Wrange O, Okret S, Radojćić M, Carlstedt-Duke J, Gustafsson J A
J Biol Chem. 1984 Apr 10;259(7):4534-41.
The activated glucocorticoid receptor (GR) from rat liver cytosol was purified by sequential chromatography on DNA-cellulose and DEAE-Sepharose. Analysis by sodium dodecyl sulfate-gel electrophoresis demonstrated a main band with Mr = 94,000 (94K band). Two minor bands with Mr = 79,000 (79K band) and 72,000 (72K band) were also seen in this preparation. Photoaffinity labeling showed that the hormone is bound to the 94K and 79K components but not to the 72K component. Immunoblotting using antibodies raised against the 94K protein demonstrated cross-reactivity between the 94K and 79K components but not with the 72K species. The 72K species could be partially separated from the 94K and 79K components by density gradient centrifugation. Limited proteolysis of the purified GR with trypsin or alpha-chymotrypsin led to degradation of the 94K and 79K components and appearance of a 39K fragment which still retained the hormone and could be bound to DNA-cellulose. The 72K component was not affected by digestion with trypsin or alpha-chymotrypsin. However, chromatography on DNA-cellulose of the alpha-chymotrypsin-treated GR resulted in elution of the 72K component in the flow-through of the column while the 39K fragment was retained on the column and eluted with 0.18 M NaCl. In the control experiment where no alpha-chymotrypsin treatment was performed, the 72K component could not be detected in the flow-through fraction but was eluted together with the 94K and 79K components at 0.18 M NaCl. These results suggest that the 72K protein might be bound to the 94K and/or 79K component. The 39K fragment did not bind antibodies raised against the 94K protein. The 39K fragment was further degraded by trypsin but not by alpha-chymotrypsin to a 27K and a 25K fragment while both still retained the ligand. These data obtained with limited proteolysis of the purified GR are in agreement with previous findings on proteolysis of the GR in crude cytosol (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865; Carlstedt-Duke, J., Okret, S., Wrange, O., and Gustafsson, J.-A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4260-4264).
通过在DNA-纤维素和DEAE-琼脂糖上进行连续层析，从大鼠肝脏胞质溶胶中纯化了活化的糖皮质激素受体（GR）。十二烷基硫酸钠-凝胶电泳分析显示出一条主要条带，其Mr = 94,000（94K条带）。在此制备物中还可见到两条次要条带，其Mr分别为79,000（79K条带）和72,000（72K条带）。光亲和标记表明该激素与94K和79K组分结合，但不与72K组分结合。使用针对94K蛋白产生的抗体进行免疫印迹，结果显示94K和79K组分之间存在交叉反应，但与72K组分无交叉反应。通过密度梯度离心可将72K组分与94K和79K组分部分分离。用胰蛋白酶或α-胰凝乳蛋白酶对纯化的GR进行有限的蛋白水解，导致94K和79K组分降解，并出现一个39K片段，该片段仍保留激素且可与DNA-纤维素结合。72K组分不受胰蛋白酶或α-胰凝乳蛋白酶消化的影响。然而，用α-胰凝乳蛋白酶处理的GR在DNA-纤维素上进行层析时，72K组分在柱的流出物中被洗脱，而39K片段保留在柱上并用0.18 M NaCl洗脱。在未进行α-胰凝乳蛋白酶处理的对照实验中，在流出级分中未检测到72K组分，而是与94K和79K组分一起在0.18 M NaCl处被洗脱。这些结果表明72K蛋白可能与94K和/或79K组分结合。39K片段不与针对94K蛋白产生的抗体结合。39K片段被胰蛋白酶进一步降解，但未被α-胰凝乳蛋白酶降解为27K和25K片段，而两者仍保留配体。通过对纯化的GR进行有限蛋白水解获得的这些数据与先前关于粗胞质溶胶中GR蛋白水解的研究结果一致（Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856 - 865; Carlstedt-Duke, J., Okret, S., Wrange, O., and Gustafsson, J.-A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4260 - 4264）。
Zotero 插件