The biosynthesis of the eicosanoids (prostaglandins, leucotrienes, and others) is controlled by the availability of arachidonic acid. Evidence is mounting that a recently discovered 85-kDa cytosolic phospholipase A2 (cPLA2) is involved in the liberation of arachidonic acid from phospholipids in cell membranes for the formation of eicosanoids. Since the eicosanoids control a large number of inflammatory processes, the understanding of the enzymatic properties and regulation of the cPLA2 may be useful for the eventual design of anti-inflammatory therapeutics. In this grant, research along the following lines is proposed. 1) Interfacial kinetics of cPLA2 acting on phospholipids in vitro will be characterized. The effects of calcium and cPLA2 phosphorylation on the interfacial kinetics will be determined. 2) The affinity of cPLA2 and its phosphorylated forms for vesicles of different phospholipid composition will be measured, and the effect of calcium on enzyme-vesicle interactions will be studied. 3) The number of calcium ions in the enzyme-vesicle-calcium ternary complex will be determined. 4) The binding of cPLA2 to anionic phospholipids at the vesicle interface will be characterized. The question of whether binding of cPLA2 to vesicles is accompanied by segregation of acidic lipids into a patch that contacts the enzyme will be addressed. 5) The action of cPLA2 on isolated cell nuclei will be examined. 6) A series of fatty acid analogs will be prepared and evaluated as selective and potent inhibitors of cPLA2. These inhibitors will be used as probes of the role of cPLA2 in cellular arachidonic acid mobilization.
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