Characterization of curaremimetic neurotoxin binding sites on membrane fractions derived from the human medulloblastoma clonal line, TE671.
Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.
Lukas
R J
RJ
D000109:Acetylcholine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D002999:Clone Cells / Q000378:metabolism*; D003472:Curare / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008527:Medulloblastoma / Q000378:metabolism*; D009538:Nicotine / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism; D014403:Tubocurarine / Q000378:metabolism
D002038:Bungarotoxins; D011950:Receptors, Cholinergic; D009538:Nicotine; D003472:Curare; D000109:Acetylcholine; D005033:Ethylmaleimide; D004229:Dithiothreitol; D014403:Tubocurarine