Studies on rat liver ATP citrate lyase and acetylCoA carboxylase

Houston, Brian (1983) Studies on rat liver ATP citrate lyase and acetylCoA carboxylase. PhD thesis, University of Glasgow.

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ATP citrate lyase and acetylCoA carboxylase catalyse the first two steps in de novo fatty acid synthesis. Both enzymes are substrates for protein kinases vivo and in vitro. The work described in the first part of this thesis was undertaken in order to study the phosphorylation of purified liver acetylCoA carboxylase. The work reported in the second part was concerned with the effect of phosphorylation on the activity of ATP citrate lyase. In addition to the work on the liver enzyme the chromatographic behaviour of mammary gland acetylCoA carboxylase was studied. In the course of this work evidence was obtained that the use of polyethylene glycol precipitation during purification results in the irreversible aggregation of the enzyme. Low amounts of acetylCoA carboxylase were found in pig liver indicating that this tissue would be a poor source of the enzyme. In general, higher levels of acetylCoA carboxylase were found in rat liver, although there was considerable variation in the amounts of activity measured. A number of techniques were investigated in attempts to purify the rat liver enzyme. Some success was achieved using polyethylene glycol precipitation or using chromatography on Phenyl-Sepharose, but these procedures were not reproducible. Under certain conditions, chromatography on DSAE cellulose resulted in the separation of two peaks of acetylCoA carboxylase activity which corresponded to polypeptides with subunit Mrs = 125000 and 2500. This was initially interpreted as the separation of proteolytically degraded acetylCoA carboxylase from the undegraded form. Subsequent study indicated that the 125000 Mr polypeptide was ATP citrate lyase and that the acetylCoA carboxylase activity associated with this enzyme was spurious and was the result of the contamination of acetylCoA by CoA. Evidence was then obtained that the purification of acetylCoA carboxylase would be difficult to achieve due to the presence in rat liver of an inhibitor of the enzyme. Rat liver ATP citrate lyase was purified using ammonium sulphate fractionation and chromatography on DEAE cellulose, Affigel Blue and Ultrogel A2 to a final specific activity of 13.4 U/mg. The enzyme was pure and free from proteolytic degradation as judged by non-denaturing and SDS-polyacrylamide gel electrophoresis. Various physicochemical propeirties of the enzyme were studied. The kinetic mechanism of ATP citrate lyase was investigated. Provided that the assays were performed in the absence of ADP, parallel double reciprocal plots were obtained when MgATP and GoA or MgATP and Mg-citrate were varied at non-saturating concentrations of the third substrate. These studies demonstrated that the kinetic mechanism is ping-pong and thus resolved the discrepancy between the ping-pong mechanism implied by enzyme labelling experiments and the sequential mechanism implied by previous kinetic studies. Purified ATP citrate lyase was phosphorylated by cAMP dependent protein kinase to the extent of 2.08 +/- 0.15 moles of phosphate/mole of ATPC L tetramer. No change in activity upon phosphorylation was detected using either the standard assay or an assay in which the substrate concentrations were approximately physiological. A kinetic study indicated that phosphorylation did not significantly alter the Vmax nor the K s for GoA or max m Mg-citrate. Phosphorylation resulted in a significant increase in the for ATP (from 90.1 +/- I.7 muM to 193.0 +/- 3.6 muM : P < 0.01). Assay conditions were developed whereby the decrease in activity which accompanies phosphorylation could be monitored directly. The physiological significance of these results is discussed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: R MS Smellie
Keywords: Molecular biology
Date of Award: 1983
Depositing User: Enlighten Team
Unique ID: glathesis:1983-72630
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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