Sheean L A, Meigs R A
Steroids. 1981 Aug;38(2):211-20. doi: 10.1016/0039-128x(81)90035-0.
Suitable incubation conditions were developed for reduced pyridine nucleotide protection and regeneration to permit quantitative assessment of the NADPH requirement for steroid aromatization by human placental microsomes. 10 mM dithiothreitol was found to protect NADP(H) from microsomal nucleotide pyrophosphatase and 2 mM nicotinamide mononucleotide was utilized to control nucleotide glycohydrolase activity. Under these assay conditions, the initial rates of aromatization obtained with restricted NADPH levels were critically dependent upon both the amount and the source of exogenous NADPH-regenerating dehydrogenase system. With excess Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, an apparent Km for NADPH of 0.20 microM was observed for aromatization which is significantly below all previous estimates of the NADPH requirement and which is at greatest only one-tenth the Km value for NADPH utilization by NADPH-cytochrome c reductase. These findings suggest a potential regulatory role for both NADPH-generating and NADPH-accepting enzymes in the support of estrogen biosynthesis.
为减少吡啶核苷酸的保护和再生以允许对人胎盘微粒体类固醇芳香化所需的NADPH进行定量评估，开发了合适的孵育条件。发现10 mM二硫苏糖醇可保护NADP(H)免受微粒体核苷酸焦磷酸酶的作用，并且使用2 mM烟酰胺单核苷酸来控制核苷酸糖苷水解酶的活性。在这些测定条件下，在有限的NADPH水平下获得的芳香化初始速率严重依赖于外源NADPH再生脱氢酶系统的量和来源。使用过量的肠系膜明串珠菌葡萄糖-6-磷酸脱氢酶，观察到芳香化的NADPH表观Km为0.20 microM，这明显低于先前所有对NADPH需求的估计，并且最大仅为NADPH-细胞色素c还原酶利用NADPH的Km值的十分之一。这些发现表明，NADPH生成酶和NADPH接受酶在支持雌激素生物合成中可能具有调节作用。
