Acute effects of repeated sprinting upon serum creatine kinase (CK), lactic dehydrogenase (LDH), and isozymal activities were studied in five collegiate runners (R_s) and six non-runners (NR_s ). After an intermittent running treadmill test, blood sampling showed three-fold mean increases in CK with no change in LDH in both groups; group differences were insignificant (p>.05). Results suggest (1) intense anaerobic exercise produces moderate enzyme elevations; (2) relatively equivalent exercise intensities are critical to enzyme responses in exercising individuals of varying fitness levels; and (3) exercise-induced enzyme release may be consequential to muscle cell membrane permeability changes from decreased intracellular high-energy phosphates.

Cytoplasmic 15-hydroxyprostaglandin dehydrogenase from swine kidney was purified to specific activity of 1.2 U per mg protein, by chromatographic techniques. Native molecular weight of enzyme was estimated at 45,000. Enzyme was inhibited by sulfhydryls, diuretics, and various fatty acids. Substrate studies indicated NAD+ specificity and ability to catabolize prostaglandins, except prostaglandin B and thromboxane B. Initial velocity studies gave intersecting plots conforming to a sequential mechanism. 15-keto-prostaglandin exhibited linear noncompetitive production inhibition with respect to either prostaglandin or NAD+; NAD yielded linear competitive production inhibition with respect to NADH. Results, and those of dead-end inhibition and alternated substrate studies, are consistent with an ordered Bi-Bi mechanism: NAD+ is added first, then prostaglandin; then 15-keto-rostaglandin is released, then NADH.

by Ng, Shuet Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 118-137. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / CONTENTS --- p.iv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter I. --- The History of Gossypol --- p..4 / Chapter II. --- The Chemistry of Gossypol --- p..7 / Chapter A. --- Enantiomers of Gossypol --- p.10 / Chapter B. --- Analysis of Gossypol --- p.11 / Chapter III. --- Metabolism and Distribution of Gossypol in Body --- p.12 / Chapter IV. --- The Effect of Gossypol --- p.14 / Chapter A. --- Effect of Gossypol on Different Animal Species --- p.14 / Chapter B. --- Effect of Gossypol on Male Reproductive Organs --- p.16 / Chapter 1. --- Testis and Epididymis --- p.16 / Chapter 2. --- Spermatozoa and Spermatogenic Cells --- p.18 / Chapter C. --- Effect of Gossypol on Reproductive Hormones --- p.20 / Chapter D. --- Effect of Gossypol on Enzymes --- p.22 / Chapter 1. --- Enzymes in Capacitation --- p.23 / Chapter 2. --- The Metabolic Enzymes --- p.24 / Chapter a. --- On Glycolysis --- p.24 / Chapter b. --- On TCA Cycle --- p.26 / Chapter c. --- On Oxidative Phosphorylation --- p.26 / Chapter 3. --- Adenyl Cyclase --- p.26 / Chapter 4. --- Na+/K+-ATPase --- p.28 / Chapter E. --- Other Effects of Gossypol --- p.29 / Chapter V. --- Toxicity of Gossypol --- p.30 / Chapter A. --- General Toxic Effect --- p.30 / Chapter B. --- The Mutagenicity --- p.32 / Chapter VI. --- Clinical Trials of Gossypol --- p.33 / Chapter VII. --- The Purpose of This Study --- p.35 / Chapter A. --- Gossypol and LDH-X --- p.35 / Chapter B. --- Effect of Gossypol on Antioxidant Defense Systems / Chapter C. --- The Influence of Gossypol on Zinc Metabolism --- p.37 / Chapter CHAPTER 2: --- EFFECT OF GOSSYPOL ON LDH-X OF COCK AND DRAKE / Chapter I. --- Introduction --- p.39 / Chapter A. --- General Description of Lactate Dehydrogenase --- p.39 / Chapter B. --- Discovery of LDH-X --- p.40 / Chapter C. --- Properties of LDH-X --- p.41 / Chapter D. --- Role of LDH-X in the Spermatozoa --- p.42 / Chapter E. --- Gossypol and LDH-X --- p.43 / Chapter II. --- Materials and Methods --- p.44 / Chapter A. --- Reagents --- p.45 / Chapter B. --- Methods --- p.45 / Chapter 1. --- The Effect of Gossypol on the Spermatozoa of Cock and Drake --- p.45 / Chapter 2. --- "Measurement of LDH-X Activities in Cock, Drake, Mouse and Rat" --- p.46 / Chapter a. --- Preparation of Samples --- p.46 / Chapter b. --- Enzyme Assay --- p.47 / Chapter III. --- Results / Chapter A. --- Effect of Gossypol on the Spermatozoa of Cock and Drake --- p.48 / Chapter 1. --- Cock --- p.48 / Chapter 2. --- Drake --- p.49 / Chapter B. --- "The LDH-X Activities of Cock, Drake, Rat and Mouse" --- p.51 / Chapter IV. --- Discussion --- p.54 / Chapter CHAPTER 3: --- "THE EFFECT OF GOSSYPOL ON THE METABOLISM OF ZINC IN THE TESTIS, HAIR AND RETINA OF HAMSTER AND MOUSE" / Chapter I. --- Introduction --- p.57 / Chapter A. --- The Physiological Role of Zinc in Animals --- p.57 / Chapter B. --- The Relationship Between Gossypol and Divalentions --- p.58 / Chapter II. --- Materials and Methods --- p.60 / Chapter A. --- Reagents --- p.60 / Chapter B. --- Treatment of Animals for the Zinc Content Study / Chapter 1. --- Detection of Zinc Content in Hamster Hair --- p.61 / Chapter 2. --- "Detection of Zinc Distribution in the Hair, Testis, and Retina in Hamster and Mouse" --- p.62 / Chapter C. --- Determination of Zinc and Iron --- p.63 / Chapter III. --- Results --- p.63 / Chapter A. --- Amount of Zinc in Hamster's Hair --- p.63 / Chapter B. --- "The Distribution of Zinc Content in Hair, Testis and Retina of Hamster and Mouse" --- p.69 / Chapter IV. --- Discussion --- p.71 / Chapter CHAPTER 4: --- EFFECT OF GOSSYPOL ON THE ANTIOXIDANT DEFENSE SYSTEM IN THE TESTIS OF HAMSTER / Chapter I. --- Introduction --- p.73 / Chapter A. --- Oxygen Radicals in Living Cells --- p.75 / Chapter B. --- Oxygen Damage of Spermatozoa --- p.76 / Chapter C. --- Antioxidant Defense System --- p.77 / Chapter 1. --- Enzymatic Antioxidants --- p.78 / Chapter 2. --- Non-Enzymatic Antioxidants --- p.78 / Chapter II. --- Materials and Methods --- p.81 / Chapter A. --- Reagents --- p.81 / Chapter B. --- Experimental Animals --- p.82 / Chapter C. --- The Effect of Scavengers on the Antifertility of Gossypol --- p.83 / Chapter 1. --- Vitamin C --- p.83 / Chapter 2. --- Selenium --- p.84 / Chapter 3. --- Vitamin E --- p.85 / Chapter D. --- Chemical Interaction of Gossypol with Vitamin C and Sodium Selenite --- p.85 / Chapter E. --- Malonaldehyde Assay --- p.85 / Chapter F. --- The Effect of Gossypol on the Antioxidant Defense Enzymes --- p.86 / Chapter 1. --- Drug Treatment --- p.86 / Chapter 2. --- Preparation of Samples for Enzyme Determination --- p.87 / Chapter 3. --- Enzyme Assays --- p.87 / Chapter a. --- Se-GSH-Px --- p.88 / Chapter b. --- Glutathione-S-Transf erase --- p.88 / Chapter c. --- Catalase --- p.89 / Chapter d. --- Superoxide Dismutase --- p.90 / Chapter III. --- Results --- p.90 / Chapter A. --- The Effect of Scavengers on the Antifertility of Gossypol --- p.90 / Chapter B. --- Chemical Interaction of Gossypol with Vitamin C and sodium selenite --- p.91 / Chapter C. --- The MDA Concentration of the Testis of Hamster after Gossypol and Scavengers Injection --- p.101 / Chapter D. --- The Antioxidant Defense Enzymes --- p.101 / Chapter IV. --- Discussion --- p.109 / Chapter CHAPTER 5: --- CONCLUSION AND GENERAL DISCUSSION --- p.113 / REFERENCES --- p.118 / Chapter APPENDIX I --- THE INFLUENCE OF DIETARY LARD ON THE ANT I FERTILITY EFFECT OF GOSSYPOL --- p.138 / Chapter APPENDIX II --- PROTOCOL FOR GOSSYPOL DETERMINATION --- p.158

This is an investigation into alcohol dehydrogenases (ADHs) from Geobacillus thermoglucosidasius. Eighteen ADHs have been studied, with seven taken for closer inspection. Characterisation was carried out to determine the industrial significance of these enzymes, starting with the substrate scope of the ADHs. The key results obtained are as follows: ADH A is the alcohol dehydrogenase domain of the bifunctional ADHE enzyme (Extance, 2012; Extance et al., 2013). It has been determined that the substrate scope, whilst restricted to linear aliphatic aldehydes, extends at least to dodecanal. Also, with a specificity constant of 167 mM-1 min-1 it appears that ADH A could prefer butanal to shorter-chain aldehydes such as ethanal and propanal with specificity constants of 38 mM-1 min-1 and 35 mM-1 min-1, respectively. Thus ADH A may have a preference for longer aldehydes than previously believed due to its native role in the production of ethanol from acetyl-coA. ADH B was previously investigated for its potential role in the production of butanol. Here it was confirmed as an NADH-dependent ADH, with a substrate scope limited to five carbon length substrates and smaller, with residual activity with C6 substrates. ADH B demonstrated activity with ethyl 4-chloroacetoacetate, an intermediate in the production of statins. Further, an estimated half-life whilst stored at 4°C of 770 days; retention of 86% activity with 10vol% ethyl acetate and 92% activity with 10vol% acetonitrile; and a specific activity of 27 U mg-1 with 3M 2-butanone are all indications that ADH B is a potentially useful enzyme for industry. The last enzyme to be previously investigated was ADH C, which in this work was confirmed to be an acetoin reductase with a very small substrate scope exclusively based around the acetoin motif, and therefore no further work was conducted. ADH D and ADH F both have broad substrate scopes including the industrially-relevant substrates, 5-norbornene-2-carboxaldehyde, 1-phenyl-1,2-propanedione, ethyl 4-chloroacetoacetate and ethyl-2-oxo-4-phenylbutyrate. ADH D is an NADPH-dependent enzyme whereas ADH F can utilise both NADH and NADPH. Both enzymes are annotated as aldo-keto reductases, which is further indicated by multiple sequence alignment with the most similar available protein sequences and crystal structures. Thus, these two enzymes are the first aldo-keto reductases to be examined from moderate thermophiles, and are tentatively assigned in the AKR family as AKR6D1 and AKR5G4 respectively. ADH D has a very low KM (≤0.1 μM) with NADPH, giving a specificity constant of 2,800,000 mM-1 min-1, substantially higher than any other noted. ADH D showed >80% activity from pH 5.0 - 8.0. The enzyme was resistant to solvents DMSO (at 5 vol%) and ethyl acetate, acetonitrile and cyclopentyl methyl ether (at 20vol%). ADH F had the broadest substrate scope of any ADH tested, with 1-phenyl-1,2-propanedione the most preferred substrate with a KM of 0.010 mM and a specificity constant of 54,000 mM-1 min-1. It greatly preferred sodium phosphate at pH 7.0, as almost any deviation resulted in a substantial loss of activity. Activity of ≥70% was recorded in 5vol% DMSO, ethyl acetate, acetonitrile, cyclopentyl methyl ether and 50vol% hexane . Both ADH D and F have optimal activities at 70 °C and both may have application in the biotechnology industry for the production of pharmaceutical intermediates and other high value chemicals. ADH E acts solely as an aldehyde reductase, with Vmax using NADH of 74, 331, 320 and 281 U mg-1 for methanal, ethanal, propanal and butanal, respectively. Activity with NADPH was limited (< 1% compared with NADH). Activity was also noted with higher aldehydes such as octanal and furfural. ADH G is an NADPH-dependent ADH utilizing aldehydes only. It has an optimal temperature of 60°C with a half-life of under two hours at that temperature. In conclusion, this thesis reports a feasibility study into the potential industrial use of specific enzymes for a variety of purposes ranging from the production of pharmaceutical intermediates to bioremediation. ADHs D and F are most likely to have use in the biotechnology industry, and ADHs B and E may be suitable for cofactor regeneration. ADH E may additionally be useful in the bioremediation industry. In addition, the anticipated biological significance of these enzymes is described.

Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD) is the most common inborn error of metabolism affecting the fatty acid oxidation pathway. The deficiency is caused by a defect in the medium-chain acyl-CoA dehydrogenase enzyme which catalyzes the first step in the oxidation of medium-chain fatty acids. Long-chain fatty acids, after being transported into the mitochondria and activated into long-chain acyl-CoAs, are sequentially broken down until they become medium-chain acyl-CoAs. Medium-chain acyl-CoAs are then broken down until they become short-chain acyl-CoAs. Short-chain acyl-CoAs are broken down until only acetyl-CoA remains. The block in the oxidation of fatty acids in those with MCADD happens once the long-chain acyl-CoAs have been oxidized to medium-chain acyl-CoAs. The medium-chain acyl-CoAs cannot be further oxidized and build up. Without the breakdown of fatty acids, individuals with MCADD cannot produce enough energy during times of increased metabolic demand. Thus, prolonged exercise, fasting, or fever can precipitate clinical symptoms once the body enters a hypoketotic hypoglycemic state. Those with MCADD typically present in the early months of life with fasting intolerance, vomiting, lethargy, and, in more serious cases, seizures. Adult presentation is rare, but should not be ruled out of a differential diagnosis, because early detection and intervention can prevent permanent brain damage and death. Because early detection can prevent the serious effects of metabolic decompensation, MCADD was added to the Newborn Screen and is tested through measuring levels of medium-chain acylcarnitines in dried blood smears by tandem mass spectrometry. Metabolic decompensation is manifested clinically through dehydration, vomiting, and acidosis. In serious cases, metabolic decompensation can progress to seizures, coma, and death. Introduction of the Newborn Screen has reduced the morbidity of the deficiency, but has not eliminated it. Those with MCADD need to be closely monitored and emergency glucose needs to be available to them in case of a hypoglycemic emergency. The Newborn Screen has been effective in finding mutations in the ACADM gene that produce a mild phenotype of MCADD. Before the Newborn Screen, the most common variant, K329E, was detected in clinically diagnosed patients. However, the screen has shown that there are about 150 variants leading to MCADD. The most common variant of the MCAD protein, K329E, has been studied and characterized in order to further understand the pathogenesis of MCADD. This mutation substitutes a lysine for a glutamic acid, introducing hindrance and the inability of the protein to form its fully functional tetrameric form. The mutant protein also has an increased sensitivity to heat denaturation. Currently, there are no pharmacological treatments for MCADD. The idea of pharmacological chaperones is explored by using the example of tetrahydrobiopterin and phenylketonuria. Future studies will need be done to find a treatment for MCADD that is curative rather than treating the symptoms of the deficiency; however, curative therapies which target the mutant enzyme may be problematic since there is a wide array of mutations that result in a defective enzyme in affected individuals.

Glucose-6-phosphate dehydrogenase (G6PD) is a cytoplasmic enzyme with an important function in cell oxidative damage prevention. Erythrocytes have a predisposition towards oxidized environments due to their lack of mitochondria, giving G6PD a major role in its stability. G6PD deficiency (G6PDd) is the most common enzyme deficiency in humans; it affects approximately 400 million individuals worldwide. The overall G6PDd allele frequency across malaria endemic countries is estimated to be 8%, corresponding to approximately 220 million males and 133 million females. However, there are no reports on the prevalence of G6PDd in Andean communities where bartonellosis is prevalent.

A multitude of microorganisms possess the ability to metabolise molecular hydrogen (H2). The major enzyme family involved in hydrogen metabolism are Hydrogenases. These enzymes catalyse the reversible conversion of molecular hydrogen to protons and electrons (H2 ↔ 2H+ + 2e-). These enzymes have the potential to be utilised for biotechnological applications such as hydrogen fuel cells, but they also represent promising drug targets for inhibition of bacterial energy metabolism both within the gastrointestinal tract and after infection. Therefore, further understanding and discoveries made in the hydrogenase field warrants progression into applied medical and biotechnological research areas. Hydrogenases are also interesting due to their phylogeny and physiology in a large number of microbial species. These enzymes are categorised by their active site architecture. One well studied, ancient group is termed the [NiFe]-hydrogenases, which all harbour a complex NiFe(CN-)2CO active site in the 'large' catalytic subunit and usually have three iron-sulfur clusters within a 'small' electron transferring partner subunit. [NiFe]-hydrogenases have undergone massive diversification, with four major phylogenetic subgroups arising. The major part of this Thesis concerns work on a Group 4 [NiFe]-hydrogenase that functions in partnership with a formate dehydrogenase as a formate hydrogenlyase (FHL). This FHL complex generates H2 and CO2 from the disproportionation of formate (CHOO- + H+ ↔ H2 + CO2). In this Thesis, genetic and biochemical characterisation of Pectobacterium atrosepticum SCRI1043, a potato pathogen, led to the identification of a novel FHL complex. The [NiFe]-hydrogenase in this organism is similar to that of Escherichia coli Hydrogenase-4, with an extended membrane domain similar to that of respiratory Complex I. Importantly, the P. atrosepticum formate dehydrogenase is selenium-free, while previously characterised FHL complexes have selenocysteine-containing formate dehydrogenases. Using genetic and biochemical approaches it was shown that the [NiFe]-hydrogenase and a formate dehydrogenase were vital for H2 production by P. atrosepticum. Using plant infection assays it was also shown that the gene encoding the formate dehydrogenase was important for full infective ability of P. atrosepticum in potato plants and tubers. The latter part of this Thesis focuses on developing genetic tools to study this novel FHL from P. atrosepticum as well as Hydrogenase-1 and -2 from E. coli.

by Lau King-Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 119-142). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / ABBREVIATIONS --- p.IV / TABLE OF CONTENTS --- p.V / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- PURIFICATION OF ADH-I & ADH-C --- p.25 / Chapter CHAPTER 3 --- "PURIFICATION & IDENTIFICATION OF ""ADH-ACTIVATING"" PROTEASE" --- p.60 / Chapter CHAPTER 4 --- ACTIVATION OF ADH-I BY COMMERCIAL PROTEASE & BY ACETIMIDYLATION --- p.90 / Chapter CHAPTER 5 --- CONCLUSION --- p.114 / REFERENCES --- p.118

Tetrahydrofolate-mediated one-carbon metabolism is required for the biosynthesis of many central metabolites, including some amino acids, nucleobases, and nucleotides, and hence dysfunction of one-carbon metabolism is associated with many human diseases and disorders. The mitochondrial glycine decarboxylase complex (GDC) is an important component of one-carbon metabolism, generating 5,10-methylene-tetrahydrofolate (5,10-CH2-H??4folate) from glycine. Previous work has shown that the genes encoding the unique sub-units of the Saccharomyces cerevisiae GDC (GCV1, GCV2 and GCV3) are regulated in response to changes in the levels of cytosolic 5,10-CH2-H??4folate (Piper et al., 2000). Given the centrality of 5,10-CH2-H??4folate to many aspects of metabolism, it was hypothesised that other genes may be regulated by the same mechanism. Using microarray analysis of S. cerevisiae under a number of conditions that affect 5,10-CH2-H??4folate levels, the ??one-carbon regulon??, a group of genes that were co-regulated with the GCV genes was identified. The one-carbon regulon corresponds closely to genes whose promoters are bound by the purine biosynthesis regulator Bas1p, but not all one-carbon regulon members are significantly purine regulated. Genetic approaches demonstrated that the one-carbon unit response and the purine response are distinct, though both depend on the presence of Bas1p. This demonstrated that the close metabolic connections of one-carbon and purine metabolism are reflected in over-lapping, but separable regulatory mechanisms. The identity of the sensor of one-carbon unit depletion remains unknown, but in the course of investigation of the candidate regulator Caf17p, it was demonstrated that Caf17p is in fact involved in Fe/S cluster protein maturation. Examination of the effects of Caf17p depletion revealed that Caf17p is required for the function and maturation of the related mitochondrial Fe/S proteins aconitase and homoaconitase, as well as the function of, but not de novo iron incorporation into, the mitochondrial radical-SAM Fe/S protein biotin synthase. Because other Fe/S proteins were unaffected, Caf17p appears to be a specialised Fe/S maturation factor. The presence of a putative H4folate binding site indicates that Caf17p may constitute a metabolic link between one-carbon and iron metabolism.

South Africa is one of the most important mining countries in the world, hosting the world's largest reserves of platinum group metals (PGMs). Even though mining is clearly an important activity in South Africa, contributing approximately US$ 7.4 billion annually to the countries' gross domestic product (GDP), the costs to the environment are not insignificant. One of the most severe environmental aspects associated with mining is the storage of mineral waste on tailings storage facilities due to their impacts on air quality, ground water quality, aesthetics and land use. It is also unknown whether the environmental effects of tailings storage facilities increase or decrease over time. The aim of this study was to determine the ecotoxicity of platinum tailings storage facilities of different ages by means of soil physical and chemical analysis, earthworm ecotoxicological studies, dehydrogenase activity and soil mesofauna studies. Samples were obtained from three platinum tailings storage facilities of different ages of which two were already rehabilitated while the third was still operational at the time this study was performed. The latter was used as a negative control for the purpose of the study. Soil samples were physically and chemically analysed. Earthworm ecotoxicological studies were conducted to determine changes in biomass, reproduction, mortality, neutral red retention times and tissue metal concentrations. Dehydrogenase activity was determined before the introduction of earthworms and manure, after introductions of manure and after introductions of earthworms and manure. Soil mesofauna were extracted and identified in order to determine species richness, diversity, abundance and functional grouping. Soil chemical analysis indicated that concentrations of certain heavy metals, especially chrome (Cr), present in platinum tailings materials could have a potential effect on microorganisms, microbial processes and earthworms. Earthworm ecotoxicological results indicated that earthworms that bioaccumulated higher levels of heavy metals showed poor hatchability of cocoons. Dehydrogenase activity indicated that earthworms play a significant role in increasing the number and biomass of soil microbes because significant increases in dehydrogenase activity were noticed after the addition of earthworms to platinum tailings materials. Results from the earthworm ecotoxicological studies, dehydrogenase activity, and soil mesofauna composition indicated that environmental impacts of tailings storage facilities did not increase with age, but is more likely to be an indication of the rehabilitation measures administered to the different tailings storage facilities. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.