Nakajo S, Sugiura M, Snajdar R M, Boehm F H, Inagami T
Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, TN 37232.
Biochem Biophys Res Commun. 1989 Oct 16;164(1):205-11. doi: 10.1016/0006-291x(89)91703-8.
Endothelin-1 (ET-1) receptor was identified on the membranes from human placenta and 66% of original binding activity in the membranes was solubilized with 0.75% (w/v) CHAPS. Binding studies of the solubilized membranes using 125I-ET-1 indicated the presence of a single class of high-affinity binding sites with an apparent Kd of 760 pM and a Bmax of 1.8 pmol/mg of protein. The binding was inhibited by addition of unlabeled ET-1 and ET-3 in dose dependent manner. The Ki values of solubilized membranes were 84 pM for ET-1 and 250 pM for ET-3, whereas particulate membranes had weaker affinities (Ki = 410 pM for ET-1, 2500 pM for ET-3). Calcium channel blockers such as nicardipine, verapamil and diltiazem did not affect the binding of 125I-ET-1. Affinity labeling of the particulate and solubilized membranes with CHAPS revealed a specific binding protein with a Mr of 32,000.
在内皮素-1（ET-1）受体是在人胎盘膜上鉴定出来的，膜中66%的原始结合活性可被0.75%（w/v）的CHAPS溶解。使用125I-ET-1对溶解后的膜进行结合研究表明，存在一类单一的高亲和力结合位点，其表观解离常数（Kd）为760 pM，最大结合容量（Bmax）为1.8 pmol/mg蛋白质。未标记的ET-1和ET-3以剂量依赖的方式抑制该结合。溶解后的膜对ET-1的抑制常数（Ki）值为84 pM，对ET-3为250 pM，而微粒膜的亲和力较弱（ET-1的Ki = 410 pM，ET-3的Ki = 2500 pM）。钙通道阻滞剂如尼卡地平、维拉帕米和地尔硫䓬不影响125I-ET-1的结合。用CHAPS对微粒膜和溶解后的膜进行亲和标记，显示出一种分子量为32,000的特异性结合蛋白。
