The primary structure of the toxin Laticauda semifasciata III, a weak and reversibly acting neurotoxin from the venom of a sea snake, Laticauda semifasciata

A weak and reversibly acting neurotoxic protein of Laticauda semifasciata venom, Laticauda semifasciata III (component LsIII), was sequenced. Component LsIII consists of 66 amino acid residues and has five disulphide bridges, one of which was located between residues 26 and 30. The weak and reversible neurotoxicity of component LsIII is discussed in relation to its structure, which falls between those of the neuro- and cardiotoxins of sea snakes and Elapidae snakes isolated and sequenced so far.     

Detection of erabutoxins in the venom of sea snake Laticauda semifasciata from The Philippines

Erabutoxins a, b and c were detected in the venom of sea snake Laticauda semifasciata from The Philippines.     

The isolation, properties and amino acid sequence of erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata

Erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata, was isolated in pure form by repeated column chromatography on CM-cellulose columns. The toxin was crystallizable and monodisperse in rechromatography, disc electrophoresis and isoelectric focusing (isoelectric point, pH9.23-9.25). The molecular weight of the toxin, as estimated by gel filtration, was 7000. The toxin showed the same lethal activity to mice (0.13mug/g body wt., intramuscular injection) and the same effect on isolated frog muscle as erabutoxins a and b, the main toxic components of the venom. The toxin inhibited the acetylcholine contracture but not the potassium chloride contracture of muscle. Erabutoxin c consisted of 62 amino acid residues, containing one fewer lysine and one more histidine than erabutoxin a and one fewer lysine and one more aspartic acid (or asparagine) than erabutoxin b. Erabutoxin c was reduced, S-carboxymethylated and hydrolysed with trypsin. The only fragment different from the corresponding fragments from erabutoxin b was hydrolysed further with pepsin. One of the peptic fragments, which was assumed to have the aspartic acid (or asparagine) residue in question at the C-terminal end, was treated with carboxypeptidase A. The C-terminal residue was found to be an asparagine. It was therefore concluded that erabutoxin c was [51-asparagine]-erabutoxin b.     

Correction of the amino acid sequences of erabutoxins from the venom of the sea snake Laticauda semifasciata.

The amino acid sequences of erabutoxins a and b were reinvestigated. The previously reported sequences of Gln-His at positions 6 and 7, and of Pro-Ser at positions 18 and 19 of erabutoxins a and b were corrected to His-Gln and Ser-Pro respectively.     

Amino acid sequences of three phospholipases A I, III and IV from the venom of the sea snake Laticauda semifasciata.

Amino acid sequences of three phospholipases A, I, III and IV, from the venom of the sea snake Laticauda semifasciata were elucidated. Each protein consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. They showed high homology among themselves, and with the other snake-venom phospholipases A and with the enzymes from mammalian pancreas. Phospholipases A III and IV were especially similar to each other, with only four differences out of their 118 amino acid residues. Phospholipase A I contained one tryptophan residue at position 64, which was important for enzymic activity, whereas III and IV did not contain tryptophan residues and their corresponding positions were occupied by leucine residues. The substitution by leucine resulted in a decreased, but definite, phospholipase A activity. The substituted enzymes have a more potent neuromuscular blocking activity. Full experimental details and evidence for the amino acid sequences of the proteins have been deposited as Supplementary Publication SUP 50118 (39 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem.J. (1981) 193, 5.     

Phospholipase A of sea snake Laticauda semifasciata venom. Isolation and properties of novel forms lacking tryptophan.

The venom gland extracts of the sea snake Laticauda semifasciata contained at least four forms of phospholipase A separable on a CM-cellulose column. They were designated as phospholipases A I-IV in the order of elution from the column. Phospholipases A I, III, and IV were isolated in a homogeneous state. They were similar to one another in amino acid composition and molecular weight (14,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholipase A I contained one tryptophan residue. whereas III and IV did not. Although all these forms had the same A2-type positional specificity, they were classified into two groups (I, and III and IV) on the basis of enzymic properties. Phospholipase A I had a higher specific activity and showed normal kinetics, whereas III and IV had approximately one-tenth of the specific activity of I and showed biphasic kinetics due to their activation by the reaction products. Phospholipase A I, the major form, seems to be identical with phospholipase A reported previously (Tu, A.T., Passey, R.B., & Toom, P.M. (1970) Arch. Biochem. Biophys. 140, 96-106), whereas the other two, III and IV, are new. Phospholipase A I became more like III and IV in enzymic properties on modification with N-bromosuccinimide.     

The neurotoxins of the sea snake Laticauda schistorhynchus.

Erabutoxins a and b, the major neurotoxins in the venom of the sea snake Laticauda semifasciata, were detected in the venom of Laticauda schistorhynchus. The identity of the toxins was confirmed on the basis of elution position on CM-cellulose column chromatography, disc electrophoretic mobility, amino acid analysis and toxicity measurement.     

Studies on sea-snake venoms. Crystallization of erabutoxins a and b from Laticauda semifasciata venom

1. The toxic principles in the venom of the sea-snake Laticauda semifasciata were separated into two components by CM-cellulose chromatography and obtained in crystalline forms. They were named ;erabutoxins a and b'. 2. The homogeneity of each toxin was shown by rechromatography, by disk electrophoresis, by ultracentrifuging, by toxicity measurements before and after repeated crystallizations and by N-terminal analysis. 3. They had molecular weights of about 7000. Both of them contained 61 (or 62) amino acid residues/molecule. The only difference between erabutoxins a and b was that one of the aspartic acid (or asparagine) residues in erabutoxin a was replaced by a histidine residue in erabutoxin b. 4. Both of the toxins had LD(50) values of 0.15mug./g. body wt. for mice and 0.07mug./g. for rats. It was shown with frog-muscle preparations that they acted on postsynaptic membrane to block neuromuscular transmission.     

The disulphide bonds of erabutoxin a, a neurotoxic protein of a sea-snake (Laticauda semifasciata) venom

Erabutoxin a was partially hydrolysed with enzymes and sulphuric acid and the resulting peptides were separated from each other by column chromatography and paper electrophoresis. From the results of amino acid analyses of the sulphur-containing peptides and their oxidized components, all four disulphide bridges in the toxin molecule were located. The disulphide bonds were found between half-cystine residues at positions 3 and 24, 17 and 41, 43 and 54, and 55 and 60 from the N-terminus.     

Amino acid sequences of two novel long-chain neurotoxins from the venom of the sea snake Laticauda colubrina.

From the venom of a population of the sea snake Laticauda colubrina from the Solomon Islands, a neurotoxic component, Laticauda colubrina a (toxin Lc a), was isolated in 16.6% (A280) yield. Similarly, from the venom of a population of L. colubrina from the Philippines, a neurotoxic component, Laticauda colubrina b (toxin Lc b), was obtained in 10.0% (A280) yield. The LD50 values of these toxins were 0.12 microgram/g body wt. on intramuscular injection in mice. Toxins Lc a and Lc b were each composed of molecules containing 69 amino acid residues with eight half-cystine residues. The complete amino acid sequences of these two toxins were elucidated. Toxins Lc a and Lc b are different from each other at five positions of their sequences, namely at positions 31 (Phe/Ser), 32 (Leu/Ile), 33 (Lys/Arg), 50 (Pro/Arg) and 53 (Asp/His) (residues in parentheses give the residues in toxins Lc a and Lc b respectively). Toxins Lc a and Lc b have a novel structure in that they have only four disulphide bridges, although the whole amino acid sequences are homologous to those of other known long-chain neurotoxins. It is remarkable that toxins Lc a and Lc b are not coexistent at the detection error of 6% of the other toxin. Populations of Laticauda colubrina from the Solomon Islands and from the Philippines have either toxin Lc a or toxin Lc b and not both of them.     

Isolation and primary structure of the major toxin from sea snake, Acalyptophis peronii, venom

The major neurotoxin from the venom of Acalyptophis peronii captured in the Gulf of Thailand was isolated. Although there are two toxic fractions in the venom, the most toxic and abundant fraction was selected for purification and chemical characterization. The LD50 of the major toxin is 0.125 micrograms/g mice, indicating an extremely toxic nature. The toxin consists of 60 amino acid residues with methionine as the amino-terminal and asparagine as the carboxy-terminal end. It contains nine half-cystine residues. There is 1 mol each of tryptophan, tyrosine, methionine, valine, aspartic acid, leucine, and alanine, and there is no phenylalanine. The molecular weight calculated from the amino acid sequence determination was 6600. The toxin replaces alpha-bungarotoxin in binding with the acetylcholine receptor, indicating that the A. peronii major neurotoxin competes with alpha-bungarotoxin for the same binding site of the acetylcholine receptor.     

The amino acid sequences of erabutoxins, neurotoxic proteins of sea-snake (Laticauda semifasciata) venom

1. Erabutoxin b was reduced, S-carboxymethylated and hydrolysed with trypsin. Seven tryptic fragments were isolated by column chromatography and paper electrophoresis. Some of the fragments were further hydrolysed with alpha-chymotrypsin, pepsin, Nagarse, Proctase A or Proctase B. The amino acid sequences of the fragment peptides were determined by subtractive Edman degradation. 2. From the tryptic digest of reduced, S-carboxymethylated and trifluoroacetylated erabutoxin b two fragments were isolated. From the amino acid composition of the fragments and from the terminal sequence studies on the reduced and S-carboxymethylated erabutoxin b, the sequence of the above seven tryptic fragments was elucidated. 3. The tryptic digestion of reduced and S-carboxymethylated erabutoxin a gave fragments, only one of which was different from the corresponding fragment from erabutoxin b. The amino acid sequence analysis of the fragment peptide showed that the only difference between erabutoxins a and b was that the former had asparagine and the latter had histidine at position 26.     

The venom and venom apparatus of the sea snake Lapemis hardwicki Gray

The venom apparatus of Lapemis hardwicki , consisting of two functional fangs, their venom glands, and associated musculature, are described. The yield of venom per snake ranged from 2.4-5.2 mg. The LD₅₀ of the crude venom varied from 0.7-1.4 mg/kg intravenously in mice. The toxicological, chemical and immunological properties of the venom are discussed.     

Isolation, properties and amino acid sequences of a phospholipase A2 and its homologue without activity from the venom of a sea snake, Laticauda colubrina, from the Solomon Islands.

A phospholipase A2, Laticauda colubrina phospholipase A2 II (LcPLA-II), and a phospholipase A2 homologue, Laticauda colubrina phospholipase A2 homologue I (LcPLH-I), were isolated from the venom of the yellow-lipped sea snake, Laticauda colubrina, from the Solomon Islands. LcPLA-II showed phospholipase A2 activity towards egg-yolk phosphatidylcholine (24 mumol/min per mg at optimal conditions at 37 degrees C) and lethal potency (LD50 45 micrograms/kg body wt. intravenously in mice). Both of the activities were lost by treatment with p-bromophenacyl bromide. LcPLH-I showed neither phospholipase A2 activity nor lethal potency at a dose of 4.5 mg/kg body wt. in mice. It was not modified by the treatment with p-bromophenacyl bromide. LcPLA-II and LcPLH-I bound Ca2+ at a 1:1 molar ratio with KCa values of 105 microM and 44 microM at pH 8.0 respectively. Elucidation of the amino acid sequences of these two proteins showed that each protein consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. The two sequences are different from each other at 22 residues and highly homologous to those from other sources. The essential histidine residue for the phospholipase A2 activity at position 48 is replaced by an asparagine residue in the homologue LcPLH-I. Details of the separation of the peptides obtained by proteinase digestions of LcPLA-II and LcPLA-I and the determination of their amino acid sequences are given in Supplementary Publication SUP 50145 (14 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.     

1.9-A resolution structure of fasciculin 1, an anti-acetylcholinesterase toxin from green mamba snake venom.

The crystal structure of fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba snake venom, has been solved by the multiple isomorphous replacement method complemented with anomalous scattering and subsequently refined at 1.9-A resolution. The overall structure of fasciculin is similar to those of the short alpha-neurotoxins and cardiotoxins, with a dense core rich in disulfide bridges and three long loops disposed as the central fingers of a hand. A comparison of these three prototypic toxin types shows that fasciculin 1 has structural features that are intermediate between those of the other two molecules. Its core region, which can be defined as a continuous stretch of conserved residues, is very similar to that of erabutoxin b, whereas the orientation of its long loops resembles that of cardiotoxin VII4. This result introduces a new element in the study of phylogenetic relationships of snake toxins and suggests that, after divergency from an ancestral gene, convergent evolution may have played an important factor in the evolution of these proteins. In fasciculin 1, several arginine and lysine residues are well ordered and relatively exposed to the solvent medium and may play a role in the binding to the peripheral site of acetylcholinesterases.