Shimizu Y, Yarborough C, Osawa Y
Endocrine Biochemistry Department, Medical Foundation of Buffalo Research Institute, NY 14203.
J Steroid Biochem Mol Biol. 1993 Mar;44(4-6):651-6. doi: 10.1016/0960-0760(93)90274-z.
In order to better understand the function of aromatase, we carried out kinetic analyses to assess the ability of natural estrogens, estrone (E1), estradiol (E2), 16 alpha-OHE1, and estriol (E3), to inhibit aromatization. Human placental microsomes (50 micrograms protein) were incubated for 5 min at 37 degrees C with [1 beta-3H]testosterone (1.24 x 10(3) dpm 3H/ng, 35-150 nM) or [1 beta-3H,4-14C]androstenedione (3.05 x 10(3) dpm 3H/ng, 3H/14C = 19.3, 7-65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1 beta-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The Ki of E1, E2, 16 alpha-OHE1, and E3 for testosterone aromatization was 1.5, 2.2, 95, and 162 microM, respectively, where the Km of aromatase was 61.8 +/- 2.0 nM (n = 5) for testosterone. The Ki of E1, E2, 16 alpha-OHE1, and E3 for androstenedione aromatization was 10.6, 5.5, 252, and 1182 microM, respectively, where the Km of aromatase was 35.4 +/- 4.1 nM (n = 4) for androstenedione. These results show that estrogen inhibit the process of androgen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogen binds to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.
为了更好地理解芳香化酶的功能，我们进行了动力学分析，以评估天然雌激素雌酮（E1）、雌二醇（E2）、16α-羟基雌酮1（16α-OHE1）和雌三醇（E3）抑制芳香化作用的能力。将人胎盘微粒体（50微克蛋白质）在37℃下与[1β-3H]睾酮（1.24×10³ dpm ³H/纳克，35 - 150纳摩尔）或[1β-3H,4-¹⁴C]雄烯二酮（3.05×10³ dpm ³H/纳克，³H/¹⁴C = 19.3，7 - 65纳摩尔）作为底物在NADPH存在的情况下孵育5分钟，分别加入和不加入作为假定抑制剂的天然雌激素。通过从底物1β位释放到水中的氚来评估芳香化酶活性。天然雌激素对雄激素芳香化表现出竞争性产物抑制作用。E1、E2、16α-OHE1和E3对睾酮芳香化的Ki分别为1.5、2.2、95和162微摩尔，其中芳香化酶对睾酮的Km为61.8±2.0纳摩尔（n = 5）。E1、E2、16α-OHE1和E3对雄烯二酮芳香化的Ki分别为10.6、5.5、252和1182微摩尔，其中芳香化酶对雄烯二酮的Km为35.4±4.1纳摩尔（n = 4）。这些结果表明雌激素抑制雄激素芳香化过程，并表明天然雌激素在体内通过产物抑制机制调节自身合成。由于天然雌激素作为竞争性抑制剂与人胎盘芳香化酶P - 450复合物的活性位点结合，天然雌激素可能会被芳香化酶进一步代谢。这表明人胎盘雌激素2 - 羟化酶活性是由芳香化酶细胞色素P - 450的活性位点催化的，这也与孕期母体血浆中儿茶酚雌激素水平升高的事实相符。雄激素和雌激素的相对亲和力及浓度将通过芳香化酶控制雌激素和儿茶酚雌激素的生物合成。
