Affinity labelling of endothelin receptor and characterization of solubilized endothelin-endothelin-receptor complex.
Chick cardiac membranes were affinity labelled by cross-linking to membrane-bound 125I-endothelin-1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I-endothelin-1-labelled material in the presence or absence of 2-mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose-dependently inhibited by the addition of unlabelled endothelin-1 (1-100 nM). Subtracting the molecular mass of 125I-endothelin-1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa. To investigate further the molecular properties of endothelin receptor, the 125I-endothelin-1-endothelin-receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)-[3H]PN200-110, a dihydropyridine derivative, and dihydropyridine-sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine-sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I-endothelin-1-endothelin-receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine-sensitive Ca2+ channels and endothelin has its own specific 50-kDa receptor.
Miyazaki
H
H
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Kondoh
M
M
Watanabe
H
H
Masuda
Y
Y
Murakami
K
K
Takahashi
M
M
Yanagisawa
M
M
Kimura
S
S
Goto
K
K
Masaki
T
T
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D016232:Endothelins; D004730:Endothelium, Vascular; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017466:Receptors, Endothelin; D013388:Succinimides / Q000378:metabolism
D000345:Affinity Labels; D016232:Endothelins; D010455:Peptides; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D013388:Succinimides; C018052:disuccinimidyl tartarate