Purification of the glucocorticoid receptor from rat liver cytosol.
Purification of the glucocorticoid receptor from rat liver cytosol.
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从大鼠肝细胞质中纯化糖皮质激素受体。
DOI：
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发表时间：
1979
影响因子：
4.8
通讯作者：
J. Gustafsson
中科院分区：
文献类型：
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作者：
O. Wrange;J. Carlstedt;J. Gustafsson
[3 H]-曲安奈德标记的糖皮质激素受体从大鼠肝胞质溶胶纯化，根据十二烷基硫酸钠凝胶电泳至85%的均匀性。它由一个分子量为89，000的亚基组成，每个分子有一个配体结合位点。纯化涉及磷酸纤维素，DNA-纤维素两次，和Sephadex G-200上的连续色谱。在DNA-纤维素上的两个层析步骤之间，受体被热激活。用5 ′-磷酸吡咯啶醛从第二个DNA-纤维素柱上亲和洗脱受体。在不同的实验中，在前三个色谱步骤中实现的纯化在60%和95%均匀度之间变化。在第二个DNA-纤维素柱上层析后，类固醇受体复合物的斯托克斯半径为6.0 nm，在0.15 M KCl中的沉降系数为3.4 S。在不存在KCl的情况下，沉降系数为3.6 S。在羟基磷灰石上浓缩后，在聚丙烯酰胺凝胶中用等电聚焦分析类固醇受体复合物。放射性与pI 5.8的主要蛋白条带集中在一起。在用胰蛋白酶进行有限蛋白水解后，放射性与主要蛋白条带一起集中在pI 6.2处，如先前对未纯化类固醇受体复合物所述。
The [3H]-triamcinolone acetonide-labeled glucocorticoid receptor from rat liver cytosol was purified to 85% homogeneity according to sodium dodecyl sulfate gel electrophoresis. It consisted of one subunit with a molecular weight of 89,000 and had one ligand-binding site per molecule. The purification involved sequential chromatography on phosphocellulose, DNA-cellulose twice, and Sephadex G-200. Between the two chromatography steps on DNA-cellulose, the receptor was heat activated. The receptor was affinity eluted from the second DNA-cellulose column with pyrodixal 5'-phosphate. The purification achieved in the first three chromatographic steps varied between 60 and 95% homogeneity in different experiments. After chromatography on the second DNA-cellulose column, the steroid.receptor complex had a Stokes radius of 6.0 nm and a sedimentation coefficient of 3.4 S in 0.15 M KCl. In the absence of KCl, the sedimentation coefficient was 3.6 S. After concentration on hydroxylapatite, the steroid.receptor complex was analyzed by isoelectric focusing in polyacrylamide gel. The radioactivity was shown to focus together with the major protein band with pI 5.8. Following limited proteolysis with trypsin, the radioactivity, together with the major protein band, focused at pI 6.2 as previously described for the unpurified steroid.receptor complex.