Kellis J T, Vickery L E
J Biol Chem. 1987 Mar 25;262(9):4413-20.
Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.
催化雄激素转化为雌激素的芳香化酶细胞色素P-450是从人胎盘微粒体中纯化得到的。该酶用胆酸钠提取，经硫酸铵沉淀分级分离，并在其底物雄烯二酮和非离子去污剂诺乃洗涤剂P-40存在下进行柱层析。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析时，该制剂呈现一条主要条带，其细胞色素P-450的比含量为11.5 nmol/mg蛋白质。纯化后的酶表现出细胞色素P-450铁离子和亚铁离子形式的典型光谱特性。完整的酶活性可用兔肝微粒体细胞色素P-450还原酶和诺乃洗涤剂P-40重建。纯化后的芳香化酶细胞色素P-450在雄烯二酮、19-羟基雄烯二酮和19-氧代雄烯二酮的芳香化反应中表现出与完整微粒体中酶相似的催化特性。睾酮和16α-羟基睾酮以与雄烯二酮相似的最大速率进行芳香化反应，所有底物的相对亲和力与微粒体中观察到的一致。我们已制备了针对纯化酶的兔抗体，这些抗体在免疫印迹上显示出相当高的特异性和敏感性。
