J. Smisterova et al., EFFECT OF PURIFICATION FOLLOWED BY SOLUBILIZATION OF RECEPTOR MATERIAL ON QUANTITATIVE RECEPTOR ASSAYS FOR ANTICHOLINERGIC DRUGS, Journal of pharmaceutical and biomedical analysis, 14(11), 1996, pp. 1425-1433
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.