Affinity chromatography of the muscarinic acetylcholine receptor.
A novel compound, 3-(2'-aminobenzhydryloxy)-tropane (ABT), and an ABT-agarose gel were synthesized and used for the purification of solubilized muscarinic receptors. ABT had a high affinity with an apparent dissociation constant (Kd) of 7 nM for the muscarinic receptors solubilized from the porcine brain by digitonin. An ABT-agarose gel was prepared by coupling ABT with epoxy-activated Sepharose 6B, and the degree of substitution to the gel was determined to be 4-5 mumol/ml of the gel by UV absorption spectrum. During affinity chromatography using 10 ml of the ABT-agarose gel and 100 ml of the digitonin-solubilized preparation, 70% of muscarinic receptors were adsorbed to the gel, in marked contrast with the adsorption of only 2% of proteins. Approximately 25% of muscarinic receptors applied to the gel were eluted biospecifically with 1 mM muscarinic ligands. The purified fraction showed a high affinity for [3H]quinuclidinyl benzylate with a Kd of 0.4 nM and similar specificity for muscarinic ligands to that of unpurified soluble receptors. The protein concentration of the purified fraction was too low to be determined accurately, but very approximately a purification of 10(3)-fold was indicated.
D000818:Animals; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004072:Digitonin; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013552:Swine
D011976:Receptors, Muscarinic; D011813:Quinuclidinyl Benzilate; D004072:Digitonin