Pratt W B, Sando J J, Nielsen C J
Adv Exp Med Biol. 1979;117:343-56. doi: 10.1007/978-1-4757-6589-2_19.
The specific glucocorticoid binding capacity of cytosol preparations is rapidly lost on incubation at 25 degrees in the absence of ligand. We have examined this process in cell-free preparations from rat thymus, rat liver and mouse fibroblasts (L 929 cells), and we have found that the unoccupied receptor is inactivated by endogenous enzymes to a form that does not bind steroids. The inactivation can be prevented by inhibitors of phosphatase action such as molybdate, fluoride and glucose-1-phosphate. On the basis of this type of evidence we propose that the receptor activity of cytosol can be rendered inactive by a dephosphorylation process. We have now been able to partially reactivate the receptor in both L cell and rat thymus cytosols in an ATP dependent manner. If fibroblast (L cell) cytosol is preincubated to permit receptor inactivation by endogenous enzyme, further inactivation can be prevented by the addition of 10 mM molybdate and reactivation of the binding capacity can be obtained by adding 5 to 10 mM ATP in addition to molybdate. ATP dependent activation is prevented with EDTA and this block is overcome by added magnesium. ADP, CTP, GTP, and UTP are inactive. After inactivating the glucocorticoid binding capacity of rat thymocyte cytosol by incubation for 45 minutes at 25 degrees, considerable reactivation is obtained by addition of dithiothreitol and ATP. This system does not absolutely require the presence of a phosphatase inhibitor in order to show activation. Thymocyte cytosol can also be activated to a steroid binding state by addition of DTT and heat-treated (90 degrees for 15 min.) cytosol from a variety of cell types. The heat-treated cytosol contains ATP, reducing equivalents, and a relatively small molecular weight heat-stable activator(s) that potentiates the reactivation process. Maximum receptor activation is obtained by adding dithiothreitol, heat-stable factor, ATP, and molybdate to the inactivated thymocyte cytosol.
在没有配体的情况下，于25摄氏度孵育时，胞浆制剂的特异性糖皮质激素结合能力会迅速丧失。我们已在来自大鼠胸腺、大鼠肝脏和小鼠成纤维细胞（L929细胞）的无细胞制剂中研究了这一过程，并且发现未被占据的受体被内源性酶失活为一种不结合类固醇的形式。磷酸酶作用抑制剂如钼酸盐、氟化物和葡萄糖-1-磷酸可防止这种失活。基于这类证据，我们提出胞浆的受体活性可通过去磷酸化过程而失活。我们现在已经能够以ATP依赖的方式使L细胞和大鼠胸腺胞浆中的受体部分重新激活。如果将成纤维细胞（L细胞）胞浆预先孵育以使其受体被内源性酶失活，那么加入10 mM钼酸盐可防止进一步失活，并且除钼酸盐外再加入5至10 mM ATP可使结合能力重新激活。EDTA可阻止ATP依赖的激活，加入镁可克服这种阻断。ADP、CTP、GTP和UTP无活性。在25摄氏度孵育45分钟使大鼠胸腺细胞胞浆的糖皮质激素结合能力失活后，加入二硫苏糖醇和ATP可获得相当程度的重新激活。该系统并非绝对需要磷酸酶抑制剂的存在才能显示激活。加入二硫苏糖醇和来自多种细胞类型的经热处理（90摄氏度15分钟）的胞浆，胸腺细胞胞浆也可被激活至类固醇结合状态。经热处理的胞浆含有ATP、还原当量以及一种相对小分子量的热稳定激活剂，其可增强重新激活过程。通过向失活的胸腺细胞胞浆中加入二硫苏糖醇、热稳定因子、ATP和钼酸盐可获得最大程度的受体激活。
