Notechis Research Articles

Effect of tiger snake venom on the liver: E. A. Bennett and I. F. Coombe (Department of Medical Laboratory of Science, Queensland Institute of Technology, Brisbane, Australia)

Effect of tiger snake venom on the liver: E. A. Bennett and I. F. Coombe (Department of Medical Laboratory of Science, Queensland Institute of Technology, Brisbane, Australia)

Studies on the venom of the peninsula tiger snake, Notechis ater niger: J. White and V. Williams (Department of Haematology, Adelaide Children's Hospital, North Adelaide 5006, South Australia)

Studies on the venom of the peninsula tiger snake, Notechis ater niger: J. White and V. Williams (Department of Haematology, Adelaide Children's Hospital, North Adelaide 5006, South Australia)

Platelet Procoagulant Properties Studied with Snake Venom Prothrombin Activators

Purified snake venom prothrombin activators were used to probe the procoagulant properties of platelet membranes. Human platelets were able to stimulate prothrombin activation by the venom activators from Oxyuranus scutellatus and Notechis scutatus, while the prothrombin activator from Echis carinatus was not affected by the presence of platelets. The prothrombin-converting activity of platelets was further studied with the venom activator from Oxyuranus scutellatus and with the factor Xa-Va complex as prothrombin activating enzymes. Stimulation of platelets with collagen, collagen plus thrombin or with the Ca-ionophore A23187 resulted in a considerable increase of platelet prothrombin converting activity probed with the factor Xa-Va complex as well as with the prothrombin activator from Oxyuranus scutellatus. The stimulatory effect of activated platelets on the rates of prothrombin activation by Oxyuranus scutellatus was similar to that determined for factor Xa-Va-catalyzed prothrombin activation. Compared to non-stimulated platelets, platelets stimulated with thrombin plus collagen exposed 20-times more procoagulant sites for as well the factor Xa-Va complex, as for the venom activator from Oxyuranus scutellatus. The actual number of procoagulant sites per platelet determined with the factor Xa-Va complex was in close agreement with the number of sites determined with the venom activator. Also the time course of appearance of procoagulant activity during platelet stimulation by collagen plus thrombin was comparable for both activator complexes. Phospholipase A2 treatment of stimulated platelets resulted in an almost complete loss of their ability to stimulate prothrombin activation by the enzyme from Oxyuranus scutellatus or by factor Xa-Va complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the structure and function of three phospholipases A 2 from the venom of Agkistrodon halys pallas

Y.-C. Chen, J. M. Maraganore, I. Reardon and R. L. Heinrikson. Characterization of the structure and function of three phospholipases A 2 from the venom of Agkistrodon halys pallas. Toxicon 25, 401 – 409. 1987.—Three monomeric phospholipases A 2 with isoelectric points 4.5, 6.9 and 9.3 were purified from the venom of Agkistrodon halys pallas. The complete amino acid sequence of the acidic enzyme and partial amino acid sequences of the neutral and basic phospholipases were determined in order to relate differences in enzymatic reactivities, pharmacologic activities and cytotoxicities to aspects of structure. Studies reported here and elsewhere demonstrate that the three phospholipases A 2 exhibit pronounced differences relative to function. The acidic enzyme maintains the highest reactivity toward hydrolysis of monolayers at the air — water interface and may share a feature in common with the acidic enzyme from A. h. blomhoffii, namely the inhibition of platelet aggregation. The neutral phospholipase A 2 designated agkistrotoxin, is characterized by potent activity as a pre-synaptic neurotoxin. Agkistrotoxin is the first single polypeptide chain, neurotoxic phospholipase A 2 to be documented with a Group II disulfide pattern and, in several respects, may be considered functionally and structurally analogous to notexin from the Australian tiger snake venom. Finally, the basic enzyme exhibits the abilities to hemolyze erythrocytes and to hydrolyze phospholipids of E. coli membranes in the presence of a bactericidal-permeability-increasing protein from neutrophil sources.

Variation in venom constituents within a single isolated population of peninsula tiger snake (Notechis ater niger)

V. Williams and J. White. Variation in venom constituents within a single isolated population of peninsula tiger snake ( Notechis ater niger). Toxicon 25, 1240 – 1243, 1987. — Venom from seven individuals in a homogeneous isolated population of Notechis ater niger on Roxby Island, off the South Australia coast, was subjected to gel filtration liquid chromatography and polyacrylamide gel electrophoresis to determine the extent of homogeneity of the venom constituent profile. The gel filtration chromatograms from six of the specimens were easily equated, while the seventh showed a slight variation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the venoms in a native and reduced state showed no detectable difference between six of the seven individuals, the remaining individual showing minor changes only in the native venom.

Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus.

The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex G-200 followed by ion-exchange chromatography on SP-Sephadex. The venom activator has an apparent molecular weight of 54,000. It consists of a heavy chain (Mr = 32,000) and a light chain (Mr = 23,000) held together by one or more disulfide bridges. The active site is located at the heavy chain region of the molecule. The venom activator contains 8 gamma-carboxyglutamic acid residues/molecule. Gel electrophoretic analysis of prothrombin activation indicates that the venom activator is capable of cleaving both the Arg 274-Thr 275 and Arg 323-Ile 324 bonds of bovine prothrombin. The order of bond cleavage appears to be random since prethrombin-2 and meizothrombin occur as intermediates during prothrombin activation. Prothrombin activation by the venom activator alone is very slow. This is explained by the unfavorable kinetic parameters for the reaction (Km for prothrombin = 105 microM, Vmax = 0.0025 nmol of prothrombin activated per min/microgram of venom activator). Phospholipids plus Ca2+ and Factor Va greatly stimulate venom-catalyzed prothrombin activation. In the presence of 50 microM phospholipid vesicles composed of 20 mol % phosphatidylserine and 80 mol % phosphatidylcholine, the Km drops to 0.2 microM, whereas there is hardly any effect on the Vmax. Factor Va causes a 3,500-fold increase of the Vmax (8.35 nmol of prothrombin activated per min/microgram of venom activator) and a 10-fold decrease of the Km (9.5 microM). The most favorable kinetic parameters are observed in the presence of both 50 microM phospholipid and Factor Va (Km = 0.16 microM, Vmax = 27.9 nmol of prothrombin activated per min/microgram of venom activator). These changes of the kinetic parameters explain the stimulatory effects of Factor Va and phospholipid on venom-catalyzed prothrombin activation. The venom activator slowly converts the Factor Xa-specific chromogenic substrates CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroani lide hydrochloride. Factor Va causes a 7-fold stimulation of chromogenic substrate conversion by the venom activator. This stimulation appears to be the result of the formation of a tight 1:1 complex between the venom activator and Factor Va.

An investigation, in vitro, of the actions of three Western Australian snakes on the blood coagulation of the dog, cat, horse and wallaby.

Venoms of the tiger snake and brown snake were procoagulant, in vitro, when tested with cat, dog, horse and wallaby plasma. In the absence of calcium and phospholipid the coagulant activity of tiger snake venom was minimal. In contrast, brown snake venom alone had marked procoagulant activity. This activity, however, was enhanced by the presence of calcium and phospholipid. Death adder venom exerted an anticoagulant effect. Apparent species' differences in susceptibility to the coagulant venoms were noted. However, the probable explanation of these differences was attributed to variation in the control values of the special studies rather than to a difference in the postulated actions of the venoms on prothrombin. A possible role for clotting studies in suspected snake bite in veterinary practice is suggested.

Can Australians identify snakes?

A study of the ability of Australians to identify snakes was undertaken, in which 558 volunteers (primary and secondary schoolchildren, doctors and university science and medical students) took part. Over all, subjects correctly identified an average of 19% of snakes; 28% of subjects could identify a taipan, 59% could identify a death adder, 18% a tiger snake, 23% an eastern (or common) brown snake, and 0.5% a rough-scaled snake. Eighty-six per cent of subjects who grew up in rural areas could identify a death adder; only 4% of those who grew up in an Australian capital city could identify a nonvenomous python. Male subjects identified snakes more accurately than did female subjects. Doctors and medical students correctly identified an average of 25% of snakes. The ability to identify medically significant Australian snakes was classified according to the observer's background, education, sex, and according to the individual snake species. Australians need to be better educated about snakes indigenous to this country.

The observation of quasi-molecular ions from a tiger snake venom component ( Mr 13 309) using 252Cf-plasma desorption mass spectrometry

A method involving fast heavy-ion bombardment of a solid sample called 252Cf-plasma desorption mass spectrometry has been used to study of a non-enzymatic, non-toxic phospholipase homolog from Australian tiger snake ( Notechis scutatus) venom. The protein consists of 119 amino acids in a single polypeptide chain cross-linked by 7 disulfide bridges. The isotopically averaged molecular mass as determined by protein sequence analysis is 13 309 atomic mass units (amu). The mass distributions were studied by means of time-of-flight measurements. Quasi-molecular ions associated to the molecule and its dimer were observed. The mass of the quasi-molecular ion corresponding to the molecule was determined to be 13 285 ± 25 amu.

Preliminary observations of satellite cells in undamaged fibres of the rat soleus muscle assaulted by a snake-venom toxin.

Satellite cells were studied in the undamaged part of the rat soleus muscle rendered partially necrotic by a subcutaneous injection of notexin, the myotoxic toxin purified from the venom of the Australian snake Notechis scutatus scutatus. Nuclei of satellite cells were twice as numerous as in the controls, with a decreasing gradient in the number of satellite cells with distance from the necrotic area. The cells were in an activated state with an increased cytoplasmic volume and prominent organelles. Occasionally, mitosis of some satellite cells was observed. Between the satellite cell and the muscle fibre, an unusually wide space was frequently seen, within which a "new" basal lamina was often visible. It is suggested that the role of the satellite cells of undamaged muscle fibres in the regeneration of necrotic muscle is worthy of more detailed investigation.

The mass of venom injected by two Elapidae: The Taipan ( Oxyuranus scutellatus) and the Australian Tiger snake ( Notechis scutatus)

Using an enzyme immunoassay technique, a new method for measuring, in vivo, the mass of venom injected during snake bite, is presented. The venom injected into mice (as prey) and the venom left on the skin surface during bites by the two Australian Elapidae, the Taipan ( Oxyuranus scutellatus) and the Tiger Snake ( Notechus scutatus) has been measured. Venom delivery patterns vary significantly between these two species. In the case of the Tiger Snake (a total of 45 bites studied) the mean mass of venom injected in a first bite was 12.7 mg (S.E. 3.4 mg, median 8.1 mg); an average mass of 0.8 mg (S.E. 0.4 mg, median 0.17 mg) was left on the skin surface. A second bite delivered by the same snake yielded a mean venom mass only 27% of the first. In the case of the Taipan (a total of 24 bites) the mean venom mass injected in the first bite was 20.8 mg (S.E. 6.4 mg); with an average of 0.9 mg (S.E. 0.5 mg) left on the skin surface. In contrast to the situation observed with Tiger Snakes, second and third bites delivered in a rapid sequence yielded increasing masses of venom. The mean delivered in the third of a sequence of three bites was 48.8 mg (S.E. 23.8 mg). The ranges of venom mass, by species and by the sequence number of the bite, are also presented. In 66 of the 69 experimental bites studied in this report, venom could be easily detected, the species identified, and the absolute mass of venom measured.

In vitro coagulant properties of venoms from Australian snakes

The procoagulant properties of venomous Australian snakes are limited to prothrombin activation. Venoms of Notechis scutatus, Tropidechis carinatus and Pseudechis porphyriacus require phospholipid, calcium and factor V to maximally activate normal prothrombin. Venoms of Pseudonaja textilis and Oxyuranus microlepidotus activate both normal prothrombin and the decarboxy form and do not require co-factors. The actions of the venom of Oxyuranus scutellatus are dependent on concentration.

PROCEEDINGS OF THE AUSTRALIAN SOCIETY FOR MEDICAL RESEARCH

PROCEEDINGS OF THE AUSTRALIAN SOCIETY FOR MEDICAL RESEARCH

Structure of presynaptic toxins: Crystallization and preliminary x-ray diffraction data on Notechis II-5, a presynaptic toxin phospholipase

The presynaptic neutrotoxin-phospholipase, Notechis II-5 from the venom of NotechisScutatus scutatus (Australian tiger snake) has been crystallized in a form suited for x-ray diffraction analysis. The crystals belong to the orthorhombic space group P21 21,21, with unit cell dimensions, a=146.1,b =43.5 and c =39.0 A. There are two molecules of Notechis II-5 in the asymmetric unit. The molecular weight is about 13,500. Notechis II-5 is highly homologous to Notexin, another presynaptic toxin from the venom of the Australian tiger snake, to bovine and porcine pancreatic phospholipases A and other venom phospholipases.

A study of the major australian snake venoms in the monkey (Macaca fascicularis): I. the movement of injected venom, methods which retard this movement, and the response to antivenoms

<h2>Summary</h2> Conscious monkeys were comfortably restrained for several hours whilst the movement and effects of injected snake venom was studied. Routine clinico-pathological studies were carried out on blood samples obtained at regular intervals and a solid phase radioimmunoassay was used to assay plasma levels of snake venoms and an individual neurotoxin. When snake venom was injected subcutaneously into the lower limb of a monkey and no first aid was applied, venom could be detected in the plasma of the animals within 15 min of the injection. Plasma venom and neurotoxin levels peaked about 60 min after the injection but signs of neurotoxicity were usually not seen until 120 min after injection. Post mortem studies showed that very little Tiger snake venom remained at the injection site, but high concentrations were found in the regional lymph nodes. Tiger snake venom components including the neurotoxin, notexin, were excreted in both the bile and urine of monkeys. The high levels of venom and neurotoxin found in the urine of monkeys parallel those found in some human snake bite victims. Recently developed first aid measures were applied to the envenomed limb of a number of monkeys. These measures consisted of the application of a firm crepe bandage to the length of the injected limb which was then immobilized with a splint. The first aid measures were found to delay the movement of all the major Australian land snake venoms and also the venom of the beaked sea snake (<i>Enhydrina schistosa</i>). A number of envenomed monkeys infused with antivenom immediately neurotoxic signs developed, barely survived even when given 10 times the amount of antivenom required to neutralize <i>in vitro</i> the dose of venom they had received. Surviving monkeys had persistent ptosis and lethargy for up to 5 d after envenoming. Failure to respond to antivenom was more likely if no effective first aid measure had been employed.

A study of the major australian snake venoms in the monkey (Macaca fascicularis): II. Myolytic and haematological effects of venoms

Rhesus monkeys were kept in a restraining apparatus and while conscious injected with the venoms of a range of Australian snakes. Although the action of restraining itself caused an elevation of the animals' plasma creatine kinase (C.K.), the rise was up to 5 times greater in envenomed monkeys. The venoms of the Tiger snake, Mulga, Beaked Sea snake, Rough-Scaled, Copperhead and Red-Bellied snakes were all powerfully myolytic, and those of the Taipan and Small-Eyed snake less so. No myolytic activity was found in the venoms of the Common Brown snake and Death Adder, and the results with Dugite, Gwardar and Small-Scaled snake venom were equivocal. Significant coagulation disturbances were produced by all venoms studied except those of the Small-Eyed and Rough-Scaled snakes. When first aid measures were used, the coagulation disturbances which developed after the release of the injected venom were significantly less than when no first aid was used. This finding, which was observed with all snake venoms studied, suggests that the procoagulant factor may be subject to some inactivation whilst immobilized at the injection site.

Phospholipase A 2 activity of notexin and its role in muscle damage

The phospholipase A 2 activity of notexin, a toxin isolated from the crude venom of Notechis scutatus scutatus, the Australian tiger snake, has been partially characterised using as substrates synthetic dipalmitoyl phosphatidyl choline and bacterial membrane fragments in which the phosphoglycerides were labelled with 1- 14C palmitic acid. The phospholipase activity was Ca 2+-dependent and inhibited by Mg 2+, Ba 2+, Sr 2+, Zn 2+ and Mn 2+; Mg 2+ was the least potent inhibitor and Ba 2+ the most potent. The enzyme was active over a wide range of temperatures and pH, and was stimulated by the presence of deoxycholate. When muscles were incubated in vitro in the presence of notexin, mean muscle fibre potential fell by 16 mV over the first 3 hr and by 60 mV over 6 hr. The depolarisation was Ca 2+-dependent. Muscles were also exposed to notexin in vivo and one day later the phospholipids extracted. Thin layer chromatography revealed the presence of lysophosphatides in the toxin damaged muscles, but not in the contralateral muscles. It was concluded that notexin is a potent phospholipase A 2, capable of hydrolysing phospholipids in micelles, in fragmented bacterial membranes, and in intact muscle fibre membranes, and that this activity represents its primary mode of attack.

Complete amino-acid sequence of a non-neurotoxic, non-enzymatic phospholipase A2 homolog from the venom of the Australian tiger snake Notechis scutatus scutatus.

The complete amino acid sequence of Notechis II-1, a non-neurotoxic, non-enzymatic phospholipase A2 homolog from the venom of the Australian tiger snake Notechis s. scutatus has been determined. The protein consists of a single chain of 119 amino acids. The main fragmentation of the reduced and S-carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease. Tryptic peptides were used to align and complete the sequence, which was determined mainly by automated Edman degradation. Notechis II-1 contains all of the residues that appear to be invariant in elapid and pancreatic phospholipases A2 except at position 30 in the sesquence, where an otherwise invariant glycine is replaced by serine.

Trypsin fails as Australian snake bite cure.

Trypsin has been claimed a new and effective treatment for venomous snake bite. We found that significant inactivation of snake venom lethal potency occurred in vitro when trypsin was incubated with venom and subsequently injected into mice. Premixing of tiger snake venom (TSV) and trypsin just before injection did not significantly increase the survival rate of mice over that of controls injected with TSV alone. Trypsin injected 10 to 30 minutes after TSV injection did not increase the survival rate of mice compared with controls. Specific antivenom was effective as an antidote when there was a 10 minute delay after venom injection. There was varying susceptibility of different venoms to trypsin inactivation in vivo.

Behavioral Thermoregulation in Australian Elapid Snakes

Behavioral thermoregulation in laboratory thermal gradients was studied in seven species of Australian snakes of the Elapidae: Acanthophis antarcticus, Austrelaps superbus, Notechis scutatus, Pseudechis porphyriacus, Pseudonaja nuchalis, P. textilis and Unechis flagellum. All species exhibited well-developed thermoregulatory behavior and controlled body temperatures with precision comparable to that reported for various heliothermic lizards. Temperature regulation is accomplished by shuttling and by adjustments in the snake's position or orientation while basking. Thermal preferenda of the various species range roughly between 30 and 35 C and are similar to those of other terrestrial, Temperate Zone snakes in which thermal preferences have been adequately assessed. Thermal preferenda of adult snakes appear to be higher than those of newborn or juveniles (in four species) and may vary with levels of critical thermal minima (in five species) and geographic distribution.