| Abstract
| - Ligand binding to the nicotinic acetylcholine receptor isstudied by surface plasmon resonance. Biotinylated bungarotoxin, immobilized on a streptavidin-coated gold film,binds nicotinic acetylcholine receptor both in detergent-solubilized and in lipid vesicle-reconstituted form withhigh specificity. In the latter case, nonspecific binding tothe sensor surface is significantly reduced by reconstituting the receptor into poly(ethylene glycol)−lipid-containing sterically stabilized vesicles. By preincubation of abulk nicotinic acetylcholine receptor sample with thecompeting ligands carbamoylcholine and decamethoniumbromide, the subsequent specific binding of the receptorto the surface-immobilized bungarotoxin is reduced,depending on the concentration of competing ligand. Thiscompetition assay allows the determination of the dissociation constants of the acetylcholine receptor−carbamoylcholine complex. A KD = 3.5 × 10-6 M for thedetergent-solubilized receptor and a KD = 1.4 × 10-5 Mfor the lipid vesicle−reconstituted receptor are obtained.For decamethonium bromide, a KD = 4.5 × 10-5 M isdetermined for the detergent-solubilized receptor. Thisapproach is of general importance for investigating ligand−receptor interactions in case of small ligand molecules bymass-sensitive techniques.
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