Affinity labeling of endothelin receptors in bovine and rat lung membranes by Nϵ9 -azidobenzoyl- 125I-endothelin-1

Abstract

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 ( K d) for ET-1 of 0.22 nM and 0.15 nM, and receptor densities ( B max) of 6.1 pmol/mg and 2.7 pmol/mg for rat and bovine lung membranes, respectively. Photo-reactive radioiodinated ET-1, N ϵ9 -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 M r 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 [Arg 8]-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.