Studies are proposed (i) on the phospholipase A2 from cobra venom (Naja naja naja) which is among the smallest and most well-studied enzymes of complex lipid metabolism and (ii) on the phospholipases which have been implicated in the generation of arachidonic acid for the biosynthesis of the prostaglandins and other eicosanoid including the leukotrienes, thromboxanes, prostacyclins, and lipoxins. Specific studies on a membrane-bound phospholipase A2 from a macrophage-like cell line P388D1 and on a lysophospholipase from human amnionic membranes are proposed. Lipolytic enzymes are unusual in that they act physiologically on substrates that are part of complex aggregated structures such as lipoprotein complexes, intracellular fat droplets, bile acid-lipid mixed micelles, or membranes, rather than on phospholipids or triglycerides which are molecularly dispersed. Both in vivo and in vitro, these enzymes act preferentially on phospholipids in lipid-water interfaces and these studies focus on its role in the mechanism of action of these enzymes. Specific studies on the cobra venom phospholipase A2 are aimed at completing the amino acid sequence and evaluating the tertiary structure of the enzyme; defining the precise role of the """"""""activator"""""""" site in the mechanism; determining whether the functionally active enzyme subunit is a monomer or dimer; determining the amino acid residues important for catalysis and activation; determining how lipid induces aggregation of the enzyme; and determining the nature of phospholipid binding to the enzyme in the interface. Specific studies on the phospholipases from human amnionic membranes and the macrophage-like cell lines are aimed at characterizing the kinetics and active sites of these two enzymes; determining the substrate specificity of these enzymes with special attention to arachidonic acid and the type of phospholipid; and investigating the effects on the purified phospholipases of drugs and other inhibitors implicated in eicosanoid control with the goal of elucidating the role phospholipases plays in vivo in eicosanoid biosynthesis.
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