Meloche S, Ong H, Cantin M, De Léan A
J Biol Chem. 1986 Feb 5;261(4):1525-8.
125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.
使用亲水性交联剂双（磺基琥珀酰亚胺）辛二酸酯，将125I标记的心房利钠因子（ANF）与牛肾上腺球状带细胞膜中的结合位点进行共价交联。在2-巯基乙醇存在下，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示，有两条表观分子量分别为68,000和114,000的蛋白带被特异性标记。未标记的ANF以剂量依赖性方式阻止这两条带的标记，在10^(-10) M时观察到显著抑制。高浓度的血管紧张素II和促肾上腺皮质激素不与125I-ANF竞争结合和交联。未标记的ANF抑制125I-ANF共价交联的剂量反应曲线与抑制其与受体结合的剂量反应曲线一致。在肝细胞膜中未观察到放射性条带。在存在蛋白酶抑制剂的情况下制备并孵育肾上腺膜的实验显示标记模式没有差异。在没有还原剂的情况下进行电泳表明，亲和标记的物种不是来自更大的二硫键连接的成分。交联剂浓度100倍的变化对两种标记物种的表观分子量没有影响，并且两种物种的标记平行增加。讨论了两种标记物种之间可能的关系。
