Selective loss of binding sites for the iodinated α‐neurotoxin I from Naja mossambica mossambica venom upon enzymatic deglycosylation of Torpedo electric organ membranes

Removal of asparagine‐linked carbohydrate chains from Torpedo marmorata electric organ membranes was found to inhibit the binding of the iodinated α‐neurotoxin I from Naja mossambica mossambica snake venom to its receptor. Optimal deglycosylation of membranes by endoglycosidase F resulted in a 55% inhibition of α‐neurotoxin‐I‐saturable binding. Under these conditions, up to 70% of concanavalin A binding was also lost, indicating an efficient removal of mannose‐rich carbohydrate chains. Saturation binding experiments at equilibrium on membranes incubated in the absence of endoglycosidase F indicated, when analyzed by Scatchard plots, the presence of two classes of high‐affinity binding sites for α‐neurotoxin I ( K d = 9 pM and 68 pM respectively) with capacities of 24 and 14 pmol/mg membrane proteins, respectively. After endoglycosidase F treatment, only the former class of binding sites ( K d = 11 pM) was recovered together with a 45% reduction in the number of total binding sites. Dissociation experiments further confirmed the presence of two types of toxin‐receptor complexes in control membranes and the selective loss of the rapidly dissociating component upon deglycosylation. The binding of α‐neurotoxin I to its receptor, deglycosylated or not, was totally inhibited by carbamoylcholine, d ‐tubocurarine or α‐bungarotoxin. These findings show that the neurotoxin binding sites present on the acetylcholine receptor can be discriminated on the basis of their differential susceptibility to the removal of asparagine‐linked carbohydrate chains.