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    The purification and immunological isolation of ATP citrate lyase from rat liver : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, New Zealand
    (Massey University, 1986) Walker, Brett Keith
    ATP CITRATE LYASE (E.C 4.1.3.8) has been purified from rat hepato­cyte cytoplasm by a combination of existing published procedures. The final purification method produced homogeneous ATPCL with specific activity of 10-16 units/mg. Antibodies were raised in rabbits against purified ATPCL eluted from reactive Blue Sepharose CL-68 or DEAE anion exchange column. The purified antibodies were tested for their specificity for ATPCL. This was accomplished by Ouchterlony double diffusion analysis and also by disruption of antibody-antigen complexes and visualizing the gene­ rated protein bands on detergent gels. The equivalence point of the purified antibody was determined by immunotitration with both purified enzyme and crude extract. The equi­valence point was later confirmed by immunotitration of radiolabelled proteins. Antibodies were then used to immunochemically isolate and quantitate the amount of (35-S) methionine or (14-C) radiolabelled ATPCL in the cytosolic fraction of rat liver. Pulse labelling of rat liver proteins in vivo and then precipitation of radiolabelled proteins demonstrated that the purified antibodies precipitated proteins other than just the ATPCL subunit. The amount of ATPCL present in the cytosolic fraction could be calculated after immu­ noprecipitation and excision of radiolabelled ATPCL subunition SOS-PAGE. The proportion of ATPCL protein to the total TCA precipitable protein could then be calculated since the immunoprecipitation was carried out under conditions of antibody excess. Radiolabelled ATPCL was then immunoprecipitated from the cytosolic fractions of rats that had been subjected to different nutritional regimes. The results of this set of experiments showed that induction of ATPCL activity resulted from an increase in immunologically reactive protein. Increasing amounts of radiolabelled immunoreactive ATPCL protein could be precipitated by antibodies as the enzyme was induced. Induction of ATPCL activity resulted from increased rate of synthesis or decreased rate of degradation of immunoreactive protein and not from the activation of pre-existing enzyme protein.
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    The regulation of bovine ATP citrate lyase promoter : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry, Massey University, 2006
    (Massey University, 2006) Yosaatmadja, Yuliana
    The synthesis of fatty acids is important for many house keeping functions such as the formation of cell membranes and as energy storage This process occurs mostly in the adipose tissues and liver of monogastric animals. The regulation of fatty acid biosynthesis in monogastric animals such as human and rat have been studied intensively. Several lines of experimental evidences have shown that fatty acid biosynthesis is dependent on the nutritional state of the animal and other hormonal influences, such as insulin and glucagon. However the molecular regulation of fatty acid biosynthesis is relatively unknown in ruminants. Ruminants are large mammals that have a predominantly herbivorous diet and therefore have a very different metabolism to monogastric animals. Although a large percentage of ruminant feed is carbohydrate, very little of these dietary carbohydrates are available for de novo fatty acid biosynthesis and therefore many of the enzymes involved in the conversion of glucose to fat such as ATP citrate lyase may be down-regulated as a mean of physiological adaptation for glucose conservation. ATP citrate lyase (ACLY) is a lipogenic enzyme that catalyses the cleavage of cytosolic citrate into acetyl CoA and oxaloacetate and it is unique to the fatty acid biosynthesis pathway The molecular regulation of the bovine ACLY gene is unknown, however approximately 10 Kb of bovine ACLY gene has been sequenced and characterised. To investigate the molecular regulation of the bovine ATP citrate lyase gene, several experimental methods were used in this study such as reporter gene assays and electrophoretic mobility shift assays.
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    Structural characterization of 3-dehydroquinate synthase II : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science majoring in Biochemistry at the Institute of Fundamental Sciences, Massey University, Turitea, Palmerston North, New Zealand
    (Massey University, 2016) Somerton, Jack
    Aromatic amino acids tryptophan, tyrosine and phenylalanine are derived from a common precursor, chorismate, which is produced by the seven-step shikimate pathway in plants, fungi, Bacteria and Archaea. In Archaea the shikimate pathway typically begins with the alternative substrates, L-aspartate semialdehyde and 6-deoxy-5-ketofructose-1-phosphate, and so requires different enzymes to catalyse the first two steps in the pathway compared to those used in the (common) bacterial pathway. The archaeal enzyme for the second step, 3-dehydroquinate synthase type 2 (DHQS2), catalyses the oxidative deamination of 2-amino-3, 7-dideoxy-D-threo-heptoulsonic acid (formed in step 1) followed by cyclisation to produce 3-dehydroquinate, at which point the alternative and common shikimate pathways converge. No DHQS2 structures have yet been determined, and because DHQS2 enzymes have little sequence homology with their DHSQ1 analogues, they may have a novel fold. Bioinformatic methods were used to predict the solubility, stability and likelihood of sequenced DHQS2s to form crystals, and the five highest ranked were chosen for structural studies. Methanococcus maripaludis, Desulfatibacillum alkenivorans, Methanospirillum hungatei, and Archaeoglobus veneficus DHQS2 open reading frames were amplified by PCR and cloned into a modified pETite32a(+) vector in order to produce recombinant protein with an N-terminal, a C-terminal or no His8-tag. Soluble recombinant DHQS2 proteins were produced in Escherichia coli DL41 (DE3), then purified by immobilized metal-ion affinity chromatography followed by size exclusion chromatography. C-terminally-tagged M. maripaludis DHQS2 with bound cofactor NAD+ crystallised in two conditions: (i) 1.0 M ammonium sulfate, 0.1 M Bis-Tris pH 5.5 with 1% (w/v) PEG 3350; and (ii) 0.1 M CAPS at pH 10.5 with 40% (v/v) 2-methyl-2,4-pentanediol, but unfortunately the crystals were not of diffraction quality. Structure prediction using bioinformatic tools and/or far and near Circular Dichroism spectroscopy indicated that recombinant M. maripaludis DHQS2 was likely to have a secondary structure dominated by α-helices and had tertiary structure; recombinant A. veneficus was likely to have a secondary structure dominated by β-strands and had tertiary structure, while recombinant D. alkenivorans, and M. hungatei were more likely to assume a molten globule structure dominated by β-strands.
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    Synthetic targets as mechanistic probes for the key biosynthetic enzyme, dehydroquinate synthase : a dissertation submitted to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Institute of Fundamental Sciences, Palmerston North
    (Massey University, 2009) Negron, Leonardo
    Dehydroquinate synthase (DHQS) catalyses the five-step transformation of the seven carbon sugar 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) to the carbacycle dehydroquinate (DHQ). Multiple studies have described in detail the mechanism of most of the steps carried out by DHQS with the exception of the final cyclisation step. In this study, (3S)-3-fluoro-DAH7P and (3R)-3-fluoro-DAH7P (fluorinated analogues of DAH7P) were produced and assayed across three phylogenetically distinct sources of DHQS in order to determine the role of the enzyme during the cyclisation step of the reaction. Incubation of (3S)-3-fluoro-DAH7P with DHQS from Escherichia coli, Pyrococcus furiosus, and Kiwifruit resulted in the production of different ratios of (6S)-6-fluoro-DHQ and 1-epi-(6S)-6-fluoro-DHQ for each enzyme. In addition, enzyme catalysis showed a slowing of reaction rates when (3S)-3-fluoro-DAH7P was used, suggesting that the fluorine at C-3 is stabilising the enol pyranose. An increase in the stabilisation of the fluoro-enol pyranose would allow release of this substrate intermediate from the enzyme to compete with the on-going on-enzyme reaction. The differences in the ratio of products formed suggest that the cyclisation occurs in part on the enzyme and that the epimeric product arises only by an abortive reaction pathway where the (3S)-3-fluoro-enol pyranose is prematurely released and allowed to cyclise free in solution. Once in solution, the (3S)-3-fluoro-enol pyranose could undergo a conformational change in the ring leading to the formation of the epimeric product. Furthermore, it is suspected that the position of fluorine influences the likely transition-state in carbacycle formation leading to the production of the epimeric product. This research has illuminated the role of the enzyme in guiding the correct stereochemistry of the product and illustrates the important molecular interplay between the enzyme and substrate.