Purification of Human Liver Cytochrome P-450 Catalyzing Testosterone 6β-Hydroxylation1

Abstract

Two forms of cytochrome P-450 (P-450 human-1 and P-450 human-2) have been purified from human liver microsomes to electrophoretic homogeneity. P-450 human-1 and P-450 human-2 differ in their apparent molecular weights (52,000 and 56,000, respectively) and Soret peak maxima in the CO-binding reduced difference spectrum (447.6 and 450.3 nm, respectively). In the reconstituted system using rat liver NADPH-cytochrome c (P-450) reductase, P-450 human-2 more effectively oxidized benzo(a)pyrene (80-fold), ethylmorphine (2-fold), and 7-ethoxy-coumarin (2-fold) than did P-450 human-1. However, P-450 human-1 showed higher testosterone 6β-hydroxylase activity, and the activity was markedly increased by the inclusion of cytochrome b₅ or spermine in the reconstituted system. Antibodies raised against P-450 human-1 inhibited more than 80% of microsomal testosterone 6β-hydroxylase activity in human liver. Immunoblotting analysis using anti-P-450 human-1 IgG revealed a single immuno-staining band near M_r 52, 000 in all human liver samples examined. The amount of immunochemically determined P-450 human-1 varied in parallel with the testosterone 6β-hydroxylase activity in human liver. These results indicate that P-450 human-1 is a major form of cytochrome P-450 responsible for microsomal testosterone 6βhydroxylation. Thus, this paper is the first report on human cytochrome P-450 responsible for testosterone 6β-hydroxylation, which is the major hydroxylation pathway in human liver microsomes.