Expression, characterization and mutational studies of human antiquitin.

• Other Authors: Chan, King Lun Michel.
• Format: Others
• Language: English; Chinese
• Published: 2007
• Subjects: Aldehyde dehydrogenase; Mutation (Biology); Aldehyde Dehydrogenase
• Online Access: http://library.cuhk.edu.hk/record=b5893424; http://repository.lib.cuhk.edu.hk/en/item/cuhk-326171

Chan, King Lun Michel. === Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. === Includes bibliographical references (leaves 111-121). === Abstracts in English and Chinese. === THESIS ASSESSMENT COMMITTEE --- p.i === ACKNOWLEDGEMENTS --- p.ii === 摘要 --- p.iv === ABSTRACT --- p.v === LIST OF ABBREVIATIONS --- p.xi === Chapter CHAPTER 1 --- INTRODUCTION === Chapter 1.1 --- Aldehyde Dehydrogenase Superfamily === Chapter 1.1.1 --- Classification and Substrate Specificities of Aldehyde Dehydrogenases --- p.1 === Chapter 1.1.2 --- Multiple Functions of Aldehyde Dehydrogenases and their Roles in Metabolism --- p.4 === Chapter 1.1.3 --- Structural Organization of Aldehyde Dehydrogenases in view of their Catalytic Mechanism --- p.7 === Chapter 1.2 --- Antiquitin === Chapter 1.2.1 --- Discovery and Plant Antiquitins --- p.15 === Chapter 1.2.2 --- Animal Antiquitins --- p.17 === Chapter 1.2.3 --- Human Antiquitin Gene Mutations and Pyridoxine-dependent Seizures --- p.19 === Chapter 1.2.4 --- Previous Findings on Seabream Antiquitin --- p.20 === Chapter 1.3 --- Aims of Study --- p.22 === Chapter CHAPTER 2 --- MATERIALS AND METHODS === Chapter 2.1 --- Materials === Chapter 2.1.1 --- "Subcloning, Expression and Purification of Human Antiquitin" --- p.24 === Chapter 2.1.2 --- Characterization of Human Antiquitin --- p.25 === Chapter 2.1.3 --- "Crystallization of Human Antiquitin, Diffraction Data Collection and Structure Determination" --- p.26 === Chapter 2.1.4 --- Mutational Studies of Human Antiquitin --- p.26 === Chapter 2.2 --- Methods === Chapter 2.2.1 --- "Subcloning, Expression and Purification of Human Antiquitin" === Chapter 2.2.1.1 --- Subcloning of the Full-length Human Antiquitin cDNA --- p.27 === Chapter 2.2.1.2 --- Bacterial Expression of Recombinant Human Antiquitin --- p.29 === Chapter 2.2.1.3 --- Purification of Human Antiquitin --- p.30 === Chapter 2.2.2 --- Characterization of Human Antiquitin === Chapter 2.2.2.1 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.31 === Chapter 2.2.2.2 --- Size-exclusion Chromatography - Multi-angle Light Scattering --- p.32 === Chapter 2.2.2.3 --- Isoelectric Focusing --- p.33 === Chapter 2.2.2.4 --- pH-rate Profile --- p.33 === Chapter 2.2.2.5 --- Stability Studies --- p.34 === Chapter 2.2.2.6 --- Substrate Specificity Study --- p.35 === Chapter 2.2.3 --- "Crystallization of Human Antiquitin, Diffraction Data Collection and Structure Determination" === Chapter 2.2.3.1 --- Crystallization of Human Antiquitin --- p.36 === Chapter 2.2.3.2 --- Diffraction Data Collection and Model Building --- p.37 === Chapter 2.2.4 --- Mutational Studies of Human Antiquitin === Chapter 2.2.4.1 --- Preparation of Mutant Plasmids --- p.39 === Chapter 2.2.4.2 --- "Expression, Purification and Kinetics Studies of Mutants" --- p.39 === Chapter CHAPTER 3 --- RESULTS === Chapter 3.1 --- "Subcloning, Expression and Purification of Human Antiquitin" --- p.43 === Chapter 3.2 --- Characterization of Human Antiquitin --- p.49 === Chapter 3.3 --- "Crystallization of Human Antiquitin, Diffraction Data Collection and Structure Determination" === Chapter 3.3.1 --- "Crystallization, Data Collection and Refinement" --- p.59 === Chapter 3.3.2 --- Human Antiquitin Structure --- p.63 === Chapter 3.4 --- Mutational Studies of Human Antiquitin --- p.75 === Chapter CHAPTER 4 --- DISCUSSION === Chapter 4.1 --- Characterization and Substrate Specificity of Recombinant Human Antiquitin --- p.83 === Chapter 4.2 --- Crystallization and Crystal Structure of Human Antiquitin === Chapter 4.2.1 --- Crystallization --- p.86 === Chapter 4.2.2 --- Overall structure --- p.86 === Chapter 4.2.3 --- Cofactor binding --- p.88 === Chapter 4.2.4 --- Substrate Binding and Catalysis --- p.91 === Chapter 4.3 --- Mutations in Human Antiquitin Gene and their Relationship with Pyridoxine-dependent Seizures --- p.95 === Chapter 4.4 --- Comparison of Antiquitin Gene and Other Aldehyde Dehydrogenases --- p.104 === Chapter CHAPTER 5 --- FUTURE PROSPECTS --- p.106 === LIST OF REFERENCES --- p.111 === APPENDIX --- p.122