Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries.
Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries.
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具有独特亚基化学计量的三种主要胰岛素受体结构的电泳解析。
DOI：
10.1073/pnas.77.12.7137
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发表时间：
1980
影响因子：
11.1
通讯作者：
Czech,MP
中科院分区：
文献类型：
--
作者：
Massague,J;Pilch,PF;Czech,MP
质膜胰岛素受体，通过共价交联与受体结合的125 I标记的胰岛素进行亲和标记，在不存在还原剂的情况下，在高度多孔的十二烷基硫酸盐/聚丙烯酰胺凝胶中电泳时显示为三种主要二硫键连接复合物（Mr 350，000，320，000和290，000）的异质群体。该模式在分析的所有大鼠和人体组织中一致。在二硫键还原时，这些受体结构中的每一个在两个连续步骤中解离。低浓度的二硫苏糖醇促进二硫键还原的第一步，其中Mr 350，000物质分裂成Mr 210，000形式，Mr 290，000物质分裂成Mr 160，000形式。相比之下，Mr 210，000和Mr 160，000受体片段均由天然Mr 320，000物质在部分还原后产生，表明结构不对称。受体还原的第二步发生在用高浓度二硫苏糖醇处理天然二硫键连接的受体复合物时。在这些条件下，Mr 350，000受体产生一个Mr 125，000亚基，表示为α，和一个Mr 90，000亚基，表示为β，而Mr 290，000受体解离成α亚基和一个Mr 49，000亚基，表示为β 1。发现Mr 320，000受体带在完全还原时由α、β和β 1亚基组成。部分还原的Mr 210，000受体片段由与β亚基二硫键连接的α亚基组成，而Mr 160，000物质由与β 1亚基二硫键连接的α亚基组成。因此，Mr 350，000、320，000和290，000的三种普遍存在的天然胰岛素受体结构的化学计量分别为（alpha）2（beta）2、（alpha）2（beta）（beta 1）和（alpha）2（beta 1）2。
Plasma membrane insulin receptors, affinity labeled by covalent crosslinking to receptor-bound 125I-labeled insulin, are shown to appear as a heterogeneous population of three major disulfide-linked complexes (Mr 350,000, 320,000, and 290,000) upon electrophoresis in highly porous dodecyl sulfate/polyacrylamide gels in the absence of reductant. This pattern is consistent in all rat and human tissues that were analyzed. Upon reduction of disulfide bonds, each of these receptor structures is dissociated in two successive steps. Low concentrations of dithiothreitol promote a first step of disulfide bond reduction in which the Mr 350,000 species splits into a Mr 210,000 form and the Mr 290,000 species splits into a Mr 160,000 form. In contrast, both the Mr 210,000 and Mr 160,000 receptor fragments are generated from the native Mr 320,000 species upon partial reduction, indicating an asymmetrical structure. The second step of receptor reduction occurs upon treatment of the native disulfide-linked receptor complexes with high concentrations of dithiothreitol. Under these conditions, the Mr 350,000 receptor yields a Mr 125,000 subunit, denoted as alpha, and a Mr 90,000 subunit, denoted as beta, whereas the Mr 290,000 receptor dissociates into the alpha subunit and a Mr 49,000 subunit, denoted as beta 1. The Mr 320,000 receptor band is found to consist of alpha, beta, and beta 1 subunits upon complete reduction. The partially reduced Mr 210,000 receptor fragment is composed of the alpha subunit disulfide-linked to the beta subunit, whereas the Mr 160,000 species consists of the alpha subunit disulfide-linked to the beta 1 subunit. Thus, the stoichiometry of the three ubiquitous native insulin receptor structures of Mr 350,000, 320,000, and 290,000 are (alpha) 2 (beta) 2, (alpha) 2 (beta) (beta 1), and (alpha) 2 (beta 1) 2, respectively.