9619859 Hendrickson This research will focus on interactions of phosphatidylinositol-specific phospholipase C (PI-PLC) and sphingomyelinase with various lipid membrane systems. The major points to be addressed are the mechanisms of action of these enzymes, substrate specificities, the way in which the enzymes interact with lipid interfaces, and the nature of activation by the lipid interface. The kinetics of these enzymes will be studied using thiophosphate substrate analogs in continuous spectrophotometric assay systems with mixed-micelles or vesicles of substrate and other phospholipids or detergents. Kinetics will also be studied with fluorescent-labeled lipids in a HPLC-based assay. Protein-lipid interactions will be studied by fluorescence spectroscopy using intrinsic tryptophan or extrinsic substrate-analog probes, gel filtration, and centrifugation techniques. The micelle and lipid bilayer vesicle systems used in these studies will be produced under carefully controlled conditions and will be characterized in order to understand their specific structures and any changes that may occur as a result of phospholipase activity. Much enzymology has been done with soluble enzymes and substrates; however, many enzymes act at membrane interfaces. The significance of this research lies in understanding the interactions of enzymes with lipid substrates at an interface. The results of this research will provide new information about the recognition processes and mechanisms for enzyme binding at an interface, the activation processes that take place at the interface, how enzyme activity is regulated, and the mechanism of catalysis.
