Identification of endothelin receptors by chemical cross-linking.
Specific binding sites for human/porcine endothelin have been found in rat brain membrane preparations using an [125I]endothelin. The apparent Kd value was 3 +/- 2 nM with a Bmax value of 2,200 +/- 200 fmol/mg protein. Chemical cross-linking of [125I]endothelin to rat brain membranes by using the cross-linking reagent disuccinimidyl suberate (DSS) revealed two specific proteins of Mr = 52,000 and Mr = 30,000. The same results were obtained by photoaffinity labeling of [125I]azidoendothelin to rat brain membranes. The labeling of the two proteins was inhibited in a dose-dependent manner by unlabeled endothelin but not by unrelated peptides. Peptide map comparisons of the Mr = 52,000 and Mr = 30,000 protein bands using Staphylococcus aureus V8 protease and papain revealed that the Mr = 30,000 band is a proteolytic degradation product of the Mr = 52,000. The apparent mol wt of the endothelin receptor is, therefore, 50,000, subtracting the mol wt of the iodinated endothelin.
Schvartz
I
I
Division of Endocrinology, Glaxo Research Laboratories, Research Triangle Park, North Carolina 27709.
Ittoop
O
O
Hazum
E
E
D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D002531:Cerebellum / Q000378:metabolism; D003432:Cross-Linking Reagents; D004305:Dose-Response Relationship, Drug; D016232:Endothelins; D004730:Endothelium, Vascular / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017466:Receptors, Endothelin; D013388:Succinimides
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D016232:Endothelins; D010455:Peptides; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D013388:Succinimides; C019358:disuccinimidyl suberate