Purification and Characterization of Recombinant Rat Hepatic CYP4F1

CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18–23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the ω-hydroxylation of leukotriene B4 with a Km of 134 μM and a Vmax of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b5. In addition, 6-trans-LTB4, lipoxin A4, prostaglandin A1, and several hydroxyeicosatetraenoic acids (HETEs) were also ω-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a Km of 18.6 μM and a Vmax of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B4, laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids.