Functional characterization of a basic D49 phospholipase A2 (LmTX-I) from the venom of the snake Lachesis muta muta (bushmaster).

The whole venom of Lachesis muta muta is preponderantly neurotoxic but moderately myotoxic on the chick biventer cervicis preparation (BCp). We have now examined these toxic activities of a basic phospholipase A(2), LmTX-I, isolated from the whole venom. LmTX-I caused a significant concentration-dependent neuromuscular blockade in the BCp. The time to produce 50% neuromuscular blockade was 14.7+/-0.75 min (30 microg/ml), 23.6+/-0.9 min (10 microg/ml), 34+/-1.7 min (2.5 microg/ml) and 39.2+/-3.6 min (1 microg/ml), (n=5/concentration; p<0.05). Complete blockade with all tested concentrations was not accompanied by inhibition of the response to ACh. At the highest concentration, LmTX-I (30 microg/ml) significantly reduced contractures elicited by exogenous KCl (20mM), increased the release of creatine kinase (1542.5+/-183.9 IU/L vs 442.7+/-39.8 IU/L for controls after 120 min, p<0.05), and induced the appearance of degenerating muscle fibers ( approximately 15%). Quantification of myonecrosis indicated 14.8+/-0.8 and 2.0+/-0.4%, with 30 and 10 microg/mlvenom concentration, respectively, against 1.07+/-0.4% for control preparations. The findings indicate that the basic PLA(2) present on venom from L. m. muta (LmTX-I) possesses a dominant neurotoxic action on isolated chick nerve-muscle preparations, whereas myotoxicity was mainly observed at the highest concentration used (30 microg/ml). These effects of LmTX-I closely reproduce the effects of the whole venom of L. m. muta in chick neuromuscular preparations.     

Neurotoxic and myotoxic actions of crotoxin-like and Crotalus durissus cascavella whole venom in the chick biventer cervicis preparation.

Crotoxin from Crotalus durissus cascavella venom was purified by a combination of molecular exclusion chromatography (Superdex 75 column) and HPLC molecular exclusion (Protein Pack 300SW column). Neurotoxic and myotoxic effects from C. durissus cascavella whole venom and its main fraction, the crotoxin-like, were studied in the chick biventer cervicis (CBC) nerve-muscle preparation. Both venom and its crotoxin showed significant (p < 0.05) blockade of neuromuscular transmission at concentrations as low as 0.2-1, 5 and 25 microg/ml, but no significant effect has been shown with a concentration of 0.04 microg/ml (n = 5 each). The time required to produce 50% neuromuscular blockade with the venom and its crotoxin was 53.6+/-8.2 and 65.9+/-4.9 min (0.2 microg/ml), 29.7+/-1.9 and 34.3+/-1.9 min (1 microg/ml), 24.8+/-1.6 and 21.1+/-1.5 min (5 microg/ml), 20.9+/-3.7 and 20.1+/-1.4 min (25 microg/ml), respectively. The addition to the incubation bath of acetylcholine (55 and 110 microM) or KCl (20.1 mM), either before or after the venom or the crotoxin induced contracture in the presence of a total blockade, in all the concentrations used. Morphological analysis showed that the damage caused by C. durissus cascavella venom is stronger than that caused by crotoxin. The myonecrotic picture was more marked at higher venom and crotoxin doses (1, 5 or 25 microg/ml). Only at 25 microg/ml concentrations of the venom and crotoxin, marked muscle fiber changes were detected. We concluded that the crotoxin-like and the whole venom from C. durissus cascavella possess a preponderant and quite potent neurotoxic action in this preparation, and a myotoxic action which is observed only at higher doses.     

Cytotoxicity of Lachesis muta muta snake (bushmaster) venom and its purified basic phospholipase A2 (LmTX-I) in cultured cells.

Human envenoming by Lachesis muta muta venom, although infrequent, is rather severe, being characterized by pronounced local tissue damage and systemic dysfunctions. Studies on the pharmacological actions of L. m. muta venom are relatively scant and the direct actions of the crude venom and its purified phospholipase A(2) (PLA(2)) have not been addressed using in vitro models. In this work, we investigated the cytotoxicity of L. m. muta venom and its purified PLA(2) isoform LmTX-I in cultured Madin-Darby canine kidney (MDCK) and in a skeletal muscle (C2C12) cell lines. As revealed by neutral red dye uptake assay, the crude venom (10 or 100 microg/ml) induced a significant decrease in cell viability of MDCK cells. LmTX-I at the concentrations tested (70-270 microg/ml or 5-20 microM) displayed no cytotoxicity in both MDCK and C2C12 cell lines. Morphometric analysis of Feulgen nuclear reaction revealed a significant increase in chromatin condensation (pyknosis), apparent reduction in the number of mitotic nuclei and nuclear fragmentation of some MDCK cells after incubation with L. m. muta venom. Monolayer exposure to crude venom resulted in morphological changes as assessed by scanning electron microscopy. The staining with TRITC-labelled phalloidin showed a marked disarray of the actin stress fiber following L. m. muta venom exposure. In contrast, LmTX-I had no effect on nucleus and cell morphologies as well as on stress fiber organization. These results indicate that L. m. muta venom exerts toxic effects on cultured MDCK cells. The LmTX-I probably does not contribute per se to the direct venom cytotoxicity, these effects are mediated by metalloproteinases/disintegrins and other components of the venom.     

Mechanism of action and determination of the best substrate for a thrombin-like enzyme from Lachesis muta muta venom by regression analysis of the kinetic parameters determined with peptidyl p-nitroanilide substrates.

The kinetic behavior of a thrombin-like enzyme from Lachesis muta muta venom has been studied with 13 tripeptidyl p-nitroanilide substrates. Eight substrates were unprotected at the N terminus and were used for the regression analysis of the experimentally determined kinetic parameters 1/Km, kcat and kcat/Km. The individual contribution of each amino acid side chain to the kinetic parameters was calculated. The amino acid sequence of the ideal substrate (D-Pro-Leu-Arg-pNA) was determined from a regression analysis for each kinetic parameter. This result was confirmed experimentally. The structural analysis of the tripeptides showed that the binding to the S3 sub-site had a small effect on Km. The binding of L-Leu to the S2 sub-site increased kcat without changing the value of Km. The analysis of the kinetic parameters revealed that, in the binding of L-Leu to the S2 sub-site, the enzyme bound the transition state configuration of the substrate/product transformation more tightly than that of the substrate.     

Inflammatory oedema induced by Lachesis muta muta (Surucucu) venom and LmTX-I in the rat paw and dorsal skin

The ability of crude venom and a basic phospholipase A₂ (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in rat paw and skin was investigated. Crude venom or LmTX-I were injected subplantarly or intradermally and rat paw oedema and dorsal skin plasma extravasation were measured. Histamine release from rat peritoneal mast cell was also assessed. Crude venom or LmTX-I induced dose-dependent rat paw oedema and dorsal skin plasma extravasation. Venom-induced plasma extravasation was inhibited by the histamine H₁ antagonist mepyramine (6 mg/kg), histamine/5-hydroxytriptamine antagonist cyproheptadine (2 mg/kg), cyclooxygenase inhibitor indomethacin (5 mg/kg), nitric oxide synthesis inhibitor l-NAME (100 nmol/site), tachykinin NK₁ antagonist SR140333 (1 nmol/site) and bradykinin B₂ receptor antagonist Icatibant (0.6 mg/kg). Platelet-activating factor (PAF) antagonist PCA4248 (5 mg/kg) had no effect. LmTX-I-induced skin extravasation was inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while l-NAME and SR140333 had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving in vivo mast cell activation and arachidonic acid metabolites. Additionally, crude venom-induced responses also involve substance P, nitric oxide and bradykinin release, whether LmTX-I-induced responses involve PAF.     

Identification of novel bradykinin-potentiating peptides and C-type natriuretic peptide from Lachesis muta venom.

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.     

Pharmacological evidence for a presynaptic action of venoms from Bothrops insularis (jararaca ilhoa) and Bothrops neuwiedi (jararaca pintada).

Whereas the presynaptic action of Crotalus durissus terrificus venom is well-established, Bothrops venoms have historically been considered to have only postsynaptic and muscular effects. However, some studies have also suggested a presynaptic action for these venoms. In this work, we used chick biventer cervicis preparations to compare the presynaptic actions of two Bothrops venoms (B. insularis and B. neuwiedi) with that of C. d. terrificus venom. At 10 microg/ml, all venoms produced irreversible blockade of the twitch tension responses, with no reduction in acetylcholine (ACh)-induced contractures and only a slight decrease in potassium induced-contractures. The times (in min) required to produce 50% neuromuscular blockade (C. d. terrificus: 16.3+/-0.7, n = 8; B. insularis: 30.0+/-1.9, n = 5; B. neuwiedi: 42.0+/-2.0, n = 8; mean +/- SEM) were significantly different among the venoms (p < 0.01). Lowering the temperature at which the experiments were done (from 37 to 24 degrees C) prevented neuromuscular blockade by the three venoms, indicating that enzyme activity may be involved in this response. At concentrations capable of causing complete neuromuscular blockade, creatine kinase release remained close to levels seen in control preparations incubated with Krebs solution alone (500-1200 IU/l). Commercial crotalic antivenom, but not bothropic antivenom, protected against the neuromuscular blockade caused by B. insularis and B. neuwiedi venoms. These observations indicate that bothropic venoms may contain components which act presynaptically in a manner similar to C. d. terrificus venom, and that at low venom concentrations a direct action on skeletal muscle does not contribute to this presynaptic neurotoxicity.     

Neuromuscular activity of Bothrops alcatraz snake venom in chick biventer cervicis preparations

Venom (10-100 μg/ml) from Bothrops alcatraz, a pitviper from the Alcatrazes Archipelago off the coast of southeastern Brazil, caused progressive, irreversible neuromuscular blockade in chick isolated biventer cervicis preparations. The venom also inhibited contractures to exogenous ACh (110 μM) and KCl (20 mM), caused myofiber damage and increased creatine kinase release. Commercial bothropic antivenom raised against mainland Bothrops species neutralized the neuromuscular activity, depending on the venom concentration.     

Appraisal of Antiophidic Potential of Marine Sponges against Bothrops jararaca and Lachesis muta Venom

Snakebites are a health problem in many countries due to the high incidence of such accidents. Antivenom treatment has regularly been used for more than a century, however, this does not neutralize tissue damage and may even increase the severity and morbidity of accidents. Thus, it has been relevant to search for new strategies to improve antiserum therapy, and a variety of molecules from natural sources with antiophidian properties have been reported. In this paper, we analyzed the ability of ten extracts from marine sponges (Amphimedon viridis, Aplysina fulva, Chondrosia collectrix, Desmapsamma anchorata, Dysidea etheria, Hymeniacidon heliophila, Mycale angulosa, Petromica citrina, Polymastia janeirensis, and Tedania ignis) to inhibit the effects caused by Bothrops jararaca and Lachesis muta venom. All sponge extracts inhibited proteolysis and hemolysis induced by both snake venoms, except H. heliophila, which failed to inhibit any biological activity. P. citrina inhibited lethality, hemorrhage, plasma clotting, and hemolysis induced by B. jararaca or L. muta. Moreover, other sponges inhibited hemorrhage induced only by B. jararaca. We conclude that Brazilian sponges may be a useful aid in the treatment of snakebites caused by L. muta and B. jararaca and therefore have potential for the discovery of molecules with antiophidian properties.     

Neurotoxic and myotoxic actions of Naja naja kaouthia venom on skeletal muscle in vitro.

The neuromuscular and skeletal muscle actions of Naja naja kaouthia snake venom were studied in mammalian (rat left hemidiaphragm) and avian (chick biventer cervicis) nerve-muscle preparations. The venom (5 and 10 micro g/ml) produced neuromuscular blockade (85% in 36.8+/-2.0min, mean+/-SEM, n=5, and 18+/-0.6min, n=3, p<0.01, respectively) in the rat preparation. That the phospholipase A(2) (PLA(2)) activity of the venom is involved in this effect was evaluated by inhibiting this enzyme with p-bromophenacyl bromide. This resulted in significantly (p<0.01) increasing the time required for 85% blockade with 5 and 10 micro g/ml to 54+/-4.6min (n=3) and 29+/-0.6min (n=3), respectively. In chick preparations, the venom (5 micro g/ml) produced neuromuscular blockade in 14.0+/-1.8min (n=5). The contractures to exogenous acetylcholine were completely inhibited by the venom, whereas those to 134 micro M KCl were partially blocked in chick preparations (n=4, n=3, respectively). The venom (5 micro g/ml) produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps) in the rat hemidiaphragm, but did not alter the resting membrane potential at 5 micro g/ml. Neostigmine (5.8 micro M) immediate and partially reversed the 85% blockade produced by venom (61%, n=3) in rat preparations, as did 4-aminopyridine (53 micro M) ( approximately 59%, n=3). The 4-aminopyridine and neostigmine also restored the m.e.p.ps to pre-venom (control) values. In rat preparations, the venom damaged 47%+/-11% and 62.7+/-3.6% of the muscle fibers at concentrations of 5 and 10 micro g/ml, respectively. For venom in which PLA(2) activity was inhibited, the corresponding values were 38+/-11.8% (5 micro g/ml) and 67+/-9.6% (10 micro g/ml). These findings suggest a post-synaptic neurotoxic action for N. n. kaouthia venom, and that inhibiting phospholipase activity of the venom reduces significantly the neuromuscular block but not the direct myotoxicity.     

Purification and characterization of a phospholipase A2 isoenzyme isolated from Lachesis muta snake venom

A new phospholipase A2 (PLA2) isoenzyme was isolated from Lachesis muta crude venom, and was named LM-PLA2-II. This enzyme was purified by gel filtration on a Sephacryl S-200 HR column followed by reverse-phase chromatography on a C2/C18 column. LM-PLA2-II consists of a single polypeptide chain with an apparent molecular mass of 18 kDa and an isoelectric point at pH 5.4. The amino terminal sequence of the enzyme revealed a high degree of homology with other PLA2s from several sources. LM-PLA2-II has a high indirect hemolytic activity and a potent inhibitory effect on platelet aggregation induced by ADP and collagen. It also produces a significant paw edema reaction in rats. The edematous response in rats was abolished by pretreatment with either indomethacin or dexamethasone, suggesting the involvement of cyclo-oxygenase. Pretreatment of LM-PLA2-II with p-bromophenacyl bromide abolished all of these actions, clearly indicating that the biological activities, including the edematogenic effect, are dependent entirely on its enzymatic activity.     

The improvement of the therapeutic anti-Lachesis muta serum production in horses.

The main features associated with pit viper envenomations include the intense local lesions such as oedema, necrosis, acute renal failure and other effects. The severity of these reactions to snakebite depends on the degree of envenomation. Lachesis muta venom (LMV) has weak lethal activity, but due to the large amount often inoculated, the effects are extremely severe and demand anti-venom with a high neutralizing capacity. LMV had the lowest neutralizing antibody induction capacity in horses when compared with that of other venoms. For example, Bothrops anti-venom serum neutralizes 180 times the equivalent LD(50) to Bothrops venom; Crotalus anti-venom neutralizes 250 LD(50) of this venom, while Lachesis anti-venom neutralizes only five LD(50) of the Lachesis toxins. To examine the reasons for this low antibody induction, the H(GP) mouse line, genetically selected for high antibody production received, at different times during immunization with sheep erythrocytes (SE), whole LMV and isolated venom fractions I-VI eluted by gel-filtration chromatography on Superdex75. The specific antibody responsiveness showed a partial, but significant suppression of the anti-SE antibody responses during the kinetics of the primary and even the secondary immunizations, after 50-100 microg of fractions IV and V administration 72-48 h before the first antigen injections. Fraction IV was then applied in a Superose 12 column and three samples were obtained. The peak IVA containing a component of Mr 27 kDa was liable with the immunosuppressive effect as made evident by its effect on the H mice anti-SE responses. Horses receiving the LMV exempt of fractions IV and V produce highly significant anti-Lachesis sera with a 45 LD(50) neutralizing activity, providing, for the first time, an efficient specific therapeutic heterologous serum for human use.     

LmrTX, a basic PLA2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity

A basic phospholipase A₂ (LmrTX) isoform was isolated from Lachesis muta rhombeata snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-5 Discovery^® Bio Wide column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of LmrTX was measured as 14.277.50 Da. The amino acid sequence showed a high degree of homology between PLA₂ LmrTX from L. muta rhombeata and other PLA₂ from snake venoms, like CB1 and CB2 from Crotalus durissus terrificus; LmTX-I and LmTX-II from Lachesis muta muta. LmrTX had PLA₂ activity in the presence of a synthetic substrate and alkylation of histidine residues significantly inhibited (P < 0.05) the enzymatic activity of LmrTX and its anticoagulant and antithrombotic activity. In this study, we examined the ability of the LmrTX in altering thrombus formation in living mouse, using a photochemically induced arterial thrombosis model. The control animals that did not receive protein injection showed a normal occlusion time, which was around 57 ± 7.8 min. LmrTX, the PLA₂ from L. muta rhombeata venom, caused a change in the occlusion time to 99 ± 10 min with doses of 7.5 μg/mice. Additionally, LmrTX showed the anticoagulant activity in vitro and ex vivo and prolonging the time aggregation in wash platelet induced by ADP and Thrombin.     

Studies on the specificity of CNF, a phospholipase A2 inhibitor isolated from the blood plasma of the South American rattlesnake (Crotalus durissus terrificus). I. Interaction with PLA2 from Lachesis muta muta snake venom.

A phospholipase A₂ inhibitor has been previously purified and cloned from the blood plasma of the South American rattlesnake, Crotalus durissus terrificus. This inhibitor, named CNF for Crotalus neutralizing factor, interacts with crotoxin, the main neurotoxin from C. d. terrificus venom, abolishing its phospholipase A₂ activity. Crotoxin is a heterodimer of an acidic subunit (CA) and a basic phospholipase A₂ (CB). CNF acts by forming a stable non-toxic complex with CB, replacing CA in the toxic CA–CB of crotoxin.

In the present investigation, we have shown that CNF has a broader specificity. It is able to inhibit the PLA₂ activity of the whole venom from the bushmaster snake (Lachesis muta muta), a species evolutionary related to Crotalus. Inhibition experiments have been carried out with four PLA₂ active components isolated from L. m. muta venom, one basic and three acidic ones. CNF inhibition is not restricted to the basic PLA₂, but extended to the three acidic forms as well.     

Isolation and functional characterization of a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom.

This article reports the purification procedure and the biochemical/functional characterization of Bp-PLA(2), a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom. It was highly purified through three chromatographic steps (ion-exchange on CM-Sepharose, hydrophobic chromatography on Phenyl-Sepharose and RP-HPLC on a C8 column). Bp-PLA(2) is a single-chain protein of 15.8kDa and pI 4.3. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 585.3U/mg. It displayed a high indirect hemolytic activity and inhibited platelet aggregation induced by collagen or ADP. It also induced in vivo edema and myotoxicity. Pretreatment of Bp-PLA(2) with BPB reduced the enzymatic activity, the inhibitory action on platelet aggregation and myotoxicity in vitro. Morphological analyses indicated that Bp-PLA(2) induced an intense edema, with visible leukocyte infiltrate and damaged muscle cells 24h after injection. Acidic myotoxic PLA(2)s from Bothrops snake venoms are still not extensively explored and knowledge of their structural and functional features will contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.     

Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom.

Two basic myotoxic PLA(2)s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA(2)s showed a M(r) around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), pI approximately 7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA(2)s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn(2+) ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115-129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA(2)s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

A phospholipase A2 isolated from Lachesis muta snake venom increases the survival of retinal ganglion cells in vitro

We have previously showed that a phospholipase A₂ isolated from Lachesis muta snake venom and named LM-PLA₂-I displayed particular biological activities, as hemolysis, inhibition on platelet aggregation, edema induction and myotoxicity. In the present work, we evaluated the effect of LM-PLA₂-I on the survival of axotomized rat retinal ganglion cells kept in vitro, as well as its mechanism of action. Our results clearly showed that treatment with LM-PLA₂-I increased the survival of ganglion cells (100% when compared to control cultures) and the treatment of LM-PLA₂-I with p-bromophenacyl bromide abolished this effect. This result indicates that the effect of LM-PLA₂-I on ganglion cell survival is entirely dependent on its enzymatic activity and the generation of lysophosphatidylcholine (LPC) may be a prerequisite to the observed survival. In fact, commercial LPC mimicked the effect of LM-PLA₂-I upon ganglion cell survival. To investigate the mechanism of action of LM-PLA₂-I, cultures were treated with chelerythrine chloride, BAPTA-AM, rottlerin and also with an inhibitor of c-junc kinase (JNKi). Our results showed that rottlerin and JNK inhibitor abolished the LM-PLA₂-I on ganglion cell survival. Taken together, our results showed that LM-PLA₂-I and its enzymatic product, LPC promoted survival of retinal ganglion cells through the protein kinase C pathway and strongly suggest a possible role of the PLA₂ enzyme and LPC in controlling the survival of axotomized neuronal cells.     

Increase of the cytotoxic effect of Bothrops jararacussu venom on mouse extensor digitorum longus and soleus by potassium channel blockers and by Na(+)/K(+)-ATPase inhibition

We investigated the myotoxicity of Bothrops jararacussu crude venom and other cytolytic agents on mouse isolated extensor digitorum longus (EDL) and soleus (SOL) muscles, which present distinct properties: EDL is a fast-twitch, white muscle with predominantly glycolytic fibers, while SOL is slow-twitch, red muscle with predominantly oxidative fibers. Muscles were exposed to B. jararacussu crude venom (25mug/ml) and other crotaline venoms (Agkistrodon contortrix laticinctus; Crotalus viridis viridis; Crotalus durissus terrificus) at the same concentration. Basal creatine kinase (CK) release to bathing solution was 0.43+/-0.06 for EDL and 0.29+/-0.06 for SOL (U g(-)(1) h(-)(1), n=36 for each muscle). Sixty minutes after exposure to B. jararacussu venom, EDL presented higher increase in the rate of CK release than SOL, respectively, 13.2+/-1.5 and 2.9+/-0.7Ug(-)(1)h(-)(1), n=10-12. Muscle denervation, despite decreasing CK content, did not affect sensitivities to B. jararacussu venom. Ouabain and potassium channel blockers (TEA; clotrimazole; glibenclamide) increased the rate of CK release by B. jararacussu in EDL and SOL muscles, decreasing and almost abolishing the different sensitivity. When we exposed EDL or SOL muscles to Naja naja, Apis mellifera venoms (25mug/ml), or Triton X-100 (0.01%), they showed similar rate of CK release. Our present data suggest that a mechanism involving intracellular calcium regulation or potassium channels may participate in the different sensitivity of EDL and SOL to B. jararacussu venom.     

Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom.

A hemorrhagic metalloproteinase has been isolated from Bothrops alternatus venom from specimens that inhabit the north-east region of Argentina. The present study aimed at evaluating the proteolytic, hemorrhagic, edematogenic and myotoxic activities of the purified metalloproteinase, in order to consider its participation on the phatophysiology of the intoxication by Bothrops alternatus venom. The hemorrhagic metalloproteinase was isolated by a combination of DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The enzyme showed a molecular mass around 55k Da, it exhibited a hemorrhagic activity with a minimal hemorrhagic dose of 1.9 microg, almost two fold minor than the whole venom (3.6 microg). The enzyme showed a weak proteolytic activity on casein (18.72 U/mg enzyme), similar to the one exhibited by the whole venom (20 U/mg venom). Besides, the ability to degrade casein could be detected by SDS-PAGE; beta-casein was the fraction that showed the higher degradation, followed by alphas(1)-casein and kappa-casein degradation. The hemorrhagic metalloproteinase rapidly hydrolysed the A alpha-chain of fibrinogen, followed by B beta-chain degradation and leaving the gamma-chain unaffected. Proteolytic activities were inhibited by EDTA whereas they were not inhibited by benzamidine and PMSF. The metalloproteinase showed several polypeptides chains after autocatalytic processing, including a chain of 28k Da, it could be the processed disintegrin-like and cysteine-rich domains. The isolated enzyme exhibited myotoxic activity with high CK levels at 6h, due to local ischemia resulting of its hemorrhagic activity, and a significant edema-inducing effect (MED=1.3 microg), corroborated both results by the histological observations of samples of gastrocnemius muscle. These findings showed that this hemorrhagic metalloproteinase, possesses high edematogenic and myotoxic activities and, in despite of exhibiting a weak proteolytic activity, it is able to degrade fibrinogen. So, this enzyme would contribute markedly to the phatophysiology of the bothropic envenomation.