Dissociation kinetics of the nuclear estrogen receptor.
Dissociation kinetics of the nuclear estrogen receptor.
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核雌激素受体的解离动力学。
DOI：
10.1021/bi00508a038
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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：建立了一种测定小牛子宫核雌激素受体解离的方法，小牛子宫核是从用[3H]雌二醇预孵育的组织切片中分离出来的。[3H]雌二醇与核雌激素受体的解离为单一的缓慢解离组分，k =（6.02 ± 0.26）× 10~（-3）min~（-1）。这种核结合的雌激素受体类似于盐提取的核雌激素受体，k = 8.8 X 10 - 3min-1，并且类似于细胞质雌激素受体的双相[3H]雌二醇解离过程的第二阶段，k = 8.68 X 10 - 3min-1。[3H]雌二醇解离的第二阶段以前被证明是细胞质雌激素受体的活化形式。在向子宫核中加入子宫胞质溶胶代替缓冲液后，核[3H]雌二醇-受体解离速率降低一半至2.83 ± 0.21 X 10 - 3min-1，表明子宫胞质溶胶中存在仅当其与染色质结合或与染色质相互作用时与受体相互作用并间接影响染色质-受体相互作用的因子。胞质溶胶因子是一种存在于子宫中的大分子，但在隔膜中不存在;它在40 ℃下稳定，但在100 ℃下10分钟不稳定，并在60 - 90%饱和度下用硫酸铵沉淀。当子宫胞液在29 ℃预孵育时，与分离的细胞核结合的雌激素受体的比例比在0 ℃时高5倍（9%对43%）。无论在0 ℃或29 ℃预孵育，吸附在分离的细胞核上的[3H]雌二醇受体的解离在29 ℃显示出相同的缓慢[3H]雌二醇解离动力学。存在0.01% Triton X-100或过高的未标记雌二醇浓度（10 - 100 µ）导致核雌激素受体的[3H]雌二醇解离动力学异常，表明高浓度类固醇可与受体上的疏水位点相互作用，对受体产生非特异性去污剂样作用。这些结果表明，核雌激素受体具有与胞浆雌激素受体活化形式相似的雌二醇解离动力学，子宫胞液中存在影响雌激素受体-核相互作用的蛋白因子。雌激素受体的细胞质非活性形式通过雌激素和温度依赖性反应的活化导致受体易位到细胞核（詹森等人，1968; Shyamala & Gorski，1969）。我们最近已经证明，雌激素受体的非活性和活性构象状态反映在两个亲和力状态的受体。[3H]雌二醇从细胞质雌激素受体的解离以两个指数分量发生。第一个或快速组分（ki）是由[3H]雌二醇从受体的非活性状态解离而产生的。细胞质雌激素受体的活化形式产生第二种或更慢的[3H]雌二醇解离组分（fc_2）。通过将两种形式的受体之间的平衡从非活性、低亲和力受体状态向受体的较高亲和力、活化状态驱动，E2结合调节受体活化过程（Weichman & Notides，1977，1979，1980）。[~3H]雌二醇解离动力学为受体的两种状态提供了灵敏的指示。在本文中，我们研究了[3H]雌二醇解离核雌激素受体的动力学，评估其与激活的细胞质雌激素受体的关系，并观察核雌激素受体-染色质的相互作用。
Willem de Boer1 and Angelo C. Notides** abstract: A method was developed for measuring the [3H] estradiol dissociation of the nuclear estrogen receptor from calf uterine nuclei that were isolated from tissue slices preincubated with [3H] estradiol. The [3H] estradiol dissoci-ation from the nuclear estrogen receptor was a single, slowly dissociating component at 29 C, k=(6.02±0.26) X 10~ 3 min™ 1. This nuclear-bound estrogen receptor was similar to the salt-extracted nuclear estrogen receptor, k= 8.8 X 10 “3 min™ 1, and to the second phase of the biphasic [3H] estradiol dissociation process of the cytoplasmic estrogen receptor, k= 8.68 X 10 “3 min'1. The second phase of the [3H] estradiol dissociation was previously shown to be the activated form of the cytoplasmic estrogen receptor. The nuclear [3H] estradiol-receptor dissociation rate was reduced by one-half to 2.83±0.21 X 10~ 3 min™ 1 after the addition of uterine cytosol to the uterine nuclei in place of the buffer, indicating the presence of a factor in the uterine cytosol that interacts with the receptor only when it is chromatin bound or interacts with the chromatin and indirectly influences the chromatin-receptor interaction. The cytosol factor is a macromolecule found in uterus, but not in diaphragm; it is stableat 40 C but unstable at 100 C for 10 min and is precipitated by ammonium sulfate at 60-90% saturation. Thefraction of the estrogen receptor that bound to isolated nuclei was 5-fold greater (9% vs. 43%) when the uterine cytosol was preincubated at 29 C than at 0 C. The dissociation of the [3H] estradiol receptor adsorbed to the isolated nuclei whether preincubated at 0 C or at 29 C showed the same, slow [3H] estradiol dissociation kinetics at 29 C. The presence of 0.01% Triton X-100 or excessively high unlabeled estradiol concentrations (10-100 µ) resulted in anomalous [3H] estradiol dissociation kinetics of the nuclear estrogen receptor, suggesting that highconcentrations of steroids can interact with hydrophobic sites on the receptor to produce a nonspecific detergent-like action on the receptor. These dataindicate that the nuclear estrogen receptor has estradiol dissociation kinetics similar to those of the activated form of the cytoplasmic estrogen receptor and there is present a protein factor in the uterine cytosol that affects the estrogen receptor-nuclear interaction. e activation of the cytoplasmic, nonactive form of the estrogen receptor by an estrogen-and temperature-dependent reaction results in the translocation of the receptor to the nucleus (Jensen et al., 1968; Shyamala & Gorski, 1969). We have recently demonstrated that the nonactive and active conformational states of the estrogen receptor are reflected in two affinity states of the receptor. The [3H] estradiol dissociation from the cytoplasmic estrogen receptor occurs as two exponential components. The first or fast component (ki) results from the [3H] estradiol dissociating from the nonactive state of the receptor. The activated form of the cytoplasmic estrogen receptor produces the second or slower [3H] estradiol dissociating component (fc_2). Estradiol binding modulates the receptor activation process by driving the equilibrium between the two forms of the receptor from the nonactive, low-affinity receptor state toward the higher affinity, activated state of the receptor (Weichman & Notides, 1977, 1979, 1980). The kinetics of [3H] estradiol dissociation provides a sensitive indicator of thetwo states of the receptor. In this paper we investigate the [3H] estradiol dissociation kinetics of the nuclear estrogen receptor, assess its relationship to the activated cy-toplasmic estrogen receptor, and observe nuclear estrogen receptor-chromatin interactions …