Photoaffinity labeling of the solubilized, partially purified muscarinic acetylcholine receptor from porcine atria by p-azidoatropine methyl iodide

Publisher Website
Google Scholar
Abstract
The synthesis of a [3H] photoaffinity analogue of the muscarinic antagonist atropine, [3H]-p-azidoatropine methyl iodide, is described. The compound appeared to bind to a single class of sites in membrane-bound, solubilized, and partially purified preparations of muscarinic receptor from porcine atria with a dissociation constant (determined by competition vs. [3H]-L-quinuclidinyl benzilate) of .apprx. 1.0 .times. 10-7 M. This value was in agreement with the Kd (8.5 .times. 10-8 M) determined by measuring the concentration dependence of covalent incorporation into a partially purified receptor preparation. Competition experiments indicated that the specific covalent labeling could be blocked by the muscarinic agonist carbamylcholine and the antagonists L-quinuclidinyl benzilate and atropine. An apparent MW of 75,000 .+-. 5000 was found for specifically labeled peptide(s) in a solubilized, partially purified receptor preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.