Tagami T, Nakamura H, Sasaki S, Imura H
Department of Internal Medicine, Kyoto University School of Medicine, Japan.
Endocrinology. 1990 Feb;126(2):1105-11. doi: 10.1210/endo-126-2-1105.
In our previous study, we raised a polyclonal antiserum against a synthetic 15 amino acid peptide encoding the putative hormone binding domain of c-erb A, which not only inhibited T3-binding to rat liver nuclear T3 receptor (NT3R) but also immunoprecipitated [125I]T3-NT3R complex. In the present study, interactions between this antiserum (4 BII-immunoglobulin G (IgG] and NT3R were further characterized. 4BII-IgG interefered T3 from binding to rat liver NT3R both in the nuclear extract and in isolated nuclei in a similar manner. Preincubation with 4BII-IgG decreased the association constant value with no effect on maximal binding capacity of NT3R. Lineweaver-Burk plot revealed competitive inhibition of T3-NT3R binding. Both the inhibition of T3 binding and immunoprecipitation of NT3R by the antiserum were similarly observed with rat liver, kidney, brain, and testis, and human kidney nuclear extract. Since the polypeptide sequence which was used for immunization is highly homologous between c-erb A alpha 1 and beta, it is likely that 4BII-IgG recognizes both c-erb A alpha 1 and beta in various tissues. On the contrary, 4BII-IgG did not interfere with T3 binding to rat liver cytosol T3-binding protein and thyroxine-binding globulin (TBG) prepared from human serum, nor immunoprecipitated these binding proteins labeled with [125I]T3. The binding protein for reverse T3 (NrT3BP) in the rat liver nuclear extract was neither immunoprecipitated nor influenced in its rT3-binding activity by 4BII-IgG. When the immune complex between the nuclear extract and 4BII-IgG was removed by antirabbit IgG goat serum, the remaining T3-binding activity was reduced by 87%, while no rT3-binding activity was immunodepleted, suggesting that NrT3BP is a protein different from NT3R. The data show that the antiserum against c-erb A peptide recognizes NT3R specifically with no distinction between difference in tissues and species. Carboxyl-terminal region of c-erb A/NT3R is critical for T3-binding.
在我们之前的研究中，我们制备了一种针对合成的15个氨基酸肽的多克隆抗血清，该肽编码c-erb A假定的激素结合结构域，它不仅抑制T3与大鼠肝细胞核T3受体（NT3R）的结合，还能免疫沉淀[125I]T3-NT3R复合物。在本研究中，进一步对该抗血清（4BII-免疫球蛋白G（IgG））与NT3R之间的相互作用进行了表征。4BII-IgG以类似的方式干扰核提取物和分离细胞核中T3与大鼠肝NT3R的结合。与4BII-IgG预孵育会降低结合常数，而对NT3R的最大结合能力没有影响。Lineweaver-Burk图显示了T3-NT3R结合的竞争性抑制。在大鼠肝脏、肾脏、大脑和睾丸以及人肾脏核提取物中，同样观察到抗血清对T3结合的抑制和NT3R的免疫沉淀。由于用于免疫的多肽序列在c-erb Aα1和β之间高度同源，4BII-IgG很可能在各种组织中识别c-erb Aα1和β。相反，4BII-IgG不干扰T3与大鼠肝细胞溶质T3结合蛋白以及从人血清制备的甲状腺素结合球蛋白（TBG）的结合，也不免疫沉淀用[125I]T3标记的这些结合蛋白。大鼠肝核提取物中反式T3的结合蛋白（NrT3BP）既不被4BII-IgG免疫沉淀，其rT3结合活性也不受影响。当用抗兔IgG山羊血清去除核提取物与4BII-IgG之间的免疫复合物时，剩余的T3结合活性降低了87%，而没有rT3结合活性被免疫去除，这表明NrT3BP是一种与NT3R不同的蛋白质。数据表明，针对c-erb A肽的抗血清特异性识别NT3R，在组织和物种差异方面没有区别。c-erb A/NT3R的羧基末端区域对T3结合至关重要。
