Purification and characterization of two forms of hepatic microsomal cytochrome P450 from untreated cynomolgus monkeys.
Two forms of cytochrome P450, referred to as P450 CMLb and P450 CMLc, were purified and characterized from hepatic microsomes of untreated cynomolgus monkeys (Macaca irus). The final preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The minimum molecular weights of P450 CMLb and P450 CMLc estimated from the mobilities on the gel were 48 and 50 kDa, respectively. The N-terminal amino acid sequences of P450 CMLb and P450 CMLc (first 13 residues) were identical, respectively, with those of P450 FI isolated from baboons and P450 MK-2, which has been characterized as a P450 3A enzyme in cynomolgus monkeys. P450 CMLb showed coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation activities. This P450 enzyme reacted with anti-P450 2A6 antibody and acts as a coumarin 7-hydroxylase in hepatic microsomes. On the basis of these results P450 CMLb was classified into the 2A subfamily. P450 CMLb is expressed constitutively as one of the minor forms of P450 in liver microsomes of untreated cynomolgus monkeys and is inducible by phenobarbital. P450 CMLc reacted with anti-P450 3A4 antibody and hydroxylated testosterone at the 6 beta-position. The testosterone 6 beta-hydroxylation activities in hepatic microsomes of cynomolgus monkeys, common squirrel monkeys, and humans were strongly inhibited by the anti-P450 CMLc antibody. These results demonstrate that this P450 enzyme is in the P450 3A subfamily.
Division of Pharmacy, Chiba University Hospital, Faculty of Medicine, Japan.
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S
Ohmori
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Horie
F P
FP
Guengerich
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Kiuchi
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Kitada
D000595:Amino Acid Sequence; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008252:Macaca fascicularis / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000494:pharmacology; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology; D012453:Saimiri; D013379:Substrate Specificity
D011285:Pregnenolone Carbonitrile; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital