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Molecular studies of snakehead fish growth hormone receptor.

by Simon Chan Siu Hoi. / Spine title varies. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 130-148). / Acknowledgments --- p.i / Table of Contents --- p.ii / List of Abbreviations --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xvi / Page / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Growth Hormone --- p.1 / Chapter 1.2 --- Growth Hormone Receptor --- p.3 / Chapter 1.2.1 --- Cytokine/Hematopoietin Receptor Superfamily --- p.3 / Chapter 1.2.2 --- Tissue Distribution of GHR --- p.6 / Chapter 1.2.3 --- Biosynthesis and Degradation of GHR --- p.7 / Chapter 1.2.4 --- Regulation of GHR Level --- p.8 / Chapter 1.2.5 --- The GHR Protein --- p.10 / Chapter 1.2.6 --- The GHR Gene --- p.15 / Chapter 1.2.7 --- GHR Dimerization --- p.16 / Chapter 1.2.8 --- Mechanism of Signaling by GHR --- p.19 / Chapter 1.2.9 --- GH Binding Protein --- p.21 / Chapter 1.2.10 --- GHR Related Dwarfism --- p.23 / Chapter 1.3 --- Objectives of the Present Investigation --- p.25 / Chapter Chapter 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Fish Growth Hormone Radioactive Labeling --- p.27 / Chapter 2.1.1 --- Preparation of Iodogen-Coated Tubes --- p.27 / Chapter 2.1.2 --- Packing of the Sephadex G-75 Column --- p.28 / Chapter 2.1.3 --- Iodination of brGH and Purification of the Iodinated brGH --- p.28 / Chapter 2.1.4 --- Determination of the Specific Radioactivity and Percentage of 125I Incorporation --- p.29 / Chapter 2.1.5 --- Reagents and Buffers Used --- p.30 / Chapter 2.2 --- Integrity of 125I-brGH --- p.30 / Chapter 2.2.1 --- HPLC of brGH --- p.31 / Chapter 2.2.2 --- HPLC of 125I-brGH after Iodination --- p.31 / Chapter 2.2.3 --- HPLC of 125I-brGH after Receptor Binding --- p.31 / Chapter 2.3 --- Preparation of Membranes from Fish Tissues --- p.32 / Chapter 2.3.1 --- Preparation of Snakehead Fish Liver Membranes --- p.32 / Chapter 2.3.2 --- Reagents and Buffers Used --- p.33 / Chapter 2.4 --- Protein Determination of Membrane Preparations --- p.34 / Chapter 2.4.1 --- The BCA Protein Reaction Scheme --- p.34 / Chapter 2.4.2 --- BCA Protein Determination Protocol --- p.34 / Chapter 2.5 --- Receptor Binding Studies --- p.35 / Chapter 2.5.1 --- Association and Dissociation Studies --- p.36 / Chapter 2.5.2 --- pH Dependence Study --- p.36 / Chapter 2.5.3 --- Membrane Protein Dependence Study --- p.37 / Chapter 2.5.4 --- Ca2+ Dependence Study --- p.37 / Chapter 2.5.5 --- Tissue Distribution Study --- p.37 / Chapter 2.5.6 --- Displacement and Specificity Studies --- p.38 / Chapter 2.5.7 --- Dithiothreitol (DTT) Dependence Study --- p.39 / Chapter 2.5.8 --- p-Chloromercuribenzene Sulfonate (PCMBS) Pretreatment: Dose Dependence Study --- p.39 / Chapter 2.5.9 --- Scatchard Analysis of the PCMBS Pretreated and Control Snakehead Fish Liver Membranes --- p.40 / Chapter 2.5.10 --- Reversibility of the PCMBS Effect --- p.40 / Chapter 2.5.11 --- Reagents and Buffers Used --- p.41 / Chapter 2.6 --- Crosslinking Studies --- p.41 / Chapter 2.6.1 --- Crosslinking Performed on Snakehead Fish Liver Membranes --- p.41 / Chapter 2.6.2 --- Crosslinking Performed on Solubilized Snakehead Fish Liver Membranes --- p.42 / Chapter 2.6.3 --- Gel Filtration Chromatography of the Crosslinked Comp)lexes --- p.43 / Chapter 2.6.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of the Crosslinked Complexes --- p.43 / Chapter 2.6.5 --- Reagents and Buffers Used --- p.45 / Chapter 2.7 --- Western Blot Analysis of Snakehead Fish Liver GHR --- p.46 / Chapter 2.7.1 --- SDS-PAGE of Snakehead Fish Liver Membranes --- p.46 / Chapter 2.7.2 --- Transfer of Proteins onto Polyvinylidene Fluoride (PVDF) Membrane --- p.46 / Chapter 2.7.3 --- Antibody Development of PVDF Membrane --- p.47 / Chapter 2.7.4 --- Reagents and Buffers Used --- p.48 / Chapter 2.8 --- Solubilization of Snakehead Fish Liver Membranes and Solubilized Receptor Binding Studies --- p.48 / Chapter 2.8.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.49 / Chapter 2.8.2 --- Solubilized Receptor Binding Assay --- p.49 / Chapter 2.8.3 --- "Solubilization of Snakehead Fish Liver Membranes: Detergent Concentration, pH, Temperature and Time Dependence" --- p.50 / Chapter 2.8.4 --- Solubilized Receptor Binding Study: Interference of Detergent --- p.50 / Chapter 2.8.5 --- Reagents and Buffers Used --- p.51 / Chapter 2.9 --- Purification of Snakehead Fish Liver GHR by Affinity Chromatography --- p.51 / Chapter 2.9.1 --- Affinity Column Preparation --- p.52 / Chapter 2.9.2 --- Snakehead Fish Liver GHR Purification --- p.52 / Chapter 2.9.3 --- Reagents and Buffers Used --- p.53 / Chapter Chapter 3 --- Results: fGH Labeling and Integrity Determination --- p.54 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Experimental Results --- p.55 / Chapter 3.2.1 --- Iodination of fGH --- p.55 / Chapter 3.2.2 --- Integrity of 125I-fGH --- p.55 / Chapter 3.3 --- Discussion --- p.61 / Chapter Chapter 4 --- Results: Membrane Receptor Binding Studies --- p.62 / Chapter 4.1 --- Introduction --- p.62 / Chapter 4.2 --- Experimental Results --- p.63 / Chapter 4.2.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.64 / Chapter 4.2.1.1 --- Association and Dissociation Studies --- p.64 / Chapter 4.2.1.2 --- pH Dependence Study --- p.67 / Chapter 4.2.1.3 --- Membrane Protein Dependence Study --- p.70 / Chapter 4.2.1.4 --- Ca2+ Dependence Study --- p.73 / Chapter 4.2.2 --- Localization and Specificity of Snakehead Fish GHR --- p.76 / Chapter 4.2.2.1 --- Tissue Distribution Study --- p.76 / Chapter 4.2.2.2 --- Displacement and Specificity Studies --- p.78 / Chapter 4.2.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.81 / Chapter 4.2.3.1 --- Effect of DTT: Concentration Dependence Study --- p.81 / Chapter 4.2.3.2 --- Effect of PCMBS: Concentration Dependence Study --- p.84 / Chapter 4.2.3.3 --- Scatchard Analysis of Control and PCMBS- pretreated Membranes --- p.86 / Chapter 4.2.3.4 --- Reversibility of the PCMBS Effect --- p.88 / Chapter 4.3 --- Discussion --- p.90 / Chapter 4.3.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.90 / Chapter 4.3.2 --- Localization and Specificity of Snakehead Fish GHR --- p.93 / Chapter 4.3.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.96 / Chapter Chapter 5 --- Results: Crosslinking and Western Blot Analysis --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.1.1 --- Crosslinking Studies --- p.101 / Chapter 5.1.2 --- Western Blot Analysis --- p.103 / Chapter 5.2 --- Experimental Results --- p.104 / Chapter 5.2.1 --- Crosslinking Studies --- p.104 / Chapter 5.2.2 --- Western Blot Analysis --- p.105 / Chapter 5.3 --- Discussion --- p.112 / Chapter Chapter 6 --- Results: Affinity Purification of Snakehead Fish Liver GHR --- p.115 / Chapter 6.1 --- Introduction --- p.115 / Chapter 6.1.1 --- Membrane Solubilization and Solubilized GHR Binding Studies --- p.115 / Chapter 6.1.2 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.116 / Chapter 6.2 --- Exp erimental Results --- p.117 / Chapter 6.2.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.117 / Chapter 6.2.2 --- Interference of Detergents in the Solubilized Receptor Binding Assay --- p.118 / Chapter 6.2.3 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.120 / Chapter 6.3 --- Discussion --- p.122 / Chapter Chapter 7 --- General Discussion --- p.125 / References --- p.130

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321889
Date January 1997
ContributorsChan, Simon Siu Hoi., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvi, 148 leaves : ill. (some col. some mounted) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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