This project deals with the isolation and characterization of phospholipases and their possible role in the mechanism of catecholamine secretion. The hypothesis is that phospholipases play a key role in the formation of lyso-compounds, free arachidonic acid, and diglyceride, and that these metabolites may regulate the expression of lipolytic enzymes, and the fusion of chromaffin granules and plasma membrane. In an attempt to test this hypothesis, PLA2, PLC and diglyceride lipase will be thoroughly characterized in bovine adrenomedullary chromaffin cells, with regard to pH, ion requirements, substrate and positional specificities, and subcellular localization. The endogenous inhibitor(s) that suppress calcium-dependent PLA2 in adrenal medulla will be characterized with regard to chemical identity; cellular and subcellular localization; and biological or pharmacological agents that modify enzyme activity. The endogenous inhibitor will be purified using high pressure liquid chromatography and its mechanism of action determined; antibodies will be generated for histochemical localization studies and for radioimmunoassay. Alterations in membrane phospholipids that accompany exocytosis will be determined by thin layer and gas liquid chromatograpy in unlabeled cells or those prelabeled with 14C-arachidonic acid. Lipid alterations will also be examined during membrane fusion in the presence and absence of endogenous inhibitors and phospholipases (A2 and C). Finally, other secretory systems will be examined for the presence of suppressed lipolysis and similar lipolytic enzymes. These studies should enhance our understanding of calcium-dependent exocytosis, and the sequence and relationship between the cascade of reactions that follows plasma membrane excitation and culminates in exocytotic secretion.
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| Bartolf, M; Franson, R C (1990) Biomembrane-modulated, lysosomal phospholipase A2 contamination of chromaffin granule ghosts. Biochim Biophys Acta 1029:196-200 |
| Bartolf, M; Franson, R C (1990) Characterization and partial purification of soluble, lysosomal phospholipase(s) A2 from adrenal medulla. Biochim Biophys Acta 1042:247-54 |
| Rosenthal, M D; Vishwanath, B S; Franson, R C (1989) Effects of aristolochic acid on phospholipase A2 activity and arachidonate metabolism of human neutrophils. Biochim Biophys Acta 1001:1-8 |
| Gamache, D A; Fawzy, A A; Franson, R C (1988) Preferential hydrolysis of peroxidized phospholipid by lysosomal phospholipase C. Biochim Biophys Acta 958:116-24 |
