Pseudechis Australis Research Articles

Ultrastructural changes induced by the myoglobinuria-producing venom from Pseudechis australis in mice kidney: P. Gopalakrishnakone, 1S. Z. J. Chen2 and M. C. E. Gwee2 (Venom and Toxin Research Group, Departments of 1Anatomy and 2Pharmacology, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 0511)

Ultrastructural changes induced by the myoglobinuria-producing venom from Pseudechis australis in mice kidney: P. Gopalakrishnakone, 1S. Z. J. Chen2 and M. C. E. Gwee2 (Venom and Toxin Research Group, Departments of 1Anatomy and 2Pharmacology, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 0511)

Characterization of toxic effects on nerve and muscle induced by three phospholipases A 2 from the venom of Pseudechis australis : M. Fatehi, 1E. G. Rowan, 1A. L. Harvey1 and J. B. Harris2 ( 1Department of Physiology & Pharmacology, Strathclyde University, Glasgow, U.K.; and 2School of Neuroscience, University of Newcastle Upon Tyne, U.K.)

Characterization of toxic effects on nerve and muscle induced by three phospholipases A 2 from the venom of Pseudechis australis : M. Fatehi, 1E. G. Rowan, 1A. L. Harvey1 and J. B. Harris2 ( 1Department of Physiology & Pharmacology, Strathclyde University, Glasgow, U.K.; and 2School of Neuroscience, University of Newcastle Upon Tyne, U.K.)

Pharmacological effects and pathological changes induced by the venom of Pseudechis australis in isolated skeletal muscle preparations

S. Z. J. Chen, P. Gopalakrishnakone and M. C. E. Gwee. Pharmacological effects and pathological changes induced by the venom of Pseudechis australis in isolated skeletal muscle preparations. Toxicon 32, 303–315, 1994.—The venom from the Australian king brown snake Pseudechis australis produced a concentration-dependent and progressive inhibition of directly and indirectly evoked twitches of the mouse hemidiaphragm: 2, 5 and 10 μg/ml of venom produced complete blockade of directly evoked twitches after 87 ± 26 min, 66 ± 28 min and 56 ± 13 min, respectively, whereas twitches elicited by indirect stimulation were blocked after 43 ± 4 min, 34 ± 2 min and 21 ± 2 min, respectively. Two, 5 and 10 μg/ml of the venom also progressively inhibited the twitch responses of the chick biventer cervicis muscle evoked by nerve stimulation: the times (min) to 90% blockade of the twitches were 127 ± 12, 41 ± 8 and 26 ± 6, respectively. Light and electron microscopic studies confirmed that the venom caused severe muscle damage and necrosis in the chick and mouse skeletal muscle. The degree of muscle damage was proportional to the concentration of venom used. The muscle degeneration was characterized by disorganized myofibrils, dilatation of the sarcoplasmic reticulum and swollen or broken-down mitochondria. Depletion and a decrease in population of storage vesicles in the neuromuscular junction were also observed. Thus, the inhibition of neuromuscular function by the venom of P. australis was a consequence of the extensive morphological damage to muscle fibres, motor nerve terminals and cytoplasmic organelles which could be attributed to a direct myotoxic action presumably mediated by the enzymatic action of phospholipase A 2 present in the venom of P. australis.

Neutralization of tiger snake ( Notechis scutatus) venom by serum from other Australian elapids

Sera from four Australian elapids and one boidid (python) were tested for their ability to protect neonatal mice against the toxic action of tiger snake ( Notechis scutatus) venom. Of the five serum samples tested, only serum from Pseudechis australis and Pseudechis porphyriacus were capable of neutralizing the tiger snake venom. In addition, neutralization was shown to be highly variable within serum taken from individual snakes of the same species ( P. porphyriacus). Previously, only viperid and colubrid snakes have been shown to possess neutralizing factors against snake venoms.

Effects of two toxic phospholipases A 2 from venom of the king brown snake, Pseudechis australis, on neuromuscular transmission : M. Fatehi, E. G. Rowan and A. L. Harvey (Department of Physiology and Pharmacology, Strathclyde University, Glasgow G1 1XW, U.K.)

Effects of two toxic phospholipases A 2 from venom of the king brown snake, Pseudechis australis, on neuromuscular transmission : M. Fatehi, E. G. Rowan and A. L. Harvey (Department of Physiology and Pharmacology, Strathclyde University, Glasgow G1 1XW, U.K.)

Pathological and pharmacological effects of the venom of the Australian king brown snake Pseudechis australis on the mouse phrenic nerve-hemidiaphragm: S. Z. J. Chen, P. Gopalakrishnakone1 and M. C. E. Gwee (Venom and Toxin Research Group (Departments of Pharmacology and Anatomy 1), Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 0511)

Pathological and pharmacological effects of the venom of the Australian king brown snake Pseudechis australis on the mouse phrenic nerve-hemidiaphragm: S. Z. J. Chen, P. Gopalakrishnakone1 and M. C. E. Gwee (Venom and Toxin Research Group (Departments of Pharmacology and Anatomy 1), Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 0511)

Neuromuscular effects of four phospholipases A 2 from the venom of Pseudechis australis, the Australian king brown snake

Four homologous single chain phospholipases A 2 (Pa-1G, Pa-5, Pa-12C and Pa-15) were tested for neuromuscular effects on chick biventer cervicis and mouse hemidiaphragm nerve-muscle preparations. The four isozymes blocked directly elicited (mouse hemidiaphragm) and indirectly elicited (mouse and chick nerve-muscle preparations) twitch responses in concentrations of 1–30 μg/ml. The order of potency seen in both types of preparations was Pa-1G = Pa-5 > Pa-12C ⪢ Pa-15. All four isozymes caused slow-onset, sustained contractures and reduction of muscle membrane potentials. In the chick preparation, responses to acetycholine, carbachol and KCI were reduced by exposure to the toxins. It is concluded that the toxins act primarily postsynaptically to depress muscle contractility, perhaps by directly damaging muscle fibres. The order of potency agrees with their phospholipase A 2 activity. Pa-1G is unusual because it is an acidic molecule, most toxic phospholipases being basic.

A comparison of the neuromuscular effects of two phospholipases A 2 from the venom of the Australian king brown snake : S. L. Geh, E. G. Rowan and A. L. Harvey (Department of Pharmacology, Faculty of Medicine, University of Malaya, 59100 Kuala Lumpur; Strathclyde Institute for Drug Research, University of Strathclyde, Glasgow, Scotland, U.K.).

A comparison of the neuromuscular effects of two phospholipases A 2 from the venom of the Australian king brown snake : S. L. Geh, E. G. Rowan and A. L. Harvey (Department of Pharmacology, Faculty of Medicine, University of Malaya, 59100 Kuala Lumpur; Strathclyde Institute for Drug Research, University of Strathclyde, Glasgow, Scotland, U.K.).

Antibacterial effects of different snake venoms: Purification and characterization of antibacterial proteins from Pseudechis australis (Australian king brown or mulga snake) venom

Venoms from 30 different snake species were tested in a disc diffusion assay for antibacterial effects against gram-positive and gram-negative bacteria. A number of venoms gave a zone of inhibition against both groups of bacteria, including Aeromonas hydrophila, an important pathogen of reptiles and amphibians. Two antibacterial components from the venom of an Australian elapid, Pseudechis australis (Australian king brown or mulga snake) were purified to homogeneity. The proteins, designated LAO1 and LAO2, had potent antibacterial properties associated with l-amino acid oxidase activity. Both had native and subunit mol. wts of 142,000 and 56,000, respectively. Antibacterial activity correlated with enzymatic activity and was eliminated with catalase. LAO1 and LAO2 had 244 and 113 units of l-amino acid oxidase activity/mg protein, respectively. Compared to tetracycline, a drug of choice for Aeromonas infections in humans, reptiles and amphibians, the in vitro antibacterial effects of LAO1 and LAO2 were respectively 70 and 17.5 times more effective (on a molar basis).

Tryptophan residues of phospholipase A 2 from the venom of an Australian elapid snake (Pseudechis australis)

S. Nishida and N. Tamiya. Tryptophan residues of phospholipase A 2 from the venom of an Australian elapid snake ( Pseudechis australis). Toxicon 29, 429–439, 1991.—Tryptophan residues 31 and 69 (Trp-31 and Trp-69) in phospholipase A 2 (Pa-11) from the venom of an Australian elapid snake, Pseudechis australis, were modified with N-bromosuccinimide (NBS) or with 2-nitrophenylsulphenylchloride (NPSC1). NBS oxidized only Trp-31, whereas NPSC1 reacted with both Trp-31 and Trp-69. Treatment of the enzyme with NBS at various pH values resulted in losses of enzymic and lethal activities. No protective effect on the oxidation with NBS was observed by the addition of calcium ion (20 mM) or lecithin (4 mM). The observations suggest that Trp-31 is exposed to the surface of the molecule, composes a part of the lipid-water interface recognition site around the active site and is essential for enzymic activity. Calcium ion addition to the solution caused a change in ultraviolet spectrum of the native enzyme Pa-11. The difference spectrum indicates that a charge effect caused a typical tryptophan blue shift in the Ca 2+-enzyme complex. Pa-11 oxidized with NBS showed a smaller ultraviolet absorption difference on the addition of Ca 2+ ion. The results show that the hypochromic effect induced upon the binding of Ca 2+ is due to perturbation of the specific tryptophan residue (Trp-31) which is involved in the active site. Dissociation constant, Kd, of the Ca 2+-enzyme complex was calculated to be 3.4 × 10 −4 M at pH 8.0.

Amino acid sequences of eight phospholipases A 2 from the venom of Australian king brown snake, Pseudechis australis

The amino acid sequences of eight phospholipases A 2 (Pa-1G, Pa-3, Pa-5, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-15) which had been isolated from the venom of Australian king brown snake ( Pseudechis australis) were elucidated. Pa-1G, Pa-3 and Pa-15 showed micro-heterogeneity at the 103rd position and Pa-5 was separated into two components, Pa-5a ([Pro-18 and Tyr-61]Pa-5) and Pa-5b ([Ser-18 and Phe-61]Pa-5). All the phospholipase A 2 molecules except Pa-1Ga and Pa-1Gb which lack the 118th residue, consisted of a single chain of 118 amino acid residues including 14 half-cystine residues and all the common residues among phospholipases A 2 from other sources. From comparison studies, Asp-50, Lys-58 and Asp-90 seem to be important for the toxicity, and we propose that the domain for the presynaptic toxicity consists of seven hydrophilic residues, i.e. Arg-43, Lys-46, Asp-50, Glu-54, Lys-58, Asp-90 and Glu-94.

Purification and properties of several phospholipases A 2 from the venom of Australian king brown snake ( Pseudechis australis)

Thirteen isoenzymes of phospholipases A 2 were purified from the venom of Australian king brown snake, Pseudechis australis. They (except phospholipase A 2 Pa-9C) showed normal properties of snake venom phospholipases A 2; the apparent mol. wts were about 13,000, the optimum pH values were around 8, calcium ion was indispensable for the enzymatic activity and the optimum calcium ion concentrations were more than 5 mM. Phospholipase A 2 Pa-9C had a lag period at the initial stage of the enzymatic reaction. The enzymatic activities determined by the titration method using 1,2-dipalmitoylglycerophosphocholine as a substrate at 37°C were 10,500 units/mg for Pa-1G and 75 units/mg for Pa-13. The lethal activities measured by i.v. injections in mice were 0.09 μg/g body wt for Pa-5 and 6.8 μg/g body wt for Pa-13. The lethal activity correlates with the enzymatic activity (correlation coefficient of 0.92), and both activities showed no relationship to the basicity of the enzyme. Pa-1G is the first acidic phospholipase A 2 (pI = 6.4) with high neurotoxicity (0.13 μg/g body wt).

Effects of chemical modifications of Pa-11, a phospholipase A 2 from the venom of Australian king brown snake ( Pseudechis australis), on its biological activities

C. Takasaki, A. Sugama, A. Yanagita, N. Tamiya, E. G. Rowan and A. L. Harvey. Effects of chemical modifications of Pa-11, a phospholipase A 2 from the venom of Australian king brown snake ( Pseudechis australis), on its biological activities. Toxicon 28, 107–117, 1990.—Pa-11, a phospholipase A 2 isolated from the venom of an Australian elapid snake Pseudechis australis, was chemically modified and its enzymic, neuromuscular and lethal activities were studied. Carboxymethylation of Met-8 gave a derivative with 2% of the enzymic activity and less than 3% of the lethal activity of native Pa-11; it had about 5% of the original ability to block directly and indirectly stimulated mouse phrenic nerve-hemidiaphragm preparations. Nitrophenylsulfenylation of tryptophanyl residues at positions 31 and 69 caused loss of all activities. Amidination of all 14 lysyl residues gave a derivative with 41% and 16% of the enzymic and lethal activities, respectively, but with less than 5% of the original neuromuscular blocking activity. Mono-carbamoylation of lysyl residues at positions 58, 63, 81 and 85 was achieved. The most abundant derivative, 58-carbamoyl-lysine Pa-11 was enzymically 130% and lethally 100% as active as native Pa-11, but it had only about 20% of the native's neuromuscular activity in vitro. 63-Carbamoyl-lysine Pa-11 had 10% of the enzymic and 20% of the lethal activities, respectively; however, it retained at least 50% of its ability to block neuromuscular transmission in vitro, while losing most of its activity to block directly stimulated muscle contractions. 81- and 85-Carbamoyl derivatives have the same enzymic and lethal activities as the original protein, but the 85 derivative had less than 10% of the native neuromuscular activity. Hence, modifications of lysine residues at positions 58, 63 and 85 seem to be particularly significant in altering the neuromuscular, but not enzymic, activity of Pa-11, perhaps by altering the ability of the toxin to bind to its target on nerve and muscle membranes. Modification at position 63 appeared to lead to a dissociation of effects on neuromuscular transmission and directly on muscle cells.

Amino acid sequence of a long-chain neurotoxin homologue, Pa ID, from the venom of an Australian elapid snake, Pseudechis australis.

Pa ID, a long-chain neurotoxin homologue, was isolated from the venom of an Australian elapid snake, Pseudechis australis, and its amino acid sequence was determined by conventional methods. Pa ID was an acidic protein (pI = 6.2) and consisted of 68 amino acid residues. It did not show binding activity to the acetylcholine receptor of an electric ray (Narke japonica) nor lethal effect on mice, though the amino acid sequence is homologous with those of long-chain neurotoxins isolated from other elapid snakes (homology, 39-51%). In the sequence of Pa ID, a structurally invariant residue (Tyr-22) and two functionally invariant residues (Val/Ala-49 and Lys/Arg-50) in snake venom neurotoxins are replaced by a cysteine, an arginine, and a methionine residue, respectively, and furthermore, four common residues in long-chain neurotoxins, Gly-17, Ala-43, Ser-59, and Phe/His-66 are replaced by a glutamic acid, a threonine, a threonine, and a valine residue, respectively. The conformational change of the protein molecule caused by these replacements and the removal of a positive charge at position 50 are probably the reasons why Pa ID has lost the lethality.

A basic phospholipase A from the venom of the australian king brown snake ( Pseudechis australis) showing diverse activities against membranes

1. 1. A basic phospholipase A (MSPA) was isolated from the venom of the Australian king brown snake, Pseudechis australis. 2. 2. MSPA had an approximate M r of 13,000 and consisted of a single polypeptide chain of 119 amino acid residues cross-linked by seven disulphide bridges. 3. 3. MSPA exhibited direct haemolytic, anticoagulant and myotoxic activities. 4. 4. Treatment of MSPA with p-bromophenacyl bromide modified a single histidine residue, resulting in complete loss of enzyme activity.

Neuromuscular effects of three phospholipases A 2 from the venom of the Australian king brown snake Pseudechis australis

—Three single chain phospholipases A 2 (Pa-10A, Pa-11 and Pa-13) isolated from Australian king brown snake ( Pseudechis australis) venom were tested for effects on neuromuscular transmission and muscle contractility on chick biventer cervicis and mouse diaphragm preparations. At 1 μg/ml (about 85 nM) and higher, Pa-10A and Pa-11 reduced responses of both preparations to indirect stimulation in a concentration-dependent manner. Responses to direct muscle stimulation were generally reduced more slowly. Pa-11 also decreased membrane potentials of chick biventer muscle fibres and caused damage visible by light microscopy. Pa-13, which is about 50 times less active as a phospholipase A 2, was also less potent in its pharmacological effects: 20 μg Pa-13 per ml were required to reduce responses of either preparation. The phospholipases A 2 also caused a slow contracture. After block of responses to nerve stimulation, responses of the chick preparation to acetylcholine, carbachol and KCl could be obtained, although they were smaller than control and highly variable in different preparations. It is concluded that Pa-10A and Pa-11 produce muscle paralysis by reducing acetylcholine release and by a direct blockade of muscle fibre contractility. Pa-13 has similar, though less pronounced, activities.

Chemical modification of phospholipase A 2 from the venom of king brown snake, Pseudechis australis : C. Takasaki, A. Yanagita, A. Sugama and N. Tamiya (Department of Chemistry, Tohoku University, Sendai, Japan)

Chemical modification of phospholipase A 2 from the venom of king brown snake, Pseudechis australis : C. Takasaki, A. Yanagita, A. Sugama and N. Tamiya (Department of Chemistry, Tohoku University, Sendai, Japan)

Isolation and amino acid sequence of a short-chain neurotoxin from an Australian elapid snake, Pseudechis australis.

A short-chain neurotoxin Pseudechis australis a (toxin Pa a) was isolated from the venom of an Australian elapid snake Pseudechis australis (king brown snake) by sequential chromatography on CM-cellulose, Sephadex G-50 and CM-cellulose columns. Toxin Pa a has an LD50 (intravenous) value of 76 micrograms/kg body wt. in mice and consists of 62 amino acid residues. The amino acid sequence of Pa a shows considerable homology with those of short-chain neurotoxins of elapid snakes, especially of true sea snakes.

Amino acid sequences of phospholipases A2 from the venom of an Australian elapid snake (king brown snake, Pseudechis australis)

Two basic phospholipases A2 (Pa-11 and Pa-13) have been isolated from the venom of an Australian elapid snake, Pseudechis australis (king brown snake). The reduced and S-carboxymethylated phospholipases A2 were digested with trypsin and the resulting peptides were purified by a combination of chromatography on a DEAE-cellulose DE-52 column and gel filtration procedures. Eleven main peptides from Pa-11 and 9 peptides from Pa-13 could account for the amino acid compositions of the respective enzyme molecules. The alignment of the tryptic peptides and unelucidated regions of the amino acid sequences of tryptic peptides were established by the analysis of the peptides obtained by chymotryptic and/or Staphylococcal protease digestions. Each phospholipase A2 consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. Although Pa-11 is enzymically 30-times as active as Pa-13 and highly toxic as compared to Pa-13, they are highly homologous in their amino acid sequences. They are also homologous to the enzymes from mammalian pancreas and the other snake venom phospholipases A2, especially to those from snakes belonging to the subfamilies Acanthophiinae and Laticaudinae.

Isolation and properties of two phospholipases A2 from the venom of an australian elapid snake (Pseudechis australis)

Two phospholipases A2 (Pa-11 and Pa-13) were purified from the venom of an Australian elapid snake (subfamily Acanthophiinae) Pseudechis australis (king brown snake) by chromatography on CM-cellulose CM-52 followed by gel filtration on a Sephadex G-75 column. The apparent molecular weights of the two phospholipases A2 (Pa-11 and Pa-13) were 14,000 and 13,500, respectively, by gel filtration analysis on a Sephadex G-75 column.Each enzyme molecule consists of a single polypeptide chain of 118 amino acid residues. The isoelectric points of Pa-11 and Pa-13 were 10.5 and 10.0, respectively. The optimum pH values of Pa-11 and Pa-13 for hydrolysis of egg-yolk phosphatidylcholine were 7.8 and 7.5, respectively. Pa-11 was lethal to mice (ld50 0.23 μg/g body weight), whereas Pa-13 showed no lethal activity at a dose level of 7.4 μg/g mouse. Each enzyme was inactivated by reaction with p-bromophenacylbromide on the sole histidine residue (Pa-11) or on one of the two histidine residues (Pa-13). Oxidation of the tryptophan residues in Pa-11 and Pa-13 with N-bromosuccinimide led to a decrease in the phospholipase A activity. A complete loss of both enzymic and lethal activities of Pa-11 was observed upon oxidation of one of the two tryptophan residues of the molecule.