by Deng Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 111-120). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / List of Abbreviations --- p.viii / List of Tables --- p.ix / List of Figures --- p.x / Chapter 1 --- Introduction / Chapter 1.1 --- Primary nitrogen metabolism in fungi --- p.1 / Chapter 1.1.1 --- Ammonium assimilation --- p.4 / Chapter 1.1.2 --- Regulation of ammonium assimilating enzymes --- p.8 / Chapter 1.2 --- Relevant central carbon metabolism in fungi --- p.11 / Chapter 1.2.1 --- Glyoxylate cycle and isocitrate metabolism --- p.11 / Chapter 1.2.2 --- GABA shunt --- p.15 / Chapter 1.3 --- Relationship between nitrogen metabolism and fungi morphogenesis --- p.15 / Chapter 1.4 --- General background of Volvariella volvacea --- p.17 / Chapter 1.5 --- Objectives of the study --- p.20 / Chapter 2 --- Materials and methods / Chapter 2.1 --- Organism --- p.22 / Chapter 2.2 --- Growth media --- p.22 / Chapter 2.2.1 --- Basal medium --- p.22 / Chapter 2.2.2 --- Solid-state cultivation --- p.23 / Chapter 2.3 --- Effect of different nitrogen sources on the mycelial growth of V volvacea in submerged culture --- p.26 / Chapter 2.4 --- Effect of different carbon and nitrogen sources and concentrations on the production of selected enzymes involved in central carbon and nitrogen metabolism --- p.27 / Chapter 2.5 --- Enzyme extraction --- p.28 / Chapter 2.6 --- Enzyme assays --- p.28 / Chapter 2.6.1 --- NAD-dependent glutamate dehydrogenase --- p.28 / Chapter 2.6.2 --- NADP-dependent glutamate dehydrogenase --- p.29 / Chapter 2.6.3 --- NAD- dependent isocitrate dehydrogenase --- p.29 / Chapter 2.6.4 --- Isocitrate lyase --- p.30 / Chapter 2.7 --- Protein determination --- p.30 / Chapter 2.8 --- Determination of optimum pH for enzyme assays --- p.31 / Chapter 2.9 --- Determination of optimum temperatures for enzyme assays --- p.31 / Chapter 2.10 --- Transfer experiments --- p.31 / Chapter 2.11 --- Enzyme stability --- p.32 / Chapter 2.12 --- Purification of NAD-dependent glutamate dehydrogenase --- p.33 / Chapter 2.12.1 --- Ammonium sulphate precipitation --- p.33 / Chapter 2.12.2 --- Ion exchange chromatography --- p.33 / Chapter 2.12.3 --- Ultrafiltrartion --- p.34 / Chapter 2.12.4 --- Gel filtration chromatography --- p.34 / Chapter 2.12.5 --- Affinity chromatography --- p.34 / Chapter 2.13 --- Electrophoresis --- p.35 / Chapter 2.13.1 --- SDS polyacrylamide gel electrophoresis --- p.35 / Chapter 2.13.2 --- Native polyacrylamide gel electrophoresis --- p.35 / Chapter 2.13.3 --- Activity staining for NAD-dependent glutamate dehydrogenase --- p.36 / Chapter 2.13.4 --- Protein staining --- p.36 / Chapter 2.14 --- NAD-dependent glutamate dehydrogenase characterization studies --- p.37 / Chapter 2.14.1 --- Effect of substrate concentration --- p.37 / Chapter 2.14.2 --- Molecular weight determination --- p.37 / Chapter 2.14.2.1 --- Molecular weight determination by gel filtration chromatography --- p.37 / Chapter 2.14.2.2 --- Molecular weight determination by native PAGE --- p.38 / Chapter 2.14.2.3 --- Protein subunit molecular weight determination by SDS- PAGE --- p.38 / Chapter 3 --- Results / Chapter 3.1 --- Effect of different nitrogen sources on the mycelial growth of V. volvacea in submerged culture --- p.39 / Chapter 3.2 --- Optimum assay conditions for NAD-dependent glutamate dehydrogenase --- p.42 / Chapter 3.3 --- Optimum assay conditions for NADP-dependent glutamate dehydrogenase --- p.46 / Chapter 3.4 --- Optimum assay conditions for NAD-dependent isocitrate dehydrogenase --- p.50 / Chapter 3.5 --- Optimum assay conditions for isocitrate lyase --- p.54 / Chapter 3.6 --- Biomass production and enzyme activities in extracts of in vegetative mycelia grown with different nitrogen and carbon sources provided at different concentrations --- p.58 / Chapter 3.6.1 --- Mycelia growth under different conditions --- p.58 / Chapter 3.6.2 --- NAD- and NADP-dependent glutamate dehydrogenases in extracts of vegetative mycelia grown with different nitrogen and carbon sources provided at different conditions --- p.58 / Chapter 3.6.3 --- NAD-dependent isocitrate dehydrogenase and isocitrate lyase in vegetative mycelia grown with different nitrogen and carbon sources provided at different conditions --- p.64 / Chapter 3.7 --- Transfer experiments --- p.67 / Chapter 3.7.1 --- Activities of glutamate dehydrogenases in extracts of myceila transferred to media containing different carbon sources --- p.67 / Chapter 3.7.2 --- Effect of different carbon sources on the glutamate dehydrogenases in submerged cultures --- p.67 / Chapter 3.8 --- Glutamate dehydrogenase activity in various parts of the fruit body during different stages of fruit body development --- p.70 / Chapter 3.9 --- Stabilization of NAD-dependent glutamate dehydrogenase activity --- p.75 / Chapter 3.10 --- Purification of NAD-dependent glutamate dehydrogenase --- p.77 / Chapter 3.10.1 --- Ammonium sulphate precipitation --- p.77 / Chapter 3.10.2 --- Partial purification by column chromatography --- p.78 / Chapter 3.10.3 --- Electrophoretic determination of the protein profiles of crude extract and partially purified samples --- p.83 / Chapter 3.11 --- Characterization of partially purified NAD-dependent glutamate dehydrogenase from V. volvacea --- p.86 / Chapter 3.11.1 --- Optimum pH and temperature --- p.86 / Chapter 3.11.2 --- Kinetic parameters --- p.86 / Chapter 3.11.3 --- Molecular weight --- p.92 / Chapter 3.11.3.1 --- Molecular weight determination by gel filtration chromatography --- p.92 / Chapter 3.11.3.2 --- Molecular weight determination by native PAGE --- p.92 / Chapter 3.11.3.3 --- Subunit molecular weight determination by SDS-PAGE --- p.92 / Chapter 4 --- Discussion / Chapter 4.1 --- Nutrient nitrogen for the growth of Volvariella volvacea --- p.97 / Chapter 4.1.1 --- Mycelial growth on simple nitrogen compounds --- p.97 / Chapter 4.1.2 --- Nutrient nitrogen in mushroom compost --- p.98 / Chapter 4.2 --- Production and regulation of selected enzymes in vegetative mycelia --- p.98 / Chapter 4.2.1 --- Production and regulation of glutamate dehydrogenases --- p.98 / Chapter 4.2.2 --- Production and regulation of isocitrate dehydrogenase and isocitrate lyase --- p.103 / Chapter 4.3 --- Glutamate dehydrogenases and fruit body development --- p.104 / Chapter 4.4 --- Purification and characterization of NAD-dependent glutamate dehydrogenase --- p.105 / Chapter 4.4.1 --- Enzyme purification --- p.105 / Chapter 4.4.2 --- Enzyme stability --- p.106 / Chapter 4.4.3 --- Enzyme properties --- p.107 / Chapter 4.5 --- Future works: nitrogen metabolism and the growth of Vohariella volvacea --- p.109 / References --- p.111

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Bibliography: leaves 205-233 / xix, 234 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.S.)--University of Adelaide, 1985

Butane-grown Pseudomonas butanovora oxidized butane by a soluble butane monooxygenase through the terminal pathway yielding 1 -butanol as the predominant product. Alcohol dehydrogenases (ADHs) involved in butane oxidation in P. butanovora were purified and characterized at the biochemical, genetic and physiological levels. Butane-grown P. butanovora expressed a type I soluble quinoprotein 1 -butanol dehydrogenase (BOH), a soluble type II quinohemoprotein 1 -butanol dehydrogenase (BDH) and an NAD���-dependent secondary ADH. Two additional NAD���-dependent secondary ADHs were also detected in cells grown on 2-butanol and lactate. BDH was purified to near homogeneity and characterized. BDH is a monomer of 66 kDa consisting of one mole of pyrroloquinoline quinone (PQQ) and 0.25 mole of heme c as the prosthetic groups. BOH was partially purified and its deduced amino acid sequence suggests a 67-kDa ADH containing a PQQ as a cofactor. BOH and BDH exhibited high activities and preference towards I -butanol and fair preference towards butyraldehyde. While BDH could not oxidize 2-butanol, BOH is capable of 2-butanol oxidation and has a broader substrate range than that of BDH. Genes encoding BOH and BDH and their deduced amino acid sequences were identified. BOH and BDH mRNAs and 1-butanol oxidation activity were induced when cells were exposed to butane. Primary C��� and C��� alcohols were the most effective inducers for boh and bdh. Some secondary alcohols, such as 2-butanol, were also inducers for BOH mRNA, but not for BDH mRNA. Insertional inactivation of boh or bdh affected unfavorably, but did not eliminate, butane utilization in P. butanovora. The P. butanovora mutant strain with both boh and bdh genes disrupted was unable to grow on butane and 1-butanol. This result confirmed the involvement of BOH and BDH in butane and 1-butanol metabolism in P. butanovora. Roles of B011 and BDH in butane and 1-butanol metabolism were further studied at the physiological level. There are no substantial differences between BOH and BDH in the mRNA expressions in response to three different 1- butanol levels tested and in their abilities to respond to 1-butanol toxicity. Different bioenergetic roles of BOH and BDH in butane and 1-butanol metabolism were suggested. A model of 1 -butanol- dependent respiratory systems was proposed where the electrons from 1 -butanol oxidation follow a branched electron transport chain. The role of BOH was suggested to function primarily in energy generation because B011 may couple to ubiquinone with the electrons being transported to a cyanide-sensitive terminal oxidase. BDH may be more important in the detoxification of 1 -butanol because the electrons from BDH may be transferred to a terminal oxidase system that is less sensitive to cyanide, which is not capable of energy generation. / Graduation date: 2003

The vascular system transports components essential to the survival of the individual and acts as a barrier to substances that may injure the organism. Atherosclerosis is a dynamic, lesion producing disease of the arterial system that compromises the functioning of the organ by occlusive and thrombogenic processes. This investigation was undertaken to elucidate some of the normal biochemical processes related to the development of atherosclerosis. A significant part of the investigation was directed toward developing and combining methods and protocols to obtain the data in a concerted manner. A postmitochondnal supernatant of bovine aorta, using mevalonate-2-�����C as the substrate, was employed in the investigation. Methods included paper, thin layer, and silica gel chromatography; gel filtration, high performance liquid chromatography (HPLC), and mass spectrometry. This current research demonstrated direct incorporation of mevalonate-2- �����C into the trans-methyiglutaconic shunt intermediates. The aorta also contains alcohol dehydrogenase activity, which converts dimethylallyl alcohol and isopentenol to dimethylacrylic acid, a constituent of the trans-methylgiutaconate Small, radioactive peptides, named Nketewa as a group, were biosynthesized using mevalonate-2-�����C as the substrate. They were shown to pass through a 1000 D membrane. Acid hydrolysis and dabsyl-HPLC analysis defined the composition of the Nketewa peptides. One such peptide, Nketewa 1, had a molecular weight of 1038 and a sequence of his-gly-val-cys-phe-ala-ser-met (HGVCFASM), with a farnesyl group linked via thioether linkage to the cysteine residue. Methods were developed for the concerted investigation of the trans-methylglutaconate shunt, the isolation of mevalonate-2-�����C labeled peptides, and characteristics of neutral and acidic metabolites of mevalonate. The question as to whether or not mevalonate was the direct precursor was answered in the affirmative. These results contribute to the understanding of the biochemistry of the vessel wall and the associated atherogenic processes. Mevalonate-derived volatile and acidic compounds may represent an alternate metabolic pathway. The prenylated Nicetewa peptide may be, as are other prenylated peptides, participants in the intracellular signaling process, release of cytokines, expansion of extracellular matrix, and calcium release. / Graduation date: 2001

The aged T cell is characterized by decreased responsiveness to stimulation. Aging is associated with reduced membrane glycerophospholipid (GPL) to cholesterol ratios so it is interesting that deletion of mitochondrial glycerol-3-phosphate acyltransferase-1 which catalyzes the first step in de novo GPL synthesis induces an aged T cell phenotype in otherwise healthy mice. GPAT-1 could regulate T cell function through three possible mechanisms: maintenance of membrane GPL ratios and membrane based signaling, providing a specific substrate for downstream signaling, or direct regulation of cellular metabolism. Therefore, the goal of this project was to determine whether these mechanisms contribute to the dysfunctional T cell phenotype observed with decreased GPAT-1 activity. T cell stimulation requires significant upregulation of metabolic processes to drive clonal expansion and cytokine production. T cell dysfunction in GPAT-1 knockout mice may be partially explained by altered metabolic function. We found that GPAT-1 KO T cells have significantly reduced basal respiration rates and spare respiratory capacity which is not compensated for by increased glycolytic metabolism suggesting an inherent metabolic defect in GPAT-1 KO T cells. To better understand mechanistically how GPAT-1 regulates T cell function we moved into the Jurkat T cell line and found that shRNA mediated knockdown of the human isoform of GPAT-1 (GPAM) recapitulated key aspects of the dysfunctional T cell phenotype we observed in the mouse including highly significant reductions in IL-2 production and altered membrane GPL to cholesterol ratios. Phosphatidic acid addition was not capable of rescuing these deficiencies suggesting that GPAT-1/GPAM activity is required for proper T cell function. This was the first time that GPAT-1 activity has been shown to be important for T cell function in a non-murine model system and strongly suggests that GPAT-1/GPAM deficiency regulates T cell function at the cellular level. We further demonstrate that phosphorylation of ZAP-70 a proximal effector of T cell activation is significantly reduced in GPAM knock down Jurkat T cells, suggesting that membrane based signaling is dysfunctional. Taken together these data suggest that GPAT-1 is necessary for regulating cellular energy demands in T cells and essential for optimal T cell activation following stimulation. / text

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