This project involves investigation of the role of lipoxygenases in the oxygenation of arachidonic acid; these reactions usually accompany cell activation and are best known for the generation of inflammatory mediators. Our experimental approach is to define a novel reaction, then examine its occurence in cells, and finally to investigate the role in cell physiology. Recently we established that 12(S)- and 15(S)-lipoxygenases can use arachidonate esterified in phospholipids as substrate for specific oxygenation, and the first part of this proposal is to examine the role of this type of reaction in the physiology of leukocytes and reticulocytes. The second part is to elucidate a novel lipoxygenase pathway which appears to proceed from an 8- hydroperoxide product, to an unstable allene oxide intermediate, and then to prostaglandins and related cyclopentanes. Lipoxygenase metabolism of cellular esters. We will investigate the 12(S)- and 15(S)- oxygenation of lipids of porcine leukocytes with the primary objective of determining the extent these reactions occur in the intact cell. In rabbit reticulocytes there is strong evidence the lipoxygenase will oxygenate cellular lipids and we will focus on the potential role of these oxygenations in breakdown of mitochondria and maturation of the cell. This will be tested by quantification and assay of endogenous lipoxygenase products during cell maturation under natural conditions in vitro. Non-cyclooxygenase prostaglandin biosynthesis. Our initial thrust in definition of the lipoxygenase-catalyzed prostaglandin biosynthesis is to elucidate the pathway from arachidonate to PGA2 to PGE2 in the sea whip coral, Plexaura homomalla. Preliminary studies have uncovered a novel intermediate in the pathway (an allene oxide) and we plan to characterize its structure, metabolism and pharmacology. With information gained in the coral studies we will proceed to explore related biochemistry in mammals and man: already there is strong suggestive evidence of related reactions (8-lipoxygenase metabolism in mouse skin), (a characteristic allene oxide hydrolysis product in rats testis). We will investigate the ramifications of this new and evolving biochemistry in mammals and man. At present it is understood that lipoxygenases can form leukotrienes and related mediators, but this does not account for many lipoxygenase pathways. We anticipate that novel compounds will be discovered in our investigations, and that we will able to extend our application of lipoxygenase metabolism to the normal physiology of cells.
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