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 cat alyze S-mephenytoin 4'-hydroxylation from hepatic microsomes of cynomo lgus monkeys. The final preparation (referred to as P450 CMLd) was app arently homogenous judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the estimated minimum molecular weigh t of this protein was 53 kDa. The N-terminal amino acid sequence of P4 50 CMLd (identified 16 residues) was identical with that of protein en coded by P450 2C9 cDNA. P450 CMLd was cross-reactive with both antibod ies raised against P450 2C11 and P450 2C9 which were purified from hep atic microsomes of male rats and humans, respectively. In hepatic micr osomes of cynomolgus monkeys, both antibodies recognized two proteins showing different mobilities on SDS-PAGE (50 and 53 kDa). P450 CMLd wa s a good catalyst for S-mephenytoin 4'-hydroxylation in a reconstitute d system. Anti-P450 2C9 antibody inhibited the activity of S-mephenyto in 4'-hydroxylase, but not the activities of R-mephenytoin 4'-hydroxyl ase and R- and S-mephenytoin N-demethylases in liver microsomes from c ynomolgus 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 monke ys. (C) 1994 Academic Press, Inc.