Interactions of phospholipases A1, A2, and phosphatidylinositol- specific phospholipase C (PI-PLC) with lipid membrane systems will be investigated with respect to the mechanisms of action, substrate specificities, interactions with lipid interfaces, and the nature of activation by the lipid interface. Kinetics will be studied using thiolester substrate analogs a continuous spectrophotometric assay with mixed-micelles, and with short- chain substrate homologs in monolayer systems. Protein-lipid interactions will be studied by fluorescence spectroscopy, and by penetration of proteins into monolayers of non-hydrolyzable phospholipids. Genetically engineered native hepatic lipase (phospholipase A1) and mutants (at the putative lipid-binding site) expressed from cDNA of rat hepatic lipase will be studied. The kinetics of PI-PLC from Baccilus cereus will be studied in micellar systems with thiophosphate ester analogs of phosphatidylinositol, and in a monolayer system. The kinetics of phospholipase A2 (PLA2 from human synovial fluid will be studied in mixed-micelle and monolayer systems. PLA2S from porcine pancreas, and snake venom (Crotalus adamanteus) will also be studied. The nature of autocatalytic derivatization of PLA2 by acyl transfer from the substrate, and its subsequent dimerization and activation, will be investigated with fluorescent-labeled substrate analogs. These are very attractive proteins with which to study protein- lipid interactions since they act preferentially at the lipid- water interface and have a functional activity which can readily be followed. Although much enzymology has been done on soluble enzymes with soluble substrates, many enzymes act at membrane interfaces. The significance of this research is the understanding of the interactions of enzymes with lipid substrates at an interface.
