David Eaker

Purification of a presynaptic neurotoxin from the venom of the Australian tiger snake Notechis scutatus scutatus

Abstract The venom of the Australian tiger snake Notechis scutatus scutatus has been fractionated by ion-exchange chromatography on Bio-Rex 70 in an ammonium acetate gradient. The main neurotoxic component, the notexin, which constituted 6 per cent of the crude venom, was thereby obtained in a high degree of purity (⩾97 per cent) in one purification step. It is a basic protein of 119 amino acid residues in a single peptide chain cross-linked by seven disulfide bridges. The formula weight is 13,574. The LD 100 is 25 μg/kg mouse. The toxin causes dyspnea and paralysis of the hind legs and in the final stages before death, the mice lie immobilized. The death follows two to three days after the administration of a lethal dose. The notexin is a presynaptic toxin which inhibits the release of acetylcholine from motor nerves. The tiger snake venom also contained four other highly active toxins, three of which were submitted to a preliminary study. The first of these toxins has about 60 amino acid residues and an LD 100 of 100 μg/kg mouse, and the second has about 70 residues and an LD 100 of 150 μg. Both toxins have probably a postsynaptic mode of action, as they appear to have all characteristics in common with the postsynaptic cobra ( Naja naja ) neurotoxins. The third toxin, which contains about 120 residues and has an LD 100 of 600 μg, causes respiratory distress characteristic for a postsynaptic neurotoxin. Sublethal doses can give rise to difficulty in breathing lasting for as long as 48 hr. It resembles the dimers formed during the freeze-drying of cobra neurotoxins. These dimers also cause dyspnea lasting for a long period, whereas the corresponding time for the monomeric cobra toxins is maximally 6 hr.

The role of phospholipase activity in the action of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (australian tiger snake)

Notexin from the venom of Notechis scutatus scutatus is a basic protein with 119 amino acid residues and seven disulfide bridges which blocks neuromuscular transmission by interfering with the release of acetylcholine from the motor nerve terminals [ 1,2] . Notexin exhibits phospholipase activity and is highly homologous to phospholipase A from porcine pancreas and various snake venoms [3-51, raising the possibility that the neurotoxicity is catalytic in nature, involving the hydrolysis of a special phospholipid structure in the nerve terminal. Furthermore, it has recently been suggested that another presynaptic snake venom neurotoxin, /I-bungarotoxin, acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals through a phospholipase A activity [6], The discovery that porcine pancreatic phospholipase AZ can be completely inactivated by the modification of a single histidine residue with p-bromophenacyl bromide [7] appeared to offer a means of assessing the role of phospholipase activity in the presynaptic blocking action of notexin. Notexin exhibited a 99.8% loss of both phospholipase A activity and lethal neurotoxicity upon treatment withp-bromophenacyl bromide. Ultraviolet difference spectroscopy and amino acid analysis showed that the inactivation was accompanied by the incorporation of 1 pbromophenacyl residue and the disappearance of 1 histidine residue per mole of protein. The modified residue was identified as histidine-48,

Purification of a neurotoxin from the venom of Naja nigricollis

A neurotoxin, called toxin α, has been isolated from the venom of Naja nigricollis by ion-exchange chromatography on Amberlite IRC-50. The yield of neurotoxin obtained corresponded to about 3% of crude venom, by weight. The neurotoxin was homogeneous by chromatography on IRC-50, free-zone electrophoresis, starch-gel electrophoresis, immunoelectrophoresis, and end-group analysis. The results of amino acid analysis, end-group analysis, and alkylation studies indicate that the neurotoxin of 61 amino acids in a single chain cross-linked by four disulfide bridges and terminated by leucine and asparagine at its amino- and carboxyl-terminal ends, respectively. The formula weight is 6787. The neurotoxin contains one residue each of tyrosine and tryptophan and its molar extinction coefficient at neutral pH is 8.7·103. The neurotoxin is strongly basic and its cathodal mobility at pH 8.7 and 1° is 5.0·10−5cm2·V−1·sec−1. The L.D.100 of the pure neurotoxin, determined by intravenous injection into mice weighing 18–20 g, is 1.8 μg, as compared with a value of 16 μg for crude venom. The preparation and some properties of the reduced and S-carboxymethylated derivative are described.

Isolation and characterization of a lethal myotoxic phospholipase A from the venom of the common sea snake Enhydrina schistosa causing myoglobinuria in mice.

Abstract A strongly myotoxic phospholipase A was isolated from the venom of Enhydrina schistosa by gel filtration on Sephadex G-75 and ion exchange chromatography on Bio-Rex 70. The myotoxin is a basic protein containing 120 amino acid residues and seven disulfide bridges. The formula weight is 13,500. The release of myoglobin diagnostic of muscle necrosis was monitored by placing injected mice on sheets of white paper. Affected mice stained the paper red. The myotoxin has an acute ld 50 of 110 μg/kg and causes myoglobinuria at doses down to 30 μg/kg. The molecule lacks trytophan, indicating that this amino acid is not required either for toxicity or catalytic activity. A single histidine residue was modified by reaction with p -bromophenacyl bromide, resulting in > 99% loss of enzymatic and toxic activities. The N-terminal sequence shows homology with nontoxic and neurotoxic phospholipases A 2 of vertebrate origin. The myotoxicity may be of greater clinical importance than the curarimimetic toxins that have received so much attention heretofore.

Effects of Sr2+ and Mg2+ on the phospholipase A and the presynaptic neuromuscular blocking actions of beta-bungarotoxin, crotoxin and taipoxin.

Summary1.β-Bungarotoxin, crotoxin and taipoxin, presynaptic neurotoxins of snake venom origin, have about the same phospholipid-splitting activities as a much less toxic cobra phospholipase A2 in the presence of Ca2+ and deoxycholate.2.Sr2+ was a much less effective activator of the enzymes than is Ca2+, the activation by Sr2+ being only 3–6% for β-bungarotoxin and crotoxin and 12% for taipoxin.3.Sr2+ also inhibited the Ca2+-activated enzymes by 80% in the cases of β-bungarotoxin and crotoxin, but only 16% in the case of taipoxin.4.Mg2+ had no significant effect on β-bungarotoxin or crotoxin, but activated taipoxin in the presence or absence of Ca2+.5.In Sr2+-Tyrode lacking Ca2+ all three toxins exhibited the same immediate depression followed by facilitation in the rat and mouse diaphragms, but the final blocking activity was only 3–10% with β-bungarotoxin and crotoxin and was 30% with taipoxin.6.In Sr2+-Tyrode, increasing in the rate of nerve stimulation had less accelerating effect on the development of neuromuscular block than in Ca2+-Tyrode for any of the toxins.7.Removal of Mg2+ from Sr2+-Tyrode did not diminish the potency of taipoxin in blocking neuromuscular transmission, suggesting that enzyme activity at the outer surface of the axolemma does not contribute to the neuromuscular blocking action.8.All of the results indicate that there are close correlations between the presynaptic activities of these toxins and their phospholipid-splitting activities in the cationic environment prevailing in the axoplasm. Apparently the final blocking effect of these toxins is due to phospholipase A action within the nerve terminal.

Sorption Effects in Gel Filtration: I. A Survey of Amino Acid Behavior on Sephadex G-10

Abstract Sephadex G-10, as supplied by the manufacturer, behaves as a weak cation exchanger. The cation-exchange capacity can be nearly eliminated by washing the gel with 1 M aqueous pyridine. The behavior of amino acids and other small solutes on pyridine-washed columns under defined conditions is very reproducible. The behavior of a solute on Sephadex G-10 fundamentally depends on its effective molecular size. Charged solutes tend to be excluded in media of low ionic strength, but the effective size and hence the degree of exclusion of such molecules can be decreased substantially by including high concentrations of small electrolytes in the medium. Salt can increase the adsorption of potentially adsorbable charged molecules in a passive way by increasing the amount of gel available to them. Salt might actively promote the adsorption of hydrophobic molecules by causing them to seek out regions within the gel where there is no salt, e.g., in the immediate vicinity of the gel matrix. Any pair of small wat...

Modification of amino groups in Naja naja neurotoxins and the preparation of radioactive derivatives.

Abstract Separation and analytical studies of carbamylated, acetylated, and guanidinated derivatives indicate that none of the amino groups present in the siamensis 3 (one α and five e) and naja 3 (one α and four e) neurotoxins (E. K ARLSSON , H. A RNBERG AND D. E AKER , Eur. J. Biochem. , 21 (1971) 1.) are essential for the curarizing action. The fully (penta)carbamylated derivative of the naja 3 toxin is 20% as active as the native molecule, while the penta- and hexacarbamyl derivatives of the siamensis 3 toxin show residual activities of only 7% and 3%, respectively, in the mouse assay. The guanidinated derivative of the siamensis 3 toxin showing five residues of homoarginine and no trace of lysine, but possessing the free α-amino group of isoleucine, is approximately half as toxic as the native toxin. Highly active (70%) and highly radioactive (0.13 mC/mg) monoacetylated derivatives can be prepared very efficiently by treating a large excess of the toxin with tritiated acetic anhydride. The six different monoacetyl derivatives of the siamensis 3 toxin are separated readily by gradient chromatography on Bio-Rex 70.

The three-dimensional structure of notexin, a presynaptic neurotoxic phospholipase A2 at 2.0 A resolution.

The three‐dimensional structure of notexin has been solved by molecular replacement methods. The structure has been refined at 2.0 Å resolution to a crystallographic R‐value of 16.5% with good stereo‐chemistry. The core of the protein is very similar to other phospholipase A2s (PLA2s) but several parts of the molecule are distinctly different. The most significant differences from PLA2s from bovine pancreas and rattlesnake occur in the stretches 56–80 and 85–89. Residue 69, which has been shown to be important for phospholipase binding, has a different conformation and different interactions than in other known PLA2s. The Cα positions for residues 86–88 differ by about 6 Å from both the bovine and the rattlesnake enzyme. The crystals contain no Ca2+ ions. Instead, a water molecule occupies the calcium site.

The three-dimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2.

The three-dimensional structures of the class II anticoagulant phospholipase A2 (PLA2) toxin RVV-VD from the venom of Russells viper, Vipera russelli russelli, and the class I neurotoxic PLA2 Notechis II-5 from the, Australian tiger snake, Notechis scutatus scutatus, were determined to 2.2 A and 3.0 A resolution, respectively. Both enzymes are monomeric and consist of 121 and 119 residues, respectively. A comparison of ten class I/II PLA2 structures showed, among other differences, that the beta-sheet of these enzymes (residues 76-83) is about 90 degrees less twisted in class I than in class II PLA2s. This, along with the insertion of some residues in the region 57-59 in class I enzymes (the elapid loop), could be the main reason for the significant difference in the anticoagulant and (presynaptic) neurotoxic properties between the two classes of PLA2. It seems apparent from sequence and structural comparisons that the toxic site of PLA2 responsible for the strong anticoagulancy of these toxins consists of a negatively charged part, Glu53, together with a positively charged ridge of lysine residues free for intermolecular interactions. These lysines differ between the two classes of PLA2.

An extracellular proteolytic enzyme from a strain of Arthrobacter II. Purification and chemical properties of the enzyme

Summary 1. A new extracellular proteolytic enzyme has been isolated from the culture medium of a strain of Arthrobacter grown in the presence of gelatin. The fractionation procedure makes use of precipitation with ammonium sulfate in the presence of cellulose, treatment with DEAE-Sephadex, gel filtration on Sephadex G-100 and crystallisation with ammonium sulfate or acetone. 2. Ultracentrifugation of the crystallized enzyme showed only one sedimenting component and its molecular weight determined by equilibrium centrifugation was approx. 22 ooo. Free electrophoresis revealed only one ultraviolet-absorbing peak with a mobility of 2.57 · 10−5 cm2 · V−1 · sec−1 at pH 8.6. 3. Twice-crystallized enzyme may contain traces of peptides which can be removed by gel filtration in aqueous acetic acid. Some of the pitfalls encountered in studies on the amino acid composition of the enzyme are discussed. 4. The enzyme contained 221 amino acid residues with a formula weight of 23 041. The amino-terminal residue was valine. The protein contains 2 disulfide bridges, a total of 12 basic amino acid residues and 29 amide groups. The molar extinction coefficient is 44.4 · 103 reflecting a high content of tyrosine and tryptophan.