Solubilization of active receptors for glucagon-like peptide-1(7-36)amide from rat lung membranes.
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)
Göke
R
R
Department of Internal Medicine, Philipps-University of Marburg, Germany.
Kolligs
F
F
Richter
G
G
Lankat-Buttgereit
B
B
Göke
B
B
D000818:Animals; D002412:Cations / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005934:Glucagon; D052216:Glucagon-Like Peptide 1; D000067757:Glucagon-Like Peptide-1 Receptor; D004763:Glucagon-Like Peptides; D006150:Guanine Nucleotides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000378:metabolism*; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D018027:Receptors, Glucagon; D012995:Solubility
D002412:Cations; D003432:Cross-Linking Reagents; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D006150:Guanine Nucleotides; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018027:Receptors, Glucagon; C055453:glucagon-like peptide 1 (7-36)amide; D004763:Glucagon-Like Peptides; D052216:Glucagon-Like Peptide 1; D005934:Glucagon