Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the alpha and beta subunits of thyrotropin are crosslinked to the receptor.
Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the alpha and beta subunits of thyrotropin are crosslinked to the receptor.
复制标题
关于促甲状腺素与其受体共价交联的进一步研究：促甲状腺素的α和β亚基均与受体交联的证据。
DOI：
10.1016/0003-9861(87)90047-6
复制
发表时间：
1987
影响因子：
3.9
通讯作者：
Nayfeh,SN
中科院分区：
文献类型：
--
作者：
McQuade,R;ThomasJr,CG;Nayfeh,SN
高纯度的促甲状腺激素α和β亚基分别被放射性碘标记，然后与其未标记的互补亚基重组。这一过程导致了[125I]促甲状腺激素(TSH)杂化分子的形成，这些杂化分子只标记在一个激素亚基上。对这两个杂化分子的结合性质的表征表明，这两个杂化分子都能与TSH受体的高亲和力和低亲和力结合组分相互作用，并且都能产生与放射性碘标记的天然TSH相似的Kd值和Bmax的非线性Scatchard图。重组的[125I]TSH分子然后用琥珀酸二丁二亚胺与TSH受体交联。经电泳法和放射自显影后，观察到两个标记的TSH受体复合体，Mr分别为68000和80000。这两个复合体表现出激素特异性和电泳迁移率，与以前使用天然[125I]TSH观察到的相同。随着琥珀酸二丁二亚胺浓度的增加，交联表明68,000和80,000个复合体的形成是顺序的，其中68,000个出现在80,000个之前。此外，无论杂交TSH的哪个TSH亚基被放射性碘标记，这两条带都被标记。这些数据有力地表明，68,000和80,000个促甲状腺激素受体复合体是与促甲状腺激素α-β二聚体交联的结果，而不是像先前假设的那样，在68,000个复合体的情况下与促甲状腺激素α-β二聚体的一个亚基或在80,000个复合体的促甲状腺激素受体二聚体的情况下交联。
Highly purified α and β-subunits of thyrotropin were individually radioiodinated and, subsequently, recombined with their unlabeled complementary subunits. This procedure resulted in the formation of [125I]thyrotropin(TSH) hybrid molecules which were labeled on only one hormone subunit. Characterization of the binding properties of these two hybrid molecules demonstrated that both yielded nonlinear Scatchard plots withKdandBmaxvalues similar to those obtained with radioiodinated native TSH and that both were capable of interaction with the high- and low-affinity binding components of the TSH receptor. The recombined [125I]TSH molecules were then crosslinked to the TSH receptor using disuccinimidyl suberate. Following electrophoresis and autoradiography, two labeled TSH-receptor complexes withMrof 68,000 and 80,000 were observed. These two complexes exhibited hormone specificity and electrophoretic mobility identical to those previously observed using native [125I]TSH. Crosslinking with increasing concentrations of disuccinimidyl suberate suggested that the formation of the 68,000 and 80,000 complexes was sequential with the 68,000 appearing before the 80,000. Furthermore, the two bands were labeled regardless of which TSH subunit of the hybrid TSH was radioiodinated. These data strongly suggest that the 68,000 and 80,000 TSH-receptor complexes are the result of crosslinking to the TSH α-β dimer and not to one subunit in the case of the 68,000 complex and to the TSH α-β dimer in the case of the 80,000 complex, as had been hypothesized previously.