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This paper reports the results of identification of powdered Bungarus muliteinctus and its adulterants (Natrix annularis; Dinodon rufozonatum and Bungarus fascitus). A comparative table of and a key to the characteristics are given.
The comparative study of the proteins of Bungarus multicinctus multicinctus and Natrix annularis was carried out by SDS-PAGE. The results showed that there were significant differences between their SDS-PAGE figures. On the basis of the study the molecular weights of their characteristic proteins were determined. The results could be regarded as a reference for identification of Bungarus multicinctus multicinctus and its confused species.
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)
Ceruleotoxin is a potent neurotoxin which was originally purified from a batch of venom labelled Bungarus caeruleus, from the Pasteur Institute. Since NOBLE et al. have shown that this batch differs in its protein composition from that of B. caeruleus provided by Miami Serpentarium, we decided to clarify this point by comparing the composition of venoms from various Bungarus species of several origins. Although individual variations exist between samples of the same species, the venom from B. multicinctus, B. caeruleus and B. fasciatus possess characteristic protein compositions which allowed us to identify the batch used to purify ceruleotoxin as a B. fasciatus venom. We identified and purified ceruleotoxin from each of the five samples of B. fasciatus venoms tested. We failed to find this neurotoxin in either B. multicinctus or B. caeruleus venoms. Purified ceruleotoxin is a slightly basic protein with an isoelectric point of 7.4 which possesses a significant phospholipase A2 activity (200 mumoles lecithin hydrolyzed per min per mg) and a high lethal potency (i.v. LD50 in mice 0.03-0.07 mg/kg). It is composed of two identical subunits of 13,000 mol. wt. which resemble pancreas and snake venom phospholipases in their amino acid composition. Like crotoxin, ceruleotoxin irreversibly blocks the postsynaptic response of Torpedo and Electrophorus electroplaques to cholinergic agonists without preventing the binding of acetylcholine to its receptor. By hydrolyzing critical lipids of the postsynaptic membrane, it stabilizes the acetylcholine receptor - ionophore assembly in a desensitized state.
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.
1. Five presynaptic toxins have been isolated in pure form from the venom of Bungarus multicinctus and Bungarus caeruleus and named beta1, beta2, beta3, beta4, and beta-ceruleotoxin. 2. They differ in electrophoretic mobility and amino acid composition, while all have the same molecular weight (22000) and are composed of two subunits of molecular weight 9000 and 12000. 3. The toxins have phospholipase A activity when assayed with both natural and synthetic phospholipids, and this activity requires the presence of Ca2+ ions. 4. beta-Bungarotoxin (beta3) binds 1 mol of Ca2+ per mol of protein and this binding induces a conformational change as detected by fluorescence measurements in the presence of the dye 8-anilino-1-naphthalene sulfonic acid. 5. The phospholipase activity of all the toxins is lost when a critical histidine residue is modified with p-bromophenacyl bromide. 6. As a result of the modification the lethality of the toxins is greatly reduced. 7. Native toxin causes a rapid decrease in amplitude of end-plate potentials, followed by a transient increase and subsequent decrease, until transmitter release is completely abolished. The modified toxin still causes the early decrease in release but toxin action does not progress to complete block. 8. The rate of blockage of transmitter release by native toxin is reduced in the presence of modified toxin. 9. It is concluded that phospholipase activity plays an important role in the action of this class of toxins at the neuromuscular junction.
A basic phospholipase A2 (PLA2) was isolated and purified from the venom of Bungarus fasciatus. Four kinds of enzymes, lysyl endopeptidase, endoproteinase Asp-N, endoproteinase Glu-C and trypsin, were employed to elucidate the complete primary structure by means of gas-phase sequencing. The amino-acid sequence reveals 118 amino-acid residues containing seven pairs of half-cystine. It has 78% and 61% structural identities with PLA2 from Bungarus multicinctus and Naja melanoleuca DE-II, respectively.
A nontoxic phospholipase A2 was purified from the venom of Indian krait (Bungarus caeruleus) by a four-step procedure involving electrophoresis, gel filtration and ion-exchange chromatography. The recovery of the enzyme activity was 37% and the purified preparation was 38 times as active as the crude venom. The purified enzyme had a molecular weight of 12,500 and the optimum pH of 7.2. The enzyme showed higher specificity toward phosphatidylethanolamine than phosphatidylcholine. The preparation was not very labile to heat and its activity was dependent on the presence of divalent cations, calcium ions being the most effective activators. The enzyme was completely inhibited by iodoacetic acid but showed high stability against 8 M urea. Purified phospholipase A2 was nontoxic at an iv dose of 5 microgram/g mouse. The high specific activity, the high yield and the nontoxic nature of the enzyme indicate that the major form of phospholipase A2 in Bungarus caeruleus venom is not associated with any toxicity and has properties somewhat similar to that of phospholipase A2 from some other venoms.
The venom of elapid snakes contains a number of small proteins that display a broad spectrum of pharmacological activities. In study on the neurotoxin from the venom gland of Bungarus multicinctus, five cDNA sequences (MNCTL1-a,MNCTL1-b,MNCTL1-c,MNCTL1-d and MNCTL2) encoding two novel proteins were obtained from the total RNA by reverse transcription-polymerase chain reaction. Among them, four sequences (MNCTL1-b,MNCTL1-c,MNCTL1-d and MNCTL2) are at the length of 505 bp, composing of a 32 bp 5'-untranslational region,a 212 bp 3'-untranslational region, and a 261 bp open reading frame which encodes a 20 amino acids of signal peptide and a 65 amino acids of mature peptide. But the sequence of MNCTL1-a has only 481 bp because of a 24 bp deletion in 3'-untranslational region. Comparison between cDNA sequences obtained here and that of cardiotoxin-like protein reported by Chang previously shows a homological value above 95.8%. Computer simulation based on deduced amino acid sequences reveals that both proteins encoded by these cDNAs and cardiotoxin-like protein share similar molecular characters, suggesting their functional similarity. This result implies that multicopy genes existing in the genome of Bungarus multicinctus encode cardiotoxin-like proteins.
Neurophysiological studies were performed in 12 patients with neuromuscular paralysis due to envenomation by the common krait (Bungarus caeruleus). All patients presented with an acute, reversible, oculofaciobulbar paresis. In addition, 7 patients had weakness of the limb muscles and 4 required assisted mechanical ventilation. Neurophysiological abnormalities included: (1) a reduction in the amplitude of the median-elicited compound muscle action potential (CMAP) in 4 patients; and (2) a decremental response (>10%) to 3-Hz repetitive nerve stimulation (RNS) in 4 patients. With 1 exception, these abnormalities were noted only in clinically weak muscles. The administration of edrophonium to 6 patients produced an insignificant increase in CMAP amplitudes as well as partial (not significant) improvement in the decremental response to 3-Hz RNS. Neurophysiological studies were performed in 2 patients before and after the administration of 20 mL of polyvalent antivenom. A decrease in amplitude of the median-elicited CMAP amplitude occurred after the administration of antivenom. In 1 patient, administered 100 mL of antivenom, the median-elicited CMAP amplitude increased and the decrement to 3-Hz RNS decreased. Neurophysiological studies can provide useful information regarding the nature, severity, and therapy of the neuroparalytic syndrome of krait envenomation.
The cDNA encoding alpha-bungarotoxin (alpha-BuTx) was constructed from the cellular RNA isolated from the venom glands of Bungarus multicintus by RT-PCR. The primers deduced from the N-, C-terminal amino acid residues and the conserved middle segment of the sequence, i.e., R25KMWC29 in alpha-BuTx could specifically amplify cDNAs encoding amino acid residues from 1 to 29 and 25 to 74 of alpha-BuTx. The complete cDNA was obtained by linking sufficient amounts of the two cDNA fragments by PCR. The amino acid sequence translated from the alpha-BuTx cDNA sequence was identical to that of native toxin. The PCR product was then cloned into pGEX vector and transformed in DH5 E. coli strain. The fused protein expressed in host strain exhibited the same antigenicity as native alpha-BuTx. This communication is the first report for the cloning and expression of a long alpha-neurotoxin from the venom glands.
The cDNA encoding the A chain of beta-bungarotoxin (beta-Bgt) was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait). The deduced amino acid sequence encoding the A chain revealed that the determined one was different from the known A chains (A1, A2, A3 and A4). Nevertheless, the amino acid sequence and cDNA sequence of the new A chain (A5) was highly homologous with those of other A chains. The A5 chain was subcloned into the expression vector pT7-7 and transformed into BL21(DE3) E. coli strain. The expressed protein was isolated from the inclusion bodies of E. coli, and the refolded A chain was purified by reversed phase high performance liquid chromatography. The purified recombinant A chain exhibited an about 16% phospholipase activity of beta-Bgt. These results strongly suggest that the A chain is an active subunit responsible for the phospholipase activity of beta-Bgt.
During the mapping and sequencing of Naja naja atra cobrotoxin and cardiotoxin 4 genes, we have found that novel small nucleolar RNAs (snoRNAs) are encoded in the first intron of the two genes. The snoRNAs in Naja naja atra were amplified from the venom glands cDNA mixtures of Naja naja atra by reverse transcription-polymerase chain reaction using the primers designed from the first intronic sequences of cobrotoxin and cardiotoxin 4 genes. Likewise, the snoRNAs in Bungarus multicinctus were also amplified by the same primers. Comparison of these snoRNA genes reveals that the regions involved in binding to 5S rRNA are highly conserved among these genes, and form 12-nt and 15-nt tracts of complementarity to phylogenetically invariant sequences in eukaryotic 5S rRNAs. The box C sequence in these snoRNAs is consensus, however, variations with the sequence of box D motif are observed. The present study is the first case of intron-encoded snoRNAs contain extended regions of perfect complementarity to mature 5S rRNA.
Twenty novel snoRNAs forming extensive sequence complementarities to mature 5S rRNA were identified from Bungarus multicinctus by reverse transcription-polymerase chain reaction. It was found that the snoRNA species were differentially transcribed in different tissues as evidenced by single stranded conformational polymorphism analysis and direct nucleotide sequence analysis. Although the diversity in the sequences of snoRNAs is observed, comparison of these snoRNA genes reveals that the regions involved in binding to 5S rRNA are highly conserved and form two 12-nt-15-nt tracts of complementarity to phylogenetically invariant sequences in eukaryotic 5S rRNAs. Nevertheless, the lower conservation of box C/D or box H/ACA in these snoRNAs was observed. Likewise, the sequences in several fish and human genes forming perfect duplexes with 5S rRNA also did not highly retain these box elements. These results may infer that the box elements are dispensable for the function of snoRNA species identified in the present study. Moreover, the novel finding of the differentially expressed snoRNA variants in B. multicinctus suggests that the snoRNA genes are selectively processed in different tissues and are likely associated with tissue-specific regulation of their host gene transcripts.
A 30 year old woman bitten by a large Sri Lankan krait (Bungarus ceylonicus) developed progressive paralytic symptoms within one hour of the bite. After seeking traditional treatment her condition deteriorated and when she arrived at hospital 6 h after the bite she was drowsy, with bilateral ptosis and signs of aspiration pneumonia (a complication of traditional treatment). She developed an anaphylactic reaction after antivenom treatment and, despite treatment, had a cardio-respiratory arrest. She was resuscitated and mechanically ventilated, but remained deeply unconscious until her death 90 h after the bite. B. ceylonicus is endemic in Sri Lanka, is common in some areas, and could be mistaken for the common krait (B. caeruleus). It is doubtful whether any existing antivenom is effective against its venom.
A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.
Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.
This is the first crystal structure of a carbohydrate induced dimer of phospholipase A(2) (PLA(2)). This is an endogenous complex formed between two PLA(2) molecules and two mannoses. It was isolated from Krait venom (Bungarus caeruleus) and crystallized as such. The complete amino acid sequence of PLA(2) was determined using cDNA method. Three-dimensional structure of the complex has been solved with molecular replacement method and refined to a final R-factor of 0.192 for all the data in the resolution range 20.0-2.1A. The presence of mannose molecules in the protein crystals was confirmed using dinitrosalicylic acid test and the molecular weight of the dimer was verified with MALDI-TOF. As indicated by dynamic light scattering and analytical ultracentrifugation the dimer was also stable in solution. The good quality non-protein electron density at the interface of two PLA(2) molecules enabled us to model two mannoses. The mannoses are involved extensively in interactions with protein atoms of both PLA(2) molecules. Some of the critical amino acid residues such as Asp 49 and Tyr 31, which are part of the substrate-binding site, are found facing the interface and interacting with mannoses. The structure of the complex clearly shows that the dimerization is caused by mannoses and it results in the loss of enzymatic activity.