Nicotinic acetylcholine receptor from rat brain. Solubilization, partial purification, and characterization.
Nicotinic acetylcholine receptor protein (nAChR) has been solubilized from rat cerebral cortices by extracting a crude membrane fraction with the nonionic detergent Triton X-100 (polyoxyethylene-p-t-octylphenol). The solubilized nAChR was partially purified by affinity chromatography (Naja naja siamensis alpha-toxin affinity arm, linked to Sepharose 4B) and characterized by binding of 125I-labeled alpha-bungarotoxin. The reaction of labeled toxin and nAChR appears to be second order with a rate constant (k1) equal to 0.38 X 10(5) M-1 S-1 at 20 degrees. The toxin-nAChR complex dissociates with a dissociation rate constant (k-1) of 1.23 X 10(-5) S-1 at 20 degrees (t 1/2 = 15.6 h). The kinetically determined dissociation constant (Kd) for the complex is 3.24 X 10(-10) M. A variety of cholinergic ligands were studied for their ability to inhibit binding of labeled toxin. The results indicate that the brain receptor is indeed nicotinic. The s20, w and v of the toxin-nAChR complex in 0.1% Triton were determined by velocity sedimentation in D2O and H2O sucrose gradients. The values are 12.9 S and 0.80 cm3 g-1. The Stokes radius of the complex determined by gel filtration equals 7.5 nm. The Mr of the complex calculated from the hydrodynamic parameters, and corrected for bound detergent, equals 357,000.
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D002846:Chromatography, Affinity; D003964:Diaphragm / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity
D002038:Bungarotoxins; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D000109:Acetylcholine