Lipoxygenase metabolism of arachidonic acid leads to formation of hydroperoxides (HPETEs) and leukotrienes (LTs). While the 5- lipoxygenase pathway of leukocytes is known to form potent LT mediators, other lipoxygenase pathways appear to stop at the HPETE product and a function has not been ascribed to most of these enzymes (including the platelet 12(S)-lipoxygenase). Nevertheless, the 12(S)-lipoxygenase has proved a useful model enzyme for the study of mechanisms of leukotriene biosynthesis, and we used it to show that a lipoxygenase catalyzes LT synthesis and that the initial reaction step is identical to an oxygenation by the enzyme. Recently, an important precedent was set with the finding that the simple product 8(R)-HETE is a natural mediator of maturation in starfish oocytes. A search in sea urchin eggs then led us to identification of a 12(R)-lipoxygenase (and 11(R)-lipoxygenase); this oxygenase activity is reported to be activated at fertilization. We now plan to continue our studies on 12- lipoxygenases and the mechanisms of LT synthesis, using the sea urchin 12(R)-lipoxygenase as a potential new """"""""window"""""""" on the role of 12-lipoxygenase pathways.
The specific aims are: 1) To purify and characterize the 11(R)- and 12(R)-lipoxygenase(s) from sea urchin eggs. 2) To establish the chiral features of LTA epoxide biosynthesis using R and S HPETE enantiomers reacted with lipoxygenase enzymes of differing specificity, initially the 12(R)- and 12(S)- lipoxygenase. 3) To examine the distribution of 12(R)-lipoxygenase, the biosynthesis and metabolism of the products, and the biological functioning of the pathway in sea urchin eggs and mammalian tissues. Comparison of the 12(R) and 12(S) lipoxygenase enzymes has a relevance to their potential role in reproductive biology and in hemostasis and inflammation. Our studies are directed at understanding the factors which regulate the lipoxygenases, the enzymatic mechanisms, and the biological role of these pathways.
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