Date of Award
Doctor of Philosophy
The effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on phospholipid metabolism in L6 rat myoblasts were examined. TPA, but not its inactive analog PDD, causes a two fold increase in the rate of incorporation of radioactive precursors into phosphatidylcholine. This effect is not blocked by the pretreatment of the cells with cycloheximide or Actinomycin D. Increased labelling is paralleled by a TPA-dependent increase in the activity of the rate-limiting enzyme, CTP:cholinephosphate cytidylyltransferase. This increased activity is apparent within two minutes and is not blocked by pre-treatment of the cells with cycloheximide. Exposure of cells causes not only increased cytidylyltransferase activity, but also a translocation of the activity from the cytosol to the endoplasmic reticulum. The same effect can be seen when L6 extracts are in vitro activated with cAMP, MgATP and exogenously added cAMP-dependent protein kinase. In vitro activation of extracts of TPA treated cells did not, however, result in further activation of cytidylyltransferase. Addition of protein kinase inhibitor to the activation mixture resulted in a partial inhibition of the in vitro activated activity. Treatment of cell extracts with agarose-bound alkaline phosphatase diminished the cytidylyltransferase activity.;Cytidylyltransferase from L6 myoblasts or from rabbit muscle has properties very different from those of the enzyme from other tissue sources. It appears only in the large "aggregated" form, and thus is not dependent on added lipid for maximum activity. In the presence of non-ionic detergents, however, it is dependent upon added lipid for activity, but this treatment does not result in the appearance of the low molecular weight form of the enzyme.;Purification of cytidylyltransferase from rabbit muscle based upon the shift in size between the two forms of the enzyme is not possible. Different techniques, ion-exchange chromatography in the presence of Triton X-100 and polyacrylamide immobilized phosphatidylglycerol chromatography, were used to achieve partial purification of the enzyme. These techniques provide up to seven hundred fold purification of the enzyme.
Hill, Stephen Arthur, "Phosphatidylcholine Metabolism In Myoblasts" (1985). Digitized Theses. 1450.