Hyder S M, Wittliff J L
James Graham Brown Cancer Center, University of Louisville, KY 40292.
J Chromatogr. 1989 Aug 4;476:455-66. doi: 10.1016/s0021-9673(01)93889-0.
High-performance hydrophobic interaction chromatography (HPHIC) was used to separate and characterize two molecular forms of estrogen receptor with a SynChropak propyl hydrophobic column (300 A pore size). The linear gradient utilized earlier with a polyether-bonded column (2 to 0 M) ammonium sulfate in 40 min, gave poor resolution with the propyl column. However, resolution was maximized with either an initial ammonium sulfate concentration of 1 M (40-min gradient) or with a two-phase gradient (2 to 0.5 M in 10 min, 0.5 to 0 M in 30 min). This indicated that the propyl column was more hydrophobic than the polyether column. Estrogen receptor separated into two isoforms, either in the presence [MI, retention time (tR) = 13-14 min; MII, tR = 20-21 min] or absence (I, tR = 21-23 min; II, tR = 31-33 min) of the estrogen receptor stabilizing reagent, sodium molybdate. Similar isoforms were observed in cytosols from human breast tumors, uterus, and MCF-7 breast cancer cells. Unlike others, MCF-7 estrogen receptor did not show MI. Since MCF-7 cells contain 90,000 dalton heat shock proteins (HSP-90), HSP-90 is probably not directly involved in MI formation. Sodium molybdate selectively interacted with isoform II and converted it to MI. All isoforms appeared to be high-molecular-weight proteins (greater than 60 A) when subsequently analyzed by high-performance size-exclusion chromatography. Interestingly, when estrogen receptor was immobilized on the stationary phase, no change was detected in either hydrophobicity or steroid-binding capacity. After 16-18 h, immobilized receptor was eluted with a slightly longer tR. During incubation on the column, component MI was converted into I and/or II. HPHIC appears to be a rapid, yet gentle procedure for isolating large receptor complexes in significant quantities with high recoveries. This allows one to discern the complicated structure-function relationships of estrogen receptor and associated non-receptor proteins and provides information about the on-column behavior of complex proteins.
采用高效疏水作用色谱法（HPHIC），使用SynChropak丙基疏水柱（孔径300 Å）对雌激素受体的两种分子形式进行分离和表征。早期在聚醚键合柱上使用的线性梯度（40分钟内硫酸铵浓度从2 M降至0 M），在丙基柱上的分离效果较差。然而，当硫酸铵初始浓度为1 M（40分钟梯度）或采用两相梯度（10分钟内从2 M降至0.5 M，30分钟内从0.5 M降至0 M）时，分离效果最佳。这表明丙基柱比聚醚柱的疏水性更强。雌激素受体可分离为两种异构体，无论是否存在雌激素受体稳定剂钼酸钠，分别为[MI，保留时间（tR）= 13 - 14分钟；MII，tR = 20 - 21分钟] 或 （I，tR = 21 - 23分钟；II，tR = 31 - 33分钟）。在人乳腺肿瘤、子宫和MCF - 7乳腺癌细胞的胞质溶胶中也观察到了类似的异构体。与其他情况不同，MCF - 7雌激素受体未显示出MI。由于MCF - 7细胞含有90,000道尔顿的热休克蛋白（HSP - 90），因此HSP - 90可能不直接参与MI的形成。钼酸钠与异构体II选择性相互作用并将其转化为MI。随后通过高效尺寸排阻色谱分析，所有异构体似乎都是高分子量蛋白质（大于60 Å）。有趣的是，当雌激素受体固定在固定相上时，其疏水性或类固醇结合能力均未检测到变化。16 - 18小时后，固定化受体的洗脱保留时间略长。在柱上孵育期间，组分MI转化为I和/或II。HPHIC似乎是一种快速且温和的方法，可大量分离具有高回收率的大型受体复合物。这使得人们能够辨别雌激素受体与相关非受体蛋白之间复杂的结构 - 功能关系，并提供有关复合蛋白在柱上行为的信息。
