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

1 Homogeneous β‐bungarotoxin, isolated from the venom of Bungarus multicinctus was radiolabelled with N ‐Succinimidyl‐[2,3‐ 3 H]propionate. Stable, di‐propionylated material was obtained which was tritiated on both subunits and had a specific radioactivity of 102 Ci/mmol. 2 After separation from unlabelled toxin by isoelectric focussing, it was shown to exhibit significant biological activity in both the peripheral and central nervous systems but had negligible phospholipase A 2 activity towards lecithin or cerebrocortical synaptosomes. 3 The labeled neurotoxin binds specifically to a single class of non‐interacting sites of high affinity ( K d = 0.6 nM) on rat cerebral cortex synaptosomes; the content of sites is about 150 fmol/mg protein. This binding was inhibited by unlabelled β‐bungarotoxin with a potency which indicates that tritiation does not alter the affinity significantly. 4 The association of toxin with its binding component and its dissociation were monophasic; rate constants observed were 7.8 × 10 5 M −1 s −1 and 5.6 × 10 −4 s −1 at 37°C, respectively. 5 β‐Bungarotoxin whose phospholipase activity had been inactivated with p ‐bromophenacyl bromide inhibited to some extent the binding of tritiated toxin but with low efficacy. Taipoxin and phospholipase A 2 from bee venom, but not Naja melanoleuca , inhibited the synaptosomal binding of toxin with low potencies in the presence, but not the absence, of Ca 2+ . 6 Toxin I, a single‐chain protein from Dendroaspis polylepis known to potentiate transmitter release at chick neuromuscular junction, completely inhibited the binding of 3 H‐β‐bungarotoxin with a K i of 0.07 nM; this explains its ability to antagonise the neuroparalytic action of β‐bungarotoxin. Other pure presynaptic neurotoxins, α‐latrotoxin and botulinum neurotoxin failed to antagonise the observed binding; likewise tityustoxin, which is known to affect sodium channels, had no effect on 3 H‐β‐bungarotoxin binding. 7 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.