Smith R G, Schwartz R J
Biochem J. 1979 Nov 15;184(2):331-43. doi: 10.1042/bj1840331.
An oestrogen receptor was isolated, characterized and purified from the nuclear fraction of the hen oviduct. The receptor sediments at 4.6 S on glycerol gradients, has an equilibrium dissociation constant (Kd) of 1.1 X 10(-10)M, an association constant (ka) of 1.4 X 10(-6) M-1.S-1, and a dissociation constant (kd) of 5 x 10(-5) s-1. The receptor chromatographed from DEAE-cellulose as a single peak at 0.15 M-KCl and was not retained by phosphocellulose. Polyacrylamide-gel electrophoresis of the receptor in the presence of sodium dodecyl sulphate demonstrated two subunits with apparent mol.wts. of 74000 and 80000. The overall purification achieved was 90000-fold by using a combination of cell fractionation, (NH4)2SO4 fractionation and affinity chromatography. This represents the first separation, isolation and purification of the highest-affinity binding component (Kd 10(-10)M) of two high-affinity oestrogen-binding proteins present in both chick and hen oviduct cytosol and nuclei. To examine directly the effect of the purified receptor on transcription a reconstituted cell-free system was used, which contained the receptor--oestradiol complex, Escherichia coli RNA polymerase, rifampicin and chromatin prepared from hormone-withdrawn chick tissue. The receptor-hormone complex at a concentration of 0.1 nM stimulated transcription of oviduct chromatin by promoting an increase of 14000 sites for RNA-chain initiation, which is similar to the number of additional sites measured in the oviducts of diethylstilboestrol-stimulated immature chicks [Tsai, Schwartz, Tsai & O'Malley (1975) J. Biol. Chem. 250, 5165-5174]. Oestradiol alone had no effect on transcription. Thus the data demonstrate that the purified nuclear oestradiol-receptor complex can regulate gene transcription in vitro in a manner similar to that observed in target cells in vivo.
从母鸡输卵管的核部分中分离、鉴定并纯化了一种雌激素受体。该受体在甘油梯度上的沉降系数为4.6S，平衡解离常数（Kd）为1.1×10⁻¹⁰M，缔合常数（ka）为1.4×10⁻⁶M⁻¹·s⁻¹，解离常数（kd）为5×10⁻⁵s⁻¹。该受体在0.15M - KCl浓度下从DEAE - 纤维素柱上以单一峰形式被洗脱，且不被磷酸纤维素保留。在十二烷基硫酸钠存在下对该受体进行聚丙烯酰胺凝胶电泳，显示出两个亚基，其表观分子量分别为74000和80000。通过细胞分级分离、硫酸铵分级分离和亲和层析相结合的方法，总体纯化倍数达到了90000倍。这代表了首次分离、鉴定和纯化出存在于雏鸡和母鸡输卵管细胞质及细胞核中的两种高亲和力雌激素结合蛋白中亲和力最高的结合成分（Kd为10⁻¹⁰M）。为了直接检测纯化后的受体对转录的影响，使用了一个重组的无细胞系统，该系统包含受体 - 雌二醇复合物、大肠杆菌RNA聚合酶、利福平以及从摘除激素的雏鸡组织制备的染色质。浓度为0.1nM的受体 - 激素复合物通过促使RNA链起始位点增加14000个来刺激输卵管染色质的转录，这与在己烯雌酚刺激的未成熟雏鸡输卵管中测得的额外位点数量相似[蔡、施瓦茨、蔡和奥马利（1975年）《生物化学杂志》250，5165 - 5174]。单独的雌二醇对转录没有影响。因此，这些数据表明纯化后的细胞核雌二醇 - 受体复合物能够在体外以类似于在体内靶细胞中观察到的方式调节基因转录。
