CHARACTERIZATION OF CYTOCHROME-P-450 2B6 IN HUMAN LIVER-MICROSOMES

A cytochrome P-450 (P-450) enzyme of the CYP2B subfamily was partially purified from human liver microsomes and characterized with respect to immunochemical properties, N-terminal amino acid sequence, and catalytic activities toward typical P-450 substrates. P-450 enzymes were monitored in chromatographic fractions by immunoblotting analysis using antibodies raised against a monkey P-450 2B, as well as several purified human P-450 enzymes. The final P-450 2B preparation thus obtained was contaminated with P450 3A4, but an N-terminal amino acid sequence matching the sequence predicted from the CYP2B6 cDNA was obtained. The apparent Mr of this protein was 48 kDa, and the migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was the same as that of the P-450 286 protein expressed in a human lymphoblast cell line. Immunoblotting analysis of 50 human liver samples revealed that the protein band considered to be P-450 2B6 was detected in only 12 samples, with four of these having relatively high levels. Several activities toward typical P-450 substrates were determined in a reconstituted monooxygenase system containing partially purified P-450 2B6 and compared with those obtained using a highly purified preparation of P-450 3A4 enzyme; we found that most of the activities were similar in these preparations, except that the partially purified P-460 2B6 showed high rates of activation of the mutagens 6-aminochrysene and 3-methoxy-4-aminoazobenzene to genotoxic metabolites in Salmonella typhimurium NM2009 strain. Antimonkey P-460 2B antibodies partially inhibited the activities toward these promutagens in liver microsomes of those human samples that contained relatively high levels of P-450 2B6. 6-Aminochrysene was also found to be converted to genotoxic metabolites by P-450 2B6 expressed in human lymphoblast cell lines; these activities were inhibited by antimonkey P-450 2B. All of these results collectively indicate that a P-450 2B protein is expressed in human liver and can be involved in monooxygenation reactions, including carcinogen activation, although the enzyme appears to be a rather minor P-450 constituent of most liver samples.