Muscarinic receptor-detergent complexes with different biochemical properties: selective solubilization, lectin affinity chromatography and ligand binding studies.
Muscarinic receptors were solubilized by nonionic, zwitterionic and ionic detergents from porcine striatum. A mixture of digitonin and gitonin (3:2) was found to be most suitable in respect to receptor yield and stability. The solubilization of muscarinic receptors by this detergent appears to be dependent on the existence of free detergent micelles. Consequently, the receptor solubilization was studied at different protein-to-detergent ratios. Based on these experiments, a double extraction procedure was developed in which the receptor is solubilized subsequent to the solubilization of other membrane proteins. After elimination of the detergent excess, the binding of the receptor-detergent complex to six immobilized lectins was studied. In accordance with previous reports, we found a considerable portion of the digitonin/gitonin solubilized receptors (one step extraction procedure) specifically bound to wheat germ agglutinin via sialic acid residues. Muscarinic receptors solubilized by a double extraction procedure (either from porcine striatum or rat brain) did not bind to the lectin. This is not owing to selective extraction or partial denaturation, and indicates that considerable portions of the glycan residues are not covalently bound to the receptor polypeptide. A GTP-insensitive heterogenous agonist binding was found only at the non-wheat germ agglutinin binding receptors. The data analysis was performed by the affinity spectra method.
D000818:Animals; D001923:Brain Chemistry; D002793:Cholic Acids; D002846:Chromatography, Affinity; D003902:Detergents / Q000378:metabolism / Q000494:pharmacology*; D004071:Digitalis Glycosides; D004072:Digitonin; D008823:Micelles; D019158:N-Acetylneuraminic Acid; D011485:Protein Binding; D051381:Rats; D011975:Receptors, Mitogen / Q000302:isolation & purification / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000302:isolation & purification* / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis; D012995:Solubility; D013501:Surface-Active Agents / Q000494:pharmacology*; D013552:Swine; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D002793:Cholic Acids; D003902:Detergents; D004071:Digitalis Glycosides; D008823:Micelles; D011975:Receptors, Mitogen; D011976:Receptors, Muscarinic; D012794:Sialic Acids; D013501:Surface-Active Agents; D014909:Wheat Germ Agglutinins; C027503:wheat germ agglutinin receptor; C029021:gitonin; D019158:N-Acetylneuraminic Acid; D004072:Digitonin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate