Solubilization and characterization of atrial muscarinic acetylcholine receptors in sucrose monolaurate.
Muscarinic acetylcholine receptors (mAChRs) can be solubilized from porcine atrial membranes in sucrose monolaurate (SM-1200) with retention of up to 80% of N-[3H]methylscopolamine ([3H]NMS) binding activity. The mAChRs solubilized by SM-1200 were relatively stable at 4 degrees C with an estimated half-life (tau 1/2) of the ligand binding activity of 19 days. Inactivation of the ligand binding activity is dependent on the incubation temperature, and tau 1/2 was estimated to be 5.7 h at 20 degrees C, 28 min at 30 degrees C, and less than 1 min at 45 degrees C. The activation energy of the receptor inactivation was estimated to be 199 kJ/mol. Ligand binding characteristics of SM-1200-solubilized mAChRs were similar to those of digitonin/cholate-solubilized receptors. Sucrose density gradient centrifugation revealed a single peak with an apparent sedimentation coefficient of 5.7 S. The solubilized atrial mAChRs were purified approximately 1000-fold by using affinity chromatography with aminobenztropine as the ligand. The purified mAChRs were reconstituted with GTP binding regulatory proteins (G(o)) in lipid vesicles, and the reconstituted vesicles showed guanine nucleotide-sensitive, high-affinity agonist binding and agonist-stimulated GTP gamma S binding in the presence of GDP. Thus, sucrose monolaurate is a new detergent in which mAChRs can be solubilized in stable form with high yield and purified up to 1000 times with retention of the binding activity with muscarinic ligands and G-proteins.
Rinken
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Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Haga
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D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D004355:Drug Stability; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006207:Half-Life; D006325:Heart Atria / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry*; D019832:N-Methylscopolamine; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012602:Scopolamine Derivatives / Q000378:metabolism; D012995:Solubility; D013395:Sucrose / Q000031:analogs & derivatives*; D013552:Swine; D013696:Temperature; D013816:Thermodynamics
D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; C018152:sucrose monolaurate; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013395:Sucrose; D011813:Quinuclidinyl Benzilate; D019204:GTP-Binding Proteins; D019832:N-Methylscopolamine