A complete initial velocity study of the 6-phosphogluconate dehydrogenase from Candida utilis in both reaction directions suggests a rapid equilibrium random kinetic mechanism with dead-end E:NADP:(ribulose 5-phosphate) and E:NADPH:(6- phosphogluconate) complexes. Initial velocity studies obtained as a function of pH and using NAD as the dinucleotide substrate for the reaction suggest that the 2'-phosphate is critical for productive binding of the dinucleotide substrate. Primary deuterium isotope effects using 3-<i-6-phosphogluconate were obtained for the 6-phosphogluconate dehydrogenase reaction using NADP and various alternative inucleotide substrates.

No description available.

An examination of oxidation products of the pterin cofactor in the formate dehydrogenase of Methanobacterium formicicum revealed that the cofactor is a 6-substituted pterin similar to the molybdopterin of xanthine oxidase. In contrast to the molybdopterin from xanthine oxidase the formate dehydrogenase cofactor was unable to complement the cofactor-deficient nitrate reductase of Neurospora crassa mutant nit-1 and possessed two phosphate groups. Incubation of oxidized formate dehydrogenase with cyanide resulted in an irreversible loss of enzyme activity which could not be restored by treatment with sulfide. Equimolar amounts of thiocyanate were released from cyanide-treated formate dehydrogenase suggesting the loss of one terminal sulfur ligand to molybdenum. These results along with electron paramagnetic resonance spectroscopy of the cyanide-inactivated formate dehydrogenase suggest that the the molybdenum ligands of the enzyme are similar to those of xanthine oxidase. The concentration of molybdenum and the level of formate dehydrogenase activity in extracts of hydrogen-grown Methanobacterium formicicum decreased by at least 10- fold when the organism was grown for several transfers in molybdenum-deficient media or with added tungstate. Immunochemical analysis showed that both subunits of the formate dehydrogenase were produced regardless of the growth condition. However the amount of formate dehydrogenase protein decreased more than 10-fold when the amount of molybdenum in the cell was low. The pterin cofactor was present in the inactive enzyme from tungstategrown cells; however the protein contained less than 0.05 molecules of molybdenum or tungsten per formate dehydrogenase. Messenger RNA specific for fdh gene was detected in high amount in cells grown without added molybdenum and in low amount in cells that contained high amounts of molybdenum. These results suggest that molybdenum is required for the synthesis of a stable formate dehydrogenase and that a molybdenum-dependent repressor may be required for the termination of fdh transcription. / Ph. D.

Chong, King Wai Isaac. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 99-104). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / Lists of Figures --- p.ix / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Overview of Glyceraldehyde 3-phosphate Dehydrogenases --- p.1 / Chapter 1.2 --- Properties And Molecular Structures of GAPDH --- p.3 / Chapter 1.3 --- Action Mechanism of GAPDH --- p.6 / Chapter 1.4 --- Novel Functions of GAPDH Unrelated to Carbohydrate Metabolism --- p.8 / Chapter 1.5 --- Effects of Heavy Metal on Enzyme Activity And Gene Expression of GAPDH --- p.10 / Chapter 1.6 --- Metal Binding Properties And Metal Binding Sites of GAPDH --- p.12 / Chapter 1.7 --- Isolation And Purification of GAPDH from Different Organisms --- p.13 / Chapter 1.8 --- Development of New Purification Method of GAPDH Using Immobilized Metal Affinity Chromatography --- p.15 / Chapter 1.9 --- Study of GAPDH from Chironomidae Larvae --- p.16 / Chapter 1.10 --- Aims of Study --- p.18 / Chapter Chapter Two: --- Methods And Materials --- p.19 / Chapter 2.1 --- Isolation of Native Chironomidae GAPDH --- p.19 / Chapter 2.1.1 --- Chemicals And Reagents --- p.19 / Chapter 2.1.2 --- Reagents --- p.19 / Chapter 2.1.3 --- Preparation of Crude Protein Extract from Chironomidae Larvae --- p.24 / Chapter 2.1.4 --- Immobilized Metal Affinity Chromatography --- p.24 / Chapter 2.1.5 --- Large Scale Preparation of Crude Protein Extract --- p.25 / Chapter 2.1.6 --- Ammonium Sulfate Fractionation --- p.25 / Chapter 2.1.7 --- Copper Affinity Column Chromatography --- p.26 / Chapter 2.1.8 --- Dye Affinity Column Chromatography --- p.26 / Chapter 2.2 --- Identification of Chironomidae GAPDH --- p.27 / Chapter 2.2.1 --- Chemicals And Reagents --- p.27 / Chapter 2.2.2 --- Reagents --- p.28 / Chapter 2.2.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.32 / Chapter 2.2.4 --- Non-Denaturing Polyacrylamide Gel Electrophoresis --- p.33 / Chapter 2.2.5 --- Protein Extraction from Coosmassie Blue Stained Polyacrylamide Gel --- p.33 / Chapter 2.2.6 --- N-terminal Amino Acid Analysis --- p.33 / Chapter 2.2.7 --- Sequence Analysis --- p.34 / Chapter 2.3 --- Kinetic Analysis of Chironomidae GAPDH --- p.34 / Chapter 2.3.1 --- Chemcials And Reagents --- p.34 / Chapter 2.3.2 --- Reagents --- p.34 / Chapter 2.3.3 --- Determination of Enzyme Concentration And GAPDH Activity --- p.35 / Chapter 2.4 --- Molecular Cloning of Chironomidae GAPDH --- p.36 / Chapter 2.4.1 --- Chemicals And Reagents --- p.36 / Chapter 2.4.2 --- Reagents --- p.37 / Chapter 2.4.3 --- RNA Extraction from Chironomidae Larvae --- p.41 / Chapter 2.4.4 --- DNase I Removal of Genomic DNA Contamination --- p.42 / Chapter 2.4.5 --- RNA Concentration Determination And RNA Agarose Electrophoresis --- p.42 / Chapter 2.4.6 --- First Strand cDNA Synthesis --- p.43 / Chapter 2.4.7 --- pRSet A B C Vectors --- p.43 / Chapter 2.4.8 --- Cloning Primer Design --- p.45 / Chapter 2.4.9 --- Polymerase Chain Reaction --- p.45 / Chapter 2.4.10 --- DNA Agarose Electrophoresis --- p.46 / Chapter 2.4.11 --- Restriction Enzyme Digestion of Insert And Plasmid --- p.46 / Chapter 2.4.12 --- Ligation of Plasmid And Insert DNA --- p.46 / Chapter 2.4.13 --- Preparation of Chemically Competent E. coli --- p.47 / Chapter 2.4.14 --- Transformation of Plasmid by Heat Shock --- p.47 / Chapter 2.4.15 --- Colony PCR --- p.48 / Chapter 2.5 --- Recombinant Protein Expression And Purification --- p.48 / Chapter 2.5.1 --- Chemicals And Reagents --- p.48 / Chapter 2.5.2 --- Reagents --- p.49 / Chapter 2.5.3 --- Protein expression by IPTG --- p.51 / Chapter 2.5.4 --- Protein purification by Nickel Affinity Column Chromatography --- p.52 / Chapter 2.5.5 --- EnterokinaseMax ´ёØ Removal of Polyhistidine Fusion Tag --- p.52 / Chapter 2.5.6 --- Western Blotting of Protein --- p.53 / Chapter Chapter Three: --- Results --- p.54 / Chapter 3.1 --- Two Affinity Chromatography Methods for GAPDH Purification --- p.54 / Chapter 3.2 --- Isolation And Purification of Native Chironomidae GAPDH --- p.54 / Chapter 3.3 --- Identification of Chironomidae GAPDH --- p.62 / Chapter 3.3.1 --- N-terminal amino acid analysis --- p.62 / Chapter 3.3.2 --- Sequence Analysis --- p.62 / Chapter 3.4 --- Molecular Cloning of Chironomidae GAPDH --- p.63 / Chapter 3.5 --- Isolation And Purification of recombinant Chironomidae GAPDH --- p.70 / Chapter 3.6 --- Protein Gel Electrophoresis Analysis of GAPDHs --- p.74 / Chapter 3.7 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.76 / Chapter 3.7.1 --- Heavy Metal Effect --- p.76 / Chapter 3.7.2 --- pH Effect --- p.76 / Chapter 3.7.3 --- Temperature --- p.77 / Chapter 3.8 --- Kinetic Analysis of GAPDHs --- p.84 / Chapter Chapter Four: --- Discussion --- p.89 / Chapter 4.1 --- New Method for The Isolation and Purification of Chironomidae GAPDH --- p.89 / Chapter 4.2 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.91 / Chapter 4.3 --- Kinetic Analysis of GAPDHs --- p.91 / Chapter 4.4 --- Zinc Activation of Chironomidae GAPDH --- p.92 / Chapter 4.5 --- Future Study --- p.93 / Chapter 4.5.1 --- Sequence Analysis Using Prediction Programmes --- p.94 / Chapter 4.5.2 --- Protein Crystallization --- p.95 / Chapter 4.5.3 --- Site-Directed Mutagenesis --- p.95 / Chapter 4.5.4 --- Biacore Surface Plasmon Resonance --- p.95 / Chapter Chapter Five: --- Conclusion --- p.98 / Chapter Chapter Six: --- References --- p.99

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Call number: LD2668 .T4 1985 J54 / Master of Science

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Diabetic retinopathy (DR) was characterized by numerous hyperglycemia-dependent cellular and pathological changes in the retina, including retinal ischemia/reperfusion (I/R) injury. To determine the role of the 2nd enzyme of polyol pathway in relation with the pathogenesis in ischemic retinopathy, SDH deficient mice, C57BL/LiA, that lacked SDH activity, was used to study the pathogenesis of diabetic retinopathy, which also included I/R injury. Wild type and SDH-deficient mice were subjected to I/R injury by transiently occluding middle cerebral artery for two hours and twenty-two hour of reperfusion. The rationale of this study was to investigate the effect by blocking the conversion of sorbitol to fructose by SDH null mutation (SDH -/-), leading to accumulation of sorbitol level and reduction of oxidative stress, as demonstrated by the polyol pathway. Results: After induction with transient MCAO, there was increase in the thickness of OLM to ILM ipsilateral SDH+/+ compared with contralateral SDH+/+ (from 84 +/- 1 to 96 +/- 2 μm) while that of ipsilateral SDH-/- compared with contralateral SDH -/- (from 77 +/- 2 to 90 +/- 2 μm) suggested that there was edema after ischemic reperfusion injury. The result showed that there was increased cellular edema in ipsilateral retina of both SDH +/+ and SDH -/- retina after transient MCAO. The level of immunoreactivity against Aquaporin-4 and nitrotyrosine in studying the presence of oxidative stress; glutamine synthetase and glutamate in studying the toxicity of astrocyte glutamate; sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) in studying the regulation Ca2+ homeostasis was determined using immunohistochemistry. For all the antibodies, there was similar immunoreactivity level between the contralateral side of both SDH+/+ and SDH -/- mice. For the SDH+/+ group, there was increase in signal in the ipsilateral retina in comparison with the contralateral one. On the other hand, for the SDH-/- group, similar result was observed. There was increase in signal and it was found more in the ipsilateral retina in comparison with the contralateral retina. Finally, in the ipsilateral retina of both SDH +/+ and SDH -/- mice, increased immunoreactivity was found in both but their difference was not statistically significant. This concluded that SDH deletion and subsequent accumulation of sorbitol metabolites did not contribute significantly in the role of pathogenesis of ischemic retinopathy especially in mice after I/R injury. / published_or_final_version / Anatomy / Master / Master of Medical Sciences