Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration.
The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.
Marchot
P
P
Laboratoire de Biochimie, Faculté de Médecine, Université d'Aix-Marseille, France.
Frachon
P
P
Bougis
P E
PE
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D004546:Elapid Venoms / Q000378:metabolism*; D004557:Electric Organ / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D009498:Neurotoxins / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014101:Torpedo; D014403:Tubocurarine / Q000378:metabolism
D002038:Bungarotoxins; D004546:Elapid Venoms; D009498:Neurotoxins; D011950:Receptors, Cholinergic; D014403:Tubocurarine