Expression and refolding of bioactive α-bungarotoxin V31 in E. coli.

In order to obtain bioactive α-bungarotoxin (αBtx) using recombinant protein technique, a codon-optimized synthetic gene was expressed in fusion with the N-terminal 10-His-tag and C-terminal Strep-tag in Escherichia coli. Further optimization through site-directed mutagenesis enabled moderate expression of the protein without the N-terminal His-tag or the C-terminal Strep-tag. Two such recombinant αBtx (rαBtx) were obtained, both with an additional methionine and a glycine at the N-terminal and one with (G4S1)2-Strep-tag at the C-terminal. The rαBtx proteins were refolded using a novel protocol, which efficiently produced final products with activity similar to its natural counterpart. The protocol could easily be scale up, which produced 0.3-1mg of pure and highly active rαBtx per liter of E. coli culture.

Nonconventional three-finger toxin BMLCL from krait Bungarus multicinctus venom with high affinity interacts with nicotinic acetylcholine receptors.

Nonconventional three-finger toxin BMLCL was isolated from B. multicinctus venom, and its interaction with different subtypes of nicotinic acetylcholine receptor (nAChR) was studied. It was found that BMLCL is able to interact with high efficiency with both α7 and muscle type nAChRs.

Isolation, complete amino acid sequence and characterization of a previously unreported post-synaptic neurotoxin - AlphaN3, from the venom of Bungarus candidus.

The Malayan krait (Bungarus candidus) is one of the medically most important snake species in Southeast Asia. The venom from this snake has been shown to posses both presynaptic and post-synaptic neurotoxins. We have isolated a previously uncharacterized post-synaptic neurotoxin - alphaN3 from the venom of B. candidus. Isolation of the toxin was achieved in three successive chromatography steps - gel filtration on a Sephadex G75 column, followed by ion exchange chromatography (Mono-S strong cationic exchanger) and a final reverse-phase chromatography step (PRO-RPC C18 column). Purified toxin alphaN3 was shown to have an apparent molecular weight of approximately 7 to 8 kDa on SDS-PAGE. The complete amino acid sequence of toxin alphaN3 was determined by Edman degradation and was found to share a high degree of homology with known post-synaptic neurotoxins (93% with alpha-bungarotoxin from Bungarus multicinctus, 50% with alpha cobratoxin from Naja kaouthia). The intravenous LD(50) of toxin alphaN3 was determined to be 0.16+/-0.09 microg/g in mice which is comparable to alpha-bungarotoxin from B. multicinctus. Experiments with isolated nerve-muscle preparations suggested that toxin alphaN3 was a post-synaptic neurotoxin that produced complete blockade of neuromuscular transmission by binding to nicotinic acetylcholine receptors.

Molecular cloning of the major lethal toxins from two kraits (Bungarus flaviceps and Bungarus candidus).

The major lethal toxins present in the venoms of the red-headed krait, Bungarus flaviceps, and the Malayan krait, Bungarus candidus, have both been purified. Each consists of two polypeptide chains, A and B, joined by a disulfide bond. In the present study, primary structures of these toxins were determined by Edman degradation and by nucleotide sequencing of the cDNA clones. Amino acid sequencing of the N-terminus and enzymatically digested peptides revealed that the A and B chains were highly homologous to those of beta-bungarotoxins (beta-Bgts) from Bungarus multicinctus, respectively. We isolated cDNA clones encoding the A and B chains from both B. flaviceps and B. candidus venom gland cDNA libraries using probes designed based on the cDNA sequence of beta-Bgt from B. multicinctus. Two isoforms of the A chain and one isoform of the B chain were obtained from B. flaviceps, and one isoform of the A chain and two isoforms of the B chain were obtained from B. candidus. Both of the two A chains from B. flaviceps are made up of 119 amino acids and comprise 15 cysteine residues, while the A chains of beta-Bgt from other Bungarus species including B. candidus comprise 13 cysteine residues. The B chains from both species are composed of 59 amino acid residues and comprise seven cysteines. In conclusion, the lethal toxin from B. flaviceps is considered to be a novel isoform of beta-Bgt, which has a different pattern of cysteine residues from known beta-Bgts.

Purification and biochemical characterization of an 11 000-dalton beta-bungarotoxin.

The chromatographic separation and biochemical characterization of a beta-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 beta-bungarotoxin was similar to that of the 22 000-dalton beta-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 larger toxin. The 11 000-dalton beta-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 microgram/g body weight. This beta-bungarotoxin exhibited Ca2+-dependent phospholipase A activity comparable to that of the 22 000-dalton beta-bungarotoxin. The enzyme exhibited phospholipid substrate specificity in the rank order of phosphatidyl-choline, phosphatidylserine, phosphatidylethanolamine, and phosphatidyl-inositol. 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 A2 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 beta-bungarotoxin, the specificity of action of the toxin is not determined by its enzyme activity.

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.

Lys-64 of the A chain is involved in the enzymatic activity and neurotoxic effect of beta-bungarotoxin.

Two beta-bungarotoxin isotoxins BM12 and BM13 were isolated from Bungarus multicinctus (Taiwan banded krait) venom by sequential chromatography on ion-exchange and reverse phase columns. The two toxins have the same A chain, but different B chains. Different phospholipase A2 activity and different potencies in inhibiting the spontaneous enhancement of spontaneous synaptic current frequency and muscle contraction were observed for BM12 and BM13. Nevertheless, modification of Lys-64 in the A chain of BM12 and BM13 similarly reduced in their phospholipase A2 activity and toxicity. The modified derivatives retained their affinity with Ca2+ and their conformation as deduced by CD. These results suggest that Lys-64 of the A chain is involved in the phospholipase A2 activity and in the neurotoxic effect of beta-bungarotoxin.

Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity.

BACKGROUND: Bleeding remains a major limitation of standard anticoagulant drugs that target the extrinsic and common coagulation pathways. Recently, intrinsic coagulation factors are increasingly being investigated as alternative targets for developing anticoagulant drugs with lower bleeding risk. OBJECTIVES: Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway and (ii) characterize and further improve the properties of the identified anticoagulants. METHODS AND RESULTS: We have isolated and sequenced a specific factor XIa (FXIa) inhibitor, henceforth named Fasxiator, from the venom of the banded krait snake, Bungarus fasciatus. It is a Kunitz-type protease inhibitor that prolonged activated partial thromboplastin time without significant effects on prothrombin time. Fasxiator was recombinantly expressed (rFasxiator), purified, and characterized to be a slow-type inhibitor of FXIa that exerts its anticoagulant activities (doubled activated partial thromboplastin time at ~ 3 µmol L(-1) ) by selectively inhibiting human FXIa in in vitro assays. A series of mutants were subsequently generated to improve the potency and selectivity of recombinant rFasxiator. rFasxiatorN17R,L19E showed the best balance between potency (IC50 ~ 1 nmol L(-1) ) and selectivity (> 100 times). rFasxiatorN17R,L19E is a competitive slow-type inhibitor of FXIa (Ki  = 0.86 nmol L(-1) ), possesses anticoagulant activity that is ~ 10 times stronger in human plasma than in murine plasma, and prolonged the occlusion time of mice carotid artery in FeCl3 -induced thrombosis models. CONCLUSION: We have isolated an exogenous FXIa specific inhibitor, engineered it to improve its potency by ~ 1000 times and demonstrated its in vitro and in vivo efficacy. These proof-of-principle data supported the further development of Fasxiator as a novel anticoagulant candidate.

Primary structure of alpha-bungarotoxin. Six amino acid residues differ from the previously reported sequence.

alpha-Bungarotoxin (alpha-BuTx) was isolated from the venom of the Formosan banded krait (Bungarus multicinctus). The amino acid sequence was determined by a combination of conventional methods. In contrast to the sequence of alpha-BuTx reported by Mebs et al. ([1971) Biochem. Biophys. Res. Commun. 44, 711-716], our results revealed the presence of Ser-Pro-Ile, Pro-His and Gln-Arg at positions 9-11, 67-68 and 71-72 from the amino-terminal, respectively, and not Ile-Pro-Ser, His-Pro and Arg-Gln as reported previously.

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 alpha-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 alpha- and beta-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 alpha-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 alpha-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 Ca2+ 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 alpha-bungarotoxin. P15 had a Ca2+-dependent phospholipase A2 activity. Lowering Ca2+ concentration in incubation media affected the phospholipase A2 activity more than binding properties and inhibition of phospholipase activity with p-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.

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. alpha-Bungarotoxin purified from this venom failed to block transmission at 50 micrograms/ml. A second neurotoxin, which we designate Toxin F, blocked transmission at 1-3 micrograms/ml and also blocked ganglionic depolarizations induced by carbachol. Toxin F was clearly distinguishable from alpha-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 alpha-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.

N,O-di and N,N,O-tri ( 3 H) acetyl -bungarotoxins as specific labelling agents of cholinergic receptors.

1. alpha-Bungarotoxin isolated from the venom of Bungarus multicinctus was acetylated with [(3)H] acetic anhydride and N-[(3)H] acetyl imidazole. Tri-N-acetyl and hexa-N-acetyl derivatives were obtained from the former, and N,O-di, N,N,O-tri and N,N,N,O-tetraacetyl derivatives from the latter reaction, respectively.2. There were parallel decreases in both neuromuscular blocking action in the phrenic nerve-diaphragm preparation of rats and depression of acetylcholine response of the rectus abdominis muscle of frogs with increased acetylation. Also, a parallel but greater decrease of toxicity in mice was found.3. N,O-Di and N,N,O-triacetyl toxins were localized mostly in the motor endplate region of the rat diaphragm, whereas a slight nonspecific binding along the whole muscle fibre in addition to the peak in the endplate region was observed with N,N,N,O-tetraacetyl and tri-N-acetyl toxins. In contrast, there was a marked nonspecific binding with hexa-N-acetyl toxin and no peak was observed at the endplate zone.4. The specific binding was saturable and irreversible. The number of toxin-receptive sites in one endplate was 1.9-2.2 x 10(7) for all of the labelled toxins irrespective of their potency.5. (+)-Tubocurarine protected effectively against the binding as well as the irreversible neuromuscular blocking effect of the toxins.6. Denervation of the rat diaphragm caused an increase of toxin-receptive sites beginning from the endplate zone at 1-2 days and then along the whole muscle fibre, reaching the maximum at about 18 days. The total receptive sites increased by about 30-fold.7. The significance of the findings is discussed and it is concluded that N,O-di and N,N,O-tri-[(3)H] acetyl alpha-bungarotoxins are specific and irreversible labelling agents for the cholinergic receptors of skeletal muscle.

Primary structure of an inactive mutant of phospholipase A2 in the venom of Bungarus fasciatus (banded krait).

From the acidic components of Bungarus fasciatus venom, a very small amount (0.16%) of a novel phospholipase A2 was obtained. Both neurotoxicity and enzyme activity were found to be lacking. Amino acid sequence study showed that it has a normal backbone of group I snake venom phospholipase A2 with 118 amino acid residues. The lack of enzyme activity was attributed to its mutation of the indispensable Asp residue to an Ala residue, i.e., the usual His-Asp47 turned out to be His-Ala47. This is the eighth isoform of phospholipase A2 found from the venom of Bungarus fasciatus. Examination of structural homology with three other isoforms revealed 66% similarity at most.

Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait).

beta-Bungarotoxin purified from the venom of Bungarus multicinctus (Formosan banded krait) contained no carbohydrate and behaved as a homogeneous protein on polyacrylamide gel electrophoresis at pH 4.1 and SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol treatment. Its molecular weight and isoelectric point were estimated to be about 21,000 by gel filtration and about 9.5 by isoelectric focusing, respectively. The toxin treated with the reducing agent was split into two polypeptide chains as revealed by SDS-polyacrylamide gel electrophoresis and their molecular weights were calculated to be about 13,000 and 7,000. The two polypeptide chains (the large one named the A chain and the small one the B chain) were isolated by gel filtration after reduction of disulfide bonds in the toxin followed by alkylation. The A chain contained 120 amino acid residues including 13 half-cystines and the B chain 60 residues including 7 half-cystines. The two chains were supposed to link by disulfide bond(s) in the intact toxin which contained no free sulfhydryl groups. The N-terminal residues of the A and B chains were asparagine and arginine and the C-terminal ones were glutamine and proline, respectively, in accordance with the results of the terminal analyses of the intact toxin.

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.

Kappa 2-bungarotoxin and kappa 3-bungarotoxin: two new neuronal nicotinic receptor antagonists isolated from the venom of Bungarus multicinctus.

Neuronal nicotinic acetylcholine receptors are recognized with high affinity by two snake venom kappa-neurotoxins, kappa-bungarotoxin and kappa-flavitoxin. Native and radiolabeled kappa-neurotoxins have been used to localize and quantitate neuronal nicotinic receptors in a variety of species. We now report the identification of two new kappa-neurotoxins. kappa 2-Bungarotoxin and kappa 3-bungarotoxin were purified from the venom of Bungarus multicinctus collected in the province of Guangdong, China. kappa-Bungarotoxin has as yet not been found in this venom, although it is the only kappa-neurotoxin to be isolated thus far from Taiwanese Bungarus multicinctus. The geographical separation of Guangdong and Taiwan might account for this evolutionary divergence within the species. Both of the new kappa-neurotoxins are potent antagonists of nicotinic transmission in the chick ciliary ganglion. kappa 3-Bungarotoxin, the least potent of the kappa-neurotoxins, produces a complete blockage of nicotinic transmission in 60 min at 250 nM. Protection experiments using the short-acting nicotinic antagonists dihydro-beta-erythroidine and (+)-tubocurarine demonstrate that kappa 2-bungarotoxin blocks transmission by binding to the acetylcholine recognition sites of neuronal nicotinic receptors. The isoelectric point of kappa 2-bungarotoxin (pI = 8.9) is similar to that of kappa-bungarotoxin and kappa-flavitoxin, but kappa 3-bungarotoxin is considerably more basic, with pI greater than 11. Partial amino acid sequences are reported for both kappa 2-bungarotoxin and kappa 3-bungarotoxin. These sequences show a high degree of homology (approximately 80%) with other kappa-neurotoxins, and allow the determination of the critical differences between the kappa-neurotoxins and the structurally related alpha-neurotoxins. For example, all 4 kappa-neurotoxins lack a tryptophanyl residue which is invariant and important for function in the alpha-neurotoxins. The kappa-neurotoxins also differ from the alpha-neurotoxins by having an invariant prolinyl residue at a critical sequence position. Heterodimers were detected consisting of one subunit each of kappa 2-bungarotoxin and kappa 3-bungarotoxin. These heterodimers, which form between any combination of two kappa-neurotoxins, appear to be physiologically active and confirm that a further distinction between kappa-neurotoxins and alpha-neurotoxins is the strong tendency of the former to self-associate in solution. The present results help to establish the definition of 'kappa-neurotoxin'. These snake toxins are now being used by a number of laboratories in physiological and biochemical experiments on neuronal nicotinic receptors from a variety of species.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of alpha-toxins from Bungarus multicinctus and Bungarus caeruleus on cholinergic responses in Aplysia neurons.

The effects of alpha-bungarotoxin from Bungarus multicinctus and Bungarus caeruleus were studied on three types of cholinergic response in Aplysia central neurones. These responses are the result of three distinct changes in ionic permeability: selective increases in permeabiliyt to Na, Cl and K, respectively. It was shown that 10(-5) M alpha-bungarotoxin from B. multicinctus completely blocks the response resulting from an increase in Cl permeability, while having no effect on either of the other two responses types (even when the toxin concentration was increased to 5 X 10(-5) M). The block of the Cl-dependent response by alpha-bungarotoxin is reversible. None of the three response types were affected by similar concentrations of alpha-toxin from B. caeruleus. Higher concentrations were not systematically tested. These results contradict reports of other authors on the action of alpha-bungarotoxin on molluscan acetylcholine (ACh) responses. Possible reasons for the discrepancies between our findings and those published by other authors are discussed.

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

The interaction of snake alpha-neurotoxins with neuronal membranes has been examined in the chick ciliary ganglion. Some, but not all, alpha-neurotoxins block nicotinic transmission in this ganglion. alpha-Bungarotoxin (ABgT), the major alpha-neurotoxin in the venom of Bungarus multicinctus, does not block transmission at high concentrations (1.2 microM) although it binds (Kd = 1 nM) 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 microM 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.

Blockade of transmission in rat sympathetic ganglia by a toxin which co-purifies with alpha-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 alpha-bungarotoxin (II2) 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 alpha-bungarotoxin fraction (II2) was achieved on Sephadex G-50. The 9000 weight component (labelled II-S2) was identical to alpha-bungarotoxin; at a concentration of 1 microgram/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 microgram/ml). BGT II-S1 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.