Lennon A M
Unité de Recherche sur la Glande Thyroide et la Régulation Hormonale, Institut National de la Santé et de la Recherche Médicale, Le Kremlin Bicêtre, France.
Eur J Biochem. 1992 Nov 15;210(1):79-85. doi: 10.1111/j.1432-1033.1992.tb17393.x.
A rat brain cytosolic 3,5,3'-triiodo-L-thyronine-(T3)-binding protein (CTBP) was purified using, successively, carboxymethyl-Sephadex, DEAE-Spherodex, T3-Sepharose-4B affinity chromatography and Sephacryl S-200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a single class of binding sites with a Ka of 1.56 nM-1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D-T3 > L-T3 > L-T4 > 3,3'-5-triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 microM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2-4 times and the Ka was 2.6 nM-1. These results show that the activity of purified cytosolic brain T3-binding protein may be modulated by NADPH, NADP or thioredoxin.
使用羧甲基葡聚糖凝胶、二乙氨基乙基琼脂糖凝胶、三碘甲状腺原氨酸琼脂糖凝胶4B亲和层析和Sephacryl S-200先后对大鼠脑胞质3,5,3'-三碘-L-甲状腺原氨酸（T3）结合蛋白（CTBP）进行纯化。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳测定的分子量为58 kDa。通过Scatchard分析确定的结合特性显示存在一类单一的结合位点，其解离常数（Ka）为1.56 nM-1，最大结合容量为7500 nmol T3/g蛋白。碘甲状腺原氨酸类似物的相对结合亲和力为：D-T3＞L-T3＞L-T4＞3,3'-5-三碘甲状腺乙酸＞反式T3。结合的最适pH值为7.5。纯化的脑CTBP可被活性炭可逆性失活。烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、烟酰胺腺嘌呤二核苷酸磷酸（NADP）和硫氧还蛋白可将结合活性恢复至高于对照的水平；这种效应呈浓度依赖性。在25 nM NADPH时观察到最大激活。NADP仅在存在1 mM二硫苏糖醇时有效；在10 nM NADP时获得最大活性。在浓度高于50 nM NADP时，结合逐渐降低。在存在1 mM二硫苏糖醇的情况下，硫氧还蛋白可激活CTBP；在4 μM硫氧还蛋白时获得最大结合。在存在NADPH、NADP或硫氧还蛋白的情况下，最大结合容量增加2至4倍，Ka为2.6 nM-1。这些结果表明，纯化的胞质脑T3结合蛋白的活性可能受NADPH、NADP或硫氧还蛋白的调节。
