Preparation of neurotoxic 3H-beta-bungarotoxin: demonstration of saturable binding to brain synapses and its inhibition by toxin I.

1. Homogeneous beta-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 A2 activity towards lecithin or cerebrocortical synaptosomes. 3. The labeled neurotoxin binds specifically to a single class of non-interacting sites of high affinity (Kd = 0.6 nM) on rat cerebral cortex synaptosomes; the content of sites is about 150 fmol/mg protein. This binding was inhibited by unlabelled beta-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 x 10(5) M-1 s-1 and 5.6 x 10(-4) s-1 at 37 C, respectively. 5. beta-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 A2 from bee venom, but not Naja melanoleuca, inhibited the synaptosomal binding of toxin with low potencies in the presence, but not the absence, of Ca2+. 6. 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 antagonise the neuroparalytic action of beta-bungarotoxin. Other pure presynaptic neurotoxins, alpha-latrotoxin and botulinum neurotoxin failed to antagonise the observed binding; likewise tityustoxin, which is known to affect sodium channels, had no effect on 3H-beta-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.

Biochemical and electrophysiological demonstrations of the actions of beta-bungarotoxin on synapses in brain.

Homogeneous beta-bungarotoxin interacts irreversibly with rat olfactory cortex and produced permanent inhibition of neurotransmission (half-time of blockade for 230 nM toxin in 25 min). Binding occurs in the absence of divalent cations, but the rate of synaptic blockade is increased by Ca2+, which activates the intrinsic phospholipase A2 activity of the toxin. Other observable actions of the toxin, seen with rat cerebrocortical synaptosomes, are an increase in the release of acetylcholine, glutamate and gamma-aminobutyrate and impairment of transmitter uptake, which are all insensitive to tetrodotoxin. Inactivation of the toxin's phospholipase activity by chemical modification with p-bromophenacyl bromide diminishes the observed concomitant efflux of the neurotransmitters and lactate dehydrogenase. Collectively, the results support the idea that the toxin binds specifically and irreversibly to component(s) on nerve terminals and this together with the resultant phospholipolysis leads eventually to synaptic blockade. Such a proposal would account for the unique toxicity of the protein relative to phospholipase A2 enzymes.

Complete purification of beta-bungarotoxin. Characterization of its action and that of tityustoxin on synaptosomal accumulation and release of acetylcholine.

beta-Bungarotoxin, a snake venom protein (molecular weight 21 000) that irreversibly blocks release of acetylcholine from nerve terminals, was purified to homogeneity by ion-exchange chromatography and isoelectric focussing. Sodium dodecyl sulphate gel electrophoresis under reducing conditions resolved two subunits of molecular weight 11 400 and 9000. In the presence of deoxycholate, it showed phospholipase activity which was activated by Ca2+ but not Sr2+.beta-Bungarotoxin and tityustoxin, a polypeptide that prolongs the opening of sodium channels, inhibited choline accumulation by synaptosomes purified from rat cortex. Both toxins also induced release of acetylcholine which was maximal in the presence of Ca2+ and showed ED50 values of 5 . 10(8) and 10(6) M, respectively. Unlike tityustoxin, beta-bungarotoxin also induced release of choline and cytoplasmic lactate dehydrogenase from synaptosomes, with similar potency, suggesting that it causes some membrane disruption, following its binding to the membrane. The effects of tityustoxin on both accumulation and release were antagonised by tetrodotoxin, which specifically blocks Na+ channels, indicating that it mediates these effects by depolarization. Thus, these toxins may prove to be useful probes for characterisation of nerve membrane components involved in triggering transmitter release.