Dissociation kinetics of the estrogen receptor immobilized by hydroxylapatite.
Dissociation kinetics of the estrogen receptor immobilized by hydroxylapatite.
复制标题
羟基磷灰石固定的雌激素受体的解离动力学。
DOI：
10.1021/bi00508a037
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发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
Notides,AC
中科院分区：
文献类型：
--
作者：
deBoer,W;Notides,AC
Willem de Boer 1和Angelo C.通知 ** 摘要：将小牛子宫内膜溶出液中的雌激素受体吸附到羟基磷灰石上，为研究雌激素受体-核复合物的[~ 3 H]雌二醇解离动力学提供了一个更简单、更可控的模型。[3 H]雌二醇在29 ℃下从羟基磷灰石固定的雌激素受体上的解离显示出双相动力学，分别具有受体非活化和活化状态的快和慢组分特征。用雌二醇交换法测得的快组分的解离速率常数（fc_ [= 0.059± 0.004min ~（-1））比胞液中游离受体的解离速率常数（fc_[= 0.059± 0.004min ~（-1））慢三分之一。0.162±0.011 min-1）。当受体与羟基磷灰石结合时，快速组分的幅度为17%，当在胞质溶胶中时为26%。[~ 3 H]雌二醇解离速率的降低和快成分的减少表明受体与羟基磷灰石的结合促进了雌激素受体的激活。在29 ℃时，慢组分的解离速率常数L· 2与受体与羟基磷灰石结合的情况没有显著差异（k.游离于胞浆中（fc_2= 7.25±0.46 × 10 ~ 3 min ~（-1）），或在与羟基磷灰石结合前被预活化（k_2= 6.93±0.52 × 10~ 3 min ~（-1））。当通过交换测量时，[3 H]雌二醇从与羟基磷灰石结合的活化雌激素受体的解离速率不受150- 1500倍过量雌二醇的影响（0.5-5 µ雌二醇），虽然大量雌二醇（10或200 μ l）使解离速率增加（k分别为12.2 × 10 ~（-3）min ~（-1）和0.088 min ~（-1））。1 µ雌二醇和200 µ孕酮的组合增加了解离速率（k= 0.067 min-1），而单独使用200 µ孕酮并不取代结合的[3 H]雌二醇或影响受体稳定性。高浓度的类固醇对受体有非特异性的洗涤作用。Triton X-100（0.01%）显著增加[3 H]雌二醇解离（k= 0.11 min-1）。使用Triton X-100分离含有受体的细胞核会影响受体的[3 H]雌二醇解离动力学，并可能影响受体与细胞核的相互作用。羟基磷灰石固定的雌激素受体在研究雌激素受体激活的动力学和机制方面是有用的，并且作为理解雌激素受体-核相互作用的工具。已经综述了特异性雌激素结合蛋白或受体的存在以及受体与由雌激素引发的组织反应的关系（Gorski等人，
Willem de Boer1 and Angelo C. Notides** abstract: The estrogen receptor from the calf uterinecytosol was adsorbed to hydroxylapatite to provide a simpler and more controlled model for investigating in vitro the [3H] estradiol dissociation kinetics of the estrogen receptor-nuclear complex. The dissociation of [3H] estradiol at 29 C from the estrogen receptor immobilized by hydroxylapatite showed biphasic kinetics with fast and slow components characteristic of the nonactivated and activated states of the receptor, respectively. The dissociation rate constant of the fast component (fc_ [= 0.059±0.004 min'1), measured by exchange with estradiol, was one-third slower than that for the receptor free in the cytosol (&_!= 0.162±0.011 min'1). The magnitude of the fast component was 17% when the receptor was bound to hydroxylapatite and 26% when in cytosol. The decrease in the rate of [3H] estradiol dissociationand the reduction in the magnitude of the fast component suggest that receptor binding to hydroxylapatite facilitates activation of the estrogen re-ceptor. The dissociation rate constant of the slower component, L· 2, at 29 C was not significantly different whetherthe re-ceptor was bound to hydroxylapatite (k. 2= 6.62±0.26 X 10r3 min'1), free in cytosol (fc_2= 7.25±0.46 X 10~ 3 min'1), or preactivated before being bound to the hydroxylapatite (k_2= 6.93±0.52 X 10~ 3 min'1). The [3H] estradiol dissociation rate from the activated estrogen receptor bound to hydroxylapatite, when measured by exchange, was not influenced by a 150-to 1500-fold excess of estradiol (0.5-5 µ estradiol), although massive quantities (10 or 200 µ) of estradiol in-creased the dissociationrate (k= 12.2 X 10'3 min'1 and 0.088 min" 1, respectively). Combinations of 1 µ estradiol plus 200 µ progesterone increased the dissociation rate (k= 0.067 min'1), while 200 µ progesterone alone did not displace the bound [3H] estradiol or affect receptor stability. High con-centrations of steroids exerted a nonspecific detergent action on the receptor. Triton X-100 (0.01%) markedly increased [3H] estradiol dissociation (k= 0.11 min'1). The use of Triton X-100 to isolate nuclei containing receptors affects the [3H] estradiol dissociation kinetics of the receptor and may influence the receptor’s interactions with the nucleus. The hydroxylapatite-immobilized estrogen receptor is useful in investigating the kinetics and mechanism of estrogen receptor activation and serves as a tool for understanding the estrogen receptor-nuclear interactions. e existence of a specific estrogen-binding protein or re-ceptor and the receptor’s relationship to the tissue responses initiated by the estrogens have been reviewed (Gorski et al.,