Very little is known about the effect of hydrofluoric acid and of the fluoride ion on enzyme systems. The purpose of this work was to determine the effect of hydrofluoric acid and of the fluoride ion on the enzyme, yeast alcohol dehydrogenase and to distinguish between the effect of the fluoride ion and of hydrofluoric acid. The rate of the enzyme reaction was followed spectrophotometrically at 340 mμ on the Cary 14 Model spectrophotometer according to the method of Racker. The data taken from the instrument recordings were plotted on two types of graphs, the Lineweaver-Burk plot and the Hanes plot. Conclusions were drawn from the calculations made on these plots. Inhibition studies were run using KCI, NaCl, KF and NaF varying in concentration from 0.001 to 0.12 M at two different pH levels. For the fluoride salts, this gave a concentration of HF which varied from 8.94 x 10ˉ⁸ to 1.07 x 10ˉ⁵ M at pH 7.5 and 8.94 x 10ˉ̄⁹ to 1.07 x 10˜⁶ M at pH 8.5 The fluoride salts showed no greater inhibition than the chloride salts at either pH. Since there is no difference in inhibition between the two types of salts, the inhibition cannot be attributed to the presence of hydrofluoric acid. If the inhibition had been due to hydrofluoric acid, we would have observed a greater inhibition with the fluoride salts than with the chloride salts since hydrochloric acid is 100% ionized.

Dihydroorotate (DRO) dehydrogenase catalyzes the oxidation of DHO to orotate in the pyrimidine biosynthetic pathway. This enzyme was originally isolated from a bacterium, Zymobacterium oroticum, which would ferment orotate as a sole source of energy. This adaptive catabolic enzyme, which catalyzes the reduction of orotate to DRO in an efficient pyridine nucleotide-linked reaction, has been extensively studied by several workers. Until recently, no study has been carried out on the enzyme which catalyzes the reaction in the biosynthetic direction. Preliminary studies have shown that the biosynthetic enzyme in Esherichia coli and a pseudomonad is not capable of reducing orotate to DRO by a pyridine nucleotide-linked reaction. These results suggested that there may be significant differences between the catabolic and biosynthetic enzymes. In the present study biosynthetic DHO dehydrogenase from Lacto-bacillus bulgaricus was investigated on the basis of physical and kinetic properties in order to compare the enzyme with the extensively studied catabolic enzyme. The stoichiometry exhibited by the DHO oxidase activity of the biosynthetic enzyme and the absorption spectrum suggest that biosynthetic DHO dehydrogenase is a flavoprotein. Thin layer chromatography of the flavins extracted from the enzyme and reactivation of apoenzymes specific for flavin mononucleotide or flavin adenine dinucleotide have shown that the enzyme contains flavin mononucleotide. The demonstration of enzyme-catalyzed sulfite autoxidation suggested that iron is present and is involved in electron transport. Inhibitor studies have shown that the enzyme contains sulfhydryl groups and the inactivation of such groups halts internal electron transport early in the sequence. Kinetic studies were carried out including the determination of the Km for dihydroorotate, Ki for orotate, and the pH optimum. The kinetic behavior of the enzyme in the presence of various inhibitors suggest that the essential sulfhydryl groups reside at or near the active site. Ammonium sulfate was found to enhance the activity of the enzyme. Evidence presented suggested that this phenomenon is probably an unspecific anion effect in which the rate constant for the breakdown of the enzyme substrate complex is directly affected. A possible scheme of the internal electron transport of biosynthetic DHO dehydrogenase was presented, using the data from this thesis and additional evidence from studies carried out by other workers on similar enzymes. A summary of the physical and kinetic properties of biosynthetic and catabolic DHO dehydrogenase was presented and a detailed comparison between the two enzymes made.

none

Hannover, Universiẗat, Diss., 2000.

Lactate dehydrogenases from an abyssal fish, a dogfish, a tidepool sculpin, and a mammal have been found to differ in their ability to bind substrate analog and coenzyme at varying temperatures and pressures. Affinities for a substrate analog are quite similar for each lactate dehydrogenase at their respective biological temperatures, suggesting temperature-dependent modification of enzyme-substrate binding for optimal function. Binding of coenzyme by the three ectothermic enzymes is less affected by changes in temperature than is coenzyme binding by the mammalian enzyme, and coenzyme binding by the abyssal fish enzyme is considerably less sensitive to high hydrostatic pressure than it is in the case of the other three lactate dehydrogenases. The total free energy change involved in binding coenzyme and substrate analog is only slightly higher for the endothermic than for the three ectothermic enzymes, but the enthalpic and entropic contributions are quite different. The ectotherms appear to have minimized the enthalpic contribution and hence minimized temperature effects on binding. The relationship between enthalpy and entropy for each of the binding interactions studied is a straight line of slope within the limits found by other workers for water-solute interactions and/or weak bond formation and is presumed to be a result of the conformational changes accompanying ligand binding. The contributions to binding of the AMP and nicotinamide subsites of the coenzyme binding site give a good estimate of many of the binding interactions of the coenzyme as a whole, and appear to compensate one another to maintain low AH and AS values for coenzyme binding to the ectothermic enzymes. This same type of compensation in volume change can be seen between the substrate and coenzyme binding sites for the abyssal fish lactate dehydrogenase, resulting in a net volume change very close to zero. The observed temperature and pressure effects on binding cannot be explained solely in terms of the types of weak bonds involved, and known homologies between dogfish and pig LDH make major differences between the active sites unlikely. Conformational changes occurring simultaneously with binding may be of considerable importance in modifying the observed responses to both temperature and pressure. / Science, Faculty of / Zoology, Department of / Graduate

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Wong, Chun Pong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 130-146). / Abstract also in Chinese. / Thesis Assessment Committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / 摘要 --- p.iv / Abstract --- p.vi / List of Abbreviations --- p.viii / List of Figures --- p.xii / List of Tables --- p.xiv / Content --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Classification of aldehyde dehydrogenase --- p.1 / Chapter 1.2 --- Structure and catalytic mechanism of aldehyde dehydrogenase --- p.4 / Chapter 1.3 --- Multiple functions of aldehyde dehydrogenase --- p.11 / Chapter 1.4 --- Background of antiquitin --- p.13 / Chapter 1.5 --- Aim of study --- p.24 / Chapter Chapter 2 --- Structural Analysis of Human Antiquitin --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.30 / Chapter 2.2.1 --- Subcloning and expression of human antiquitin and its mutants --- p.30 / Chapter 2.2.2 --- Purification of human antiquitin and its mutants --- p.31 / Chapter 2.2.3 --- Kinetic properties of human antiquitin and its mutants --- p.32 / Chapter 2.2.4 --- Inhibitor studies of human antiquitin --- p.33 / Chapter 2.2.5 --- X-ray crystallography of human antiquitin ternary complex --- p.34 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- "Subcloning, expression and purification of human antiquitin and its mutants" --- p.36 / Chapter 2.3.2 --- Kinetic properties of human antiquitin and its mutants --- p.41 / Chapter 2.3.3 --- Inhibitor studies of human antiquitin --- p.44 / Chapter 2.3.4 --- X-ray crystallography of human antiquitin ternary complex --- p.47 / Chapter 2.4 --- Discussion --- p.56 / Chapter 2.4.1 --- Substrate specificity of recombinant human antiquitin --- p.56 / Chapter 2.4.2 --- Pyridoxine-dependent seizures and mutations in human antiquitin gene --- p.63 / Chapter 2.4.3 --- X-ray crystallography of human antiquitin ternary complex --- p.76 / Chapter Chapter 3 --- Functional Analysis of Human Antiquitin --- p.79 / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Materials and Methods --- p.83 / Chapter 3.2.1 --- Cell culture --- p.83 / Chapter 3.2.2 --- Transfection of HEK293 cells with siRNA --- p.83 / Chapter 3.2.3 --- Total protein extraction --- p.84 / Chapter 3.2.4 --- Total RNA extraction --- p.85 / Chapter 3.2.5 --- Real-time PCR assay --- p.86 / Chapter 3.2.6 --- Stress responsiveness of transfected HEK293 cells --- p.87 / Chapter 3.2.7 --- Cell growth analysis of transfected HEK293 cells --- p.87 / Chapter 3.2.8 --- Cell cycle profile analysis of transfected HEK293 cells --- p.88 / Chapter 3.2.9 --- Programmed cell death analysis of transfected HEK293 cells --- p.89 / Chapter 3.2.10 --- Confocal immunofluorescence microscopic analysis of transfected HEK293 cells --- p.89 / Chapter 3.2.11 --- Subcellular fractionation of transfected HEK293 cells --- p.90 / Chapter 3.2.12 --- Western blot analysis of transfected HEK293 cells --- p.90 / Chapter 3.3 --- Results --- p.93 / Chapter 3.3.1 --- Condition optimization for siRNA transfection in HEK293 cells --- p.93 / Chapter 3.3.2 --- Knock down of human antiquitin at protein and mRNA levels in HEK293 cells --- p.93 / Chapter 3.3.3 --- Stress responsiveness of transfected HEK293 cells --- p.99 / Chapter 3.3.4 --- Cell growth in transfected HEK293 cells --- p.102 / Chapter 3.3.5 --- Cell cycle profile analysis of transfected HEK293 cells --- p.107 / Chapter 3.3.6 --- Western blot analysis of cell cycle regulatory proteins of transfected HEK293 cells --- p.107 / Chapter 3.3.7 --- Programmed cell death analysis of transfected HEK293 cells --- p.111 / Chapter 3.3.8 --- Confocal immunofluorescence microscopic analysis of transfected HEK293 cells --- p.113 / Chapter 3.3.9 --- Subcellular fractionation of transfected HEK293 cells --- p.116 / Chapter 3.4 --- Discussion --- p.118 / Chapter 3.4.1 --- Lack of response of human antiquitin towards hyperosmotic stress --- p.118 / Chapter 3.4.2 --- Involvement of human antiquitin in cell growth --- p.119 / Chapter 3.4.3 --- Subcellular localization of human antiquitin --- p.124 / Chapter 3.4.4 --- Study of physiological function of human antiquitin using siRNA technique --- p.125 / Chapter Chapter 4 --- Future Prospects --- p.128 / References --- p.130

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

Thesis (Doctoral)--Ludwig-Maximilians-Universität zu München, 1935.