SOLUBILIZED BENZODIAZEPINE RECEPTORS FOR USE IN RECEPTOR ASSAYS

In the development of non-radioactive receptor assays for benzodiazepi nes, employing fluorescent ligands, it was observed that the fluoresce nce measurements were hampered by the background fluorescence of the r eceptor preparation. This receptor preparation is a brain tissue homog enate in which the benzodiazepine receptors are membrane-bound. To min imize the influence of the receptor material on the fluorescence detec tion, the benzodiazepine receptors were solubilized with 0.5% sodium d eoxycholate. The binding characteristics of the receptors were examine d after solubilization and compared with membrane-bound receptors. The K-d and B-max values for membrane-bound receptors were 1.20 nM and 1. 01 pM mg(-1) protein and for solubilized receptors they were 4.1 nM an d 0.54 pM mg(-1) protein respectively. Inhibition curves with the benz odiazepine antagonist flumazenil and the agonist lorazepam revealed th at their affinities for the solubilized receptor as compared to the me mbrane-bound receptor were also reduced from 0.67 nM to 3.2 nM and fro m 1.49 nM to 8.4 nM respectively. The detection limits for the two ben zodiazepines, however, were not affected by the solubilization. Furthe rmore, three different methods to separate the fraction of free labell ed ligand and the fraction bound to the solubilized receptor were comp ared, namely polyethylene glycol precipitation/filtration, ion exchang e filtration and charcoal adsorption. Polyethylene glycol precipitatio n/filtration gave the highest yield for the bound fraction and the bes t reproducibility.