Notides A C, Hamilton D E, Auer H E
J Biol Chem. 1975 May 25;250(10):3945-50.
The rate of the 4 to 5 S estrogen-binding protein (EBP) in vitro transformation was measured by sucrose gradient centrifugation analysis. The temperature-activated 4 to 5 S EBP transformation is found to be highly reproducible without loss of [3H]estradiol-binding activity in a buffer containing an excess of [3H]estradiol, 40 mM Tris, 1 mM dithiothreitol, and 1 M urea at pH 7.4. The presence of [3H]estradiol is necessary for the 4 to 5 EBP transformation. A kinetic analysis of the 4 to 5 EBP transformation shows that it is a bimolecular reaction, the dimerization of the 4 S EBP with a second (similar or dissimilar) monomer or subunit. In buffers containing 0.4 M KCl the apparent second order rate constant is 2.3 plus or minus 0-2 times 10-7 M minus 1 min minus 1 at 28 degrees. The reaction is independent of the initial receptor concentration, suggesting that the 4 S EBP is dissociated into monomeric units in buffers of high ionic strength. In buffers without KCl or with 0.1 M KCl the apparent second order rate constant of receptor transformation increases with decreasing receptor concentration. This suggests that the 4 S EBP is associated weakly with another macromolecule (or macromolecules) in buffers of low ionic strength. The rate of 4 to 5 S EBP transformation shows a 200-fold increase between 0 and 35 degrees. The Arrhenius energy of activation is 21.3 kcal mol minus 1 in buffer without KCl and 19.1 kcal mol minus 1 in buffer with 0.4 M KCl. Following the temperature-activated dimerization, the avidity of binding between the 4 S EBP and its complementary subunit is increased, 0.4 M KCl can no longer cause dissociation, and the 5 S EBP dimer appears. This kinetic analysis indicates that the avidity of binding between the subunits of the estrogen receptor is modulated by estradiol, temperature, and ionic strength. We propose that these interactions of the estrogen receptor's subunits reflect conformational changes involved in receptor activation.
通过蔗糖梯度离心分析来测定4到5S雌激素结合蛋白（EBP）的体外转化率。发现在含有过量[³H]雌二醇、40mM Tris、1mM二硫苏糖醇和pH 7.4的1M尿素的缓冲液中，温度激活的4到5S EBP转化具有高度可重复性，且不会损失[³H]雌二醇结合活性。[³H]雌二醇的存在对于4到5 EBP的转化是必需的。对4到5 EBP转化的动力学分析表明，这是一个双分子反应，即4S EBP与第二个（相似或不同的）单体或亚基二聚化。在含有0.4M KCl的缓冲液中，在28℃时表观二级速率常数为2.3±0.2×10⁻⁷M⁻¹min⁻¹。该反应与初始受体浓度无关，这表明4S EBP在高离子强度缓冲液中解离为单体单元。在不含KCl或含有0.1M KCl的缓冲液中，受体转化的表观二级速率常数随受体浓度降低而增加。这表明4S EBP在低离子强度缓冲液中与另一种（或多种）大分子弱结合。4到5S EBP的转化率在0到35℃之间增加了200倍。在不含KCl的缓冲液中，阿累尼乌斯活化能为21.3 kcal mol⁻¹，在含有0.4M KCl的缓冲液中为19.1 kcal mol⁻¹。在温度激活的二聚化之后，4S EBP与其互补亚基之间的结合亲和力增加，0.4M KCl不再能导致解离，5S EBP二聚体出现。这种动力学分析表明，雌激素受体亚基之间的结合亲和力受到雌二醇、温度和离子强度的调节。我们提出，雌激素受体亚基的这些相互作用反映了受体激活过程中涉及的构象变化。
