Dayani N, McNaught R W, Smith R G
Scott Department of Urology, Baylor College of Medicine, Houston, TX 77030.
J Steroid Biochem. 1988;30(1-6):219-24. doi: 10.1016/0022-4731(88)90096-9.
Two steroid binding states of an estrogen receptor each with different equilibrium constants (Kd values) Rx (Kd = 0.06 nM) and Ry (Kd = 0.8 nM) have been identified and characterized in the hen and estrogen-stimulated chick oviduct. A third non-estrogen binding form of the receptor, designated Rnb, has also been identified. These three forms of the receptor are interconvertible and appear to have a common molecular weight of approx. 66,000 under denaturing conditions. Hydroxytamoxifen binds preferentially and with high affinity to Rx (Kd 0.03 nM) and the conversion of Rx to Ry which is mediated by gamma phosphoryl group of ATP is also inhibitable by hydroxytamoxifen. Thus receptor interconversion, which may have general application to hormone action, potentially explains agonist/antagonist activity. The conversion of the non-estrogen binding form of the receptor (Rnb) to the lower affinity receptor (Ry) in chick oviduct cytosol is catalyzed by a reaction requiring the loss of the terminal phosphoryl moiety from ATP. There is a specific requirement for Mg2+. We now describe that ammonium sulfate fractionation of the cytosol allows the separation of the receptor entities from the "activating factor" (Fy) that catalyzes the conversion of Rnb to Ry. In the presence of gamma [32P]-ATP at 30 degrees C the purified non-steroid binding form of the receptor is phosphorylated. Phosphoamino acid analysis using Partisil-10 SAX anion exchange resin demonstrates that a serine is phosphorylated; and quantitation of the phosphorylation is indicative of one phosphoserine/receptor molecule. Treatment of the receptor with the partially purified activating factor to induce estradiol binding causes a dramatic reduction in phosphorylation.
在母鸡和雌激素刺激的鸡输卵管中，已鉴定并表征了雌激素受体的两种类固醇结合状态，每种状态具有不同的平衡常数（解离常数Kd值），即Rx（Kd = 0.06 nM）和Ry（Kd = 0.8 nM）。还鉴定出了受体的第三种非雌激素结合形式，称为Rnb。受体的这三种形式是可以相互转化的，并且在变性条件下似乎具有约66,000的共同分子量。羟基他莫昔芬优先且高亲和力地与Rx结合（Kd为0.03 nM），并且由ATP的γ磷酸基团介导的Rx向Ry的转化也可被羟基他莫昔芬抑制。因此，受体的相互转化可能普遍适用于激素作用，这可能解释了激动剂/拮抗剂活性。鸡输卵管细胞质中受体的非雌激素结合形式（Rnb）向低亲和力受体（Ry）的转化是由一个需要ATP末端磷酸基团丢失的反应催化的。对Mg2+有特定需求。我们现在描述，细胞质的硫酸铵分级分离允许将受体实体与催化Rnb向Ry转化的“激活因子”（Fy）分离。在30℃下存在γ[32P]-ATP时，纯化的受体非类固醇结合形式会被磷酸化。使用Partisil-10 SAX阴离子交换树脂进行的磷酸氨基酸分析表明，一个丝氨酸被磷酸化；磷酸化的定量分析表明每个受体分子有一个磷酸丝氨酸。用部分纯化的激活因子处理受体以诱导雌二醇结合会导致磷酸化显著减少。
