Isolation of human cardiac endothelin receptors by a peptide-receptor mobility shift assay

1. A peptide-protein mobility shift assay has been developed, using native polyacrylamide-gel electrophoresis, that enables the isolation of de-natured receptor proteins from small amounts of human cardiac tissue. 2. Radiolabelled endothelin-1 and related peptides were used to identify and isolate endothelin receptors from partially purified membrane extracts of human atrial tissue. 3. Binding analysis using radiolabelled endothelin-1 gave an equilibrium dissociation constant (Kd) of 2 nmol/l, similar to results from binding experiments conducted directly on tissue. 4. Peptide-receptor complexes were electroeluted from native gels and dissociated. Receptor material was characterized by dot-immunobinding analysis of eluates using an antibody raised against a predicted human endothelin receptor sequence.