title: Solubilized benzodiazepine receptors for use in receptor assays.
abstract: In the development of non-radioactive receptor assays for benzodiazepines, employing fluorescent ligands, it was observed that the fluorescence measurements were hampered by the background fluorescence of the receptor preparation. This receptor preparation is a brain tissue homogenate in which the benzodiazepine receptors are membrane-bound. To minimize the influence of the receptor material on the fluorescence detection, the benzodiazepine receptors were solubilized with 0.5% sodium deoxycholate. The binding characteristics of the receptors were examined after solubilization and compared with membrane-bound receptors. The Kd and Bmax 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 and 0.54 pM mg-1 protein respectively. Inhibition curves with the benzodiazepine antagonist flumazenil and the agonist lorazepam revealed that their affinities for the solubilized receptor as compared to the membrane-bound receptor were also reduced from 0.67 nM to 3.2 nM and from 1.49 nM to 8.4 nM respectively. The detection limits for the two benzodiazepines, however, were not affected by the solubilization. Furthermore, three different methods to separate the fraction of free labelled ligand and the fraction bound to the solubilized receptor were compared, namely polyethylene glycol precipitation/filtration, ion exchange filtration and charcoal adsorption. Polyethylene glycol precipitation/filtration gave the highest yield for the bound fraction and the best reproducibility.
authors.affiliation: Groningen Institute for Drug Studies (GIDS), University Centre for Pharmacy, Department of Analytical Chemistry and Toxicology, The Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Janssen
authors.forename: M
authors.initials: M
authors.lastname: Stegeman
authors.forename: K
authors.initials: K
authors.lastname: Ensing
authors.forename: R A
authors.initials: RA
authors.lastname: de Zeeuw
mesh_terms: D000327:Adsorption; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002606:Charcoal / Q000737:chemistry; D005374:Filtration; D005442:Flumazenil / Q000378:metabolism; D018757:GABA Modulators / Q000378:metabolism; D066298:In Vitro Techniques; D008140:Lorazepam / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence
chemical_list: D018757:GABA Modulators; D011963:Receptors, GABA-A; D002606:Charcoal; D011092:Polyethylene Glycols; D005442:Flumazenil; D008140:Lorazepam