Hashizume K, Miyamoto T, Ichikawa K, Yamauchi K, Kobayashi M, Sakurai A, Ohtsuka H, Nishii Y, Yamada T
Department of Gerontology, Endocrinology and Metabolism, Shinshu University School of Medicine, Matsumoto, Japan.
J Biol Chem. 1989 Mar 25;264(9):4857-63.
The NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine(T3)-binding protein (CTBP) has been purified over 30,000-fold from rat kidney by using charcoal extraction, Mono Q-Sepharose, Blue Sepharose CL-6B, and Sephacryl S-200 column chromatography. Purified CTBP had a sedimentation coefficient of 4.7 S, Stokes radius of 32.5A, and calculated molecular weight of 58,000. The apparently homogeneous protein consisted of a single polypeptide chain with Mr of 58,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Scatchard analysis of T3 binding showed that NADPH increases maximal binding capacity without changes in the affinity constant (Ka = 2.43 X 10(9) M-1). Double reciprocal analysis of NADPH and binding capacity gave maximal binding capacity of 16,400 pmol/mg of CTBP, Mr = 58,000. The order of affinity of iodothyronine analogues to purified CTBP was as follows: L-T3 = D-T3 greater than triiodothyroacetic acid greater than L-thyroxine. [125I]T3 bound to purified CTBP spontaneously dissociated from CTBP at 20 degrees C (t 1/2 = 22 min) in the absence of NADPH, whereas the dissociation was not observed in the presence of NADPH. The optimal pH for T3 binding was 7.2-7.5 Na+, K+, Ca2+, and Mg2+ (0-200 mM) did not influence T3 binding to CTBP. The purified CTBP did not bind to DNA and was not adsorbed to concanavalin A-Sepharose.
利用活性炭提取、单Q-琼脂糖凝胶柱层析、蓝色琼脂糖凝胶CL-6B柱层析和Sephacryl S-200柱层析，已从大鼠肾脏中纯化出依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的胞质3,5,3'-三碘-L-甲状腺原氨酸（T3）结合蛋白（CTBP），纯化倍数超过30000倍。纯化后的CTBP沉降系数为4.7 S，斯托克斯半径为32.5 Å，计算分子量为58000。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，这种明显均一的蛋白质由一条分子量为58000的单多肽链组成。T3结合的Scatchard分析表明，NADPH可增加最大结合容量，而亲和常数不变（Ka = 2.43×10⁹ M⁻¹）。NADPH与结合容量的双倒数分析得出CTBP（Mr = 58000）的最大结合容量为16400 pmol/mg。碘甲状腺原氨酸类似物与纯化后的CTBP的亲和顺序如下：L-T3 = D-T3＞三碘甲状腺乙酸＞L-甲状腺素。在没有NADPH的情况下，[¹²⁵I]T3与纯化后的CTBP结合后在20℃时会自发从CTBP上解离（半衰期 = 22分钟），而在有NADPH存在时未观察到解离现象。T3结合的最适pH为7.2 - 7.5，Na⁺、K⁺、Ca²⁺和Mg²⁺（0 - 200 mM）不影响T3与CTBP的结合。纯化后的CTBP不与DNA结合，也不被伴刀豆球蛋白A-琼脂糖吸附。
