Affinity labeling of endothelin receptors in bovine and rat lung membranes by N epsilon 9-azidobenzoyl-125I-endothelin-1.
Endothelin-1 (ET-1) is a potent, vasoconstrictive peptide isolated from culture media of vascular endothelial cells. The binding of ET-1 to membrane preparations from rat and bovine lung was studied using radioiodinated ET-1 (125I-ET-1). With both membrane preparations, 125I-ET-1 showed saturable binding to a single class of high affinity sites. Scatchard analysis of the binding data gave dissociation constants (Kd) for ET-1 of 0.22 nM and 0.15 nM, and receptor densities (Bmax) of 6.1 pmol/mg and 2.7 pmol/mg for rat and bovine lung membranes, respectively. Photo-reactive radioiodinated ET-1, N epsilon 9-azidobenzoyl-125I-ET-1, was synthesized and purified as a mono-reactive affinity labeling reagent. This reagent was used for affinity labeling of ET-1 receptor in bovine and rat lung membranes. Photoaffinity labeling followed by sodium dodecyl sulfate gel electrophoresis and autoradiography gave a radiolabeled protein band with an apparent Mr of 34,000 in both membrane preparations. The labeling of this protein band was inhibited by cold ET-1 in a concentration-dependent manner. Labeling was not abolished by unrelated peptides such as angiotensin II and [Arg8]-vasopressin, or by structurally related bee venom apamin. These results indicate that the ET-1 receptor or its ligand binding subunit consists of a 34,000 Da polypeptide.
Kundu
G C
GC
Department of Heart and Hypertension Research, Cleveland Clinic Foundation Research Institute, OH 44195.
Misono
K S
KS
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D016232:Endothelins / Q000138:chemical synthesis / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D008168:Lung / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017466:Receptors, Endothelin
D000345:Affinity Labels; D001386:Azides; D016232:Endothelins; D007457:Iodine Radioisotopes; C071618:N(epsilon)-9-azidobenzoyliodoendothelin-1; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin