Preparation of Neurotoxic 3H-β-Bungarotoxin: Demonstration of Saturable Binding to Brain Synapses and Its Inhibition by Toxin I

Abstract

Homogeneous .beta.-bungarotoxin, isolated from the venom of Bungarus multicinctus was radiolabeled with N-succinimidyl-[2,3-3H]propionate. Stable, di-propionylated material was obtained which was tritiated on both subunits and had a specific radioactivity of 102 Ci/mmol. After separation from unlabeled toxin by isoelectric focusing, it was shown to exhibit significant biological activity in both the peripheral nervous system and CNS but had negligible phospholipase A2 activity towards lecithin or cerebrocortical synaptosomes. The labeled neurotoxin binds specifically to a single class of noninteracting sites of high affinity (Kd = 0.6 nM) on rat cerebral cortex synaptosomes; the content of sites is .apprx. 150 fmol/mg protein. This binding was inhibited by unlabeled .beta.-bungarotoxin with a potency which indicates that tritiation does not alter the affinity significantly. The association of toxin with its binding component and its dissociation were monophasic; rate constants observed were 7.8 .times. 105 M-1 s-1 and 5.6 .times. 10-4 s-1 at 37.degree. C, respectively. .beta.-Bungarotoxin whose phospholipase activity was inactivated with p-bromophenacyl bromide inhibited to some extent the binding of tritiated toxin but with low efficacy. Taipoxin and phospholipase A2 from bee venom, but not Naja melanoleuca, inhibited the synaptosomal binding of toxin with low potencies in the presence, but not absence, of Ca2+. Toxin I, a single-chain protein from Dendroaspis polylepis known to potentiate transmitter release at chick neuromuscular junction, completely inhibited the binding of 3H-.beta.-bungarotoxin with a Ki of 0.07 nM; this explains its ability to antagonize the neuroparalytic action of .beta.-bungarotoxin. Other pure presynaptic neurotoxins, .alpha.-latrotoxin and botulinum neurotoxin failed to antagonize the observed binding; likewise tityustoxin, which is known to affect Na channels, had no effect on 3H-.beta.-bungarotoxin binding. Trypsinization of synaptosomes completely destroyed the binding activity, suggesting that the binding component is a protein; the functional role of the latter is discussed in relation to the specificity of toxin binding.