University of Bath PHD Studies on the alpha-bungarotoxin binding component in human brain. Whyte, J. Award date: 1985 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 i Ta \ D'--- V ^ STUDIES ON THE a-BUNGAROTOXIN BINDING COMPONENT IN HUMAN BRAIN submitted by J. Whyte for the degree of Ph.D. of the University of Bath 1985 COPYRIGHT Attention is drawn to the fact that the copyright of this thesis rests with the author. This copy of the thesis has been supplied on condition that anyone who consults it is under­ stood to recognize that its copyright rests with the author and that no quotation from the thesis and no information derived from it may be published without the prior written consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. ProQuest Number: U362950 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U362950 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 CONTENTS List of Figures (i) List of Tables (v) Acknowledgements (viii) Summary (ix) Publications (xi) Abbreviations (xii) Section 1 INTRODUCTION 1) Cholinergic Transmission in the Nervous System 1 2) Characterization of the Nicotinic Acetylcholine 8 Receptor 3) Nicotinic Acetylcholine Receptors in the Central 12 Nervous System, Are a-Neurotoxins Suitable as Probes? 4) Properties of the Central Nicotinic Acetylcholine 15 Receptor 5) Properties of a-Bungarotoxin Binding in the Central 24 Nervous System 6) Regional Distribution of a-Bungarotoxin Binding Sites 26 7) Immunological Characteristics of the a-Bungarotoxin 29 Binding Component 8) Subcellular Fractionation of Brain 32 9) Subcellular Localization of a-Bungarotoxin Binding 33 Sites 10) Developmental Aspects of a-Bungarotoxin Binding 36 Section 2 MATERIALS AND METHODS Page no. 1) Preparation and Characterization of a-Bungarotoxin 38 (a) Materials 38 (b) Chromatographic Separation of Radiolabelled 39 a-Bungar o t oxin (c) The a-Bungarotoxin Binding Assay 39 (d) Centrifugation Assay 40 (e) Filtration Assay 40 (f) Ammonium Sulphate Precipitation Assay 40 (g) DEAE Cellulose Assay 40 (h) Assessment of the Biological Activity of 42 Radiolabelled a-Bungarotoxin (i) Assessment of the Immunological Activity of 42 Radiolabelled a-Bungarotoxin 2) Subcellular Fractionation of Brain 44 (a Materials 44 (b Fractionation of Human Foetal Brain Homogenate 44 (c Fractionation of Rat Brain Homogenate 47 (d Determination of Protein 47 (e Determination of Choline Acetyltransferase Activity 47 (f Determination of Acetylcholinesterase Activity 48 (g Determination of Lactate Dehydrogenase Activity 49 (h Determination of Succinate Dehydrogenase Activity 50 (i Determination of Adenylate Cyclase Activity 51 (j Binding of [^^^1,1 a-Bungarotoxin to Subcellular 53 fractions (k Binding of [^^-QNB to Subcellular Fractions 53 (1 Preparation of Subcellular Fractions for Electron 54 Microscopy Page no, 3) Quantitative Studies of a-Bungarotoxin Binding 56 (a) Materials 56 (b) Preparation of a Crude Detergent Extract 56 (c) Determination of the Intrinsic Dissociation 56 Constant (d) Determination of the Association Rate Constant 57 (e) Determination of the Dissociation Rate Constant 59 (f) Determination of the IC^^ Values of Various Drugs 60 4) Regional Binding Studies of Brain and Spinal Cord 62 (a) Autoradiography 62 (b) Materials 62 (c) Rat Brain Intraventriculan Injections 63 (d) Preparation of Human Foetal Spinal Cord Sections 64 (e) Binding of -a-Bungarotoxin to Rat Brain 65 Regions (f) Binding of [^^^l]-a-Bungarotoxin to Human Foetal 65 Brain Regions (g) The Preparation of Human Foetal Brain Tissue for 65 Electron Microscopy 5) Binding of Anti-(nAChR) Antibodies to Human Foetal Brain 67 Purification of anti-(nAChR) Antibodies (a) Materials 67 (b) Extraction of nAChR from Muscle 67 (c) Determination of the a-Bungarotoxin Binding 68 Activity of Human Muscle Detergent Extract (d) Preparation of an a-Bungarotoxin Affinity Column 68 page no, (e) Radioimmunoassay for Anti (-nAChR) Antibodies 69 (f) Purification of Anti-(Receptor) Antibodies 69 (g) Affinity Purification of Anti-(Receptor) Anti- 70 bodies (h) Binding of Anti (-nAChR) Antibodies to the 71 a-Bungarotoxin Binding Component in Human Foetal Brain 6) a-Bungarotoxin Binding Protein Purification 72 7) List of Buffers 73 Section 3 RESULTS 1) Preparation and Properties of -Labelled 74 a-Bungarotoxin Fractions (a) Biological Activity of Radiolabelled a-Bungaro- 74 toxin (b) The Production of Anti-(a-Bungarotoxin) Antibodies 80 (c) The Immunological Activity of [^^^I_j-a-Bungaro- 80 toxin (d) a-Bungarotoxin Binding Assays: The Separation 80 of Toxin Receptor Complexes from Free Toxin (e) Dependence of Binding of a-Bungarotoxin to 81 the Particulate Fraction on the Concentration of Protein (f) The Stability of a-Bungarotoxin Binding Activity 81 (g) a-Bungarotoxin Binding Activity During Development 91 page no, 2) The Subcellular Fractionation of Brain 96 (a) Recovery of Protein 97 (b) Recovery of Enzyme Activity in Foetal Brain 98 (c) Recovery of Enzyme Activity in Rat Brain 99 (d) Recovery of Ligand Binding Sites in Rat and Human 100 Foetal Brain (e) Subcellular Fractionation of Human Foetal and 101 Rat Brain: Figure Legend (f) Examination of Subcellular Fractions at the 131 Electron Microscope Level Plates 1-5 133 3) Quantitative Studies of a-Bungarotoxin Binding in 134 Human Foetal Brain (a) Binding of -a-Bungarotoxin at Equilibrium 134 (b) Determination of the Association Rate Constants 140 (c) Determination of the Dissociation Rate Constants 143 (d) Calculation of the Intrinsic Dissociation Constant 149 (e) Regional Distribution of a-Bungarotoxin in 155 Rat Brain (f) Regional Distribution of -a-Bungarotoxin 159 Binding in Human Foetal Brain Plates 6-7 162 Page no, 2) Attempted Purification of the a-Bungarotoxin 189 Binding Conyonent 3) The Subcellular Fractionation of Brain 193 4) Quantitative Studies of a-Bungarotoxin Binding 202 (a) Determination of the Intrinsic Dissociation 204 Constant (b) Determination of the Association Constant 204 (c) Determination of the Dissociation Constant 206 (d) The Effect of Various Drugs on the Binding 209 of -a-Bungarotoxin to Human Foetal Brain 5) Binding of Anti-(nAChR) Antibodies to Human Foetal 213 Brain (a) Purification of Anti-(nAChR) Antibodies 213 (b) Recognition of the Human Neuronal a-Bungarotoxin 215 Binding Component by anti-(nAChR) Antibodies from Myasthenic Plasma REFERENCES 219 (i) List of Figures Page no, Figure 1.1 The Structure of Some Cholinergic Agonists 3 1.2 The Structure of Some Nicotinic Compounds 4 1.3 The Structure of Some Muscarinic Antagonists 5 1.4 A Diagrammatic Representation of the Cholinergic 7 Synapse 2.1 Subcellular Fractionation Scheme 46 2.2 The Human Brain at Approximately Four Months 66 Gestational Age 2.3 List of Buffers 73 3.1 The Elution Profile of the Radiolabelled 75 Derivatives of lodinated a-Bungarotoxin from CM-52 Cellulose 3.2 The Quantitation of the Concentration of a- 76 Bungarotoxin in the Mono-Iodinated Derivative of Radiolabelled Toxin 3.3 The Production of Anti-(a-Bungarotoxin) Antibodies 83 3.4 The Binding of «“Bungarotoxin to Membranes 85 from Human Foetal Brain as a Function of Protein Concentration 3.5 The Binding of -a-Bungarotoxin to the Crude 86 Detergent Extract as a Function of Protein Concentration 3.6 The Stability of Detergent Solubilized a-Bungaro- 89 toxin Sites in Human Foetal Brain (ii) Page no. 3.7(a) The Relationship Between Crown-Rump Length 92 and the Gestation Period During the Development of the Human Foetus (b) The Relationship Between Brain Weight and 92 Gestational Age During the Development of the Human Foetus 3.8(a) The Concentration of a-Bungarotoxin Binding Sites 94 in Human Brain (b) The Binding of -a-Bungarotoxin to the 94 Particulate Fraction of Foetal Human Brain During Development 3.9 Protein, Human Foetal Brain 104 3.10 Protein, Rat Brain 106 3.11 Choline Acetyltransferase, Human Foetal Brain 109 3.12 Choline Acetyltransferase, Rat Brain 110 3.13 Acetylcholinesterase,