MECHANISMS OF PHOSPHOLIPASE A2 REGULATION

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The long-term objective of this research project is to understand the molecular mechanisms of cellular regulation of phospholipase A2 (PLA2). PLA2 is the first enzyme in the biosynthesis of prostaglandins, leukotrienes and platelet activating factor. Excess PLA2 activity is implicated in a number of diseases including rheumatoid arthritis inflammatory hyperemia, inflammatory problems associated with burns and trauma, eczema, psoriasis, pancreatitis, trypanosomiasis, amoebiasis, myocardial infarction, ischemia, alveolar proteinasis, demyelinating disease, septic shock and infectious abortions (see Ref. 12). Other aspects of inflammation such as pain and pyrexia and some allergic manifestations could also conceivably be controlled by regulating PLA2 activity. An understanding of the mechanisms of cellular regulation of PLA2 would greatly aid in the design of strategies for treating these disorders. The work proposed in this application will develop a model system for investigating various possible aspects of cellular PLA2 regulation. This model will include the use of the monomeric (asp-49) PLA2 from the venom of the American Cottonmouth Water Moccasin (Agkistrodon piscivorus piscivorus) and unilamellar phospholipid vesicles. Specifically the membrane structural properities required for rapid PLA2 activation will be determined. Various fluorescent probes of membrane structure will be used to monitor time-- dependent changes that correlate with PLA2 activation. Three models of vesicles that can promote apparently instantaneous PLA2 activation will be carefully examined from a structural perspective in attempt to develop a set of membrane structural criteria for rapid PLA2 activation. These models will be unilamellar vesicles of small radius, large unilamellar vesicles submitted to osmotic shock and large unilamellar vesicles containing a critical concentration of lysophospholipid and fatty acid (which will presumably serve as a stable analog of the structural changes occurring in lipid vesicles during the time course of PLA2 activation and vesicle hydrolysis). Finally, possible cellular regulators of PLA2 such as diacylglycerol or certain polypeptides (thionine, cardiotoxin, mellitin) will be examined for their ability to induce PLA2 activation. The relationship between such activation and possible induced membrane structural changes will be determined. Methodology for this project will primarily involve enzyme activity assay (pH stat) and fluorescence spectroscopy. Differential scanning calorimetry, electron microscopy and [3lP] NMR will he used as necessary to characterize vesicle structure.