Hashizume K, Kobayashi M, Miyamoto T
Endocrinology. 1986 Aug;119(2):710-9. doi: 10.1210/endo-119-2-710.
Extraction of rat kidney cytosol with 10% charcoal at 4 C inactivated specific T3 binding. The decreased T3 binding in extracted cytosol could be restored by addition of boiled kidney cytosol. Three different factors (a, b, and c) which could increase T3 binding were identified by Sephadex G-50 column chromatography of boiled cytosol. Two factors (b and c) were eluted as relatively small molecules. Factor a was present in small amounts. Factor c was neutralized by incubation with EDTA, but factor b was not. Factor b was not destroyed by trypsin, protease, DNase, or RNase, but was destroyed by alkaline phosphatase. Factor b was destroyed by incubation with nicotinamide adenine dinucleotide phosphate (NADPH)-dependent glutathione reductase in the presence of oxidized glutathione. Although T3 binding to charcoal-extracted cytosol protein was not influenced by reduced glutathione or dithiothreitol, it was markedly increased by NADPH. Maximal activation induced by 50 microM NADPH was not further increased by further addition of endogenous factor b. The elution position of NADPH in gel chromatography corresponded to the elution position of factor b. Factor b or NADPH increased maximal binding capacity without changes in affinity constant. These observations suggest that T3-binding protein in cytosol is present in inactive and active forms and that the active form is generated by NADPH, which is present as one of the activators in cytosol. The effect of these cytosolic T3-binding proteins on nuclear T3 binding in vitro was also studied. In the absence of cytosolic T3-binding protein, [125I]T3 binding to nuclear receptor was decreased by unlabeled T3 in a concentration-dependent manner. In the presence of inactive form of cytosolic T3-binding protein, nuclear [125I]T3 binding was slightly diminished. In the presence of NADPH and cytosolic T3-binding protein, however, the amount of [125I]T3 bound to nuclei markedly decreased, which was associated with an increase of cytosolic [125I]T3 binding. NADPH alone did not influence nuclear T3 binding. These results suggest that T3 binding to nuclear receptor is regulated by an active form of cytosolic T3-binding protein in vitro.
在4℃下用10%的活性炭提取大鼠肾胞质溶胶会使特异性T3结合失活。提取的胞质溶胶中T3结合的减少可通过添加煮沸的肾胞质溶胶来恢复。通过对煮沸的胞质溶胶进行葡聚糖凝胶G - 50柱色谱分析，鉴定出三种可增加T3结合的不同因子（a、b和c）。两种因子（b和c）以相对较小的分子形式被洗脱。因子a含量较少。因子c与EDTA孵育后被中和，但因子b未被中和。因子b不被胰蛋白酶、蛋白酶、DNA酶或RNA酶破坏，但被碱性磷酸酶破坏。在氧化型谷胱甘肽存在的情况下，因子b与依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的谷胱甘肽还原酶孵育后被破坏。虽然T3与经活性炭提取的胞质溶胶蛋白的结合不受还原型谷胱甘肽或二硫苏糖醇的影响，但NADPH可使其显著增加。50μM NADPH诱导的最大激活作用不会因进一步添加内源性因子b而进一步增强。NADPH在凝胶色谱中的洗脱位置与因子b的洗脱位置相对应。因子b或NADPH增加了最大结合能力，而亲和力常数没有变化。这些观察结果表明，胞质溶胶中的T3结合蛋白以无活性和活性形式存在，活性形式由NADPH产生，NADPH作为胞质溶胶中的激活剂之一存在。还研究了这些胞质T3结合蛋白在体外对核T3结合的影响。在没有胞质T3结合蛋白的情况下，未标记的T3以浓度依赖的方式降低了[125I]T3与核受体的结合。在存在无活性形式的胞质T3结合蛋白时，核[125I]T3结合略有减少。然而，在存在NADPH和胞质T3结合蛋白时，与核结合的[125I]T3量显著减少，这与胞质[125I]T3结合的增加相关。单独的NADPH不影响核T3结合。这些结果表明，在体外，T3与核受体的结合受胞质T3结合蛋白活性形式的调节。
