Solubilization and characterization of muscarinic receptors from bovine brain.
This study compared the capacity of different detergents to solubilize the muscarinic cholinergic receptor (mAChR) from bovine brain, evaluated various procedures for the measurement of [3H]-L-quinuclidinyl benzilate [( 3H]-L-QNB) binding to solubilized receptors, and examined some physical and chemical characteristics of the soluble material. An active form of the mAChR was solubilized using digitonin (1%), Triton X-100 (0.5%), and a digitonin-cholate mixture (1%, 0.1%). Values of maximal binding (Bmax) were 2.01, 0.47, and 0.68 pmoles/mg protein, respectively. Comparison of equilibrium dialysis, charcoal adsorption, and polyethylene glycol precipitation indicated that these methods differ in their estimation of Bmax. A decrease in [3H]-L-QNB binding to digitonin solubilized receptors occurred upon dilution or incubation at 37 degrees. The half-life at 37 degrees C was 25 min., but was increased by glycerol. Antagonist binding to digitonin solubilized receptors was saturable and of high affinity. Agonist binding had Hill coefficients less than 1 and was increased by micromolar concentrations of cupric ions.
D000818:Animals; D001923:Brain Chemistry; D002217:Carbachol / Q000378:metabolism; D002417:Cattle; D002540:Cerebral Cortex / Q000032:analysis; D019826:Cholic Acid; D002793:Cholic Acids; D002846:Chromatography, Affinity; D003300:Copper / Q000494:pharmacology; D004072:Digitonin; D017830:Octoxynol; D011092:Polyethylene Glycols; D011699:Putamen / Q000032:analysis; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D012995:Solubility; D013696:Temperature
D002793:Cholic Acids; D011976:Receptors, Muscarinic; D011092:Polyethylene Glycols; D011813:Quinuclidinyl Benzilate; D003300:Copper; D002217:Carbachol; D017830:Octoxynol; D019826:Cholic Acid; D004072:Digitonin