Ichikawa K, DeGroot L J
Mol Cell Endocrinol. 1987 May;51(1-2):135-43. doi: 10.1016/0303-7207(87)90127-4.
The nuclear thyroid hormone receptors isolated from cultured human hepatoma cells (Hep G2) were characterized and compared with those from cultured human fibroblasts and rat liver. The Hep G2 nuclear thyroid hormone receptors had an affinity constant (Ka) of 2.1 X 10(10) M-1 and maximal binding capacity (MBC) of 21.0 fmol/100 micrograms DNA for T3 in assays performed on isolated nuclei. 16% of nuclear receptors were released into the media during incubation and had the same Ka. Salt-extracted receptors had a Ka of 1.8 X 10(10) M-1 and MBC of 0.1 pmol/mg protein for T3. Density gradient sedimentation and gel filtration chromatography revealed a sedimentation coefficient of 3.4 S and Stokes radius of 34 A. From these values, a molecular weight of 49,000 and total frictional ratio (f/f0) of 1.4 were calculated, suggesting an asymmetrical shape of the receptor molecule. Heat inactivation occurred with t1/2 of 28.1, 18.0, and 7.9 min at 38, 43, 45 degrees C, respectively. Isoelectric focusing (IEF) of Hep G2 nuclear receptors demonstrated T3 binding proteins at pH 5.3-5.5, 5.7, and 5.9. Evidence that these are nuclear thyroid hormone receptors includes the following: Triiodothyroacetic acid was the most potent competitor of [125I]T3 binding to these proteins followed by L-T3, and L-T4. Cytosolic protein, human serum, and fetal calf serum failed to show the same T3 binding proteins. Ka of these proteins measured by T3 displacement was 1.1-3.2 X 10(9) M-1. Human fibroblast nuclear extract showed similar T3 binding pattern in IEF, except for a slight difference in pI of an acidic band.(ABSTRACT TRUNCATED AT 250 WORDS)
对从培养的人肝癌细胞（Hep G2）中分离出的甲状腺激素核受体进行了表征，并与从培养的人成纤维细胞和大鼠肝脏中分离出的受体进行了比较。在对分离的细胞核进行的测定中，Hep G2甲状腺激素核受体对T3的亲和常数（Ka）为2.1×10¹⁰ M⁻¹，最大结合容量（MBC）为21.0 fmol/100微克DNA。在孵育过程中，16%的核受体释放到培养基中，且具有相同的Ka。盐提取的受体对T3的Ka为1.8×10¹⁰ M⁻¹，MBC为0.1 pmol/mg蛋白质。密度梯度沉降和凝胶过滤色谱显示沉降系数为3.4 S，斯托克斯半径为34 Å。根据这些值，计算出分子量为49,000，总摩擦比（f/f0）为1.4，表明受体分子呈不对称形状。在38、43、45℃时，热失活的半衰期分别为28.1、18.0和7.9分钟。Hep G2核受体的等电聚焦（IEF）显示在pH 5.3 - 5.5、5.7和5.9处有T3结合蛋白。这些是甲状腺激素核受体的证据包括：三碘甲状腺乙酸是[¹²⁵I]T3与这些蛋白结合的最有效竞争者，其次是L - T3和L - T4。胞质蛋白、人血清和胎牛血清未显示相同的T3结合蛋白。通过T3置换测量的这些蛋白的Ka为1.1 - 3.2×10⁹ M⁻¹。人成纤维细胞核提取物在IEF中显示出相似的T3结合模式，只是酸性条带的等电点略有差异。（摘要截于250字）
