Cloning, sequencing, heterologous expression, and characterization of murine cytochrome P450 3a25*(Cyp3a25), a testosterone 6beta-hydroxylase.
A full-length cDNA clone encoding a novel form of the cytochrome P450 3A subfamily (Cyp3a-25) has been isolated from a mouse liver cDNA library. The sequence contained 2010 base pairs and encoded a protein with 503 amino acids. The amino acid sequence shared greater identities with rat CYP3A18 (90%) and golden hamster CYP3A10 (81%) sequences than with known mouse sequences (Cyp3a-11, Cyp3a-13, Cyp3a-16, and Cyp3a-41 [68--70%]). CYP3A25 was expressed in the Escherichia coli PCWori(+) expression vector following slight modifications of the N- and C-terminals of the cDNA. The purified CYP3A25 was recognized on an immunoblot by CYP3A1 antibody and has a molecular weight of 50 kD. CYP3A25 was catalytically active in the 6 beta-hydroxylation of testosterone and the N-demethylation of benzphetamine and erythromycin. It was demonstrated by RT-PCR that the CYP3A25 mRNA is present in both fetal and adult tissues, including liver, lung, intestines, kidney, and brain. Northern blotting demonstrated that expression is greatest in the liver and small intestine.
Dai
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NIEHS, National Institute of Health, Research Triangle Park, NC 27709, USA.
Bai
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Hodgson
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Rose
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D000375:Aging; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics*; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014162:Transfection
D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C113764:Cyp2c13 protein, rat; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating