Affinity labelling of endothelin receptor and characterization of solubilized endothelin–endothelin‐receptor complex

H. Miyazaki, M. Kondoh, H. Watanabe, Y. Masuda, K. Murakami, M. Takahashi, Masashi Yanagisawa, S. Kimura, K. Goto, T. Masaki

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23 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)125-129
Number of pages5
JournalEuropean Journal of Biochemistry
Issue number1
StatePublished - Jan 1990

ASJC Scopus subject areas

  • Biochemistry


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