Madison L D, Rosenzweig S A, Jamieson J D
J Biol Chem. 1984 Dec 10;259(23):14818-23.
The binding of 125I-cholecystokinin-33 (125I-CCK-33) to its receptors on rat pancreatic membranes was decreased by modification of membrane protein sulfhydryl groups. Sulfhydryl modifying reagents also caused an accelerated release of bound 125I-CCK-33 from its receptor. Because of the presence of an essential sulfhydryl group(s) in CCK receptor binding we studied the application of the heterobifunctional (SH,NH2) cross-linker, m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), to affinity label 125I-CCK-33 binding proteins on rat pancreatic plasma membranes. Analysis of the cross-linked products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that this heterobifunctional cross-linker affinity labeled a major Mr = 80,000-95,000 protein previously identified as part of the CCK receptor on the basis of affinity labeling using homobifunctional and heterobifunctional photoreactive cross-linkers. Additional proteins of Mr greater than 200,000, and Mr = 130,000-140,000 were affinity labeled using MBS. The efficiency of the cross-linking reaction between 125I-CCK-33 and its membrane binding proteins with MBS was significantly greater than that obtained with NH2-directed homobifunctional reagents such as disuccinimidyl suberate. The efficiency of cross-linking could be dramatically improved by reduction of membrane proteins with low-molecular weight thiols prior to binding and cross-linking. The differential labeling patterns of the CCK binding proteins obtained with chemical cross-linkers of similar length but different chemical reactivity underscores the need for caution in predicting native receptor structure from affinity labeling data alone. Using the same pancreatic plasma membrane preparation and 125I-insulin, the Mr = 125,000 alpha-subunit of the insulin receptor was affinity labeled using MBS as cross-linker, demonstrating its utility in identifying other peptide hormone receptors.
膜蛋白巯基的修饰可降低125I-胆囊收缩素-33（125I-CCK-33）与大鼠胰腺细胞膜上其受体的结合。巯基修饰试剂还导致结合在受体上的125I-CCK-33加速释放。由于CCK受体结合中存在必需的巯基，我们研究了异双功能（SH，NH2）交联剂间马来酰亚胺苯甲酰基N-羟基琥珀酰亚胺酯（MBS）在亲和标记大鼠胰腺质膜上125I-CCK-33结合蛋白中的应用。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和放射自显影分析交联产物，结果显示这种异双功能交联剂亲和标记了一种主要的分子量为80,000 - 95,000的蛋白质，该蛋白质先前已基于使用同双功能和异双功能光反应性交联剂的亲和标记被鉴定为CCK受体的一部分。使用MBS还亲和标记了分子量大于200,000以及分子量为130,000 - 140,000的其他蛋白质。125I-CCK-33与其膜结合蛋白之间用MBS进行交联反应的效率显著高于用诸如辛二酸二琥珀酰亚胺酯等氨基导向的同双功能试剂所获得的效率。在结合和交联之前用低分子量硫醇还原膜蛋白可显著提高交联效率。使用长度相似但化学反应性不同的化学交联剂获得的CCK结合蛋白的差异标记模式强调了仅根据亲和标记数据预测天然受体结构时需谨慎。使用相同的胰腺质膜制剂和125I-胰岛素，以MBS作为交联剂亲和标记了胰岛素受体的分子量为125,000的α亚基，证明了其在鉴定其他肽激素受体方面的实用性。
