Sando J J, La Forest A C, Pratt W B
J Biol Chem. 1979 Jun 10;254(11):4772-8.
The specific glucocorticoid binding capacity in cytosols prepared from L929 mouse fibroblasts (L cells) is inactivated with a half-life of approximately 2 h at 25 degrees C. As previously published, this inactivation can be prevented with 10 mM molybdate and markedly slowed by addition of other phosphatase inhibitors such as glucose 1-phosphate and fluoride. We have now found that ATP (5 to 10 mM) also slows the rate of this inactivation. After extensively inactivating the receptor by preincubating cytosol at 25 degrees C for 4 and preventing further inactivation by addition of molybdate, addition of ATP results in reactivation of the steroid binding capacity. Maximal reactivation of 40 to 70% is achieved with 5 to 10 mM ATP. The activation is temperature-dependent and specific for ATP. ADP, GTP, CTP, and UTP do not cause activation and preliminary results indicate no effect of cyclic nucleotides in this system. If activation is prevented by addition of 10 mM EDTA to the cytosol, addition of 3 to 10 mM magnesium permits ATP-dependent activation of the binding capacity. The level of reactivation can be enhanced by addition of a heat-stable factor prepared from the same L cell supernatant. These results support the proposal that L cell glucocorticoid receptors can be activated to the glucocorticoid binding state by an ATP-dependent phosphorylation mechanism.
从L929小鼠成纤维细胞（L细胞）制备的胞质溶胶中的特异性糖皮质激素结合能力，在25℃下以约2小时的半衰期失活。如先前发表的那样，这种失活可被10 mM钼酸盐阻止，并且通过添加其他磷酸酶抑制剂如葡萄糖1-磷酸和氟化物可使其显著减慢。我们现在发现，ATP（5至10 mM）也能减慢这种失活的速率。在25℃下将胞质溶胶预孵育4小时使受体广泛失活，并通过添加钼酸盐阻止进一步失活后，添加ATP会导致类固醇结合能力重新激活。5至10 mM ATP可实现40%至70%的最大重新激活。这种激活是温度依赖性的，且对ATP具有特异性。ADP、GTP、CTP和UTP不会引起激活，初步结果表明该系统中环状核苷酸没有作用。如果通过向胞质溶胶中添加10 mM EDTA来阻止激活，添加3至10 mM镁可使ATP依赖的结合能力激活。添加从相同L细胞上清液制备的热稳定因子可增强重新激活的水平。这些结果支持了L细胞糖皮质激素受体可通过ATP依赖的磷酸化机制被激活至糖皮质激素结合状态的提议。
