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CARDIOVASCULAR

Catalytic Activity and Isoform-Specific Inhibition of Rat Cytochrome P450 4F Enzymes

Departments of Biopharmaceutical Sciences (F.X., D.L.K.) and Pharmaceutical Chemistry (P.R.O.d.M., D.L.K.), University of California San Francisco, San Francisco, California; and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.)

Arachidonic acid is -hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome P450 (P450) 4A isoforms are generally considered the major arachidonic acid -hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B₄ (LTB₄) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a K_s of 25 to 59 µM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the -hydroxylation of LTB₄ with a K_m of 24 and 31 µM, respectively, and CYP4F5 had minor activity in LTB₄ metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the -hydroxylation of arachidonic acid with an apparent k_cat of 9 and 11 min^–1, respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid -hydroxylase activity. The P450 -hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed -hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify P450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.

Address correspondence to: Dr. Deanna L. Kroetz, Department of Biopharmaceutical Sciences, 513 Parnassus, Box 0446, San Francisco, CA 94143-0446. E-mail: deanna{at}itsa.ucsf.edu

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