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Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

Thesis (M. Phil.)--University of Hong Kong, 1993.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (MSc)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

One of the major physiological responses to insulin secretion is the activation of lipogenesis in target tissues (principally fat and liver). As acetyl-CoA carboxylase (ACC) is the rate limiting enzyme in fatty acid synthesis, the mechanisms involved in the short term regulation of this enzyme represent a pertinent model system for determining elements involved in amplification of the signals produced in response to stimulation of cells with lipogenic and counter regulatory hormones. The regulation of mammalian ACC by hormones is a complex phenomenon involving interplay between allosteric and covalent mechanisms. While the effects of adrenaline and glucagon are well characterised, the mechanism of regulation by insulin has still to be defined and formed the focus for the work presented in this thesis. To study the role of phosphorylation in the response of ACC to insulin, the site-specific phosphorylation of the enzyme observed following exposure of intact cells to insulin has been reproduced in vitro. These studies for the first time describe the conditions required to achieve distribution of [³²P] in vitro among sites of acetyl-CoA carboxylase, very similar to that seen after hormone treatment of intact cells and employing endogenous polyamine-sensitive kinase(s). No corresponding increase in catalytic activity was detected following phosphorylation, in vitro, of insulin directed phosphorylation sites on purified rat liver acetyl-CoA carboxylase in these studies. Subsequently, ACC was phosphorylated by an exogenous protein kinase from maturation activated sea-star oocytes which led to high stoichiometric incorporation of ³²P into the unique site (I-site) phosphorylated in intact cells in response to insulin (0.3 mol phosphate / mol 240,000 kD subunit). Again no change in ACC activity was observed following I-site phosphorylation. The peptide containing the I-site was separated from other tryptic phosphopeptides by reverse phase HPLC and then sequenced. Phosphorylation of serine 1186 was determined to be the major phosphorylation site of ACC in response to insulin. The amino acid sequence corresponding to the peptide containing Ser 1186 is located in the putative "hinge" region of ACQ which is some 300 amino acids towards the C-terminal of the biotin binding site (Lys-784). Subsequent re-evaluation of the kinetic properties of acetyl-CoA carboxylase during purification has led to the identification of a fraction containing low Mr inhibitor(s) and an apparently novel protein activator present in rat liver. Affinity purified rat liver acetyl-CoA carboxylase can be activated 2-3 fold at physiological citrate concentrations (0.1-0.5mM) by the addition of the heat and pro tease-sensitive cytosolic protein. The ACC activator has been extensively purified (though not yet to homogeneity) from a 100,000 g supernatant fractions from rat liver extract, by a combination of ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. From these results we concluded that the activator is a protein and the native molecular weight in solution is estimated to be approximately 75 kDaltons. A popular hypothesis regarding the short term regulation of ACC involves a phosphorylation-dephosphorylation mechanism resulting in inhibition and activation respectively. A number of experiments have been carried out in order to test the hypothesis that the activator preparation may contain protein phosphatase activity directed towards ACC. The results strongly suggest that under the assay conditions described for the expression of activation of catalytic activity of ACC, there is little or no apparent dephosphorylation. Indeed, the most purified preparations of ACC activator do not contain any detectable phosphatase activity towards the model substrates histone III-S and casein. The activation of ACC occurs rapidly, in a time dependent manner (within 20 min at 37°C) and involves protein-protein interaction which is antagonized by avidin. The interactions between ACC, avidin and activator protein suggest that the activator not only induces conformational change at the active site of ACC but may also bind in such a way as to be displaced (perhaps directly) by avidin. From the data presented it is concluded that this acetyl-CoA carboxylase activator protein represents a novel factor which may be involved in the short term regulation of ACC activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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