A fundamental aspect of the cellular response to hormonal and other stimuli is the activation of arachidonic acid metabolism. Many current studies focus on the mechanism of release of free arachidonic acid. This is generally considered to be essential to the activation of the cyclo-oxygenase and lipoxygenase pathways of metabolism. However we believe there is one specific case in which this assumption is not warranted. In our opinion there is a distinct possibility that an arachidonate ester is the substrate of the 5-lipoxygenase of leukocytes. There are aspects of leukotriene synthesis via the 5-lipoxygenase which differ markedly from prostaglandin biosynthesis and the other lipoxygenase pathways. In essence, leukocytes will not readily convert exogenous arachidonic acid to leukotrienes. Even after addition of an appropriate stimulus (ionophore A23187) there is evidence that the endogenous substrate is used preferentially. A straight forward, and experimentally untested, explanation for these findings is that free arachidonic acid is not the 5-lipoxygenase substrate. We propose to test the hypothesis that there can be direct oxygenation of esterfied forms of arachidonate in leukocytes. The hypothesis will be tested by four approaches. We will activate leukocytes with A23187 and use specifically designed chromatographic methods to isolate oxygenated esters (with rigorous controls for autoxidation and re-esterification of products). Secondly we will synthesize radiolabeled esters and examine their metabolism in leukocytes. We will use both intact cells (with delivery of radiolabeled substrates in liposomes) and lyzed cells (with emulsified substrate). Thirdly we will use H2(18)0 media to permit mass spectrometric analysis of hydrolysis/esterification cycles in the turnover of arachidonate. With this technique we will establish if esterification is an obligatory step in the conversion of exogenous arachidonic acid to 5-lipoxygenase products in leukocytes. Finally, in order to substantiate a precedent for our hypothesis of lipoxygenase-catalyzed oxygenation of esters, we will analyze the structures of products from a recently described reaction of soybean lipoxygenase-1 with polyunsatured fatty acids esterified in phosphatidylcholine. Leukotrienes are potent mediators of inflammatory reactions. Unravelling of the initial step in their biosynthesis is essential to our understanding of these processes. The possibility that the 5-lipoxygenase utilizes an arachidonate ester substrate has received no prior experimental examination. Such a finding would add a new dimension to our concepts of activation of arachidonate metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK035275-03
Application #
3233544
Study Section
Biochemistry Study Section (BIO)
Project Start
1985-04-01
Project End
1988-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
3
Fiscal Year
1987
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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