alpha-Bungarotoxin-luciferin as a bioluminescent probe for characterization of acetylcholine receptors in the central nervous system.
Firefly luciferin, a substrate for luciferase-catalyzed light emission, has been covalently linked to alpha-bungarotoxin (alpha-BTX) and shown to be an extremely sensitive nonradioactive probe to study nicotinic acetylcholine receptors. The concentration of nicotinic receptors was measured in several regions of the rat brain, with highest concentrations present in hypothalamus and cerebral cortex. Using rat cerebral cortex homogenate, it was determined that alpha-BTX-luciferin binding is saturable with a high affinity (apparent KD = 0.43 nM) and low capacity (42 fmol/mg of protein). The specificity of alpha-BTX-luciferin binding was demonstrated by the ability of various cholinergic agonists and antagonists to inhibit binding. alpha-BTX, lobeline (a cholinergic agonist) and d-tubocurarine (a nicotinic antagonist) are the most potent inhibitors of specific alpha-BTX-luciferin binding (inhibition constant, KI = 0.12, 60, and 60 nM, respectively). Scopolamine, a muscarinic antagonist, is the least potent inhibitor examined (KI greater than 10 mM). The sensitivity of this assay to detect alpha-BTX-luciferin is approximately 3 X 10(-17) mol and allows us to measure alpha-BTX-luciferin binding to tissue samples containing less than 10 micrograms of protein. This represents an approximate 50-fold increase of sensitivity over conventional ligand-receptor assays using 125I-alpha-BTX. These results provide the basis for an ultrasensitive assay using nonradioactive probes in the quantitation of nicotinic acetylcholine receptors. Conjugation of luciferin to other molecules will lead to the development of sensitive bioluminescent immuno- and receptor assays.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D007031:Hypothalamus / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases; D008163:Luminescent Measurements; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014018:Tissue Distribution
D011950:Receptors, Cholinergic; D008156:Luciferases