PURIFICATION FROM LIVER-MICROSOMES FROM UNTREATED CYNOMOLGUS MONKEYS OF CYTOCHROME P450 CLOSELY RELATED TO HUMAN CYTOCHROME P450 2B6

A cytochrome P450 (P450) (referred to as P450CMLa) was purified and characterized from hepatic microsomes from untreated cynomolgus monkeys (Macaca irus). The final preparation was electrophoretically homogeneous and its estimated minimum molecular mass was 49.5 kDa. The amino-terminal amino acid sequence of the protein (first 34 residues) closely resembled that of the protein encoded by the 2B6 cDNA from humans (94%). This protein was cross-reactive with antibodies raised against P450 2B1 (P450 b), P450 2B11 (P450 PBD-2), and P450GP-1, which were purified from hepatic microsomes from phenobarbital-pretreated rats, beagle dogs, and guinea pigs, respectively. Also, the antibody raised against P450CMLa was able to cross-react with P450 2B1, P450 2B11, and P450GP-1. P450CMLa was capable of catalyzing benzphetamine N-demethylation and testosterone 16beta-hydroxylation in a reconstituted system. Anti-P450CMLa antibody inhibited the activity of testosterone 16beta-hydroxylase but not the activities of testosterone 2beta- and 6beta-hydroxylases in liver microsomes from cynomolgus monkeys. The content of P450CMLa, as estimated by immunoblot analysis, was 70 pmol/mg (about 5% of total P450). The protein immunoreactive with the anti-P450CMLa antibody was also present in liver microsomes from Japanese monkeys, baboons, common marmosets, and common squirrel monkeys. In liver microsomes from common squirrel monkeys, the content of protein immunoreactive with the anti-P450CMLa antibody and the activity of testosterone 16beta-hydroxylase were effectively increased by pretreatment with phenobarbital. The antibody against P450CMLa strongly inhibited the activity of testosterone 16beta-hydroxylase in liver microsomes not only from untreated cynomolgus monkeys but also from phenobarbital- and pregnenolone 16alpha-carbonitrile-pretreated common squirrel monkeys. These results indicated that the P450CMLa purified here is very similar to the forms of P450 classified into the 2B subfamily, in its amino-terminal amino acid sequence, catalytic activities, and immunochemical properties.