Kozuka M, Ito T, Hirose S, Lodhi K M, Hagiwara H
Department of Biological Sciences, Tokyo Institute of Technology, Japan.
J Biol Chem. 1991 Sep 5;266(25):16892-6.
Endothelin receptor was purified from bovine lung by a rapid and simple two-step procedure: 1) solubilization with the detergent 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and digitonin and 2) affinity chromatography using biotinylated endothelin and avidin-agarose. Starting from 3.5 kg of bovine lung, about 200 micrograms of pure receptor were obtained. Microsequencing of tryptic fragments of the purified protein revealed a high sequence similarity with the rat endothelin ETB receptor that has very recently been cloned by expression cloning and shown to be nonselective in terms of the ligand specificity. Purification of the receptor in the presence of low (1 mM) and high (50 mM) concentrations of EDTA yielded, as a major form, 34- and 52-kDa species, respectively, indicating that the lower Mr species (34 kDa) is a proteolytic product of the 52-kDa species. Interestingly, this metal proteinase-mediated limited proteolysis did not affect the ligand binding properties of the receptor.
1）用去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐和洋地黄皂苷进行增溶；2）使用生物素化的内皮素和抗生物素蛋白-琼脂糖进行亲和层析。从3.5千克牛肺开始，获得了约200微克的纯受体。对纯化蛋白的胰蛋白酶片段进行微量测序显示，其与大鼠内皮素ETB受体具有高度的序列相似性，该受体最近通过表达克隆被克隆出来，并在配体特异性方面显示为非选择性的。在低浓度（1 mM）和高浓度（50 mM）的乙二胺四乙酸（EDTA）存在下纯化受体，分别产生了主要形式为34 kDa和52 kDa的蛋白，这表明较低分子量的蛋白（34 kDa）是52 kDa蛋白的蛋白水解产物。有趣的是，这种金属蛋白酶介导的有限蛋白水解并不影响受体的配体结合特性。
