The phosphoinositide-specific phospholipase C (PI-PLC) isozymes comprise a family of proteins which catalyze the cleavage of phosphoinositides to yield the products diacylglycerol (DG) and the corresponding inositol phosphate headgroup. These enzymes play an essential role in the regulation of a wide variety of biological processes ranging from exocytosis to cell division (1-2). We propose to explore the role of the membrane/solution interface in regulation of the PI-PLC isozymes. We propose that the PI-PLC isozymes possess an interfacial recognition site or region, analogous to extracellular lipases, that is essential for stable association with the substrate surface and is distinct from the catalytic site. It is this region that is key to control to these intracellular lipases by calcium-mobilizing receptors. The immediate goals of this study are as follows:1)Experiments to separate and identify both the interfacial recognition region and the catalytic site will be performed. A combination of site-specific chemical reagents and suicide substrate inhibitors will be employed to label the active site. The interfacial recognition region will be tagged using lipid soluble, photoreactive crosslinking reagents. The activity of chemically modified PI-PLC and fragments of this enzyme towards the monomeric PtdIns analogs and PtdIns aggregates will serve as a method of functionally dissecting the catalytic site from an interfacial recognition region. Mapping of these sites using both monoclonal antibodies directed against the protein and sequence-specific antipeptide antibodies is also proposed. 2)We will perform steady state kinetic experiments using molecularly disperse PtdIns analogs. These studies will allow us to separate the effects of calcium, diacylglycerol and phospholipid modulators upon catalysis from effects on the enzyme's interaction with aggregated substrate surfaces. 3)We will study the interaction of the 85 kDa PI-PLC isozyme with artificial phospholipid vesicles and the membranes of human red blood cells. Our experiments are designed to test the hypothesis that the purified PI-PLC can act at the membrane surface in the """"""""scooting"""""""" mode through an interfacial recognition site on the protein, provided that sufficient calcium is available. We will use the kinetics of intervesicle exchange as well as direct binding methodologies to determine the requirements and dynamics of this PLC isozyme.
