Structure of the mouse glucocorticoid receptor: rapid analysis by size-exclusion high-performance liquid chromatography.
Structure of the mouse glucocorticoid receptor: rapid analysis by size-exclusion high-performance liquid chromatography.
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小鼠糖皮质激素受体的结构：通过尺寸排阻高效液相色谱进行快速分析。
DOI：
10.1021/bi00356a038
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Vedeckis,WV
中科院分区：
文献类型：
--
作者：
LaPointe,MC;Chang,CH;Vedeckis,WV
路易斯安那州新奥尔良市路易斯安那州立大学医学中心生物化学和分子生物学教研室，70112-10-7收到Ą985；修订稿件收到1985-12-2日摘要：用凝胶排斥高效液色谱分离从小鼠ATT-20细胞中提取的转化糖皮质激素受体(GC-R)中未转化的糖皮质激素受体(GC-R)。以200 mM磷酸二氢钾为洗脱剂，可在15-20分钟内实现GC-R各形态的有效分离。未转化的GC-R从Stokes半径为8.2-8.6 nm的色谱柱洗脱出来，钼酸盐稳定的GC-R、纯化的未转化的GC-R和交联的胞质GC-R也是如此。GC-R经硫酸铵沉淀、KCl处理或G-25层析洗脱后，其相对分子质量为5.7-6 nm。此外，用0.3Mkc1或2 mM钨酸钠从细胞核中提取的GC-R或经两次DNA-纤维素层析纯化的GC-R的Rs为5.5-6.3 nm。在存在或不存在20 mM Na2Mo04的情况下，这些数据都是相同的，这表明钼酸盐不会导致聚集产生比天然受体更大的Rs值。胞浆的立管蔗糖梯度超速离心可产生三种GC-R形式：9.1·S、5.2·S和3.8S。GC-R形式的顺序分析采用高效液相色谱法和立管转子超速离心法，反之亦然，可以在很短的时间内(共2-3小时)测定分子量。从含有Mo042™的蔗糖梯度中提取的未转化的9.IS物种的Rs为8.6 nm(Mr 310K-340K)，而5.2个S和3.8个S的Rs均为6 nm(Mr分别为115K-140K和96K-100K)。相反，如果通过KCI处理或G-25层析产生的6 nm形式的受体从高效液相色谱中汇集，那么它随后以3.54.6 S物种的形式沉淀在含有Mo042“的梯度上。这可能表明，5.2 S转化的GC-R是不稳定的，并在高效液相色谱过程中解离成其成分，尽管过程很快。根据计算的三种GC-R形式的分子量，表明未转化的寡聚体GC-R在转化过程中解离为单体亚基，中间形式不是相同的单体亚基的二聚体(jTLucocorticoid是与反应细胞中特定受体蛋白相互作用的生理调节因子(Yamamoto&Alberts，1976))。激素与受体结合后，糖皮质激素受体(GC-R)1复合体转变为DNA结合部分。转化的受体与DNA上的受体位置相互作用，影响特定基因的转录。
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112 Received October 7, Ą985; Revised Manuscript Received December 2, 1985 abstract: Gel-exclusion high-performanceliquid chromatography (HPLC) has been used to separate the untransformed from the transformed glucocorticoid receptor (GC-R) extracted from mouse AtT-20 cells. With 200 mM potassium phosphate as the eluent, an efficient separation of the forms of the GC-R is attained in 15-20 min. The untransformed cytosolicGC-R elutes from the columnwith a Stokes radius {Rs) of 8.2-8.6 nm, as do the molybdate-stabilized GC-R, the purified untransformed GC-R, and the cross-linked cytosolic GC-R. GC-R transformed in vitro by either ammonium sulfate precipitation, KC1 treatment, or G-25 chromatography elutes with an Rs of 5.7-6 nm. Also, GC-R extracted from the nucleus with either 0.3 Mkc1 or 2 mM sodium tungstate, or purified by two cycles of DNA-cellulose chromatography, has an Rs of 5.5-6.3 nm. The data are identical either in the presence or in the absence of 20 mM Na2Mo04, suggesting that molybdate is not causing aggregation to produce a larger Rs value than that of the native receptor. Vertical tube rotor sucrose gradient ultracentrifugation of cytosol produces three forms of the GC-R: 9.1 S, 5.2 S, and 3.8 S. Sequential analysis of the GC-R formsby HPLC and vertical tube rotor ultracentrifugation and vice versa allows for the hydrodynamic determination of molecular weight within a very short time period (2-3 h total). The untransformed 9. IS species of GC-R pooled from Mo042™-containing sucrose gradients has an Rs of 8.6 nm (Mr 310K-340K) while both the 5.2 S and 3.8 S forms have an Rs of 6 nm (Mr 115K-140K and 96K-100K, respectively). Conversely, if the 6-nm form of the receptor, generated either by KC1 treatment or by G-25 chromatography, is pooled from HPLC, then it subsequently sediments on Mo042"-containing gradients as a 3.5-4.6 S species. This probably indicates that the 5.2 S transformed GC-R is unstable and dissociates into its constituents during HPLC, despite the rapidity of the process. From the calculated molecular weight for each of the three GC-R forms, it is suggested that the untransformed oligomeric GC-R dissociates into monomeric subunits during transformation and that the intermediate form is not a dimer of identical monomeric subunits.(jTlucocorticoids are physiological regulators which interact with specific receptor proteins in responsive cells (Yamamoto & Alberts, 1976). After the hormone binds to the receptor, the glucocorticoid receptor (GC-R) 1 complex is transformed to a DNA-binding moiety. The transformed receptor interacts with acceptor sites on the DNA to affect specific gene tran-scription.