Purification and characterization of bovine lung endothelin receptor.
Purification and characterization of bovine lung endothelin receptor.
复制标题
牛肺内皮素受体的纯化和表征。
DOI：
10.1016/s0021-9258(18)55386-2
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Hiromi Hagiwara
中科院分区：
文献类型：
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作者：
M. Kozuka;Teizo Ito;Shigehisa Hirose;K. M. Lodhi;Hiromi Hagiwara
本文报道了一种从牛肺组织中分离纯化内皮素受体的简便方法：（1）用3-[（3-胆酰胺丙基）二甲氨基]-1-丙磺酸盐和毛地黄皂苷增溶，（2）用生物素化内皮素和亲和素-琼脂糖亲和层析。从3.5公斤牛肺开始，获得约200微克纯受体。纯化的蛋白质的胰蛋白酶片段的微测序揭示了与大鼠内皮素ETB受体的高度序列相似性，该受体最近通过表达克隆被克隆，并且显示在配体特异性方面是非选择性的。在低（1 mM）和高（50 mM）浓度的EDTA存在下的受体的纯化产生，作为一个主要的形式，34-和52-kDa的物种，分别，表明较低的Mr物种（34 kDa）是52-kDa的物种的蛋白水解产物。有趣的是，这种金属蛋白酶介导的有限的蛋白水解并不影响受体的配体结合特性。
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.