Schmidt T J, Miller-Diener A, Webb M L, Litwack G
J Biol Chem. 1985 Dec 25;260(30):16255-62.
Thermal "activation" or "transformation" of rat hepatic [6,7-3H]triamcinolone acetonide (TA)-receptor complexes purified in the unactivated state to near homogeneity (Grandics, P., Miller, A., Schmidt, T. J., Mittman, D., and Litwack, G. (1984) J. Biol. Chem. 259, 3173-3180) has been further investigated. The data generated in reconstitution experiments demonstrate that warming (25 degrees C for 30 min) of the purified unactivated complexes promotes their activation as judged by an increase in DNA-cellulose binding, but to a lower extent than that observed after warming of glucocorticoid-receptor complexes in crude cytosols. However, maximal DNA-cellulose binding capacity can be detected in reconstituted systems (also heated at 25 degrees C for 30 min) consisting of purified unactivated [3H]TA-receptor complexes and a cytoplasmic "stimulator(s)." This cytoplasmic factor(s), which does not copurify with the receptor, is heat-stable (90 degrees C for 30 min), excluded from Sephadex G-25, and trypsin-sensitive and stimulates DNA-cellulose binding in a dose-dependent manner. The ability of Na2MoO4 to block thermal activation of the highly purified receptor complexes suggests that this transition metal anion interacts directly with the receptor protein itself. The fact that the cytoplasmic stimulator(s) enhances DNA-cellulose binding of the [3H]TA-receptor complexes without increasing the proportion of those complexes eluted in the activated (low salt) position from DEAE-cellulose is consistent with a proposed two-step model of in vitro activation. During the Na2MoO4-sensitive Step 1, elevated temperature (25 degrees C for 30 min) may directly alter the conformation of the purified receptor complexes (i.e. subunit dissociation or disaggregation), resulting in the appropriate shift in the elution profile of the [3H]TA-receptor complexes on DEAE-cellulose but only in a minimal (approximately 2-3-fold) increase in the binding of these complexes to DNA-cellulose. During the Na2MoO4-insensitive and temperature-independent Step 2, a heat-stable cytoplasmic protein(s) may interact with these thermally activated [3H]TA-receptor complexes and enhance their ability to bind to DNA-cellulose without further increasing the percentage of those complexes which elute from DEAE-cellulose in the activated position. In crude cytosols these two steps would presumably occur simultaneously, and addition of Na2MoO4 prior to warming would block Step 1 and hence Step 2 would not occur.
已对以未活化状态纯化至接近均一的大鼠肝脏[6,7 - ³H]曲安奈德（TA）受体复合物的热“激活”或“转化”进行了进一步研究（格兰迪斯，P.，米勒，A.，施密特，T. J.，米特曼，D.，和利特瓦克，G.（1984年）《生物化学杂志》259，3173 - 3180）。重组实验产生的数据表明，纯化的未活化复合物在25℃温育30分钟会促进其激活，这可通过与DNA - 纤维素结合的增加来判断，但程度低于粗制胞质溶胶中糖皮质激素受体复合物温育后的增加程度。然而，在由纯化的未活化[³H]TA受体复合物和一种胞质“刺激物”组成的重组系统（同样在25℃加热30分钟）中可检测到最大的DNA - 纤维素结合能力。这种胞质因子不与受体共纯化，对热稳定（90℃ 30分钟），不能通过葡聚糖凝胶G - 25柱，对胰蛋白酶敏感，并以剂量依赖方式刺激与DNA - 纤维素的结合。钼酸钠阻断高度纯化的受体复合物热激活的能力表明，这种过渡金属阴离子直接与受体蛋白本身相互作用。胞质刺激物增强[³H]TA受体复合物与DNA - 纤维素的结合，但不增加从DEAE - 纤维素柱上以活化（低盐）位置洗脱的那些复合物的比例，这一事实与提出的体外激活两步模型一致。在钼酸钠敏感的第一步中，升高温度（25℃ 30分钟）可能直接改变纯化受体复合物的构象（即亚基解离或解聚），导致[³H]TA受体复合物在DEAE - 纤维素柱上的洗脱图谱发生适当变化，但这些复合物与DNA - 纤维素的结合仅轻微增加（约2 - 3倍）。在钼酸钠不敏感且与温度无关的第二步中，一种热稳定的胞质蛋白可能与这些热激活的[³H]TA受体复合物相互作用，并增强它们与DNA - 纤维素结合的能力，而不会进一步增加从DEAE - 纤维素柱上以活化位置洗脱的那些复合物的百分比。在粗制胞质溶胶中，这两步可能同时发生，在温育前加入钼酸钠会阻断第一步，因此第二步不会发生。
