This research will focus on interactions of phospholipases A1, A2, and phosphatidylinositol-specific phospholipase C(PI-PLC) with lipid membrane systems. These enzymes play key roles in the production of bioactive lipids (prostaglandins, leukotrienes, platelet activating factor), in signal transduction across membranes, and the release of glycosylphosphatidylinositol-anchored proteins from membranes. Major points to be addressed are the mechanisms of action, substrate specificities, interactions with lipid interfaces, and the nature of the activation by the lipid interface. Kinetics will be studied using thiolester substrate analogs in continuous spectrophotometric assay systems with mixed- micelles of detergent and substrate, with vesicles of anionic phospholipids, and in monolayer systems. Protein-lipid interactions will be studied by fluorescence spectroscopy, and by penetration of proteins into monolayers of non-hydrolyzable phospholipids. The kinetics of recombinant PI-PLC from Bacillus cereus will be studied in micellar systems using a continuous spectrophotometric assay with thiophosphate ester substrate analogs. Minimal substrate requirements will be determined. A comparison of phosphoinositide phosphotransferase (production of inositol 1,2-cyclophosphate) and inositol 1,2-cyclophosphate phosphohydrolase (production of inositol-1phosphate) activities will be made. Recombinant hepatic lipase (phospholipase A1) and mutants (at the putative lipid-binding and active sites), and recombinant placental phospholipase A2 will also be studied. //
