I. Structure/Function Studies of the Bee Venom Phospholipase A2. The understanding of the mode of action of phospholipase A2 (PLA2) continues to be of medicinal interest as this enzyme is though to control the release of arachidonic acid from the phospholipid pool for the biosynthesis of the eicosanoids. Continued studies on the enzymology of PLA2 will be carried out. Using the recently determined high resolution X-ray crystal structure of the PLA2 from bee venom as a guide, a number of site-directed mutagenesis studies will be conducted to understand the role of certain amino acid residues in PLA2 catalysis. A major focus will be directed at a number of positively charged residues that lie on the proposed membrane binding surface of the enzyme. The role of these residues in promoting the catalytically productive binding of enzyme to the vesicle interface will be studied by mutating these residues to neutral or negatively charged amino acids. These studies may suggest novel ways to design PLA2 inhibitors that function by interfering with catalytically meaningful binding rather than by binding to the catalytic site. II. Purification and Characterization of a Novel PLA2 From Human Platelets. An intracellular PLA2 bound to the plasma membrane of human platelets has been found. This enzyme is distinct from a low MW secretable PLA2 found in platelet granules and from a high MW PLA2 that is found in platelet cytosol. These studies will contribute to a full understanding of the PLA2s present in platelets and aid in the understanding of the enzymology of arachidonic acid release in these cells. III. Kinetic Studies of a High MW PLA2 From Rat Kidney. Recently a family of high MW, 110 kDa, PLA2s have been isolated from rat kidney, bovine platelets, rat macrophages, and a human monocytic cell line. It has been suggested that this enzyme acts preferentially on phospholipids with arachidonic acid in the 2-position. The reported subtracted specificity of the enzyme from rat kidney will be examined under conditions in which it is tightly bound to the vesicle surface. In this way, the true substrate specificity of the enzyme can be examined without the distortion caused by a differential binding of the enzyme to vesicles composed of different pure phospholipids. The results of these studies will be used to design and synthesize more potent inhibitors of the high MW PLA2s that. can be used in in vitro studies on the role of these enzymes in arachidonic acid generation.

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National Heart, Lung, and Blood Institute (NHLBI)
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Physical Biochemistry Study Section (PB)
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University of Washington
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