Evaluation of A Purification Procedure for the Muscarinic Receptor for the Purpose of Quantitative Receptor Assays of Anticholinergics

Abstract

For the purpose of quantitative receptor assays, a three-step solubilization procedure including three optimization sets for muscarinic receptor from calf striatum was developed. The first step includes the extraction of the P2-pellet with n-hexane and consequently with 2 M NaCl. By the latter, 39% of non-receptor proteins was extracted. The resulting pellet (NaCl-pellet), enriched in muscarinic receptors by a factor of 1.5-1.7, was solubilized with 1% digitonin. The binding parameters of the solubilized receptor were determined for the tertiary 3H-dexetimide (3H-DEX) and the quaternary 3H-N-methylscopolamine (3H-NMS). The resulting receptor density measured with 3H-dexetimide was lower (43.3% of that for the NaCl-pellet) than that for 3H-N-methyl-scopolamine (56.7%). The treatment with digitonin preserved the high affinity for 3H-N-methylscopolamine (Kd = 0.645 nM), however the affinity of 3H-dexetimide decreased after solubilization (Kd = 8.526 nM). The use of solubilized receptors in combination with hydrophilic 3H-NMS allows to increase the ratio specific/non-specific binding, since the non-specific binding for this ligand to the solubilized preparation is lower when compared with membrane-bound receptors. The above solubilization procedure was found preferable over directly solubilizing the P2-pellet since (a) the receptor density for 3H-NMS was higher for the solubilized NaCl-pellet by a factor of about 1.7, and (b) the treatment of the P2-pellet with digitonin resulted in a lowering of the Kd to 2.422 nM. However, with respect to the plasma effect on the ligand binding, both solubilized preparations give similar results. The use of the solubilized NaCl-pellet or the P2-pellet can considerably improve the quantitative receptor assays of plasma samples. Unlike the membrane-bound receptor, a high volume of plasma, such as 400 microliters, can be added to the assay without any influence on the 3H-DEX binding when solubilized preparation is used.