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P450 ARACHIDONICACID EPOXYGENASE

441

for a relatively long time (45 min). (3) At this point, increase the solvent strength in small steps (1-2%) until the separation and elution times are optimized. By this procedure, variations on the basic elution conditions summarized in Table I can be applied for the determination of a wide variety of fatty acid and eicosanoid to- and (to-1)-hydroxylations catalyzed by c y t o c h r o m e s P450. Acknowledgments This work was supported by National Institutes of Health Grants ES 03771 (RTO) and GM 31296(BSSM). J.E.C. was a Parker B. Francis Fellow in pulmonary research during the time in which these studies were performed. B.S.S.M. is The Robert A. Welch Foundation Professor in Chemistry at the University of Texas Health Science Center at San Antonio.

[42] C y t o c h r o m e P 4 5 0 A r a c h i d o n i c A c i d E p o x y g e n a s e : Stereochemical Characterization of Epoxyeicosatrienoic Acids By J O R G E H. C A P D E V I L A , E L I Z A B E T H D I S H M A N , A R M A N D O KARARA,

and J. R. FALCK Introduction The c y t o c h r o m e P450 epoxygenase catalyzes the N A D P H - d e p e n d e n t epoxidation o f arachidonic acid (AA) to 5,6-, 8,9-, 11,12-, and 14,15-cisepoxyeicosatrienoic acids (EETs) according to the following stoichiomettic relationship: AA + NADPH + 02 H ÷ --, EET + NADP÷ + H20 The epoxygenase regio- and stereoselectivity o f oxygenation is under the control o f the c y t o c h r o m e P450 protein catalyst.l'2 The potential functional significance of this metabolic pathway has been highlighted by (a) the potent biological activities of the E E T s 3 and (b) the demonstration of a role for the hemoprotein in the in vivo metabolism of endogenous arachidonic acid. 1 The enantioselective nature o f the endogenous E E T pools 1 A. Karara, E. Dishman, I. Blair, J. R. Falck, and J. H. Capdevila, J. Biol. Chem. 264, 19822 (1989).

2j. H. Capdevila, A. Karara, D. Waxman, M. V. Martin, J. R. Falck, and F. P. Guengerich, J. Biol. Chem. 265, 10865 (1990). 3 F. A. Fitzpatrick and R. C. Murphy, Pharmacol. Rev. 40, 229 (1989), and references therein. METHODS IN ENZYMOLOGY,VOL. 206

Copyright © 1991by Academic Press, Inc. All rights of reproduction in any form reserved.

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ENZYME ASSAYS

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present in rat liver and human kidney established the epoxygenase as an additional member of the "arachidonate metabolic cascade. ''L4 We describe methodology developed in our laboratories for the regioand stereochemical characterization of the cytochrome P450 epoxygenase metabolites generated in vitro by enzymatic incubations or present in vivo in extracts isolated from whole organs.

In Vitro Studies Product Isolation, Purification, and Derivatization

Studies of the enzymology of EET formation are performed by incubating a source of the hemoprotein (i.e., isolated microsomal fractions or a purified cytochrome P450 form) with radiolabeled arachidonic acid in the presence of NADPH and air. After termination, the reaction products are extracted into an organic solvent and analyzed by reversed-phase highperformance liquid chromatography (HPLC). A detailed description of the methodology utilized for isolation of the microsomal fractions, preparation of the [1-14C]arachidonic acid substrate, incubation, product extraction, ~md HPLC analysis has recently been published) The reader is referred to that publication for further details. The mixture of EETs generated by incubates containing purified forms of cytochrome P450 or microsomal fractions, NADPH, and [1-a4C]arachidonic acid is extracted into an equal volume of ethyl ether and, after solvent evaporation, initially resolved by reversed-phase HPLC on a 5/xm Dynamax Microsorb C18 column (4.6 × 250, Rainin Instruments Co., Woburn, M A ) ) The dry extract is dissolved in a small volume of ethanol (-

Cytochrome P450 arachidonic acid epoxygenase: stereochemical characterization of epoxyeicosatrienoic acids.

[42] P450 ARACHIDONICACID EPOXYGENASE 441 for a relatively long time (45 min). (3) At this point, increase the solvent strength in small steps (1-2...
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