Solubilization and identification of human placental endothelin receptor.
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.
Nakajo
S
S
Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, TN 37232.
Sugiura
M
M
Snajdar
R M
RM
Boehm
F H
FH
Inagami
T
T
D001345:Autoradiography; D001667:Binding, Competitive; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003902:Detergents; D016232:Endothelins; D004730:Endothelium, Vascular / Q000378:metabolism; D005260:Female; D006801:Humans; D010455:Peptides / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D017466:Receptors, Endothelin; D012995:Solubility
D002121:Calcium Channel Blockers; D003432:Cross-Linking Reagents; D003902:Detergents; D016232:Endothelins; D010455:Peptides; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin