Large scale preparation and characterization of membrane-bound and detergent-solubilized muscarinic acetylcholine receptor from pig atria.
The muscarinic acetylcholine receptor (mAcChR) has been prepared from pig atrial membranes by new large scale procedures which result in 30-40 fold enrichment of the receptor in the membrane-bound state and a further three fold enrichment during solubilization. The membrane-bound receptor was prepared by differential and sucrose density gradient centrifugation in 25 mM imidazole, 1 mM EDTA, pH 7.4. A double extraction procedure using a mixed digitonin/cholate detergent was used to solubilize the receptor at a 60-70% yield. The membrane and solubilized preparations had specific activities of 3.5-5 and 8-12 pmol [3H]L-quinuclidinyl benzilate (QNB) binding sites per mg of protein, respectively. The presence of imidazole, which behaved as a weak muscarinic ligand, stabilized the receptor during solubilization and storage. Both the membrane-bound and detergent-solubilized mAcChR bound antagonists at a single class of sites and agonists at two subclasses of QNB sites. The proportion of high affinity agonist sites in the solubilized receptor was about 1/3 that in the membrane receptor. [3H]Propylbenzilylcholine mustard covalently labeled a single prominent atropine-sensitive component with an apparent molecular weight of 70-74,000 on SDS-polyacrylamide gels for both the membrane and solubilized receptor.
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D003902:Detergents; D066298:In Vitro Techniques; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D011813:Quinuclidinyl Benzilate; D011976:Receptors, Muscarinic / Q000302:isolation & purification*; D013552:Swine; D013696:Temperature
D003902:Detergents; D011976:Receptors, Muscarinic; D011813:Quinuclidinyl Benzilate; D001285:Atropine