The long-term objective of this research project is to understand the molecular mechanisms of regulation of phospholipase A2 (PLA2). PLA2 is the first enzyme in the biosynthesis of prostaglandins, leukotrienes, thromboxanes and platelet-activating factor from phospholipid precursors. Excess PLA2 activity is implicated in a number of diseases including inflammatory diseases, cardiovascular disease and several infectious diseases. 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 by which the activity of the various species of PLA2 (e.g. intracellular and extracellular) may be modulated would greatly aid in the design of strategies for treating these disorders. This work will focus on one of the proposed mechanisms of PLA2 regulation: the hypothesis that PLA2 may be activated by specific alterations in the structure and dynamics of biological membranes. PLA2 purified from the venom of Agkistrodon piscivorus piscivorus and bilayers of phosphatidylcholines and/or phosphatidylglycerols will be used for most of the studies. This enzyme exhibits high structural similarity to the extracellular PLA2 associated with several inflammatory disease states. Since the enzyme is activated by the products of phospholipid hydrolysis, fatty acid and lysophospholipid, these agents will be studied as models of how molecules that perturb the membrane may activate PLA2. The interactions of each molecule with PLA2 and with the bilayer and the specific role of each on the kinetics of PLA2 activation will be examined under several experimental conditions and will be correlated with the effects of these agents on the properties of the bilayer. The relationship of calcium, fluidity, charge and heterogeneity of composition in the bilayer to the activation of PLA2 induced by the reaction product will also be investigated. Once the hypothesis is well-defined in the model system, its general applicability will be tested using platelet PLA2 and by similar characterization of an agent thought to activate PLA2 physiologically (diacylglycerol). The methods used in this project will include enzyme activity assay and protein fluorescence spectroscopy. Characterization of the effects of agents on membrane structure will include spectroscopy of fluorescent probes and electron microscopy. In summary, these experiments will provide information concerning the molecular details of how bilayer structure relates to PLA2 activity.
