAChR 85
G-proteins 85
Total lipids 87
Membranes with mAChRs but not G-proteins 88
Membranes with G-proteins but not mAChRs 88
3 Reconstitution 89
Reconstitution of mAChRs and G-proteins into phospholipid
vesicles 89
Reconstitution of mAChRs or G-proteins into membranes 90
4 Functional assay of reconstituted vesicles 91
Guanine nucleotide-sensitive high-affinity agonist binding 91
Guanine nucleotide binding 93
GTPase activity 93
5 A model for the interaction between mAChRs and
G-proteins 94
6 Regulation of mAChR—G-protein interaction 96
References 97
5 Polyphosphoinositide turnover 99
K G Oldham
1 Introduction 99
2 Column chromatography of inositol phosphates 100
3 Experimental design: use of labelled myo-inositol in
studies of phosphatidyl inositol turnover: factors
affecting sensitivity and the selection of optimum
conditions for experiments 103
Sensitivity, blanks, storage, and stability 104
What specific activity [3H]inositol and what radioactive
concentration should be used? 105
Increasing percentage incorporation 108
Advantages of using high specific activity [3H]inositol 109
4 Quantitative assays of D-myo-inositol 1,4,5-triphosphate 109
Sample preparation 110
xi
Con ten Is
Assay protocol 1 ] 2
Separation procedure 112
Presentation of results 113
Reproducibility 114
Specificity 114
Acknowledgements 115
References 115
6 Measurement and control of intracellular
calcium 117
A M Gurney
1 Introduction 117
Receptors and calcium 11 7
Methods of studying calcium acti ity 117
Properties of calcium indicators: v/hat to look for 118
2 Optical indicators 120
Principles 120
Fluorescent indicators 121
Absorbent dyes 130
Luminescent photoproteins 133
Instrumentation for measuring optical signals 136
Calcium imaging 139
3 Non-optical methods for measuring intracellular calcium 139
Ca2+-selective microelectrodes 139
Nuclear magnetic resonance 141
4 Control of intracellular calcium 141
Photolabile calcium probes 141
Comparison of available molecule : 143
Incorporation into cells 146
Instrumentation 146
Calibration 147
Applications 149
Acknowledgements 152
References 152
Appendix: addresses of some suppliers 153
7 Molecular pharmacology of ion channels using
the patch clamp 155
A M Gurney
1 Introduction 155
2 General description of the technique 155
What is patch clamping? 155
xii
Conten t s
Preparations suitable for patch clamping 156
The recording circuitry 156
3 Setting-up to patch clamp 161
Equipment 161
Problem solving 163
4 Glass micropipettes for patch-clamp recording 165
Choice of pipette glass 165
Fabricating pipettes 165
Filling pipettes 168
5 Patch-clamp experiments 170
Recording solutions 170
Patch-clamp configurations 170
How to obtain the configurations 171
6 Patch-clamp studies of receptor pharmacology 175
Acknowledgements 177
References 177
Appendix: addresses of some suppliers 178
8 Phosphate-labelling studies of receptor tyrosine
kinases 181
L C Mahadevan and J C Bell
1 Introduction and scope 181
2 Criteria for establishing receptor kinase activity 181
Demonstration of ligand-binding activity 182
Stimulation of tyrosine kinase activity 182
Molecular structural criteria 183
3 Strategies for demonstrating receptor tyrosine kinase
activity 184
[32P]Phosphate labelling studies in intact cells 184
[ -32P]ATP labelling studies in vitro 184
Distinguishing tyrosine-phosphorylated proteins from serine- and
threonine-phosphorylated proteins 185
Demonstration of receptor tyrosine kinases 186
4 Methods of 32P-labelling proteins in vivo and in vit ro 188
Safe handling of radioactivity 188
Labelling intact cells with [32P]phosphate 188
In vitro membrane phosphorylation assay 190
5 Preparation of antiphosphotyrosine antibodies 194
Coupling of phosphotyrosine to carrier proteins 194
Immunization of rabbits with phosphotyrosine conjugates 195
Affinity purification of antiphosphotyrosine antibodies 196
Immunoprecipitation studies using antiphosphotyrosine antibodies 198
xiii
Conten t s
6 Identification of phosphotyrosine 199
Proteolytic elution of protein from gel slices 199
Electroelution of protein from gel slices 200
Two-dimensional thin layer electrophoresis 200
Acknowledgements 200
References 201
Appendix I Receptor binding studies, a brief
outline 203
E C Hulme
1 Introduction 203
Basic principles 203
2 The ligand off-rate may determine the assay method 205
3 Criteria for receptor-specific binding 206
4 Tracer ligand saturation curve 206
Receptor preparation 207
Setting up the binding curve 208
Replicate incubations 208
5 Preliminary analysis and interpretation 210
6 Separation methods: particulate preparations 213
Filtration assays 213
Centrifugation assays 213
7 Soluble preparations 214
Gel filtration 214
Filtration on polyethyleneimine-treated filters 214
Precipitation by polyethylene glycol 215
Charcoal adsorption 215
References 215
Index 217
Contents lists of related volumes 221
xiv
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| id | DE-604.BV008061067 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T17:13:41Z |
| institution | BVB |
| isbn | 019963095X 0199630941 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-005304560 |
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| owner_facet | DE-20 DE-29 DE-11 DE-B768 |
| physical | XVIII, 224 S. graph. Darst. |
| publishDate | 1990 |
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| publisher | IRL Press at Oxford Univ. Press |
| record_format | marc |
| series2 | The practical approach series |
| spelling | Receptor-effector coupling a practical approach ed. by E. C. Hulme Oxford u.a. IRL Press at Oxford Univ. Press 1990 XVIII, 224 S. graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series Literaturangaben Cellular signal transduction G proteins GTP-Binding Proteins physiology Hormone receptors Research Methodology Neurotransmitter receptors Research Methodology Receptors, Cell Surface Receptors, Neurotransmitter Signal Transduction GTP-bindende Proteine (DE-588)4248354-2 gnd rswk-swf Hormon (DE-588)4025864-6 gnd rswk-swf Hormonrezeptor (DE-588)4138900-1 gnd rswk-swf Praktikum (DE-588)4127380-1 gnd rswk-swf Neurotransmitter-Rezeptor (DE-588)4171601-2 gnd rswk-swf Hormonrezeptor (DE-588)4138900-1 s DE-604 Neurotransmitter-Rezeptor (DE-588)4171601-2 s Hormon (DE-588)4025864-6 s Praktikum (DE-588)4127380-1 s 1\p DE-604 GTP-bindende Proteine (DE-588)4248354-2 s 2\p DE-604 Hulme, E. C. Sonstige oth HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=005304560&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Receptor-effector coupling a practical approach Cellular signal transduction G proteins GTP-Binding Proteins physiology Hormone receptors Research Methodology Neurotransmitter receptors Research Methodology Receptors, Cell Surface Receptors, Neurotransmitter Signal Transduction GTP-bindende Proteine (DE-588)4248354-2 gnd Hormon (DE-588)4025864-6 gnd Hormonrezeptor (DE-588)4138900-1 gnd Praktikum (DE-588)4127380-1 gnd Neurotransmitter-Rezeptor (DE-588)4171601-2 gnd |
| subject_GND | (DE-588)4248354-2 (DE-588)4025864-6 (DE-588)4138900-1 (DE-588)4127380-1 (DE-588)4171601-2 |
| title | Receptor-effector coupling a practical approach |
| title_auth | Receptor-effector coupling a practical approach |
| title_exact_search | Receptor-effector coupling a practical approach |
| title_full | Receptor-effector coupling a practical approach ed. by E. C. Hulme |
| title_fullStr | Receptor-effector coupling a practical approach ed. by E. C. Hulme |
| title_full_unstemmed | Receptor-effector coupling a practical approach ed. by E. C. Hulme |
| title_short | Receptor-effector coupling |
| title_sort | receptor effector coupling a practical approach |
| title_sub | a practical approach |
| topic | Cellular signal transduction G proteins GTP-Binding Proteins physiology Hormone receptors Research Methodology Neurotransmitter receptors Research Methodology Receptors, Cell Surface Receptors, Neurotransmitter Signal Transduction GTP-bindende Proteine (DE-588)4248354-2 gnd Hormon (DE-588)4025864-6 gnd Hormonrezeptor (DE-588)4138900-1 gnd Praktikum (DE-588)4127380-1 gnd Neurotransmitter-Rezeptor (DE-588)4171601-2 gnd |
| topic_facet | Cellular signal transduction G proteins GTP-Binding Proteins physiology Hormone receptors Research Methodology Neurotransmitter receptors Research Methodology Receptors, Cell Surface Receptors, Neurotransmitter Signal Transduction GTP-bindende Proteine Hormon Hormonrezeptor Praktikum Neurotransmitter-Rezeptor |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=005304560&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT hulmeec receptoreffectorcouplingapracticalapproach |