Venom Of Bungarus Multicinctus Research Articles

Kappa-flavitoxin: isolation of a new neuronal nicotinic receptor antagonist that is structurally related to kappa-bungarotoxin

A peptide, termed kappa-flavitoxin ( k-flavitoxin), has been purified from the venom of the red-headed krait, Bungarus flaviceps, by low- and high-pressure liquid chromatography. k-Flavitoxin has apI 8.8 and an apparent molecular weight on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of 6500 Da. k-Flavitoxin is a inhibitor of nicotinic transmission in autonomic ganglia, producing a complete and long-lasting blockade at doses as low as 50 nM. Intracellular recordings reveal a selective blockade of neuronal nicotinic receptors by the toxin, with no effects on other active or passive properties of neuronal membranes. k-Flavitoxin shares a number of pharmacological and biochemical properties with k-bungarotoxin, purified from the venom of Bungarus multicinctus. The two peptides exhibit considerable homology in their amino acid sequences. Radiolabeled k-flavitoxin binds to a nicotinic site in ciliary ganglia previously identified by k-bungarotoxin, which appears to be associated with the neuronal nicotinic receptor. This site is not recognized by α-bungarotoxin, which also does not block nicotinic transmission in this ganglion.

Amino acid sequence of toxin F, a snake venom toxin that blocks neuronal nicotinic receptors

The amino acid sequence was determined for toxin F, a component of Bungarus multicinctus venom that blocks nicotinic transmission in the chick ciliary ganglion and the rat superior cervical ganglion. Toxin F was purified by a procedure that includes preparative isoelectric focusing and ion exchange chromatography. Seventy nanomolar toxin F blocks nicotinic transmission in the chick ciliary ganglion; however, the toxin only weakly blocks the binding of 125I-α-bungarotoxin to membranes derived from Torpedo californica electroplax (IC 50 = 1 μM). These data raise the possibility that toxin F may preferentially recognize neuronal nicotinic receptors. Toxin F focused identically on an isoelectric focusing gel with samples of two similar toxins, bungarotoxin 3.1 and χ-bungarotoxin. The sequence of toxin F is identical with that recently reported for χ-bungarotoxin. When the N-terminal portion of bungarotoxin 3.1 was sequenced, it was found to be identical to the other two toxins. These and other data suggest that the 3 toxins are, in fact, the same.

Immunological cross-reactivity of phospholipase A2 from snake venoms.

The immunological cross-reactivity of phospholipase A2 (PEA2) from the venoms of snakes with a variety of family, genus and species was investigated by immunodifffusion, precipitin reaction, enzyme-linked immunosorbent assay (ELISA) and inhibition test. PEA2 from the venoms of same species of snakes, such as Taiwan cobra (Naja naja atra) and Indian cobra (Nala naja) showed a considerable degree of antigenic resemblance, whereas no immunological cross-reactivity was observed among the PLA2 enzymes from a different family and genus. However, a partial cross-reactivity was observed between the antibody against Naja naja atra PLA2 and PLA2 from the venom of Bungarus multicinctus, and CMS-5, the acidic PLA2 from Nafa nigricollis venom by ELISA. Both PEA2 enzymes from Naja naja atra and Naja naja venoms exhibit three common antigenic sites, althougi the latter had lower affinity toward the antibody against the former PLA2.

Isolation of a polypeptide from the venom of Bungarus multicinctus that binds to ganglia and blocks the ganglionic transmission in mammals

A 15,000 dalton polypeptide purified from Bungarus multicinctus venom (which normally copurifies with α-bungarotoxin) was characterized biochemically and its biological effects were studied. This polypeptide, P15, had an aminoacid composition and molecular weight different from those of both α- and β-bungarotoxin. It inhibited the ganglionic transmission in the guinea-pig hypogastric nerve-vas deferens preparation and did not block, even at very high concentrations, the neuromuscular transmission in the rat phrenic nerve-diaphragm preparation. In the same preparations α-bungarotoxin was unable to block the response at the ganglionic synapse while it was fully active in blocking the neuromuscular transmission. However, a pretreatment of the vas deferens preparation with α-bungarotoxin prevented the inhibitory effect of P15. 125I-Labeled P15 showed a specific and saturable binding to rat superior cervical ganglia homogenate and to a Torpedo postsynaptic membrane fraction. The binding of P15 to ganglia was inhibited by curare. The binding was Ca 2+ dependent. The density of binding sites was of 300 fmol/mg of protein in the ganglion and 500 fmol/mg of protein in Torpedo membranes. The amount of P15-binding sites in ganglia was not modified by denervation, indicating that P15 binds to postsynaptic receptors. The binding of 125I-labeled P15, both in ganglia and Torpedo membranes, was inhibited by α-bungarotoxin. P15 had a Ca 2+-dependent phospholipase A 2 activity. Lowering Ca 2+ concentration in incubation media affected the phospholipase A 2 activity more than binding properties and inhibition of phospholipase activity withp-bromophenacyl bromide did not affect the activity of P15 on vas deferens preparation, suggesting that the phospholipase activity is not necessary for the activity of P15 on nicotinic receptors. Our results suggest that P15 toxin may be a specific and valuable probe for studying the ganglionic nicotinic receptor.

Binding characteristics of the bungarotoxin fraction II-S1 to rat brain membranes

Bungarotoxin fraction II-S1 (designated BGT II-S1), isolated from the venom of Bungarus multicinctus, appears to affect nicotinic transmission in rat sympathetic ganglia through its phospholipase activity. On the other hand, the present investigation suggests that other modes of interaction of this toxin with nervous tissue may also exist as, in a rat brain membrane preparation, binding of this toxin to a specific binding site can be demonstrated. In a buffer containing calcium, binding of [ 125I]BGT II-S1 saturated with a B max of approximately 16 fmol/mg protein and a K d of 5 nM. This site did not appear to be directly linked to the nicotinic acetylcholine recognition site as the binding was not displaced by nicotinic agents; however, α-bungarotoxin, which interacts with a nicotinic-like site in neural tissue, did affect the binding and, conversely, BGT II-S1 inhibited the binding of [ 125I]α-bungarotoxin.

Kappa-Bungarotoxin. Self-association of a neuronal nicotinic receptor probe.

kappa-Bungarotoxin is a postsynaptic neurotoxin purified from the venom of the elapid snake Bungarus multicinctus. The amino acid sequence of this basic polypeptide reveals a single chain containing 66 amino acids having a Mr of 7,313. kappa-Bungarotoxin is a potent antagonist of nicotinic cholinergic transmission in avian and murine autonomic ganglia, a characteristic which distinguishes the toxin from other postsynaptic neurotoxins isolated from snake venoms. The self-association of kappa-bungarotoxin has now been examined using molecular sizing columns, sedimentation velocity, and sedimentation equilibrium. The results demonstrate that, under physiological solvent conditions, kappa-bungarotoxin exists as a dimer (Mr = 14,000 +/- 3,000) of identical subunits. kappa-Bungarotoxin monomers are not observed at toxin concentrations typically used in electrophysiological experiments (0.5-22 micrograms/ml), indicating that the dimer may be physiologically active. Denaturation with sodium dodecyl sulfate or urea dissociates kappa-bungarotoxin dimers into monomers. Significant amounts of monomers are also produced under nondenaturing conditions of high ionic strength and high pH. However, complete reassociation of nondenatured monomers occurs following return to a physiological buffer. The unique pharmacological spectrum of kappa-bungarotoxin may be due in part to its strong tendency to self-associate.

Characterization of monoclonal antibodies against beta-bungarotoxin and their use as structural probes for related phospholipase A2 enzymes and presynaptic phospholipase neurotoxins.

Hybridoma lines secreting monoclonal antibodies against a phospholipase-inactive derivative of the presynaptic neurotoxin, beta-bungarotoxin, have been established. These antibodies, either of the IgG1 or IgG2b isotype with affinities in the range 1-2 X 10(8) 1/mol, recognized a single immunodominant region of native beta-bungarotoxin, most probably located on the A (phospholipase homologue) chain of the toxin. Using plate-adsorbed radioimmunoassay procedures, antibodies reacted with native beta-bungarotoxin and other beta-bungarotoxin isotoxins as well as with the non-toxic phospholipase A also present in Bungarus multicinctus venom. Other phospholipase A enzymes and presynaptic phospholipase neurotoxins did not show any competition with beta-bungarotoxin in the radioimmunoassay. Globulin fractions of monoclonal antibodies partially inhibited the phospholipase activity of beta-bungarotoxin.

Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion

Bungarus multicinctus venom was fractionated by ion exchange chromatography and the various fractions were assayed for their ability to block synaptic transmission through the chick ciliary ganglion. α-Bungarotoxin purified from this venom failed to block transmission at 50μg/ml. A second neurotoxin, which we designate Toxin F, blocked transmission at 1–3μg/ml and also blocked ganglionic depolarizations induced by carbachol. Toxin F was clearly distinguishable from α-bungarotoxin on the basis of molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric point. Binding assays revealed that 125I-labeled toxin F bound to two sites in the ciliary ganglion: one site that was shared by α-bungarotoxin and toxin F and another site that was recognized solely by toxin F. Carbachol and d-tubocurarine displaced only that [ 125I]toxin F bound to the shared site and had no effect on [ 125I]toxin F bound to the site recognized by toxin F alone. The results suggest that toxin F blocks synaptic transmission in the chick ciliary ganglion by a postsynaptic mechanism. Further study is required to determine whether this effect of toxin F is mediated through a direct interaction with ganglionic nicotinic receptors.

Kappa-Bungarotoxin: a probe for the neuronal nicotinic receptor in the avian ciliary ganglion

The interaction of snake α-neurotoxins with neuronal membranes has been examined in the chick ciliary ganglion. Some, but not all, α-neurotoxins block nicotinic transmission in this ganglion. α-Bungarotoxin (ABgT), the major α-neurotoxin in the venom of Bungarus multicinctus, does not block transmission at high concentrations (1.2 μM) although it binds ( K d = 1nM ) to a pharmacologically nicotinic site in the ganglion. A toxin (kappa-bungarotoxin, KBgT) has been purified from the venom of Bungarus multicinctus. KBgT has a molecular weight of 6500 daltons and a pI of 9.1 KBgT is a potent inhibitor of nicotinic transmission in the ciliary ganglion, producing a reversible (overal several hours) blockade at 75 nM. Pre-exposure of ganglia to 1.2 μM ABgT does not prevent the effects of KBgT, indicating that the blockade occurs at a site distinct from that recognized by ABgT. Binding of [ 125I]KBgT to ciliary ganglia reveals two binding sites: one which has previously been characterized by [ 125I]ABgT and one which is not identified by [ 125I]ABgT. Both of these [ 125I]KBgT binding sites are blocked following pre-treatment of ganglia with the irreversible nicotinic affinity agent bromoacetylcholine. A two-site model is proposed to account for these observations. One site (the ABgT binding site) is seen by both ABgT and KBgT, and has as yet no physiological function associated with it. The second site is recognized only by the physiologically active KBgT, and may represent binding of the toxin to the physiologically detected nicotinic receptor.

Increased sodium ion conductance through nicotinic acetylcholine receptor channels in PC12 cells exposed to nerve growth factors

22Na+ uptake in response to cholinergic stimulation was measured in PC12 cells grown in media containing either beta-nerve growth factor (NGF) from mouse submaxillary gland or a component of Bungarus multicinctus venom (fraction 9B) which has nerve growth factor activity. Our results showed that each of these nerve growth factors increased the Na+ conductance capacity of ACh receptor-linked ion channels in PC12 cells. Compared to untreated cells, Na+ uptake was enhanced by about 40% in cells exposed for 24 to 30 hr to either 50 ng/ml of beta-NGF or to 5 micrograms/ml of fraction 9B. This cholinergically stimulated Na+ flux increased with time and reached a level 2- to 3-fold higher than that of untreated cells after 4 to 6 days' exposure to NGFs. The generation of neurites in response to NGFs from either source occurred with the same time course as the increase in Na+ channel conductance. Growth factor-enhanced Na+ uptake remained for several days after NGFs were removed from cells and neurites were lost. Na+ flux into untreated and into 9B- or beta-NGF-treated cells was inhibited to a greater extent by a nicotinic than by a muscarinic antagonist. Dibutyryl cyclic AMP did not cause an increase in Na+ uptake in response to cholinergic agonists at concentrations which stimulated neurite-like process formation, and dibutyryl cyclic AMP did not potentiate the effects of NGFs.

Amino acid sequence of beta 2-bungarotoxin from Bungarus multicinctus venom. The amino acid substitutions in the B chains.

beta 2-Bungarotoxin (beta 2-toxin) was isolated from the venom of Bungarus multicinctus by means of CM-Sephadex C-25 column chromatography, Sephadex G-75 gel filtration and CM-Sephadex C-25 column rechromatography. beta 2-Toxin consisted of two dissimilar polypeptides, a (120 amino acid residues) and B (60 amino acid residues) chains, crosslinked by an interchain disulfide bond. The neurotoxicity (LD50) and phospholipase activity of beta 2-toxin were 0.029 micrograms/g of mouse and 48.9 units/mg of toxin, respectively, and both the activities were slightly weaker than those (0.019 micrograms/g and 60.9 units/mg) of beta 1-bungarotoxin (beta 1-toxin). beta 2-Toxin was reduced and carboxymethylated and then its RCM-A and -B chains were separated. Each RCM-chain was maleylated and then digested with TPCK-trypsin. The tryptic peptides were sequences by manual Edman degradation or the dansyl-Edman method, and the total alignment of the tryptic peptides from each RCM-chain was deduced based on the amino acid sequences of the A and B chains of beta 1-toxin. The amino acid sequence of the B chain of beta 2-toxin differed from that of the B chain of beta 1-toxin by 22 amino acid substitutions, while those of their A chains were identical. We concluded that the variation in the amino acid sequence of the B chains did not significantly affect the neurotoxicity of the beta-toxins. The amino acid sequences of the B chains of the two beta-toxins were homologous to those of proteinase inhibitors from snake venoms and mammalian pancreas, but no inhibitory activity of the two beta-toxins on proteinases was observed.

Amino acid sequences of three beta-bungarotoxins (beta 3-, beta 4-, and beta 5- bungarotoxins) from Bungarus multicinctus venom. Amino acid substitutions in the A chains.

The two most basic beta-bungarotoxins (beta 3- and beta 4-toxins) and another, less neurotoxic beta-bungarotoxin (beta 5-toxin) were purified from Bungarus multicinctus venom, by a combination of CM-Sephadex C-25 column chromatography and Sephadex G-75 gel filtration. The three toxins consisted of two dissimilar polypeptides (A chain, 120 amino acid residues; B chain, 60 residues). The LD50 values of the beta 3- and beta 4-toxins were 0.066 micrograms and 0.072 micrograms/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. beta 5-Toxin was weaker in neurotoxicity (LD50, 0.13 micrograms/g of mouse) than the others, and its phospholipase activity was 47.6 units/mg of toxin. Each toxin was separated into RCM-A and RCM-B chains after reduction and S-carboxymethylation. The RCM-polypeptides were maleylated and digested with TPCK-trypsin. The tryptic peptides were sequenced with manual Edman degradation or the dansyl-Edman method. The final alignment of the tryptic peptides from the respective RCM-polypeptides was deduced on the basis of the amino acid sequences of the A and B chains of beta 1-bungarotoxin (beta 1-toxin). The amino acid sequences of the A chains of the beta 3- and beta 4-toxins were identical but differed from those of the A chains of the beta 1- and beta 2-toxins by 4 amino acid substitutions in the COOH-terminal portions (residues 109-120) and substitution at position 87. The amino acid sequences of the B chains of the beta 3- and beta 4-toxins differed from each other, but they were identical with those of the B chains of the beta 1- and beta 2-toxins, respectively. The amino acid sequence of the A chain of beta 5-toxin differed from that of the A chain of beta 1-toxin by consecutive substitutions in residues 55-60 and substitutions at positions 23, 87, and 89. The amino acid sequence of the B chain of beta 5-toxin was identical with those of the B chains of beta 1- and beta 3-toxin. From our results on the effects of the amino acid displacements found in the A chains on the neurotoxicity, it was concluded that the COOH-terminal portion in the A chains was not essential to their neurotoxicity, whereas the region of residues 55-60 in the A chains appeared to participate in the constitution of the neurotoxically active site of the beta-toxins.

Blockade of transmission in rat sympathetic ganglia by a toxin which co-purifies with α-bungarotoxin

Bungarus multicinctus venom was fractionated into its toxin components using ion-exchange chromatography on CM-Sephadex. According to previous reports, rechromatography of fraction II on a CM cellulose column yields chemically homogenous α-bungarotoxin (II 2) of molecular weight 9000. However, in our hands, using the identical purification procedure, two discrete proteins of molecular weight 9000 and 15,000 were obtained as demonstrated by SDS gel electrophoresis. Subsequent fractionation of this α-bungarotoxin fraction (II 2) was achieved on Sephadex G-50. The 9000 weight component (labelled II-S2) was identical to α-bungarotoxin; at a concentration of 1 μg/ml it blocked transmission at the neuromuscular junction but did not block nicotinic responses in rat sympathetic ganglia. Very different properties were exhibited by II-SI, the 15,000 molecular weight component; it inhibited ganglionic transmission but was ineffective at the neuromuscular junction at the same concentration (1 μg/ml). BGT II-SI was equipotent in blocking the ganglionic action potential in the presence or absence of eserine; thus, it is not acting as an acetylcholinesterase by increasing acetylcholine breakdown. In the presence of toxin, [ 3H]choline incorporation into ganglionic acetylcholine during preganglionic stimulation was not altered, suggesting that the toxin did not block transmission by a presynaptic mechanism. Thus, the site of action of the toxin appears to be postsynaptic although it did not affect depolarization of the ganglia induced by carbachol.

Internalization of alpha-bungarotoxin on neurons induced by a neurotoxin that blocks neuronal acetylcholine sensitivity

A protein neurotoxin (Bgt 3.1) present as a minor component in the venom of Bungarus multicinctus has been shown previously to block acetylcholine (ACh) sensitivity on chick ciliary ganglion (CG) neurons in cell culture. Alpha-bungarotoxin (Bgt. 2.2) binds to the neurons but does not block ACh sensitivity; the function of the Bgt. 2.2 binding site is unknown. The present studies demonstrate that Bgt 3.1 can induce the rapid internalization of Bgt 2.2 bound on the surface of CG and sympathetic neurons. The rapid internalization of bound Bgt 2.2 caused by Bgt. 3.1 can be seen with fluoresence microscopy using rhodamine-labeled Bgt 2.2 as the probe and by immunological techniques using anti-Bgt 2.2 antiserum to locate the bound 125I-Bgt 2.2. The rapid internalization is blocked by low temperature or by high concentrations of Bgt 2.2 and is not induced by Bgt 2.2 itself or by small cholinergic ligands. Bound 125I-Bgt 2.2 is released into the medium as degraded material after internalization is induced. Bgt 3.1 does not induce internalization of Bgt 2.2 bound to skeletal myotubes in culture nor does it induce the internalizaton of rhodamine-labeled nerve growth factor bound to sympathetic neurons, suggesting that its effect on neuronally bound Bgt 2.2 might be a specific one. Competition binding studies suggest that Bgt 3.1 may trigger the internalization of bound Bgt 2.2 by direct interaction with a Bgt 2.2 binding site. The effect of Bgt 3.1 on neuronal ACh sensitivity, however, does not depend on internalization of Bgt 2.2 binding sites since full inhibition of ACh sensitivity is still achieved by Bgt 3.1 under conditions where internalization is blocked. Neurons may have more than one class of Bgt 2.2 on the neurons. The internalization of Bgt 2.2 binding sites induced by Bgt 3.1 provides an unusual opportunity to study cellular mechanisms by which neurons can regulate the number and distribution of their surface components.

Effect of beta-bungarotoxin on the release of endogenous amino aids from the sensorimotor cortex.

beta-Bungarotoxin, a snake neurotoxin purified from the venom of Bungarus multicinctus, caused a significant increase in the in vivo release of glutamate from the superfused sensorimotor cortex of awake animals. A smaller effect on GABA release was observed, but no change was detected in the release of six other amino acids measured. The effects on glutamate and GABA release were entirely blocked by tetrodotoxin (1 micrometer) and were reversible when the cortical tissue was washed with saline.

Purification and characterization of phospholipase A from Bungarus multicinctus venom.

Phospholipase A was purified 16.3-fold in terms of specific activity from the venom of Bungarus multicinctus by ion exchange chromatography on a CM-Sephadex C-25 column followed by gel filtration on a Sephadex G-75 column. The overall enzyme yield was 3.5% from the crude venom. The molecular weight and isoelectric point of the purified enzyme were estimated to be 14,000 and 7.9 by electrophoresis on SDS-polyacrylamide gel and on polyacrylamide gel for isoelectric focusing, respectively. The enzyme was a single polypeptide chain consisting of 118 amino acids. Its N- and C-terminal residues were asparagine and glutamine, respectively. The enzyme was stable in the pH range of 2--10 and retained full activity after incubation for 24 h at 37 degrees C. The optimum hydrolysis of egg yolk phosphatidyl choline was observed at pH 8.5 and the specific activity was 1,450 units per mg of the enzyme. The enzyme was inactivated by treatment with p-bromophenacyl bromide, accompanied by the loss of one of two histidine residues.

Inhibition of neuronal acetylcholine sensitivity by α-toxins from Bungarus multicinctus venom

Bungarus multicinctus venom contains several alpha-toxins in addition to the widely used alpha-bungarotoxin (Bgt 2.2). We have found that two of the alpha-toxins (Bgt 3.1 and 3.3) inhibit neuronal acetylcholine (AcCho) sensitivity when tested on ciliary ganglion neurons in cell culture. Over 90% of the AcCho sensitivity recorded in response to iontophoretic application of AcCho was blocked when the neurons were incubated with either of the toxins at 10(-7) M for 1 hr at 37 degrees C. The blockade could be partially reversed by incubating the neurons for 1-2 hr in medium lacking the toxins. The neurons also had a high-affinity binding site for Bgt 2.2, as indicated by binding studies with rhodamine-labeled Bgt 2.2. Concentrations of Bgt 2.2(10(-7) M) that should be nearly adequate to saturate the high-affinity site, however, had no detectable effect on AcCho sensitivity of the neurons. Higher concentrations of Bgt 2.2(10(-5) M) produced a partial inhibition of AcCho sensitivity, suggesting either that the neurons had two classes of binding sites for Bgt 2.2 (with the low-affinity site affecting AcCho sensitivity) or that the preparation of Bgt 2.2 contained minor components (e.g., Bgt 3.1 or 3.3) that were responsible for the blockade. The mechanisms by which Bgt 3.1 and 3.3 inhibit neuronal AcCho sensitivity remain unknown. If they bind specifically to the AcCho receptor, they will be useful agents for studying the distribution and regulation of this membrane component.

Purification and biochemical characterization of an 11 000-dalton β-bungarotoxin

The chromatographic separation and biochemical characterization of a β-bungarotoxin is described. This toxin is isolated as the most basic eluting protein of Bungarus multicinctus venom when separated by column chromatography on CM-Sephadex C-25. The protein migrated as a single band on pH 4.3 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of this toxin was estimated to be 10 000 ± 1000 by analytical sedimentation analysis. This value was consistent with the electrophoretic mobility of the toxin in SDS-polyacrylamide gels. The amino acid composition of this 11 000-dalton β-bungarotoxin was similar to that of the 22 000-dalton β-bungarotoxin previously reported (Lee et al. (1972) J. Chromatogr. 72, 71–82; Kelly, R.B. and Brown, III, F.R. (1974) J. Neurobiol. 5, 135–150; Kondo et al. (1978) J. Biochem. Tokyo 83, 91–99), suggesting that the 11 000-dalton toxin may be one of the polypeptide chains of the large toxin. The 11 000-dalton β-bungarotoxin was toxic to mice when injected intravenously. Animals that received lethal doses exhibited hyperexcitability followed by ataxia, convulsions, and death. The minimum lethal dose was 0.12 μg/g body weight. This β-bungarotoxin exhibited Ca 2+-dependent phospholipase A activity comparable to that of the 22 000-dalton β-bungarotoxin. The enzyme exhibited phospholipid substrate specificity in the rank order of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. The enzyme activity was destroyed by boiling for 3 min at pH 8.6. In addition, an enzymatically inactive quantity of the 11 000-dalton toxin, equivalent to five times the minimum lethal dose of enzymatically active toxin, was not lethal when injected into mice. To test whether phospholipase A activity is responsible for lethality, bee venom phospholipase A 2 was injected into mice at similar and greater concentrations with no toxic effect. Thus, while phospholipase A activity may be required for the lethal effect of the 11 000-dalton β-bungarotoxin, the specificity of action of the toxin is not determined by its enzyme activity.

Beta-Bungarotoxin. Separation of two discrete proteins with different synaptic actions.

beta-Bungarotoxin, a specific presynaptic blocking agent, was prepared in two stages from the crude venom of Bungarus multicinctus by ion-exchange chromatography on the weakly acidic ion exchanger, CM-Sephadex, and on the strongly acidic ion exchanger, sulphopropyl-Sephadex. By these procedures it was purified to a single protein, which was shown by reduction to contain two polypeptide chains with mol.wts. of less than 15000. During purification of beta-bungarotoxin three other proteins were isolated. Two of these proteins have similar molecular weights, subunit structure and physiological properties to the major protein component. This latter is referred to as beta-bungarotoxin, since it has the same physiological properties as those described for unpurified beta-bungarotoxin by other workers. The first protein has very different physiological effects and biochemical properties from beta-bungarotoxin. This protein has a single class of polypeptide chains with an apparent molecular weight that is lower than the main beta-bungarotoxin protein, and appears to block synaptic transmission by a predominantly postsynaptic effect. It has been suggested [Oberg & Kelly (1976) J. Neurobiol. 7, 129-141] that the action of beta-bungarotoxin depends on its phospholipase A activity; however, in this preparation of the toxin less than 50 muunits of phospholipase A activity were detected (1 unit of activity is the amount of enzyme forming 1 mumol of L-alpha-phosphatidylcholine/min per mg of protein).

Similarities of beta-bungarotoxin and phospholipase A2 and their mechanism of action.

Abstract— β‐Bungarotoxin, a presynaptic neurotoxin isolated from the venom of Bungarus multicinctus, has been shown to initially cause an increase in the frequency of miniature endplate potentials and subsequently block neuromuscular transmission by inhibiting nerve impulse induced release of acetylcholine. In rat brain synaptosomes it causes a Ca2+‐dependent release of acetylcholine together, with a strong inhibition of the high affinity choline uptake system. In this report we demonstrate that β‐bungarotoxin acts as a phospholipase A2 (phosphatide 2‐acyl hydrolase, EC 3.1.1.4), liberating fatty acids from synaptic membrane phospholipids. It also exhibits a striking similarity in a number of neurochemical properties with that of a purified phospholipase A2 from Naja naja siamensis. In addition, both agents produce a marked depolarization of synaptosomal preparations as measured by a fluorescent dye. We propose that disruption of the membrane phospholipids by phospholipase activity can lead to depolarization of the synaptosomal preparation which promotes both transmitter release and inhibition of the energy‐dependent high affinity choline uptake system. With this decreased supply of choline, the acetylcholine content of the cell would be gradually depleted leading to a decrease in transmission.