Purification and characterization of constituent testosterone 2 alpha-hydroxylase (cytochrome P450(2)alpha) from mouse liver.
Hepatic microsomal testosterone/androstenedione 2 alpha-hydroxylase (i.e., cytochrome P450(2)alpha) 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 2 alpha-hydroxylase activity. The catalytic activity of the purified cytochrome P450(2)alpha, exhibiting a high degree of regioselectivity and stereospecificity, was basically restricted to the 2 alpha-hydroxylation of testosterone and androstenedione; representing > 96% and > 92% of these respective metabolites. Polyclonal antibodies against cytochrome P450(2)alpha exhibited a dose-dependent and very selective inhibition of testosterone 2 alpha-hydroxylation. The specific cytochrome P450 content of the purified cytochrome P450(2)alpha fraction was 12.06 nmol/mg protein. The specific testosterone 2 alpha-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 P450(2)alpha 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 P450(2)alpha is a member of the murine cytochrome P450 2d family.
Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, Philadelphia 19104-6048.
M C
MC
Sharma
B H
BH
Shapiro
D000595:Amino Acid Sequence; D000728:Androgens / Q000378:metabolism; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003429:Cross Reactions; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D005260:Female; D006900:Hydroxylation; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism*; D015262:Xenobiotics / Q000378:metabolism
D000728:Androgens; D007527:Isoenzymes; D015262:Xenobiotics; D013739:Testosterone; D000735:Androstenedione; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase