EFFECT OF PURIFICATION FOLLOWED BY SOLUBILIZATION OF RECEPTOR MATERIAL ON QUANTITATIVE RECEPTOR ASSAYS FOR ANTICHOLINERGIC DRUGS

In order to optimize quantitative receptor assays for anticholinergics , the different receptor preparations resulting from the purification and the solubilization of the P2 pellet From the calf striatum were ev aluated. The dissociation constants for two chemically different antic holinergics, the tertiary amine scopolamine and the quaternary amine o xyphenonium, were calculated from inhibition studies of H-3-NMS bindin g in buffer and plasma. The K-d values for both anticholinergics were similar for all the membrane-bound receptor preparations (unpurified a nd the purified P2 pellet) either in buffer or in plasma. More pronoun ced differences were observed between the membrane-bound and solubiliz ed receptors. By introducing the solubilized receptor as well, differe nces between the individual anticholinergics appeared. On the one hand : for scopolamine, a gain in sensitivity of 1.5-2.8 in plasma was obse rved for the solubilized receptor. On the other hand, in the case of o xyphenonium, a dramatic loss in sensitivity (by a factor of about 24) was observed with the solubilized receptor, as compared to the membran e-bound receptor, in buffer. Very interestingly, however, when the sol ubilized receptor was used in plasma, a lowering of the K-d value was found for both antichloinergics, i.e. the assays became more sensitive . Such an effect (not observed for the membrane-bound receptor) could be obtained only when the percentage of digitonin present in the assay was at least 0.12% (w/v) or higher.