Sharma M C, Shapiro B H
Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, Philadelphia 19104-6048.
Arch Biochem Biophys. 1995 Jan 10;316(1):478-84. doi: 10.1006/abbi.1995.1063.
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.
从雌性CD-1小鼠中纯化出肝微粒体睾酮/雄烯二酮2α-羟化酶（即细胞色素P450(2)α）。通过在417nm处吸光的血红素以及细胞色素P450含量、对针对雌性特异性大鼠细胞色素P450 2C12的单克隆抗体的反应性和睾酮2α-羟化酶活性，监测从Fractogel、DEAE-琼脂糖凝胶和羟基磷灰石柱洗脱液中的蛋白质纯化情况。纯化后的细胞色素P450(2)α的催化活性具有高度的区域选择性和立体特异性，基本上仅限于睾酮和雄烯二酮的2α-羟化反应；分别占这些相应代谢产物的>96%和>92%。针对细胞色素P450(2)α的多克隆抗体对睾酮2α-羟化反应表现出剂量依赖性且非常有选择性的抑制作用。纯化后的细胞色素P450(2)α组分的比细胞色素P450含量为12.06nmol/mg蛋白质。纯化后的蛋白质的比睾酮2α-羟化酶活性为14nmol/min/nmol细胞色素P450，约为相应微粒体的60倍。细胞色素P450(2)α的表观亚基分子量为51,000，在十二烷基硫酸钠聚丙烯酰胺凝胶上该蛋白质呈现为一条带。氨基末端序列分析表明细胞色素P450(2)α是小鼠细胞色素P450 2d家族的成员。
