Mouse Liver Testosterone 15 wHydroxylase ( Cytochrome P - 450

Testosterone 15a-hydroxylase (cytochrome P4501a,) was purified from female 129/J mouse liver microsomes based on its specific activities in the eluates from the columns of octylamino-Sepharose 4B, hydroxylapatite, DEAE-Bio-Gel A, and CM52 chromatography. The 150-hydroxylation activity was five times higher in female than in male 129/J mouse liver microsomes. The specific cytochrome P-450 content of purified P-4501s, fraction was 14.5 nmol/mg of protein. The Soret peak of the reduced cytochrome P-450CO complex was 451 nm. The apparent subunit molecular weight of P-4501s, was 48,000, and the protein appeared as only one major band on sodium dodecyl sulfate-polyacrylamide gels. The specific activity of testosterone 15a-hydroxylation reconstituted with the purified P-45016, was 94 nmol/min/nmol of cytochrome P-450 and 1349 nmol/min/mg of protein, and these were about 65and lOOO-fold higher, respectively, than the activity of solubilized microsomes. The purified P-4501e, exhibited high regioselectivity and stereospecificity for testosterone hydroxylation. More than 95% of the testosterone metabolites formed by the purified P-4501s, was 15a-hydroxytestosterone. Virtually 100% of mouse liver microsomal testosterone 15a-hydroxylation activity can be accounted for by the purified P-45015,. The P-45015, fraction was able to catalyze benzphetamin N-demethylation, 7-ethoxycoumarin 0-de-ethylation, aniline 4-hydroxylation, benzo(a)pyrene 3-hydroxylation, acetanilide 4-hydroxylation, and lauric acid (11 + 12)-hydroxylation at various turnover rates, indicating broad substrate specificity of the P-45015, for the oxidations of xenobiotics. This is in sharp contrast to high regioselectivity and stereospecificity for testosterone hydroxylation.