Large Scale Preparation and Characterization of Membrane-Bound and Detergent-Solubilized Muscarinic Acetylcholine Receptor from Pig Atria

Abstract

The muscarinic acetylcholine receptor (mAcChR) has been prepared from pig a trial 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 (QNR) 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.