EVALUATION OF A PURIFICATION PROCEDURE FOR THE MUSCARINIC RECEPTOR FOR THE PURPOSE OF QUANTITATIVE RECEPTOR ASSAYS OF ANTICHOLINERGICS .B. THE SOLUBILIZED RECEPTOR

For the purpose of quantitative receptor assays, a three-step solubili zation procedure including three optimization sets for muscarinic rece ptor from calf striatum was developed. The first step includes the ext raction of the P2-pellet with n-hexane and consequently with 2 M NaCl. By the latter, 39% of non-receptor proteins was extracted. The result ing 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 H-3-dexe timide (H-3-DEX) and the ary quaternary H-3-N-methylscopolamine (H-3-N MS). The resulting receptor density measured with H-3-dexetimide was l ower (43.3% of that for the NaCl-pellet) than that for H-3-N-methyl-sc opolamine (56.7%). The treatment with digitonin preserved the high aff inity for H-3-N-methylscopolamine (K-d = 0.645 nM), however the affini ty of H-3-dexetimide decreased after solubilization (K-d = 8.526 nM). The use of solubilized receptors in combination with hydrophilic H-3-N MS allows to increase the ratio specific/non-specific binding, since t he non-specific binding for this ligand to the solubilized preparation is lower when compared with membrane-bound receptors. The above solub ilization procedure was found preferable over directly solubilizing th e P2-pellet since (a) the receptor density for 3H-NMS was higher for t he solubilized NaCl-pellet by a factor of about 1.7, and (b) the treat ment of the P2-pellet with digitonin resulted in a lowering of the K-d 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 impro ve the quantitative receptor assays of plasma samples. Unlike the memb rane-bound receptor, a high volume of plasma, such as 400 mu l, can be added to the assay without any influence on the H-3-DEX binding when solubilized preparation is used.