HYDROXYSTEROID DEHYDROGENASES IN HUMAN PLACENTAL CYTOSOL

我们发现17b-雌二醇脱氢酶(17-ED)和 20A-羟基类固醇脱氢纤维素酶(20-HSD)活性的协同纯化 人足月胎盘的胞浆。此外，胎盘分级研究 将这两种活动定位到105,000×g胞浆。最后，亲和力 使用16a-溴乙酰氧基孕酮的烷基化研究表明 氧化还原酶活性存在于单一蛋白质的一个活性部位。 因此，我们假设类固醇17b-和20a-氧化还原是有效的。 在单一酶上的单一催化(活性)位置。亲和力 烷基化类似物和酶生成的亲和烷化剂(“自杀” 底物“)雌烯(18碳)和孕烯(21碳)系列， 亲和标记核苷酸类似物将用于同源 酶的探索：1.两种酶的活性是否同时存在 并在实验条件下进行相同的修改以计算出 灭活和重新激活酶(S)。2.是否对应 “亲和标记探针”定位酶内相同的氨基酸。 活性中心，因为雌激素和孕激素的类似物确实 在一个蛋白质上的一个共同的位置上相互作用。这些研究将允许 对双功能酶活性论题的肯定或否定。这个 拟议的研究将从结构上表征酶(S)，通过 调节胎儿-胎盘-子宫单位内的类固醇激素水平， 被认为参与了引发分娩的事件。他们会 进一步阐明允许结合和催化的结构成分 在酶活性中心的底物。如果17-ED和20-HSD活动 在人类胎盘胞浆中确实存在于单一位置，这将 为人类的双功能提供了第一个完整的证据， 类固醇激素的立体特异性氧化还原。

We have found that 17b-Estradiol Dehydrogenase (17-ED) and 20a-Hydroxysteriod Dehydrogrenase (20-HSD) activites co-purify from the cytosol of human term placenta. Further, placental fractionation studies localize both activities to the 105,000 x g cytosol. Finally, affinity alkylation studies using 16a-bromoacetoxyprogesterone suggest that both oxidoreductase activities exist at one active site on a single protein. Thus, we hypothesize that steroid 17b- and 20a-oxidoreduction are effected at a single catalytic (active) site on a single enzyme. Affinity alkylating analogs and enzyme-generated affinity alkylators ("suicide substrates") of the estrene (18 carbon) and pregnene (21 carbon) series, and affinity labeling nucleotide analogs will be used with homogeneous enzyme to explore: 1. Whether both enzyme activities are simultaneously and identically modified during experimental conditions calculated to inactivate and reactivate the enzyme(s). 2. Whether corresponding "affinity radiolabeling probes" map identical amino acids within the enzyme active center because both the estrogen and progestin analogs are indeed interacting at a common locus on one protein. These studies will allow affirmation or denial of the bifunctional enzyme activity thesis. The proposed studies will structurally characterize enzyme(s) which, by regulating steroid hormone levels within the fetal-placental-uterine unit, are believed to participate in the events which initiate labor. They will further elucidate structural components which permit binding and catalysis of substrates at an enzyme active center. If 17-ED and 20-HSD activities in human placental cytosol do indeed reside at a single site, this will provide the first well-documented evidence in man for bifunctional, stereospecific oxidoreduction of steriod hormones.