Isolation and amino acid sequence of a neurotoxic phospholipase A from the venom of the Australian tiger snake Notechis scutatus scutatus.

The complete amino acid sequence of notechis 5, a neurotoxic phospholipase A from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The main fragmentation of the 119-residue peptide chain was accomplished by digesting the reduced and S-carboxymethylated derivative of the protein with a staphylococcal protease specific for glutamoyl bonds. Tryptic peptides were used to align and complete the sequence of the four staphylococcal protease peptides. The sequence was determined by Edman degradation by means of the direct phenylthiohydantoin method. Notechis 5 differs in seven positions from the recently elucidated sequence of the presynaptic neurotoxin notexin from the same venom. Notechis 5 has a 50% higher specific prospholipase A activity than notexin when assayed against egg yolk but is only one-third as toxic.     

Notechis 11'2, a non-toxic phospholipase A2 from the venom of Notechis scutatus scutatus.

Previously, we deduced the amino acid sequence of a novel phospholipase-A2-like protein (PLA2) from the nucleotide sequence of a cDNA isolated from a library prepared from the venom gland of the Australian elapid Notechis scutatus scutatus. The corresponding protein has now been identified, purified from the venom and named Notechis 11'2. Its complete amino acid sequence has been determined by automated Edman degradation of both the whole protein and peptides generated by Staphylococcus aureus protease digestion and chemical cleavage at a tryptophan residue. As predicted from its sequence which contains all the residues putatively required for PLA2 activity, Notechis 11'2 exhibits an esterase activity, preferentially against neutral phospholipids. However, despite its sequence homology with other highly toxic PLA2 present in the venom of Notechis scutatus scutatus, notechis 11'2 has no lethal activity. This observation further supports the view that the lethal activity of PLA2 from Notechis scutatus scutatus is not due to the esterasic activity only.     

The N-terminal amino group essential for the biological activity of notexin from Notechis scutatus scutatus venom

Notexin from Notechis scutatus scutatus snake venom was modified with trinitrobenzenesulfonic acid, and the major trinitrophenylated (TNP) derivative was separated by high-performance liquid chromatography. Modification resulted in the incorporation of only one TNP group on the N-terminal alpha-amino group. The TNP derivative showed a precipitous decrease in enzymatic activity and lethal toxicity, whereas the antigenicity remained unchanged. However, trinitrophenylation did not significantly affect the secondary structure of the toxin molecule as revealed by the CD spectra. The results, that the modification reaction was accelerated by the Ca2+ and that the TNP derivative retains its affinity for Ca2+, indicate that the N-terminal alpha-amino group did not participate in the Ca2(+)-binding. The TNP derivative could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated notexin are almost the same as those of native notexin. These results suggest that the N-terminal alpha-amino group is essential for the phospholipase A2 activity and lethal toxicity of notexin, and that incorporation of the TNP group on the N-terminal alpha-amino group might give rise to a distortion of the active conformation of notexin.     

Amino acid sequence of a postsynaptic neurotoxin from the venom of the Australian tiger snake Notechis scutatus scutatus.

Although 60 percent of the protein in tiger snake (Notechis scutatus scutatus) venom consists of the basic per-synaptically neurotoxic and myotoxic phospholipases notexin and Notechis II-5 and other phospholipase homologs such as Notechis II-1, several post-synaptic "curaremimetic" neurotoxins are present in small amounts. The major one of these is a typical "long" neurotoxin containing 73 amino acids in a single peptide chain cross-linked by five disulfide bridges. The formula weight calculated from the amino acid sequence is 8,051. The LD50 for intravenous injection into mice is 125 micrograms/kg.     

Chemical modification of notexin fromNotechis scutatus scutatus (Australian tiger snake) venom with pyridoxal-5′-phosphate

Notexin fromNotechis scutatus scutatus snake venom was subjected to Lys modification with pyridoxal 5′-phosphate (PLP), and one major modified derivative was purified on a cation-exchanger SP-8HR column. The results of amino acid analysis and sequence determination revealed that only 2 Lys residues at positions 82 and 115 out of 11 Lys residues in notexin were modified. The incorporation of PLP into the protein was accompanied by the loss of 53% lethal toxicity, but the modified notexin showed an about 1.2-fold increase in enzymatic activity. However, the secondary structure of the toxin molecule did not significantly change after modification with PLP as revealed by the CD spectra, and the antigenicity of PLP derivative remained unchanged. The modified derivative retained its affinity for Ca²⁺, indicating that the modified Lys residues did not participate in Ca²⁺ binding. These results indicate that modification of Lys residues causes a differential effect on the enzymatic activity and lethal toxicity of notexin, and suggest that notexin might possess two functional sites, one responsible for the catalytic activity and the other associated with its lethal effect.     

Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake).

The complete amino acid sequence of notexin, a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The protein consists of a single chain of 119 amino acids cross-linked by seven disulfide bridges and has a formula weight of 13,578. The main fragmentation of the peptide chain was accomplished with a staphylococcal protease specific for glutamoyl bonds. A cyanogen bromide fragment and tryptic peptides were used to align the five major staphylococcal protease peptides. The sequence was determined by Edman degradation using the direct phenylthiohydantoin method and with carboxypeptidase A. Notexin is shown to be homologous to both porcine pancreatic phospholipase A and a phospholipase A from the venom of Naja melanoleuca.     

Tryptophan 110, a residue involved in the toxic activity but not in the enzymatic activity of notexin

We prepared two derivatives of notexin, a phospholipase A2 from Notechis scutatus scutatus venom, by modifying the protein with 2-nitrophenylsulfenylchloride, a tryptophan-specific reagent. One derivative was modified at both tryptophans 20 and 110 whereas the other was modified at tryptophan 20. Evidence based on circular dichroic analysis and antigenicity towards a notexin-specific monoclonal antibody indicated that derivatization at both tryptophans did not affect the tertiary structure of notexin. Concomitant modification of tryptophans 20 and 110 induced a marked decrease in the capacity of notexin to kill mice and to block neuromuscular transmission in the chick biventer cervicis preparation, whereas selective modification at tryptophan 20 had no effect on the lethal properties of notexin. This implies that the decrease in the lethal properties of notexin after derivatization was due to modification at tryptophan 110. However, the diderivatized notexin retained full enzymatic activity, implying that neither tryptophan 20 and tryptophan 110 are involved in the catalytic function of the molecule. We conclude that notexin harbours two functional sites. One of them corresponds to the enzymatic site, whereas the other, which includes tryptophan 110, provides specific toxic characteristics to notexin. By reference to previous crystallographic studies, the relative spatial positions of elements involved in toxicity and the catalytic site, we propose a possible orientation of notexin with respect to its putative membrane-bound target.     

Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus

Neutralizing antibodies were raised in mice against notexin, the most toxic phospholipase A₂ (PLA₂) from Notechis scutatus scutatus venom, without the necessity of detoxifying the toxin prior to immunization. Using a sensitive radioimmunoassay we demonstrated that anti-notexin antibodies recognized (i) the parent antigen, (ii) closely related isoforms of notexin and (iii) venoms from Notechis genus snakes. In contrast, they failed to recognize other purified PLA₂ or PLA₂-containing venoms from other origins. Substitutions or chemical modifications occurring in the C-terminal part of the polypeptide chain of notexin altered the binding affinity for antibodies, implying that this region constitutes an antigenic domain of notexin.     

The effect of several viper venoms on prothrombin Padua.

The effect of four viper venoms (Oxyuranus scutellatus, Notechis scutatus scutatus, Echis carinatus, Naja nigricollis) on prothrombin Padua has been studied. Using Oxyuranus scutellatus venom and Notechis scutatus scutatus venom, prothrombin activity resulted to be moderately decreased similarly to what observed with other one-stage and two-stage methods. On the contrary, using Echis carinatus venom a normal level was obtained. No clotting was observed using the Naja nigricollis venom, regardless of the concentration used. The normal level of factor II obtained with Echis carinatus venom as compared with the low levels obtained with the other venoms, suggests that it acts on a different site of the prothrombin molecule.     

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.     

MECHANISM OF ACTION OF NOTEXIN AND NOTECHIS 11-5 ON SYNAPTOSOMES

The neurochemical activity of notexin and notechis II-5 was investigated using a synaptosomal preparation of rat cerebral cortices. In preparations preincubated with [³H]choline in order to label acetylcholine, the toxins caused a rapid release of the transmitter which was calcium-dependent. The toxins were also potent inhibitors of high affinity choline uptake. Both agents produced a marked depolarization of the synaptosomal preparation as measured by a fluorescent dye and at high concentrations lysed the preparation. At a concentration of 0.1 μM, notexin and notechis II-5 caused a 50% increase in the efflux of lactate dehydrogenase activity. These results, together with electron microscopic observations, indicated that the toxins disrupt the synaptosomal membranes presumably by their inherent phospholipase activity. The release of acetylcholine and inhibition of choline uptake, together with the depolarization of synaptosomal membranes noted in this study, could explain the observed electrophysiological effects of these toxins.     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

Effect of presynaptic neurotoxin notechis II-5 from tiger snake venom on the motor nerve endings of mice]

The neurotoxin notechis II-5 (N-II-5) from tiger snake venom (Notechis scutatus) induces three-phasic changes in miniature end-plate potential (MEPP) frequency recorded in the mouse diaphragm muscle: an initial fall of frequency followed by increase and decrease in MEPP frequency up to complete blockade. The effect of N-II-5 was enhanced with rising of the solution temperature from 20 to 30 and 35 degrees C. Removal of Ca2+ from the solution prevented the presynaptic effect of N-II-5. After washing out of the muscle from N-II-5 with Ca-free solution, addition of Ca2+ to the solution provoked the development of the effect typical of the effect of typical of the toxin. In the presence of N-II-5 an increase in K+ concentration in the solution up to 20 mM did not result in a sharp rise of MEPP frequency characteristic of depolarized nerve terminals. The agents that raise Ca2+ axoplasmic concentration not on account of depolarization of nerve terminals (hypertonic solution, ionofor A23187) preserved the capacity for increasing MEPP frequecy. It is suggested that the presynaptic effect of N-II-5 is related to its phospholipase activity and can be explained by disturbance of the activity of release sites rather than by depletion of transmitter stores.     

Inhibition of red cell Ca2+-dependent K+ channels by snake venoms

We have investigated the effects of several snake venoms on the Ca2+-dependent K+ channels of human red cells. A heat-resistant component of the venom of the snake Notechis scutatus irreversibly inhibited Ca2+-dependent K+ transport with a Ki value of 0.1-0.2 micrograms/ml. Metabolic changes of the cells modified the maximal effect of the venom. Binding of the venom required extracellular Ca2+ and was quick, but development of full inhibition required additional time. The effects of the venoms from Notechis scutatus and Leiurus quinquestriatus were additive, suggesting that both venoms act through different mechanisms. Venoms of the snakes Vipera russelli russelli and Oxyuranus scutellatus also inhibited Ca2+-dependent K+ transport with the same characteristics as the Notechis scutatus venom.     

Biochemical and functional characterization of an L-amino acid oxidase isolated from Bothrops pirajai snake venom

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.     

Purification and some properties of low-molecular-weight proteins of Aspidelaps scutatus (shield or shield-nose snake) venom

1. Fourteen low-molecular-weight proteins from Aspidelaps scutatus venom were purified by gel filtration and ion exchange chromatography. 2. The proteins (toxins) have i.v. LD50 values ranging from 0.12 to 54 micrograms/g mouse. 3. The amino-terminal sequences of the proteins show that 12 of the 14 proteins were pure. 4. The proteins were preliminarily assigned as phospholipases A2, long neurotoxins and cytotoxin homologues.     

Snake venom toxins--I. The primary structure of a long neurotoxin S4C6 from Aspidelaps scutatus (shield or shield-nose snake) venom

1. Long neurotoxin S4C6 from Aspidelaps scutatus venom was purified by gel filtration and ion exchange chromatography (Joubert, 1987). 2. It contains 68 amino acids including 10 half-cystines. The toxicity of toxin S4C6 was determined and a LD50 of 0.13 +/- 0.04 micrograms/g mouse was found. 3. The complete primary structure of long neurotoxin S4C6 has been elucidated. In the toxin the 10 structurally invariant amino acids of the neurotoxins and cytoxins and the five functionally invariant amino acids of the neurotoxins are conserved.     

Respective effects of anabolic/androgenic steroids and physical exercise on isometric contractile properties of regenerating skeletal muscles in the rat

We examined the respective effects of anabolic-androgenic steroids and physical exercise on the contractile properties of regenerating fast and slow hindlimb skeletal muscles. Degeneration/regeneration of the left extensor digitorum longus muscles (EDL) and soleus of young Wistar male rats was induced by a snake venom (Notechis scutatus scutatus) injection. During muscle regeneration, experimental rats were either treated with nandrolone (NAN, nortestosterone, im, 2 mg X kg(-1) X week(-1), or endurance exercised on a treadmill (EXE, 60 min x day(-1), 10-40 m X min(-1). Twenty-one days after injury, isometric contractile properties of regenerating muscles were studied in situ. Neither the nandrolone treatment nor the physical exercise program was able to change significantly muscle contraction parameters both in twitch and tetanus in both regenerating EDL and soleus (p > 0.05). However, we observed a greater peak twitch tension in NAN versus grouped control and EXE EDL (p < 0.01). In conclusion, endurance exercise program or anabolic-androgenic steroid (nortestosterone) treatment did not significantly improve isometric contractile properties of regenerating slow and fast muscles in the male young rats.     

Purification, some properties of two phospholipases A2 (CM-I and CM-II) and the amino-acid sequence of CM-II from Aspidelaps scutatus (shield or shield-nose) venom

Two phospholipases A2, CM-I and CM-II, from Aspidelaps scutatus venom were purified by gel filtration followed by ion-exchange chromatography on CM-cellulose. The enzymes consist of 119 amino acids including fourteen half-cystines. The complete primary structure of CM-II has been determined. The sequence and the invariant amino acid residues resemble those of the phospholipase A2 from the genus Naja. The toxicity of the enzymes is comparable to those encountered for the phospholipases A2 from African cobra venoms. The phospholipase A2 (CM-II) contains two histidine residues which are located at position 20 and the reactive site (histidine-47) of the enzyme.