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Fluorescein isothiocyanate labeled α-cobratoxin: Interaction with acetylcholine receptor from Electrophorus electricus
KAN
FLUORESCEIN ISOTHIOCYANATE LABELED a-COBRATOXIN : INTERACTION WITH ACETYLCHOLINE RECEPTOR FROM ELECTROPHORUS ELECTRICUS S . Kang and A . Maeltcke Max-Planck-Institut for ErnNhrungsphysiologie,
Dortmund, W-Germany
Interaction of monofluoresceinated a-cobratoxin with acetylcholine receptor from Electrop h orus electricus is accompanied by large changes in the toxin's fluorescence . These were employed to probe the following properties : 1) Fluorescent a-cobratoxtn competes with u8labeled toxin for binding to the receptor . Only one class of binding sites with Kp~1 .4x10- M was observed in fluorescence binding studies . The number of binding sites for fluorescent toxin and trttlated toxin was identical . Association of fluorescent toxin and recep~or conf rmed to the rate law of second order reactions with the rate constant being kp~4x10 M- lmin -~ . Dissociation was slightly biphasic with the rate constant being of the order of 4x10 - 3min -1 . The affinity of receptor Increased during long periods of incubation but without significant changes of the toxin's fluorescence . 2) Low molecular weight cholinergic ligands compete with fluorescent toxin for receptor binding . Binding of ligands and neurotoxin is mutually exclusive at equilibrium, the ratio of ligand to neurotoxin sites being 0 .5 . For both, a representative agonist (carbamylcholine) and a representative antagonist (tubocurarine) a single class of nontnteracting sites was detected in competition binding studies . 3) When high concentration of competing small ligands are added to a solution of fluorescent toxin-receptor complexes, there is a transient increase in the rate of complex dissociation . The effectiveness of cholinergic ligands to induce accelerated dissociation varied with the Kp-value of their binary complexes with the receptor and was different for agonists and antagonists . The data support a model for receptor-ligand interaction with different binding sites for toxin and small ligands . Mutually exclusive binding at equilibrium is the result of structural rearrangements by which sites for one class of ligands are converted to sites of very low affinity upon binding of other ligands . Accelerated dissociation is then due to the transient formation of ternary complexes of receptor, toxin and small ligand and can only occur when receptor-toxin complexes are suddenly exposed to high concentrations of small ligands .
FLUORESCEIN ISOTHIOCYANATE LABELED a-COBRATOXIN : INTERACTION WITH ACETYLCHOLINE RECEPTOR FROM ELECTROPHORUS ELECTRICUS S . Kang and A . Maeltcke Max-Planck-Institut for ErnNhrungsphysiologie,
Dortmund, W-Germany
Interaction of monofluoresceinated a-cobratoxin with acetylcholine receptor from Electrop h orus electricus is accompanied by large changes in the toxin's fluorescence . These were employed to probe the following properties : 1) Fluorescent a-cobratoxtn competes with u8labeled toxin for binding to the receptor . Only one class of binding sites with Kp~1 .4x10- M was observed in fluorescence binding studies . The number of binding sites for fluorescent toxin and trttlated toxin was identical . Association of fluorescent toxin and recep~or conf rmed to the rate law of second order reactions with the rate constant being kp~4x10 M- lmin -~ . Dissociation was slightly biphasic with the rate constant being of the order of 4x10 - 3min -1 . The affinity of receptor Increased during long periods of incubation but without significant changes of the toxin's fluorescence . 2) Low molecular weight cholinergic ligands compete with fluorescent toxin for receptor binding . Binding of ligands and neurotoxin is mutually exclusive at equilibrium, the ratio of ligand to neurotoxin sites being 0 .5 . For both, a representative agonist (carbamylcholine) and a representative antagonist (tubocurarine) a single class of nontnteracting sites was detected in competition binding studies . 3) When high concentration of competing small ligands are added to a solution of fluorescent toxin-receptor complexes, there is a transient increase in the rate of complex dissociation . The effectiveness of cholinergic ligands to induce accelerated dissociation varied with the Kp-value of their binary complexes with the receptor and was different for agonists and antagonists . The data support a model for receptor-ligand interaction with different binding sites for toxin and small ligands . Mutually exclusive binding at equilibrium is the result of structural rearrangements by which sites for one class of ligands are converted to sites of very low affinity upon binding of other ligands . Accelerated dissociation is then due to the transient formation of ternary complexes of receptor, toxin and small ligand and can only occur when receptor-toxin complexes are suddenly exposed to high concentrations of small ligands .