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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.

Information available on the molecular mechanism of neurotransmitter release is minimal. However, it has been demonstrated that purified preparations of brain synaptosomes are extremely useful for biochemical studies on the uptake and release of neurotransmitters (de Belleroche & Bradford, 1972; Wonnacott & Marchbanks, 1976). Moreover, electrophysiological and pharmacological studies have shown that 8-bungarotoxin and tityustoxin, polypeptides from snake and scorpion venoms respectively, block neurotransmission by acting primarily on the presynaptic nerve membrane and specifically affecting transmitter release (Chang et al., 1973; Warnick er al., 1976). 8-Bungarotoxin causes an initial increase in transmitter release followed by complete blockade, which is probably due to an inability to release the contents of synaptic vesicles (Chang et al., 1973); at higher concentrations it causes swelling of mitochondria in the axon terminals (Chen & Lee, 1970; Sen et al., 1976). In contrast, tityustoxin increases release due to depolarization of the nerve terminal, by opening or prolonging the closing of the sodium channels (its action is inhibited by tetrodotoxin); this is followed by complete inhibition of release, probably due to ultrastructural changes in the nerve (Warnick e f al., 1976; Diniz et al., 1974). In the present communication we describe the effects of highly purified preparations of 8-bungarotoxin and tityustoxin on the uptake and release of two putatative neurotransmitters, glutamate and y-aminobutyrate by using purified synaptosomes from brain cortex. 8-Bungarotoxin was purified from the venom of Bungarus multicinctus by chromatography on CM-Sephadex ((2-50) with a convex gradient (0.05-0.9~) of ammonium acetate; complete purification was achieved by preparative isoelectric focusing on Sephadex G-75. Its molecular weight, as determined by gel filtration, was 23000; n o detectable phospholipase activity was present (Lawrence et a/., 1974). After polyacrylamide-gel electrophoresis or isoelectric focusing of the purified toxin, under native conditions, a single protein species was obtained. On dodecyl sulphate/polyacrylamidegelelectrophoresis the toxin gave two polypeptidechains withmol.wts. 12000and 14000. Its toxicity was demonstrated by intraperitoneal injection into mice (LDSo 0.01 ,ug/g body wt .) and also by measuring its ability t o block neuromuscular transmission in frog nerve-sartorius muscle preparation (OS,~g/ml produced complete blockade in 6.5 h at 20°C). Tityustoxin was purified from the venom of the Brazilian yellow scorpion (Tityus serrulatus) and characterized as previously described (Diniz et al., 1974). Its LD,, measured in mice was 0.1 pg/g body weight. Synaptosomes from rat brain cortex were purified by sucrose-density-gradient centrifugation and their respiration rates measured by the methods described by Bradford (1969). Suspensions of synaptosomes (2-4mg of protein/ml) were made in Krebs phosphate medium(or the latter solution without calcium and with0.2m~-EGTA)containing ['4C]glutam...

Homogeneous β‐bungarotoxin, isolated from the venom of Bungarus multicinctus was radiolabelled 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 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. 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 β‐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 × 105 M−1 s−1 and 5.6 × 10−4 s−1 at 37°C, respectively. 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 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+. Toxin I, a single‐chain protein from Dendroaspis polylepis known to potentiate transmitter release at chick neuromuscular junction, completely inhibited the binding of 3H‐β‐bungarotoxin with a Ki 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 3H‐β‐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.

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