Active and inactive forms of 3,5,3'-triiodo-L-thyronine (T3)-binding protein in rat kidney cytosol: possible role of nicotinamide adenine dinucleotide phosphate in activation of T3 binding.
Active and inactive forms of 3,5,3'-triiodo-L-thyronine (T3)-binding protein in rat kidney cytosol: possible role of nicotinamide adenine dinucleotide phosphate in activation of T3 binding.
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大鼠肾细胞质中 3,5,3-三碘-L-甲状腺氨酸 (T3) 结合蛋白的活性和非活性形式：烟酰胺腺嘌呤二核苷酸磷酸在 T3 结合激活中的可能作用。
DOI：
10.1210/endo-119-2-710
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发表时间：
1986
期刊：
影响因子：
4.8
通讯作者：
T. Miyamoto
中科院分区：
文献类型：
--
作者：
K. Hashizume;M. Kobayashi;T. Miyamoto
用10%炭提取大鼠肾细胞质，以4c灭活特异性T3结合。通过加入煮熟的肾细胞质，可以恢复提取细胞质中T3的结合。用Sephadex G-50柱层析法鉴定了3个能增加T3结合的因子a、b、c。两个因子（b和c）被洗脱为相对较小的分子。因子a是少量存在的。EDTA能中和因子c，但因子b不能。因子b不被胰蛋白酶、蛋白酶、DNase或RNase破坏，但被碱性磷酸酶破坏。在氧化谷胱甘肽存在的情况下，与烟酰胺腺嘌呤二核苷酸磷酸（NADPH）依赖性谷胱甘肽还原酶孵育可破坏因子b。虽然T3与炭提取细胞质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显著增加了其结合。内源因子b的加入并没有进一步提高50微米NADPH诱导的最大活化，NADPH在凝胶层析中的洗脱位置与因子b的洗脱位置相对应，因子b或NADPH在不改变亲和常数的情况下增加了最大结合能力。这些观察结果表明，细胞质中t3结合蛋白有活性和非活性两种形式，活性形式是由NADPH产生的，NADPH作为细胞质中的激活剂之一存在。我们还研究了这些胞质T3结合蛋白对体外核T3结合的影响。在缺乏胞质T3结合蛋白的情况下，[125I]T3与核受体的结合以浓度依赖的方式被未标记的T3所降低。在胞质T3结合蛋白失活形式存在时，核[125I]T3结合轻微减弱。然而，在NADPH和胞质T3结合蛋白存在的情况下，[125I]T3结合细胞核的数量明显减少，这与胞质[125I]T3结合增加有关。NADPH不影响T3的结合。这些结果表明，在体外，T3与核受体的结合受到一种活性形式的细胞质T3结合蛋白的调节。
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.