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J Biol Chem. 1984 Jan 25;259(2):1265-71.

Mouse liver testosterone 15 alpha-hydroxylase (cytochrome P-450(15) alpha). Purification, regioselectivity, stereospecificity, and sex-dependent expression.


Testosterone 15 alpha-hydroxylase (cytochrome P-450(15) alpha) 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 15 alpha-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-450(15) alpha fraction was 14.5 nmol/mg of protein. The Soret peak of the reduced cytochrome P-450-CO complex was 451 nm. The apparent subunit molecular weight of P-450(15) alpha was 48,000, and the protein appeared as only one major band on sodium dodecyl sulfate-polyacrylamide gels. The specific activity of testosterone 15 alpha-hydroxylation reconstituted with the purified P-450(15) alpha was 94 nmol/min/nmol of cytochrome P-450 and 1349 nmol/min/mg of protein, and these were about 65- and 1000-fold higher, respectively, than the activity of solubilized microsomes. The purified P-450(15) alpha exhibited high regioselectivity and stereospecificity for testosterone hydroxylation. More than 95% of the testosterone metabolites formed by the purified P-450(15) alpha was 15 alpha-hydroxytestosterone. Virtually 100% of mouse liver microsomal testosterone 15 alpha-hydroxylation activity can be accounted for by the purified P-450(15) alpha. The P-450(15) alpha fraction was able to catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-de-ethylation, aniline 4-hydroxylation, benzo(alpha)pyrene 3-hydroxylation, acetanilide 4-hydroxylation, and lauric acid (11 + 12)-hydroxylation at various turnover rates, indicating broad substrate specificity of the P-450(15) alpha for the oxidations of xenobiotics. This is in sharp contrast to high regioselectivity and stereospecificity for testosterone hydroxylation.

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