Solubilization and characterization of endothelin-1 receptors in rat cardiac tissue

Biochem Biophys Res Commun. 1990 Jan 15;166(1):299-307. doi: 10.1016/0006-291x(90)91945-o.

Abstract

Adult rat cardiac endothelin-1 (ET-1) receptors were solubilized with 0.5% digitonin and then characterized. The receptors retained binding activity after solubilization. Binding was saturable (KD of 0.065 +/- 0.004 nM, Bmax of 94.6 +/- 4.5 fmol/mg protein; Hill coefficient of 0.987 +/- 0.017 n = 6) and pH dependent, with the binding increasing as the pH was decreased from 10 to 4, but decreasing dramatically as pH dropped to 2. Specifically bound [125I]-ET-1 was not dissociated by 2 x 10(-7) M unlabelled ET-1, but was dissociated by pH 10 and 2. Returning the pH to 7.4 restored the binding activity of the receptors. Unlabelled ET-1 (10(-12) - 10(-7) M) and sarafotoxin S6b(10(-12) - 10(-7) M) competed with [125I]-ET-1 for binding to the receptors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding, Competitive
  • Cell Membrane / metabolism
  • Digitonin
  • Endothelins
  • Endothelium, Vascular
  • Female
  • Hydrogen-Ion Concentration
  • Kinetics
  • Myocardium / metabolism*
  • Peptides / metabolism
  • Rats
  • Rats, Inbred Strains
  • Receptors, Cell Surface / isolation & purification*
  • Receptors, Cell Surface / metabolism
  • Receptors, Endothelin

Substances

  • Endothelins
  • Peptides
  • Receptors, Cell Surface
  • Receptors, Endothelin
  • Digitonin