The eicosanoids, a collection of oxygenated derivatives of polyunsaturated fatty acids, have been ascribed important roles in a broad variety of physiological and pathological processes including inflammation, immune surveillance, sleep induction, stroke, myocardial infarction, hormone release, and carcinogenesis. The first committed step in the biosynthesis of each of the eicosanoids, the incorporation of molecular oxygen into a fatty acid, is catalyzed by a fatty acid oxygenase. The most prominent mammalian fatty acid oxygenase is the cyclookygenase activity of prostaglandin H synthase, whose product is subsequently converted into prostaglandins, thromboxanes and prostacyclins. Two important characteristics of prostaglandin generation by the cyclooxygenase are the feedback amplification of the reaction rate, mediated by product hydroperoxide, and the self-catalyzed inactivation. These characteristics are shared by other fatty acid oxygenases and may be important in regulation of eicosanoid biosynthesis. Prostaglandin H synthase can be isolated in quantities sufficient for detailed physical characterization and currently is more convenient to study than other mammalian fatty acid oxygenases. The overall goal is to relate the catalytic and regulatory behavior of the synthase to its reaction mechanism, and to the structure of the synthase protein and the native membrane environment.
The specific aims are to analyze the pure ovine synthase to: a) characterize the mechanism by which the peroxidase activity also present in the synthase enhances the reaction dynamics of the cyclooxygenase: (b) elucidate the mechanisms for self-catalyzed inactivation of the synthase; and c) characterize the functional domains of the synthase responsible for its interactions with activators, substrates, inhibitors, and phospholipid. The methodologies to be used include: analysis of cyclooxygenase and peroxidase activities, and computer simulation of these activities; protein and polypeptide purification and characterization; UV-vis spectrophotometry; circuLar dichroism and magnetic circular dichroism; and electron paramagnetic resonance.
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