Characterization of three highly purified cytochromes P-450 from hepatic microsomes of adult male rats.

Three hepatic microsomal cytochromes P-450 (P-450f, P-450g, and P-450h) have been purified to electrophoretic homogeneity from both untreated and ethanol-treated adult male rats. By all criteria examined, the hemoproteins isolated from untreated rats are indistinguishable from the corresponding enzymes purified from rats administered ethanol. Highly purified cytochromes P-450f, P-450g and P-450h are characterized by minimum Mr of 51,000, 50,000, and 51,000, respectively, and unique coordinates in two-dimensional isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gels. The CO-reduced spectral maxima of cytochromes P-450f and P-450g are at 447-448 nm, and the peak of cytochrome P-450h is at 451 nm. Cytochrome P-450h is a versatile catalyst exhibiting high activity toward benzphetamine, hexobarbital, and estradiol-17 beta and moderate activity toward benzo[alpha]pyrene and zoxazolamine. In contrast, cytochromes P-450f and P-450g have low metabolic activity for these substrates. The three hemoproteins catalyze the metabolism of testosterone with different regio- and stereospecificities and overall rates. Both cytochromes P-450f and P-450h catalyze the hydroxylation of testosterone at the 16 alpha-position; however, cytochrome P-450h also oxidizes the steroid at the 2 alpha- and 17 beta-position (androstenedione formation). Testosterone is oxidatively metabolized at the 6 beta-, 15 alpha- and an unknown position by cytochrome P-450g. Peptide maps, generated by proteolytic or chemical digestion of the hemoproteins, indicate that cytochromes P-450f, P-450g, and P-450h differ structurally from each other and five previously characterized rat hepatic microsomal cytochromes P-450 (P-450a, P-450b, P-450c, P-450d, and P-450e). Cytochromes P-450f, P-450g, and P-450h do not react with antibodies directed against these inducible hemoproteins by Ouchterlony immunodiffusion in the presence of detergent; however, in the absence of detergent, cytochrome P-450f cross-reacts weakly with anti-P-450b. Results of this study indicate that rat hepatic microsomal cytochromes P-450 are composed of at least four hemoproteins with CO-reduced absorbance maxima between 447-448 nm. Furthermore, a minimum of four microsomal cytochromes P-450 are now known to 16 alpha-hydroxylate testosterone.