Reed K C, Ohno S
J Biol Chem. 1976 Mar 25;251(6):1625-31.
The rapid and sensitive assay of 1beta,2beta-3H-androgen aromatization by measurement of 3H2O release (Thompson, E.A., Jr., and Siiteri, P.K. (1974) J. Biol. Chem. 249, 5364-5372) has been analyzed to determine its applicability to initial rate studies. It was found that aromatization is the sole reaction catalyzed by lyophilized placental microsomes that causes a loss of tritium from position 1 or 2 of androstenedione and testosterone. Tritium is, however, removed from position 2 of the estrogen products, presumably in 2-hydroxylation, but this does not invalidate use of the assay for initial rate measurements; it was therefore used to characterize the catalytic properties of aromatase. Aromatization by the freeze-dried preparation was stimulated by K+, EDTA, and dithiothreitol, and was maximally active at pH 7.5 TO 8.0. With incubation conditions optimized for these factors, the apparent Km for NADPH is approximately 1 muM. The maximum velocity of androstenedione aromatization exceeds that of testosterone, and the affinity of the substrate binding site is higher for the former substrate, the apparent Km values being 0.1 muM and 0.4 muM, respectively. Mutual competition experiments with the androgen substrates showed that each gives simple competitive inhibition of the other's aromatization; furthermore, the apparent Ki values for each are in close agreement with their respective Km values. Androst-1,4,6-triene-3,17-dione competitively inhibits the aromatization of both androstenedione and testosterone, the apparent Ki, in both cases being 0.2 muM. It is concluded that the two androgen substrates are aromatized at a single, identical site.
通过测量³H₂O释放来快速灵敏地检测1β,2β-³H-雄激素芳香化作用（小汤普森，E.A.，和西泰里，P.K.（1974年）《生物化学杂志》249卷，5364 - 5372页），已对其进行分析以确定其在初始速率研究中的适用性。结果发现，芳香化作用是冻干胎盘微粒体催化的唯一反应，该反应会导致雄烯二酮和睾酮1位或2位的氚丢失。然而，雌激素产物的2位会去除氚，推测是在2-羟基化过程中，但这并不影响该检测用于初始速率测量；因此，它被用于表征芳香化酶的催化特性。冻干制剂的芳香化作用受到钾离子、乙二胺四乙酸和二硫苏糖醇的刺激，在pH 7.5至8.0时活性最高。在针对这些因素优化孵育条件后，NADPH的表观Km约为1μM。雄烯二酮芳香化作用的最大速度超过睾酮，底物结合位点对前一种底物的亲和力更高，表观Km值分别为0.1μM和0.4μM。雄激素底物的相互竞争实验表明，每种底物都会对另一种底物的芳香化作用产生简单的竞争性抑制；此外，每种底物的表观Ki值与其各自的Km值非常接近。雄甾-1,4,6-三烯-3,17-二酮竞争性抑制雄烯二酮和睾酮的芳香化作用，两种情况下的表观Ki均为0.2μM。结论是，两种雄激素底物在单个相同位点进行芳香化。
