42 research outputs found
EVALUATION OF A PURIFICATION PROCEDURE FOR THE MUSCARINIC RECEPTOR FOR THE PURPOSE OF QUANTITATIVE RECEPTOR ASSAYS OF ANTICHOLINERGICS .A. THE MEMBRANE-BOUND RECEPTOR
EVALUATION OF A PURIFICATION PROCEDURE FOR THE MUSCARINIC RECEPTOR FOR THE PURPOSE OF QUANTITATIVE RECEPTOR ASSAYS OF ANTICHOLINERGICS .A. THE MEMBRANE-BOUND RECEPTOR
The presented purification procedure for the muscarinic receptor from calf striatum includes the extraction of lipids with hexane in the first step and the removal of 39% of non-receptor proteins with 2 M NaCl in the second step. The simplicity of such an approach to the purification of the receptor warrants its use in the routine practice for quantitative purposes. The high affinity binding of tertiary H-3-dexetimide (H-3-DEX) and quaternary H-3-N-methylscopolamine (H-3-NMS) is preserved after the removal of irrelevant lipids and proteins from the P2-pellet
METHODOLOGICAL ASPECTS OF QUANTITATIVE RECEPTOR ASSAYS
Receptor assays occupy a particular position in the methods used in bioanalysis, as they do not exploit the physico-chemical properties of the analyte. These assays make use of the property of the analyte to bind to the specific binding site (receptor) and to competitively replace a labelled ligand from the same binding site. The amount of labelled ligand replaced is a measure of the amount as well as the affinity of the analyte. Thus, receptor assays offer additional information about the biological (pharmacological) activity of the analyte by distinguishing the compounds on the basis of their specific binding rather than specific molecular structure (chromatographic and non-chromatographic methods). This paper, starting with the general principles of receptor-ligand interaction, focuses on the application of ligand-binding techniques to the quantitative analysis. The factors which influence the sensitivity and the specificity of quantitative receptor assays, as well as the main directions in the improvement of the receptor preparation by using the solubilized and purified receptor are discussed. In order to enhance the use of these assays in routine practice, the development of solid-phase receptor assays is considered
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 evaluated. The dissociation constants for two chemically different anticholinergics, the tertiary amine scopolamine and the quaternary amine oxyphenonium, were calculated from inhibition studies of H-3-NMS binding in buffer and plasma. The K-d values for both anticholinergics were similar for all the membrane-bound receptor preparations (unpurified and the purified P2 pellet) either in buffer or in plasma. More pronounced differences were observed between the membrane-bound and solubilized receptors. By introducing the solubilized receptor as well, differences between the individual anticholinergics appeared. On the one hand: for scopolamine, a gain in sensitivity of 1.5-2.8 in plasma was observed for the solubilized receptor. On the other hand, in the case of oxyphenonium, a dramatic loss in sensitivity (by a factor of about 24) was observed with the solubilized receptor, as compared to the membrane-bound receptor, in buffer. Very interestingly, however, when the solubilized 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
EVALUATION OF A PURIFICATION PROCEDURE FOR THE MUSCARINIC RECEPTOR FOR THE PURPOSE OF QUANTITATIVE RECEPTOR ASSAYS OF ANTICHOLINERGICS .B. THE SOLUBILIZED RECEPTOR
Oil Mill Op SC
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 H-3-dexetimide (H-3-DEX) and the ary quaternary H-3-N-methylscopolamine (H-3-NMS). The resulting receptor density measured with H-3-dexetimide was lower (43.3% of that for the NaCl-pellet) than that for H-3-N-methyl-scopolamine (56.7%). The treatment with digitonin preserved the high affinity for H-3-N-methylscopolamine (K-d = 0.645 nM), however the affinity of H-3-dexetimide decreased after solubilization (K-d = 8.526 nM). The use of solubilized receptors in combination with hydrophilic H-3-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 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 improve the quantitative receptor assays of plasma samples. Unlike the membrane-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