Purification of the muscarinic acetylcholine receptor from porcine atria.

Abstract

The muscarinic acetylcholine receptor from porcine atria has been purified 100,000-fold to homogeneity by solubilization in digitonin/cholate and sequential chromatography on wheat germ agglutinin-agarose, diethylaminoethylagarose, hydroxylapatite, and 3-(2'-aminobenzhydryloxy)tropane-agarose. The yield of purified receptor was 4.3% of that found in the membrane fraction, and the purified receptor bound 11.1-12.8 nmol of L-[3H]quinuclidinyl benzilate per mg of protein, corresponding to a binding component Mr of 78,400-90,000. The purified receptor preparation consisted of two polypeptides in approximately equimolar amounts when examined on silver-stained sodium dodecyl sulfate/polyacrylamide gels. The larger polypeptide (Mr 78,000 on 8% polyacrylamide gels) was specifically alkylated with [3H]propylbenzilylcholine mustard, whereas the smaller polypeptide (Mr 14,800) was not labeled. The possibility that the small polypeptide is a contaminant fortuitously appearing in equimolar amounts with the large polypeptide cannot be ruled out at this time. The purified preparation was highly stable, with no measurable change in the number of ligand binding sites or the gel pattern after 1 month's storage on ice. Scatchard analysis showed a single class of binding sites for the antagonist L-[3H]quinuclidinyl benzilate with a dissociation constant of 61 +/- 4 pM. Equilibrium titration experiments demonstrated that the antagonist L-hyoscyamine displaced L-[3H]quinuclidinyl benzilate from a single class of sites (Kd = 475 +/- 30 pM), whereas the agonist carbamoylcholine interacted at two populations of sites (53% +/- 3% high affinity, Kd = 1.1 +/- 0.3 microM; 47% +/- 3% low affinity, Kd = 67 +/- 14 microM). The ligand binding data were very similar to that for the membrane-bound receptor, suggesting that the receptor has not been altered radically during purification.

Chemical composition and cardiovascular effects induced by the essential oil of Cymbopogon citratus DC. Stapf, Poaceae, in rats

“…It is established that the stimulation of cardiac muscarinic receptors from sinoatrial node by vagal action induces intense bradycardia, which can be followed by hypotension due to a decrease in cardiac output (Peterson et al, 1984). It is also well known that activation of endothelial muscarinic receptors induces intense vasodilatation due to release of endothelium-derived relaxing factors, mainly NO and PGI 2 (Furchgott & Zawadzki, 1980;Moncada & Higgs, 1993).…”

The effect of ionic strength on cardiac muscarinic receptors

“…We know that in the heart, the major part of the receptor undergoes a similar conversion to the L type in the presence of certain guanine nucleotides, such as GTP or GppNHp (Berrie et al, 1979;Wei & Sulakhe, 1979;Rosenberger et al, 1980) and further that the solubilized receptor seems to contain a single population of agonist-antagonist binding polypeptides with a molecular weight ofabout 87K (Birdsall et al, 1979b;Peterson et al, 1984;Haga & Haga, 1985;Shirakawa & Tanaka, 1985). Further, it appears that agonists change the degree of association of these subunits.…”

Two molecular weight forms of muscarinic acetylcholine receptors in the avian central nervous system: switch in predominant form during differentiation of synapses.

“…Various studies using NaDodSO4/PAGE and target-site analysis have indicated only a single form of the muscarinic receptor, with estimates of apparent molecular weight ranging from 70,000 to 96,000 (20,28,29), although possible heterogeneity recently has been noted (30). These studies were done with receptors from mature tissue.…”

“…Although minor peaks of radioactivity above Mr 100,000 and below Mr 60,000 were occasionally present, the two major peaks were observed in every preparation. The average apparent molecular weight of the two receptor forms for 42 (20) and bovine brain (21). Binding was completely eliminated by treatment at 30'C for 24 hr with endoglycosidase D (0.01 unit/ml) and endoglycosidase H (0.5 ug/ml), indicating that most, if not all, N-acetylglucosamine and sialic acid residues occurred on N-linked oligosaccharide chains.…”