AbstractThe preceding paper (B. Gemzik, D. Greenway, C. Nevins, and A. Parkinson (1992). Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1.J. Biochem. Toxicol, 7(43–52).) described the regulation of two rat liver microsomal proteins (50‐ and 51‐kDa) recognized by antibody against P450 3A1. It was also shown that changes in the levels of the 51‐kDa 3A protein were usually paralleled by changes in the rate of testosterone 2β‐, 6β‐, and 15β‐hydroxylation. The present study demonstrates that age‐ and sex‐dependent changes in the 50‐kDa protein were paralleled by changes in the rate of digitoxin oxidation to digitoxigenin bisdigitoxoside. Induction or suppression of the 50‐kDa protein by treatment of rats with various xenobiotics were also paralleled by changes in the rate of digitoxin oxidation. These results suggest that, contrary to previous assumptions, the conversion of digitoxin to digitoxigenin bisdigitoxoside and the conversion of testosterone to 2β‐, 6β‐ and 15β‐hydroxytestosterone are primarily catalyzed by different forms of P450 3A. Further evidence for this coclusion was obtained from studies in which the suicide inhibitor, chloramphenicol, was administered to mature female rats previously treated with pregnenolone‐16α‐carbonitrile (PCN), which induces both the 50‐kDa and the 51‐kDa protein. Treatment of mature female rats with PCN alone caused a marked increase (16‐ to 18‐fold) in the 6β‐hydroxylation of testosterone and the rate of digitoxin oxidation. Treatment of PCN‐induced rats with chloramphenicol caused a ∼70% decrease in liver microsomal testosterone 6β‐hydroxylation, but had no effect on the rate of conversion of digitoxin to digitoxigenin bisdigitoxoside. The oxidation of testosterone by purified 3A1 (a 51‐kDa protein) was also inhibited by chloramphenicol in a time‐ and reduced nicotinamite adenine dinucleotide phosphate (NADPH)‐dependent manner. In addition to testosterone and chloramphenicol, purified 3A1 also metabolized trole‐andomycin, but it was unable to convert digitoxin to digitoxigenin bisdigitoxoside. Testosterone inhibited the microsomal oxidation of digitoxin, but digitoxin did not inhibit testosterone oxidation. This suggests that testosterone is a substrate for the 3A enzyme that metabolizes digitoxin, but that this form of P450 3A does not contribute significantly to testosterone oxidation by rat liver microsomes. We propose that the 2SbT‐, 6β‐, and 15β‐hydroxylation of testosterone by rat liver microsomes is primarily catalyzed by the 51‐kDa 3A proteins (either 3A1 or 3A2 depending on the source of microsomes)