Purification and characterization of the hepatic CYP2C and 3A isozymes from phenobarbitone pretreated rhesus monkey

Hepatic P450s, named M-3 and M-4 were purified from phenobarbitone pretreat ed rhesus monkey. These demonstrated polypeptide molecular mass of 50 and 5 2.5 kDa and specific content of 12 and 20 nmol P450/mg protein, respectivel y. Both the isozymes demonstrated low spin state of heme. Antibodies raised against M-3 inhibited the activity of aminopyrine, erythromycin and ethylm orphine N-demethylase in the microsomes obtained from PB pretreated rhesus monkey by 76, 40 and 35%, respectively. M-4 did the same by 69, 85 and 79%, respectively. These observations indicated M-3 and M-4 to be the members o f CYP2C and 3A subfamilies, respectively. These results were substantiated by the observations that M-3 metabolized aminopyrine whereas M-4 metabolize d aminopyrine, erythromycin and ethylmorphine in the reconstituted system. Microsomal lipids and cytochrome b(5) enhanced the rate of these reactions. Further confirmation to the identity of these isozymes was provided by N-t erminal amino acid sequences. The first 10 N-terminal amino acid residues o f M-3 were 90% similar to CYP2C20 and 2C9 and that of M-4 were 100 and 90% similar to CYP3A8 and 3A5, respectively. In conclusion, two isozymes of hep atic P450 purified from PB pretreated rhesus monkey belong to CYP2C and 3A subfamilies.