Takasuka T, Akiyama N, Horii I, Furuichi Y, Watanabe T
Department of Molecular Genetics, Nippon Roche Research Center, Kanagawa.
J Biochem. 1992 Jun;111(6):748-53. doi: 10.1093/oxfordjournals.jbchem.a123830.
There are at least two types of endothelin receptors, ETA and ETB, present in various tissues. We found that although biotinylated ET-1 could bind to both ETA and ETB receptors, the stability of the formed ligand-receptor complexes was different. When the preformed complexes of receptor (solubilized from canine brain and lung membranes) and biotinylated ET-1 were subjected to avidin agarose column chromatography, most of the ETA activity was recovered in the pass-through fraction and the remainder was recovered in the 0.5 M KSCN eluate as ligand-free forms. On the other hand, the ETB activity bound firmly to the avidin agarose column was eluted with 1.5 M KSCN. The detection of the complex of 125I-ET-1 and its receptor by SDS-PAGE run at a low temperature was only possible with the ETB fractions and the complex of 125I-ET-1 and ETA was unstable during the separation. These results suggest that the conformation of the ligand binding sites of canine ETA and ETB as well as the stability of their ligand-receptor complexes to SDS are significantly different. Similar observations were also obtained for human ETA and ETB receptors.
在各种组织中至少存在两种类型的内皮素受体，即ETA和ETB。我们发现，尽管生物素化的ET-1能与ETA和ETB受体都结合，但所形成的配体-受体复合物的稳定性有所不同。当受体（从犬脑和肺膜中溶解出来）与生物素化的ET-1预先形成的复合物进行抗生物素蛋白琼脂糖柱层析时，大部分ETA活性在穿透部分中回收，其余部分以无配体形式在0.5M KSCN洗脱液中回收。另一方面，牢固结合在抗生物素蛋白琼脂糖柱上的ETB活性用1.5M KSCN洗脱。只有用ETB部分才能通过在低温下进行的SDS-PAGE检测到125I-ET-1与其受体的复合物，而125I-ET-1与ETA的复合物在分离过程中不稳定。这些结果表明，犬ETA和ETB的配体结合位点的构象以及它们的配体-受体复合物对SDS的稳定性存在显著差异。在人ETA和ETB受体上也得到了类似的观察结果。
