CLONING AND EXPRESSION OF MURINE CYP2CS AND THEIR ABILITY TO METABOLIZE ARACHIDONIC-ACID

Five murine cytochrome P450 (CYP) 2C cDNAs were cloned and characteriz ed, including four new members of this subfamily: CYP2C37, CYP2C38, CY P2C39, and CYP2C40. The cDNAs ranged from 1716 to 1812 bp in length an d encoded polypeptides of 490 amino acid residues except for CYP2C40, which contained an additional glutamic acid residue at the carboxyl te rminus. The amino acid identity of the murine CYP2Cs ranged from 69 to 92%, while the overall amino acid identity was 60%; however, within t he six putative substrate recognition sites the identity was only 25 t o 41%, suggesting possible differences in substrate specificity and pr oduct profiles. The CYP2C cDNAs were expressed in Escherichia coli fol lowing modification of the N-terminus. All five recombinant CYP2Cs met abolized arachidonic acid, but with different metabolic profiles and c atalytic rates. Based on coelution with authentic standards on reverse -phase HPLC, the major metabolites were tentatively identified as foll ows: CYP2C29 and CYP2C39 produced 14,15-cis-epoxyeicosatrienoic acid ( EET); CYP2C37 produced 12-hydroxyeicosatetraenoic acid (HETE); CYP2C38 produced 11,12-EET; and CYP2C40 produced an unidentified metabolite t hat coeluted with 16-,17-, and 18-HETEs. The turnover numbers for CYP2 C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 were 0.34, 1.12, 5.15, 0.5 1, and 0.15 nmol/nmol/min, respectively. Reverse transcriptase-polymer ase chain reaction demonstrated the presence of CYP2C29 mRNA in liver as well as in extrahepatic tissues including brain, kidney, lung, hear t, and intestine. CYP2C38 and CYP2C40 were found in liver, brain, kidn ey, and intestine, with trace amounts in lung and heart, while CYP2C37 and CYP2C39 appeared to be liver specific. (C) 1998 Academic Press.