Characterization of monkey cytochrome P450, P450 CMLd, responsible for S-mephenytoin 4-hydroxylation in hepatic microsomes of cynomolgus monkeys.
We isolated a new form of cytochrome P450 (P450) which was able to catalyze S-mephenytoin 4'-hydroxylation from hepatic microsomes of cynomolgus monkeys. The final preparation (referred to as P450 CMLd) was apparently homogenous judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the estimated minimum molecular weight of this protein was 53 kDa. The N-terminal amino acid sequence of P450 CMLd (identified 16 residues) was identical with that of protein encoded by P450 2C9 cDNA. P450 CMLd was cross-reactive with both antibodies raised against P450 2C11 and P450 2C9 which were purified from hepatic microsomes of male rats and humans, respectively. In hepatic microsomes of cynomolgus monkeys, both antibodies recognized two proteins showing different mobilities on SDS-PAGE (50 and 53 kDa). P450 CMLd was a good catalyst for S-mephenytoin 4'-hydroxylation in a reconstituted system. Anti-P450 2C9 antibody inhibited the activity of S-mephenytoin 4'-hydroxylase, but not the activities of R-mephenytoin 4'-hydroxylase and R- and S-mephenytoin N-demethylases in liver microsomes from cynomolgus monkeys. From these lines of evidence we conclude that P450 CMLd is classified into the P450 2C subfamily and acts as one of the S-mephenytoin 4'-hydroxylases in hepatic microsomes of cynomolgus monkeys.
Ohmori
S
S
Division of Pharmacy, Chiba University Hospital, Faculty of Medicine, Chiba University, Japan.
Chiba
K
K
Nakasa
H
H
Horie
T
T
Kitada
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D065731:Cytochrome P-450 CYP2C19; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008252:Macaca fascicularis; D008617:Mephenytoin / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D008617:Mephenytoin