Kasuga M, van Obberghen E, Yamada K M, Harrison L C
Diabetes. 1981 Apr;30(4):354-7. doi: 10.2337/diab.30.4.354.
125I-insulin was specifically cross-linked to membranes of human cultured lymphocytes (IM-9 line) using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and autoradiography of this preparation revealed a major 125I-labeled band with an apparent mol wt of 130,000 and minor bands of about 300,000 and 95,000. Labeling of these bands was inhibited by incubation of membranes with either unlabeled insulin or autoantibodies to the insulin receptor. The bands were also observed after the 125I-insulin cross-linked preparation was solubilized and immunoprecipitated with a panel of autoantibodies to the insulin receptor. However, immunoprecipitation of the 125I-insulin-receptor complex was inhibited by preincubation with excess unlabeled insulin. Finally, 125I-Fab fragments of mol wt 50,000 prepared from anti-receptor antibodies and cross-linked to membranes were resolved into a major complex of mol wt 180,000 and a minor band of 125,000. Neither band was observed when 125I-Fab fragments were cross-linked to membranes in the presence of an excess of unlabeled insulin. These findings indicate that autoantibodies to the insulin receptor are directed against the insulin binding subunits of an oligomeric receptor.
使用辛二酸二琥珀酰亚胺酯将¹²⁵I-胰岛素特异性交联至人培养淋巴细胞（IM-9系）的膜上。在还原条件下进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，并对该制剂进行放射自显影，结果显示一条主要的¹²⁵I标记带，其表观分子量为130,000，还有约300,000和95,000的次要带。用未标记的胰岛素或胰岛素受体自身抗体孵育膜可抑制这些带的标记。在用一组胰岛素受体自身抗体对¹²⁵I-胰岛素交联制剂进行溶解和免疫沉淀后，也观察到了这些带。然而，用过量未标记胰岛素预孵育可抑制¹²⁵I-胰岛素受体复合物的免疫沉淀。最后，由抗受体抗体制备的分子量为50,000的¹²⁵I-Fab片段与膜交联后，可解析为一个主要的分子量为180,000的复合物和一个分子量为125,000的次要带。当在过量未标记胰岛素存在下将¹²⁵I-Fab片段与膜交联时，未观察到这两条带。这些发现表明，胰岛素受体自身抗体针对的是寡聚受体的胰岛素结合亚基。
