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Purification and characterization of glutathione s-transferase from chironomidae larvae (red bloodworm).

by Yuen Wai Keung. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 99-112). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Abbreviations --- p.vi / Table of Contents --- p.viii / Chapter chapter one --- introduction --- p.1 / Chapter 1.1 --- Glutathione S-transferase --- p.2 / Chapter 1.1.1 --- Introduction --- p.2 / Chapter 1.1.2 --- Classification of mammalian GST --- p.2 / Chapter 1.1.3 --- Classification of insect GST --- p.7 / Chapter 1.1.4 --- Substrate specificity --- p.11 / Chapter 1.2 --- The chironomidae --- p.13 / Chapter 1.2.1 --- Biology and life history of chironomidae --- p.13 / Chapter 1.3 --- Chironomidae larvae --- p.16 / Chapter 1.3.1 --- Bloodworm t --- p.6 / Chapter 1.3.2 --- Sources of chironomidae larvae --- p.17 / Chapter 1.4 --- Aim of research --- p.18 / Chapter chapter two --- materials and methods --- p.20 / Chapter 2.1 --- Screening of GST in different subcellular fractions --- p.21 / Chapter 2.1.1 --- Preparation of mitochondria --- p.21 / Chapter 2.1.2 --- Preparation of microsomes --- p.22 / Chapter 2.1.3 --- Preparation of cytosol --- p.22 / Chapter 2.2 --- Assay for GST activity --- p.23 / Chapter 2.2.1 --- Activity Units --- p.23 / Chapter 2.3 --- Protein assay --- p.23 / Chapter 2.4 --- Preparation of glutathione-affinity column --- p.25 / Chapter 2.5 --- Purification of cytosolic GSTs --- p.26 / Chapter 2.5.1 --- Preparation of cytosol --- p.26 / Chapter 2.5.2 --- Chromatography on Sephadex G25 --- p.26 / Chapter 2.5.3 --- Affinity Chromatography --- p.26 / Chapter 2.5.3.1 --- Specific elution of GSTs --- p.26 / Chapter 2.5.3.2 --- Non-specific elution of GSTs --- p.27 / Chapter 2.5.4 --- Fast Protein Liquid Chromatography with Mono Q --- p.27 / Chapter 2.6 --- Determination of molecular mass --- p.29 / Chapter 2.6.1 --- Subunit molecular mass --- p.29 / Chapter 2.6.2 --- Native molecular mass --- p.31 / Chapter 2.7 --- Isoelectric focusing PAGE --- p.31 / Chapter 2.8 --- Enzyme activities and kinetic studies --- p.34 / Chapter 2.8.1 --- Optimum pH --- p.34 / Chapter 2.8.2 --- Heat inactivation assay --- p.34 / Chapter 2.8.3 --- Km and Vmax --- p.34 / Chapter 2.8.4 --- Substrate specificity --- p.35 / Chapter 2.8.5 --- Glutathione peroxidase activity --- p.38 / Chapter 2.9 --- N-terminal amino acid sequence analysis --- p.39 / Chapter 2.9.1 --- Semidry electroblotting --- p.39 / Chapter 2.9.2 --- Staining of proteins on PVDF membrane --- p.40 / Chapter 2.9.3 --- N-terminal amino acid sequence analysis --- p.40 / Chapter 2.9.4 --- On-membrane deblocking of protein --- p.40 / Chapter 2.9.5 --- BLAST search --- p.41 / Chapter chapter three --- results --- p.42 / Chapter 3.1 --- Screening of GST in different subcellular fractions --- p.43 / Chapter 3.2 --- Purification of cytosolic GSTs by chromatography --- p.45 / Chapter 3.2.1 --- Sephadex G25 column --- p.45 / Chapter 3.2.2 --- GSH affinity column --- p.45 / Chapter 3.2.3 --- Mono-Q column --- p.45 / Chapter 3.3 --- Determination of molecular mass --- p.53 / Chapter 3.3.1 --- Subunit molecular mass --- p.53 / Chapter 3.3.2 --- Native molecular mass --- p.53 / Chapter 3.4 --- Isoelectric point determination --- p.53 / Chapter 3.5 --- Enzymes activities and kinetic studies --- p.57 / Chapter 3.5.1 --- Optimum pH --- p.57 / Chapter 3.5.2 --- Thermostability --- p.57 / Chapter 3.5.3 --- Km and Vmax --- p.57 / Chapter 3.5.4 --- Substrate specificity --- p.76 / Chapter 3.5.5 --- Glutathione peroxidase Activity --- p.76 / Chapter 3.6 --- N-terminal amino acid sequence analysis --- p.83 / Chapter chapter four --- discussion --- p.89 / Chapter 4.1 --- GST in different subcellular fractions --- p.90 / Chapter 4.2 --- Purification of cytosolic GST --- p.91 / Chapter 4.3 --- Physical properties --- p.93 / Chapter 4.3.1 --- Subunit molecular mass --- p.93 / Chapter 4.3.2 --- Native molecular mass --- p.93 / Chapter 4.3.3 --- Isoelectric point --- p.95 / Chapter 4.4 --- Kinetic properties --- p.94 / Chapter 4.4.1 --- Optimum pH --- p.94 / Chapter 4.4.2 --- Thermostability --- p.95 / Chapter 4.4.3 --- Km and Vmax --- p.95 / Chapter 4.4.4 --- Substrate specificity --- p.96 / Chapter 4.4.5 --- Glutathione peroxidase activity --- p.96 / Chapter 4.5 --- N-terminal amino acid sequence data --- p.97 / Chapter 4.6 --- Conclusion --- p.98 / references --- p.99

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323224
Date January 2000
ContributorsYuen, Wai Keung., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xi, 112 leaves : ill. ; 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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