Dissociation of guanosine 5'-[gamma-thio]triphosphate from guanine-nucleotide-binding regulatory proteins in native cardiac membranes. Regulation by nucleotides and muscarinic acetylcholine receptors

Eur J Biochem. 1992 Mar 1;204(2):725-31. doi: 10.1111/j.1432-1033.1992.tb16687.x.


Binding of the poorly hydrolyzable GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to purified guanine-nucleotide-binding regulatory proteins (G proteins) has been shown to be nonreversible in the presence of millimolar concentrations of Mg2+. In porcine atrial membranes, binding of [35S]GTP[S] to G proteins was stable in the presence of 1 mM Mg2+. However, either large dilution or, even more strongly, addition of unlabelled guanine nucleotides, in the potency order, GTP[S] greater than GTP greater than or equal to guanosine 5'-[beta,gamma-imino]triphosphate greater than GDP greater than or equal to guanosine 5'-[beta-thio]diphosphate greater than GMP, markedly enhanced the observed dissociation, with 20-30% of bound [35S]GTP[S] being released by unlabelled guanine nucleotide within 20 min at 25 degrees C. Most interestingly, dissociation of [35S]GTP[S] was rapidly and markedly stimulated by agonist (carbachol) activation of cardiac muscarinic acetylcholine receptors. Carbachol-stimulated release of [35S]GTP[S] was strictly dependent on the presence of Mg2+ and an unlabelled guanine nucleotide. Although having different potency and efficiency in releasing [35S]GTP[S] from the membranes by themselves, the guanine nucleoside triphosphates and diphosphates studied, at maximally effective concentrations, promoted the carbachol-induced dissociation to the same extent, while GMP and ATP were ineffective. GTP[S]-binding-saturation experiments indicated that one agonist-activated muscarinic acetylcholine receptor can cause release of bound GTP[S] from three to four G proteins. The data presented indicate that binding of GTP[S] to G proteins in intact membranes, in contrast to purified G proteins, is reversible, and that agonist-activated receptors can even, either directly or indirectly, interact with GTP[S]-bound G proteins, resulting in release of bound guanine nucleoside triphosphate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autoradiography
  • Carbachol / pharmacology
  • Cations, Divalent
  • Chromatography, Thin Layer
  • GTP-Binding Proteins / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism*
  • Magnesium / metabolism
  • Myocardium / metabolism*
  • Nucleotides / physiology*
  • Receptors, Muscarinic / physiology*
  • Swine


  • Cations, Divalent
  • Nucleotides
  • Receptors, Muscarinic
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Carbachol
  • GTP-Binding Proteins
  • Magnesium