Cloning and expression of murine CYP2Cs and their ability to metabolize arachidonic acid.
Five murine cytochrome P450 (CYP) 2C cDNAs were cloned and characterized, including four new members of this subfamily: CYP2C37, CYP2C38, CYP2C39, and CYP2C40. The cDNAs ranged from 1716 to 1812 bp in length and encoded polypeptides of 490 amino acid residues except for CYP2C40, which contained an additional glutamic acid residue at the carboxyl terminus. The amino acid identity of the murine CYP2Cs ranged from 69 to 92%, while the overall amino acid identity was 60%; however, within the six putative substrate recognition sites the identity was only 25 to 41%, suggesting possible differences in substrate specificity and product profiles. The CYP2C cDNAs were expressed in Escherichia coli following modification of the N-terminus. All five recombinant CYP2Cs metabolized arachidonic acid, but with different metabolic profiles and catalytic rates. Based on coelution with authentic standards on reverse-phase HPLC, themajor metabolites were tentatively identified asfollows: 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 that coeluted with 16-,17-, and 18-HETEs. The turnover numbers for CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 were 0.34, 1.12, 5.15, 0.51, and 0.15 nmol/nmol/min, respectively. Reverse transcriptase-polymerase chain reaction demonstrated the presence of CYP2C29 mRNA in liver as well as in extrahepatic tissues including brain, kidney, lung, heart, and intestine. CYP2C38 and CYP2C40 were found in liver, brain, kidney, and intestine, with trace amounts in lung and heart, while CYP2C37 and CYP2C39 appeared to be liver specific.
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, North Carolina 27709, USA.
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Luo
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Zeldin
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Blaisdell
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Hodgson
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Goldstein
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism
D007527:Isoenzymes; D012333:RNA, Messenger; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase