Purification and Characterization of Constituent Testosterone 2α-Hydroxylase (Cytochrome P4502α) from Mouse Liver

Hepatic microsomal testosterone/androstenedione 2α-hydroxylase (i.e., cytochrome P4502α was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-sephacel, and hydroxylapatite columns at heme absorbing 417 nm and by cytochrome P450 content, reactivity to a monoclonal antibody against female-specific rat cytochrome P450 2C12, and testosterone 2α-hydroxylase activity. The catalytic activity of the purified cytochrome P4502α, exhibiting a high degree of regioselectivity and stereospecificity, was basically restricted to the 2α-hydroxylation of testosterone and androstenedione; representing >96% and >92% of these respective metabolites. Polyclonal antibodies against cytochrome P4502α exhibited a dose-dependent and very selective inhibition of testosterone 2α-hydroxylation. The specific cytochrome P450 content of the purified cytochrome P4502α fraction was 12.06 nmol/mg protein. The specific testosterone 2α-hydroxylase activity of the purified protein was 14 nmol/min/nmol cytochrome P450, which was about 60-fold higher than the respective microsomes. The apparent subunit molecular weight of cytochrome P4502α was 51,000 and the protein appeared as a single band on sodium dodecyl sulfate polyacrylamide gels. The amino-terminal sequence analysis indicates that cytochrome P4502α is a member of the murine cytochrome P450 2d family.