Dunaway H E, Hutchens T W, Besch P K
J Chromatogr. 1985 Jun 26;327:221-35. doi: 10.1016/s0021-9673(01)81652-6.
The physicochemical properties of unliganded steroid receptor proteins remain largely unknown primarily due to receptor lability in the absence of specific ligand, especially during prolonged biochemical analyses. We have utilized high-performance size-exclusion chromatography (HPSEC) as a rapid means of investigating the structural properties of cytosolic estrogen receptor proteins in both the presence and absence of [3H]estradiol-17 beta. Cytosols prepared from immature calf uteri were analyzed by HPSEC on an Altex TSK-3000 SW column (600 mm X 7.5 mm I.D.) either before or after incubation with 10 nM [3H]estradiol-17 beta. Postcolumn detection of previously unliganded receptor was accomplished by incubation of fractions (0.38 ml) with 10 nM [3H]estradiol-17 beta for 2-18 h at 0 degrees C. Receptor-bound steroid was separated from free steroid by incubation with small pellets of hydroxylapatite. Nonspecific binding of [3H]estradiol-17 beta in parallel fractions was estimated using an unlabelled competitor (diethylstilbestrol) specific for the estrogen receptor. In low ionic strengths the receptor exists as a single, relatively stable, large form (retention time 34 min). The elution properties of this receptor configuration do not depend on bound steroid ligand. Analysis of receptor at elevated ionic strengths in the presence and absence of steroid ligand suggests that the salt-induced dissociation of receptor components to smaller forms (retention time 47 min) may be partially steroid-dependent. Characterization of receptor in 6 M urea demonstrates the presence of intermediate-sized receptor components (retention time 36-38 min). Analyses of receptor in 6 M urea-0.4 M potassium chloride suggests an inhibition of the more extensive salt-induced dissociation event seen in 0.4 M potassium chloride alone. Furthermore, the intermediate-sized receptor forms seen under these conditions (retention time 41-42 min) are generated in a steroid-dependent manner. The preparation of different molecular forms of biologically active, unliganded estrogen receptor by HPSEC should help further our investigations into the molecular mechanism(s) by which steroid hormones exert their receptor-mediated effects on target cells.
未结合配体的类固醇受体蛋白的物理化学性质在很大程度上仍不清楚，主要是因为在缺乏特异性配体时受体不稳定，尤其是在长时间的生化分析过程中。我们利用高效尺寸排阻色谱法（HPSEC）作为一种快速手段，来研究在存在和不存在[3H]雌二醇-17β的情况下，胞质雌激素受体蛋白的结构性质。从未成熟小牛子宫制备的胞质溶胶，在与10 nM [3H]雌二醇-17β孵育之前或之后，在Altex TSK-3000 SW柱（600 mm×7.5 mm内径）上通过HPSEC进行分析。通过将馏分（0.38 ml）与10 nM [3H]雌二醇-17β在0℃下孵育2 - 18小时，来完成对先前未结合配体的受体的柱后检测。通过与小颗粒羟基磷灰石孵育，将受体结合的类固醇与游离类固醇分离。使用对雌激素受体特异的未标记竞争剂（己烯雌酚）来估计平行馏分中[3H]雌二醇-17β的非特异性结合。在低离子强度下，受体以单一、相对稳定的大形式存在（保留时间34分钟）。这种受体构型的洗脱特性不依赖于结合的类固醇配体。在存在和不存在类固醇配体的情况下，对高离子强度下的受体进行分析表明，盐诱导受体成分解离为较小形式（保留时间47分钟）可能部分依赖于类固醇。在6 M尿素中对受体进行表征表明存在中等大小的受体成分（保留时间36 - 38分钟）。在6 M尿素 - 0.4 M氯化钾中对受体进行分析表明，单独在0.4 M氯化钾中观察到的更广泛的盐诱导解离事件受到抑制。此外，在这些条件下看到的中等大小的受体形式（保留时间41 - 42分钟）是以类固醇依赖的方式产生的。通过HPSEC制备具有生物活性的未结合配体的雌激素受体的不同分子形式，应该有助于进一步研究类固醇激素对靶细胞发挥其受体介导作用的分子机制。
