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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK035275-04
Application #
3233542
Study Section
Biochemistry Study Section (BIO)
Project Start
1985-04-01
Project End
1993-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37203
Song, W C; Baertschi, S W; Boeglin, W E et al. (1993) Formation of epoxyalcohols by a purified allene oxide synthase. Implications for the mechanism of allene oxide synthesis. J Biol Chem 268:6293-8
Song, W C; Funk, C D; Brash, A R (1993) Molecular cloning of an allene oxide synthase: a cytochrome P450 specialized for the metabolism of fatty acid hydroperoxides. Proc Natl Acad Sci U S A 90:8519-23
Takahashi, Y; Glasgow, W C; Suzuki, H et al. (1993) Investigation of the oxygenation of phospholipids by the porcine leukocyte and human platelet arachidonate 12-lipoxygenases. Eur J Biochem 218:165-71
Song, W C; Brash, A R (1991) Purification of an allene oxide synthase and identification of the enzyme as a cytochrome P-450. Science 253:781-4
Brash, A R; Hughes, M A; Hawkins, D J et al. (1991) Allene oxide and aldehyde biosynthesis in starfish oocytes. J Biol Chem 266:22926-31
Hughes, M A; Brash, A R (1991) Investigation of the mechanism of biosynthesis of 8-hydroxyeicosatetraenoic acid in mouse skin. Biochim Biophys Acta 1081:347-54
Song, W C; Brash, A R (1991) Investigation of the allene oxide pathway in the coral Plexaura homomalla: formation of novel ketols and isomers of prostaglandin A2 from 15-hydroxyeicosatetraenoic acid. Arch Biochem Biophys 290:427-35
Kuhn, H; Brash, A R (1990) Occurrence of lipoxygenase products in membranes of rabbit reticulocytes. Evidence for a role of the reticulocyte lipoxygenase in the maturation of red cells. J Biol Chem 265:1454-8
Brash, A R; Baertschi, S W; Harris, T M (1990) Formation of prostaglandin A analogues via an allene oxide. J Biol Chem 265:6705-12
Brash, A R; Hawkins, D J (1990) High-performance liquid chromatography for chiral analysis of eicosanoids. Methods Enzymol 187:187-95

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