The study of the regulating factor on cooperative steroid binding with estrogen receptor

雌激素与去连接受体蛋白二聚体的相互作用：化学交联和铜(II)离子处理后，高亲和力构象的稳定和合作类固醇结合的丧失。1.在没有结合配体的情况下，去连接的雌激素受体蛋白被定量地转变为其二聚体(即134 kDa)，DNA结合构型。用化学交联法对去连接受体的二聚体结构进行表征，未连接的DNA结合受体蛋白在没有配体结合的情况下，用[~3H]他莫昔芬氮杂环丙啶共价标记，在有无还原剂的情况下变性后用十二烷基硫酸钠-聚丙烯酰胺梯度凝胶电泳法进行鉴定。3.在100 MU的铜(II)离子存在下，观察到类似的结果。而在相同浓度下，锌(II)离子对雌激素受体蛋白没有影响。这些结果表明，在没有结合激素和共价化学交联后，雌激素受体蛋白作为二聚体是稳定的。去连接的雌激素受体蛋白与化学交联剂和铜(II)离子的相互作用消除了类固醇的协同结合。此外，我们还认为，铜离子可能是影响雌激素受体与类固醇协同结合的因素之一。

Interaction of estrogen with unliganded receptor protein dimers : Stabilization of high affinity conformation and a loss of cooperative steroid binding after chemical crosslinking and treatment with Cu (II) ions.1.The unliganded estrogen receptor proteins were shown to be quantitatively transformed to their dimeric (i.e., 134 kDa), DNA binding configuration ever in the absence of bound ligand. The dimeric structure of the unliganded receptor was demomstrated by chemical crosslinking ; unliganded, DNA-binding receptor proteins, covalently crosslinked without prior ligand binding, were covalently labeled with [^3H] tamoxifen aziridine and evaluated by SDS-polyacrylamide gradient gel electrophoresis after denaturation in the presence and absence of reducing agents.2.The steroid-binding characteristics of unliganded receptor proteins were evaluated in the presence and absence of receptor-bound DNA.Positive cooperativity (Hill coefficient>1.3) was observed for the unliganded receptor protein at all concentrations above 1 nM.However, after covalent crosslinking with dithiobis (succinimidylpropionate) the cooperative steroid binding was completely eliminated (Hill coefficient=1.07).3.Similar results were obtained in the presence of 100 muM Cu(II) ions.Zn(II) ions, however, were without effect at equivalent concentrations.These results demonstrate that estrogen receptor proteins were stable as dimers even in the absence of bound hormone and after covalently chemical crosslinking. The interaction of unliganded estrogen receptor proteins with chemical crosslinking reagents and Cu (II) ions eliminated the cooperative steroid binding. Furthermore, it is suggested that Cu (II) ions influence as one of the regurating factor on cooperative steroid binding with estrogen receptor.