Im J H, Meezan E, Rackley C E, Kim H D
J Biol Chem. 1983 Apr 25;258(8):5021-6.
The noncovalently associated 125I-insulin-receptor complex was isolated from human erythrocyte membranes after allowing 125I-insulin to interact with the membranes followed by extraction of the 125I-insulin-receptor complex with Triton X-102 or, alternatively, by complete solubilization of the membranes with sodium dodecyl sulfate (SDS), removal of SDS, and then treatment of the solubilized sample with 125I-insulin. Sepharose CL-6B column chromatography of the 125I-insulin-receptor complex obtained by both of the above procedures yielded a highly radioactive 140,000-Da complex which was dissociated into small peptides when subjected to SDS-polyacrylamide gel electrophoresis. In contrast, when the 125I-insulin-treated membrane sample was extracted with Triton X-102, purified by DEAE-Sephacel ion exchange chromatography, covalently cross-linked with disuccinimidyl suberate, and then subjected to SDS-polyacrylamide gel electrophoresis, a highly radioactive component with Mr = 53,000 was obtained. On the other hand, when the Triton X-102-solubilized membrane receptor sample was fractionated by DEAE-Sephacel ion exchange chromatography, complexed with 125I-insulin, covalently crosslinked, and then applied to a Sepharose CL-6B column, a 95,000-Da complex with high specific radioactivity was obtained. Upon SDS-polyacrylamide gel electrophoresis, the 95,000-Da complex was dissociated into a 53,000-Da component which appeared identical with that obtained from the receptor complex described above which was obtained by direct interaction of the membranes with 125I-insulin.
让¹²⁵I-胰岛素与人体红细胞膜相互作用，随后用 Triton X-102 提取¹²⁵I-胰岛素受体复合物，或者用十二烷基硫酸钠（SDS）使膜完全溶解，去除 SDS，然后用¹²⁵I-胰岛素处理溶解后的样品，之后从人体红细胞膜中分离出非共价结合的¹²⁵I-胰岛素受体复合物。通过上述两种方法获得的¹²⁵I-胰岛素受体复合物经琼脂糖 CL-6B 柱层析，得到一种高放射性的 140,000 道尔顿复合物，该复合物在进行 SDS-聚丙烯酰胺凝胶电泳时会解离成小肽段。相比之下，用 Triton X-102 提取经¹²⁵I-胰岛素处理的膜样品，通过 DEAE-琼脂糖离子交换层析纯化，用辛二酸二琥珀酰亚胺酯进行共价交联，然后进行 SDS-聚丙烯酰胺凝胶电泳，得到一个 Mr = 53,000 的高放射性组分。另一方面，当用 DEAE-琼脂糖离子交换层析对经 Triton X-102 溶解的膜受体样品进行分级分离，与¹²⁵I-胰岛素复合，进行共价交联，然后应用于琼脂糖 CL-6B 柱时，得到一个具有高比放射性的 95,000 道尔顿复合物。经 SDS-聚丙烯酰胺凝胶电泳，95,000 道尔顿复合物解离成一个 53,000 道尔顿的组分，该组分与上述通过膜与¹²⁵I-胰岛素直接相互作用获得的受体复合物中得到的组分相同。
