Hashizume K, Ichikawa K, Kobayashi M
Endocrinol Jpn. 1984 Jun;31(3):311-20. doi: 10.1507/endocrj1954.31.311.
The effect of calcium ion on 3,5,3'-triiodothyronine (T3) binding to rat kidney outer mitochondrial membranes was examined in vitro. The outer mitochondrial membranes were prepared by using a discontinuous sucrose density gradient centrifugation. The membrane fraction, which is enriched with monoamine oxidase activity, contained specific binding sites for T3. Scatchard analysis of T3 binding to outer mitochondrial membranes gave an association constant (Ka) of 0.53 X 10(10)M-1. The binding of [125I]-T3 to the membranes was inhibited by the addition of CaCl2(0.25 X 10(-4)--2.5 X 10(-3)M). 50% inhibition was obtained by 0.75 X 10(-4)M CaCl2 in the presence of 0.1 mM EGTA. When outer mitochondrial membranes were solubilized with Triton X-100, four main T3 binding activities were isolated by a gel filtration study. On the other hand, the binding of [125I]-T3 to the solubilized T3 receptors derived from outer mitochondrial membranes was not strongly inhibited by calcium. When outer mitochondrial membranes were preincubated in the presence of 1 mM calcium, the number of T3 binding sites in the membranes was decreased, and this was associated with an increase in the number of T3 binding sites in the supernatants of the incubation mixture. Scatchard analysis showed that the number of T3 binding sites in the membranes is decreased by calcium ion without any change in the association constant. In studies with gel filtration of receptors which are released by Ca2+ from outer mitochondrial membranes, three main T3 binding activities were isolated. Mg2+, Mn2+, Zn2+ and Cu2+ did not affect T3 binding to outer mitochondrial membranes. The results indicate that calcium ion regulates T3 binding to the outer mitochondrial membrane through the release of T3 receptors from the membranes.
体外研究了钙离子对3,5,3'-三碘甲状腺原氨酸（T3）与大鼠肾外线粒体膜结合的影响。采用不连续蔗糖密度梯度离心法制备肾外线粒体膜。富含单胺氧化酶活性的膜组分含有T3的特异性结合位点。对T3与肾外线粒体膜结合进行Scatchard分析，得到的缔合常数（Ka）为0.53×10¹⁰M⁻¹。添加CaCl₂（0.25×10⁻⁴ - 2.5×10⁻³M）可抑制[¹²⁵I]-T3与膜的结合。在0.1 mM乙二醇双四乙酸（EGTA）存在下，0.75×10⁻⁴M CaCl₂可产生50%的抑制作用。用Triton X-100溶解肾外线粒体膜后，通过凝胶过滤研究分离出四种主要的T3结合活性。另一方面，钙离子对溶解的源自肾外线粒体膜的T3受体与[¹²⁵I]-T3的结合没有强烈抑制作用。当肾外线粒体膜在1 mM钙存在下预孵育时，膜中T3结合位点的数量减少，这与孵育混合物上清液中T3结合位点数量的增加有关。Scatchard分析表明，膜中T3结合位点的数量因钙离子而减少，而缔合常数没有任何变化。在用凝胶过滤法研究由Ca²⁺从肾外线粒体膜释放的受体时，分离出三种主要的T3结合活性。镁离子、锰离子、锌离子和铜离子不影响T3与肾外线粒体膜的结合。结果表明，钙离子通过使T3受体从膜上释放来调节T3与肾外线粒体膜的结合。
