Solubilization of active receptors for glucagon-like peptide-1(7-36)amide from rat lung membranes

Am J Physiol. 1993 Feb;264(2 Pt 1):L146-52. doi: 10.1152/ajplung.1993.264.2.L146.

Abstract

We report on a protocol that allows the solubilization of active glucagon-like peptide (GLP)-1-(7-36)amide receptors from rat lung membranes. Digitonin-solubilized GLP-1(7-36)amide binding proteins from lung membranes most effectively, whereas (3-[(3-cholamidopropyl)- dimethylamino]-1-propane-sulfonate was less affective, and octyl-beta-glucoside, Triton X-100 and Lubrol PX were almost ineffective. Solubilization of binding activity was optimal at a digitonin concentration of 1%, a protein-to-detergent ratio of 1:10, and a pH between 7.0 and 8.0. Binding of GLP-1(7-36)amide to solubilized receptors was dependent on the concentration of solubilized protein. The presence of certain mono- and divalent cations was crucial for binding of GLP-1(7-36)amide to solubilized receptors. Scatchard analysis of the binding data revealed a single class of binding sites with dissociation and maximum binding constant values of 0.40 +/- 0.20 nM and 80.0 +/- 26.0 fmol/mg protein for membrane bound and 7.0 +/- 0.6 microM and 12.0 +/- 6.0 nmol/mg protein for solubilized receptors, respectively. In cross-linking experiments 125I-labeled GLP-1(7-36)amide was covalently attached to GLP-1(7-36)amide receptors on lung membranes. The apparent molecular mass of the solubilized receptor was 55,000 Da. This was proven in another experiment when receptor was consecutively cross-linked after solubilization. Nonhydrolyzable GTP analogues (GTP gamma S or GDP beta S) were unable to reduce GLP-1(7-36)amide-binding at solubilized receptors. This argues that the receptor is solubilized as a single protein and not as a receptor-G protein complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cations / pharmacology
  • Cell Membrane / metabolism
  • Cross-Linking Reagents / pharmacology
  • Glucagon
  • Glucagon-Like Peptide 1
  • Glucagon-Like Peptide-1 Receptor
  • Glucagon-Like Peptides
  • Guanine Nucleotides / pharmacology
  • Hydrogen-Ion Concentration
  • Lung / metabolism*
  • Male
  • Peptide Fragments / metabolism*
  • Peptide Fragments / pharmacology
  • Rats
  • Receptors, Cell Surface / metabolism
  • Receptors, Glucagon*
  • Solubility

Substances

  • Cations
  • Cross-Linking Reagents
  • Glp1r protein, rat
  • Glucagon-Like Peptide-1 Receptor
  • Guanine Nucleotides
  • Peptide Fragments
  • Receptors, Cell Surface
  • Receptors, Glucagon
  • glucagon-like peptide 1 (7-36)amide
  • Glucagon-Like Peptides
  • Glucagon-Like Peptide 1
  • Glucagon