Kinetic properties of human placental aromatase. Application of an assay measuring 3H2O release from 1beta,2beta-3H-androgens.
Kinetic properties of human placental aromatase. Application of an assay measuring 3H2O release from 1beta,2beta-3H-androgens.
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人胎盘芳香酶的动力学特性。
DOI：
10.1016/s0021-9258(17)33694-3
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发表时间：
1976
期刊：
影响因子：
--
通讯作者：
S. Ohno
中科院分区：
文献类型：
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作者：
K. Reed;S. Ohno
通过测量3H2O释放来快速和灵敏地测定1β，2β-3 H-雄激素芳构化(Thompson，E.A.，Jr，和Siiteri，P.K.(1974)J.Biol.化学。249,5364-5372)进行了分析，以确定其对初始速率研究的适用性。研究发现，芳构化是冷冻干燥的胎盘微粒体催化的唯一反应，导致雄烯二酮和睾酮1或2位的氚损失。然而，氚被从雌激素产物的第二位移走，推测是在2-羟化反应中，但这并不意味着使用该分析来测量初始速率；因此，它被用来表征芳香酶的催化特性。K+、EDTA和二硫苏糖醇对冻干制剂的芳构化反应有促进作用，在pH 7.5~8.0范围内活性最高。在对这些因素进行优化的培养条件下，NADPH的表观Km约为1微米。雄烯二酮芳构化的最大速度超过睾酮，底物结合部位的亲和力较高，表观Km值分别为0.1um和0.4um。与雄激素底物的相互竞争实验表明，每种雄激素底物对对方的芳构化都有简单的竞争抑制作用；而且，每种雄激素底物的表观KI值与各自的Km值非常接近。雄酮-1，4，6-三烯-3，17-二酮竞争性地抑制雄烯二酮和睾酮的芳构化，表观KI在两种情况下都是0.2um。结论是这两种雄激素底物是在一个相同的位置芳香化的。
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.