Purification of human liver cytochrome P-450 catalyzing testosterone 6 beta-hydroxylation.
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-ethoxycoumarin (2-fold) than did P-450 human-1. However, P-450 human-1 showed higher testosterone 6 beta-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 6 beta-hydroxylase activity in human liver. Immunoblotting analysis using anti-P-450 human-1 IgG revealed a single immuno-staining band near Mr 52,000 in all human liver samples examined. The amount of immunochemically determined P-450 human-1 varied in parallel with the testosterone 6 beta-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 6 beta-hydroxylation. Thus, this paper is the first report on human cytochrome P-450 responsible for testosterone 6 beta-hydroxylation, which is the major hydroxylation pathway in human liver microsomes.
Department of Pharmacology, School of Medicine, Keio University, Tokyo.
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S
Kawano
T
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Kamataki
T
T
Yasumori
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Yamazoe
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Kato
D000293:Adolescent; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006900:Hydroxylation; D008099:Liver / Q000201:enzymology*; D008297:Male; D011817:Rabbits; D013045:Species Specificity; D013055:Spectrophotometry, Infrared; D013739:Testosterone / Q000378:metabolism*
D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System