Currie R A, Cidlowski J A
J Steroid Biochem. 1982 Mar;16(3):419-28. doi: 10.1016/0022-4731(82)90055-3.
The physiochemical properties, size, shape, and surface charge, have been determined for the cytoplasmic glucocorticoid receptor complex (GR) in randomly growing HeLa S3 cells. Sucrose density gradient and gel exclusion chromatographic analysis have shown GR to undergo a marked reduction in size and shape when analyzed under increasing KCl concentration conditions. Analysis of GR prepared in a hypotonic buffer solution revealed a large, 7-8 S species with Ve/Vo ratio of 1.15, and a Stokes radius of congruent to 95 A. Increasing the KCl concentration in the analysis buffer to 0.15 resulted in reduction in GR size and shape to a 4.5 S species with the following properties: Ve/Vo = 1.38. Stokes radius = 69 A. and calculated Svedberg molecular weight = 132,000. A limit to the observed decrease in GR size and shape was obtained under hypertonic, 0.4 M KCl, conditions; a 3.75 S form was observed to elute from the gel exclusion column with a Ve/Vo = 1.40. Stokes radius = 65 A. and a calculated Svedburg molecular weight = 102,000. Exposure to higher KCl conditions, 0.6 M and 0.8 M, resulted in no further decrease in GR size or shape. Ion exchange chromatographic analysis of cytoplasmic GR revealed heterogeneous populations of GR with apparent differences in surface charge. GR binding to DEAE cellulose revealed a predominant form which eluted at 0.15 M KCl (Form I). Under both hypotonic and hypertonic conditions small populations of GR forms were observed to elute from DEAE at or approximately 0.1 M KCl (Form III), and or approximately 0.3 M KCl (Form II), respectively. Binding of GR to hydroxylapatite (HAP) confirmed the heterogenous nature of GR. Cytoplasmic GR partitioned into at least 3 forms om HAP. The predominant GR form eluted from HAP at 0.1 M K2HPO4 (Form I); secondary, and tertiary forms eluted at 0.125 M (Form II), and 0.15 M K2POH4 (Form III), respectively. Glucocorticoid receptors were observed to elute from columns of phosphocellulose, and DNA cellulose in the void volume. Two receptor forms were observed with isoelectric focusing of HeLa S3 cytoplasm in Sephadex IEF G-75 gels. The primary, and secondary species had pI values of 7.5 and 6.3, respectively. These results demonstrate that randomly growing HeLa S3 cells contain heterogeneous population of GR in the cytoplasmic compartment. Randomly growing HeLa S3 cell cytoplasm appears to contain two "unactivated" receptor species (Forms I and II) that differ in their overall surface charge properties; a third activated species (Form III) appears to arise from heat induced "activation" during cell homogenization, and is more basic in nature.
已对随机生长的海拉S3细胞中的细胞质糖皮质激素受体复合物（GR）的物理化学性质、大小、形状和表面电荷进行了测定。蔗糖密度梯度和凝胶排阻色谱分析表明，在不断增加的氯化钾浓度条件下进行分析时，GR的大小和形状会显著减小。在低渗缓冲溶液中制备的GR分析显示，有一个大的7-8S物种，其Ve/Vo比率为1.15，斯托克斯半径约为95埃。将分析缓冲液中的氯化钾浓度增加到0.15会导致GR大小和形状减小为4.5S物种，具有以下特性：Ve/Vo = 1.38，斯托克斯半径 = 69埃，计算得到的斯维德伯格分子量 = 132,000。在高渗的0.4M氯化钾条件下，观察到GR大小和形状减小存在一个限度；观察到一种3.75S的形式从凝胶排阻柱上洗脱下来，其Ve/Vo = 1.40，斯托克斯半径 = 65埃，计算得到的斯维德伯格分子量 = 102,000。暴露于更高的氯化钾条件（0.6M和0.8M）下，GR的大小或形状没有进一步减小。细胞质GR的离子交换色谱分析显示，GR群体存在异质性，表面电荷存在明显差异。GR与二乙氨基乙基纤维素的结合显示出一种主要形式，在0.15M氯化钾时洗脱（形式I）。在低渗和高渗条件下，分别观察到少量GR形式在约0.1M氯化钾（形式III）和约0.3M氯化钾（形式II）时从二乙氨基乙基纤维素上洗脱下来。GR与羟基磷灰石（HAP）的结合证实了GR的异质性。细胞质GR在HAP上至少分为3种形式。从HAP上洗脱的主要GR形式在0.1M磷酸氢二钾时（形式I）；次要和第三形式分别在0.125M（形式II）和0.15M磷酸二氢钾时（形式III）洗脱。观察到糖皮质激素受体从磷酸纤维素柱和DNA纤维素柱的空体积中洗脱下来。在Sephadex IEF G-75凝胶中对海拉S3细胞质进行等电聚焦观察到两种受体形式。主要和次要物种的pI值分别为7.5和6.3。这些结果表明，随机生长的海拉S3细胞在细胞质区室中含有异质性的GR群体。随机生长的海拉S3细胞质似乎含有两种“未活化”的受体物种（形式I和II），它们的整体表面电荷性质不同；第三种活化物种（形式III）似乎是在细胞匀浆过程中由热诱导的“活化”产生的，性质更碱性。
