Hagiwara H, Kozuka M, Eguchi S, Shibabe S, Ito T, Hirose S
Department of Biological Sciences, Tokyo Institute of Technology, Japan.
Biochem Biophys Res Commun. 1990 Oct 30;172(2):576-81. doi: 10.1016/0006-291x(90)90712-v.
Bovine lung endothelin receptors were solubilized in a non-aggregated state and characterized in terms of their minimal functional size and chemical nature of the ligand-binding site. A variety of detergents and their combinations were tested for their efficiency to solubilize endothelin receptors from bovine lung plasma membranes, and a combination of 0.4% digitonin and 0.25% CHAPS was found to be very effective in obtaining highly dispersed receptor solution. Gel filtration of the CHAPS/digitonin-solubilized receptors revealed the presence of two receptor species eluting at the positions corresponding to 34 and 52 kDa. These values were in good agreement with those estimated by affinity labeling and SDS-polyacrylamide gel electrophoresis, establishing that they represent minimal functional units. Chemical modification of ligand-occupied and free receptors with p-chloromercuriphenylsulfonic acid revealed that both 34 and 52 kDa receptors have SH group(s) essential for the receptor activity in their ligand-binding sites.
牛肺内皮素受体以非聚集状态溶解，并根据其最小功能大小和配体结合位点的化学性质进行表征。测试了多种去污剂及其组合从牛肺质膜中溶解内皮素受体的效率，发现0.4%洋地黄皂苷和0.25% CHAPS的组合在获得高度分散的受体溶液方面非常有效。对CHAPS/洋地黄皂苷溶解的受体进行凝胶过滤，结果显示存在两种受体，分别在对应于34 kDa和52 kDa的位置洗脱。这些值与通过亲和标记和SDS-聚丙烯酰胺凝胶电泳估计的值非常一致，表明它们代表最小功能单位。用对氯汞苯磺酸对结合配体和游离的受体进行化学修饰，结果表明34 kDa和52 kDa的受体在其配体结合位点均具有对受体活性至关重要的巯基。
