The surface membrane of embryonic muscle cells is involved in the processes of intercellular recognition and cell fusion. The proposed research is concerned with the role of cell surface phospholipids in myoblast development, and specifically with the alteration in phospholipid metabolism caused by treatment of myoblasts with phospholipase C. Degradation of phosphatidylcholine is increased in myoblasts treated with this enzyme and incorporation of biosynthetic precursors into phosphatidylcholine is dramatically increased, indicating the existence of a regulatory mechanism by which myoblasts sense the need for new phosphatidylcholine to replace that which has been degraded. The proposed research is concerned with the elucidation of that regulatory mechanism. The research will involve a detailed study of the regulation of CTP: phosphocholine cytidylyltransferase, the rate-controlling enzyme in phosphatidylcholine biosynthesis. The enzyme will be purified from chick muscle and will be used for cell-free experiments on the ability of the enzyme to bind to and be activated by phospholipid surfaces. An antibody will be prepared to cytidylyltransferase and used to determine quantitatively the levels of enzyme in homogenates and subcellular fractions from cells grown in the presence and absence of phospholipase C. Cytidylyltransferase activity will also be studied in intact cells in which the phospholipid composition has been altered by growth in the presence of choline analogues. The subcellular sites of the cytidylyltransferase and CDP-choline: diacylglycerol cholinephosphotransferase will be determined. Rates of endocytosis will be determined in myoblasts to determine if endocytosis could be involved in the transfer of information about plasma membrane damage to the cellular interior. Pulse-chase experiments and enzymatic analysis will also be performed to determine whether CDP-choline or phosphatidylcholine is the direct precursor of sphingomyelin in these cells.
Showing the most recent 10 out of 13 publications
