In vitro hemolytic activity of Bothrops lanceolatus (fer-de-lance) venom

Bothrops lanceolatus venom contains a variety of enzymatic and biological activities. The present work investigated the hemolytic activity of this venom and its phospholipase A2 (PLA2). Bothrops lanceolatus venom (6.7 µg/mL) caused indirect hemolysis of cow, horse, rat and sheep erythrocytes, with horse erythrocytes being the most sensitive; no direct hemolysis was observed. Hemolysis in sheep erythrocytes was concentration-dependent (5-11.7 µg/mL) and markedly attenuated by heating the venom for 30 minutes at ≥ 40°C and by the PLA2 inhibitor p-bromophenacyl bromide. An acidic PLA2 (5 µg/mL) purified from B. lanceolatus venom also caused hemolysis. This PLA2 showed immunoprecipitin lines with antivenom against B. lanceolatus, which suggests that the enzymatic and hemolytic activities of this enzyme may be neutralized during antivenom therapy. These results indicate that B. lanceolatus venom and its PLA2 can cause hemolysis in vitro.(AU)

In vitro hemolytic activity of Bothrops lanceolatus (fer-de-lance) venom

Bothrops lanceolatus venom contains a variety of enzymatic and biological activities. The present work investigated the hemolytic activity of this venom and its phospholipase A2 (PLA2). Bothrops lanceolatus venom (6.7 µg/mL) caused indirect hemolysis of cow, horse, rat and sheep erythrocytes, with horse erythrocytes being the most sensitive; no direct hemolysis was observed. Hemolysis in sheep erythrocytes was concentration-dependent (5-11.7 µg/mL) and markedly attenuated by heating the venom for 30 minutes at 40°C and by the PLA2 inhibitor p-bromophenacyl bromide. An acidic PLA2 (5 µg/mL) purified from B. lanceolatus venom also caused hemolysis. This PLA2 showed immunoprecipitin lines with antivenom against B. lanceolatus, which suggests that the enzymatic and hemolytic activities of this enzyme may be neutralized during antivenom therapy. These results indicate that B. lanceolatus venom and its PLA2 can cause hemolysis in vitro.

Bothrops jararaca venom gland transcriptome: Analysis of the gene expression pattern

Bothrops jararaca is a pit viper responsible for the majority of snake envenoming accidents in Brazil. As an attempt to describe the transcriptional activity of the venom gland, ESTs of a cDNA library constructed from B. jararaca venom gland were generated and submitted to bioinformatics analysis. The results showed a clear predominance of transcripts coding for toxins instead of transcripts coding for proteins involved in cellular functions. Among toxins, the most frequent transcripts were from metalloproteinases (52.6%), followed by serine-proteinases (28.5%), C-type lectins (8.3%) and bradykinin-potentiating peptides (BPPs) (6.2%). Results were similar to that obtained from the transcriptome analysis of B. insularis, a phylogenetically close sister of B. jararaca, though some differences were observed and are pointed out, such as a higher amount of the hypotensive BPPs in B. insularis transcriptome (19.7%). Another striking difference observed is that PIII and PII-classes of metalloproteinases are similarly represented in B. jararaca in contrast to B. insularis, in which a predominance of PIII-class metalloproteinase, which present a more intense hemorrhagic action, is observed. These features may, in part, explain the higher potency of B. insularis venom. The results obtained can help in proteome studies, and the clones can be used to directly probe the genetic material from other snake species or to investigate differences in gene expression pattern in response to factors such as diet, aging and geographic localization.

Pharmacological and histopathological characterization of Bothrops lanceolatus (Fer de lance) venom-induced edema.

OBJECTIVE: Bothrops venoms cause local edema, pain, hemorrhage and necrosis. In this study, we investigated the ability of Bothrops lanceolatus venom to cause edema in rat hind paws and examined the mediators involved. MATERIALS AND METHODS: Hind paw edema was induced in male Wister rats by the subplantar injection of venom (12.5-100 microg/paw) in the absence and presence of antagonists. Edema was quantified by hydroplethysmometry at 0.25, 0.5, 2, 4, 6 and 24 h post-injection and was expressed as the percentage increase relative to the contralateral (control) paw. The ability of the venom to release histamine from rat peritoneal mast cells was also assessed. RESULTS: Venom caused dose- and time-dependent edema that was maximal within 15 min but disappeared after 24 h and was accompanied by hemorrhage. Dexamethasone (1 mg/kg, s.c.), methysergide (6 mg/kg, i.p.), HOE 140 (0.6 mg/kg, i.v.) and mepyramine (6 mg/kg, i.p.) significantly ( p < 0.05) reduced edema formation, whereas indomethacin (10 mg/kg, i.p.) was ineffective. Dialysis did not affect venom-induced edema. Venom (1, 10 and 30 microg/ml) caused a concentration-dependent release of histamine (13 +/- 1%, 61.9 +/- 4.6% and 73.6 +/- 2.4%, respectively; n = 5) from rat peritoneal mast cells in vitro. Histological analysis confirmed the presence of edema, hemorrhage and neutrophil infiltration. Pretreating the venom with EDTA partially inhibited the edema and hemorrhage, but did not affect the migration of neutrophils. CONCLUSIONS: B. lanceolatus venom produced dose- and time-dependent edema in rat paws. This edema was not dependent on low molecular weight substances in the venom, but was partially dependent on a hemorrhagin and also involved the release of arachidonic acid metabolites, bradykinin, histamine and serotonin.

Comparative evaluation of immunological and structural similarities of snake venom C-type lectin proteins.

Antibodies raised against denatured and native forms of bothrojaracin were used to analyze the immunological similarities compared to the structural and biological features of five C-type lectin proteins from snake venom (bothrojaracin, botrocetin, Factor IX/X binding protein (FIX/Xbp), convulxin and Bothrops jararaca lectin). Anti-denatured-bothrojaracin antibodies, which recognize mainly linear epitopes, cross-reacted with botrocetin, FIX/Xbp and convulxin, as expected for homologous proteins. On the other hand, anti-native-bothrojaracin antibodies, which mostly interact with conformational epitopes, exhibited a higher degree of selectivity. These results show that differences exist at the surface of these proteins and that they should be related to their different biological activities, while they share a common and similar scaffold.

Pharmacological characterization of the rat paw edema induced by Bothrops lanceolatus (Fer de lance) venom.

The inflammatory response induced by Bothrops lanceolatus venom (BLV) in the rat hind-paw was studied measuring paw edema. Non-heated BLV (75microg/paw) caused a marked paw edema accompanied by intense haemorrhage whereas heated venom (97 degrees C, 30s; 12.5-100microg/paw) produced a dose- and time-dependent non-haemorrhagic edema. The response with heated BLV was maximal within 15min disappearing over 24h. Heated venom was then routinely used at the dose of 75microg/paw. The prostacyclin analogue iloprost (0.1microg/paw) potentiated by 125% the venom-induced edema. The histamine H(1) receptor antagonist mepyramine (6mg/kg) or the serotonin/histamine receptor antagonist cyproheptadine (6mg/kg) partially inhibited BLV-induced edema whereas the combination of both compounds virtually abolished the edema. The lipoxygenase inhibitor BWA4C (10mg/kg), but not the cyclooxygenase inhibitor indomethacin (10mg/kg), significantly inhibited the edema (35% reduction; P<0.05). Dexamethasone (1mg/kg) also markedly (P<0.001) reduced venom-induced edema. The bradykinin B(2) receptor antagonist Hoe 140 (0.6mg/kg) reduced by 30% (P<0.05) the venom induced edema, whereas the angiotensin-converting enzyme inhibitor captopril (300microg/paw) potentiated by 42% (P<0.05) the edema. Bothrops lanceolatus antivenon (anti-BLV) reduced by 28% (P<0.05) the venom-induced edema while intravenous administration of antivenom failed to affect the edema. In conclusion, BLV-induced rat paw edema involves mast cell degranulation causing local release of histamine and serotonin, a phenomenon mediated mainly by kinins and lipoxygenase metabolites. Additionally, the use of a specific Bothrops lanceolatus antivenom, given subplantarily or intravenously, revealed to be little effective to prevent BLV-induced edema.

Coagulant and anticoagulant activities of Bothrops lanceolatus (Fer de lance) venom.

Bothrops lanceolatus venom contains caseinolytic, phospholipase, esterase and haemorrhagic activities. We have investigated the coagulant and anticoagulant actions of B. lanceolatus venom on human citrated plasma and on purified plasma components. Although B. lanceolatus venom up to 50 microg/ml was unable to clot citrated plasma, at concentrations > or = 5 microg/ml the venom dose-dependently clotted purified human fibrinogen, indicating the presence of a thrombin-like enzyme. Human plasma (final concentration > or = 12.5%) dose-dependently inhibited the venom-induced fibrinogen clotting. This finding suggested that endogenous plasma protease inhibitors can affect the venom's action on fibrinogen. To investigate this possibility, B. lanceolatus venom was incubated with different plasma protease inhibitors and the activity on fibrinogen tested. alpha(2)-Macroglobulin and alpha(1)-antitrypsin did not interfere with the coagulant activity of the venom whereas the antithrombin-III/heparin complex partially inhibited this activity. A non-toxic, acidic phospholipase A(2) purified from B. lanceolatus venom prolonged the activated partial thromboplastin time in human plasma from 39.7+/-0.5 s (control with saline) to 60.2+/-0.9 s with 50 microg of PLA(2) (p<0.001), suggesting an anticoagulant activity associated with this enzyme. This anticoagulant activity may account for some of the effects of the venom on blood coagulation.

Bothrops lanceolatus (Fer de lance) venom induces oedema formation and increases vascular permeability in the mouse hind paw.

The ability of snake venoms to increase vascular permeability and to induce oedema through the release of pharmacologically active substances is well known. We have studied the oedema and vascular permeability induced by Bothrops lanceolatus venom in male Swiss white mice. Paw oedema was induced by the subplantar injection of B. lanceolatus venom (125-1000 ng/paw) and was quantified as the increase in paw weight. Changes in vascular permeability were assessed by measuring the amount of Evans blue dye extravasation. The oedema and the increase in vascular permeability were maximal within 2 h and had resolved after 24 h. The administration of the vasodilator iloprost (20 ng/paw) immediately after B. lanceolatus venom potentiated the oedema and the increase in vascular permeability by approximately four-fold. Pretreating the mice with indomethacin, dexamethasone, NDGA or BW A4C inhibited the venom-induced oedema and the increase in vascular permeability. In contrast, histamine, serotonin and PAF-acether antagonists (mepyramine, cyproheptadine and WEB 2086, respectively) were ineffective. Histological examination showed that B. lanceolatus venom (250 ng and 500 ng/paw) caused thickening of the inner dermal layers which was accompanied by extensive intercellular spaces indicative of oedema. In addition, there was a marked infiltration of inflammatory cells, particularly neutrophils, into the underlying muscle layer. The latter, however, remained morphologically unaffected during the 3 h of observation. Venom doses larger than 500 ng/paw produced intense haemorrhage. These results indicate that B. lanceolatus venom induces oedema and increases vascular permeability in the mouse hind paw. The principal mediators of this inflammatory response are cyclooxygenase and lipoxygenase products.

A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech.

The salivary complex of the leech Haementeria depressa produces potent anticoagulant components. Among them, a protein named lefaxin inhibits factor Xa (FXa). Lefaxin was purified to homogeneity from dissected salivary complexes by gel filtration in Sephadex G-150 followed by two ion exchange chromatography steps in Mono-Q. Inhibition of FXa by lefaxin was demonstrated by the inhibition of its amidolytic activity, measured with chromogenic substrate S-2765 (apparent K(I) of 4 nM), and of its ability to inhibit thrombin generation in the prothrombinase complex (EC50 of 40 nM). Lefaxin has a molecular weight of 30 kDa and an isoelectric point of 5.7. It is made of a polypeptide chain whose N-terminal sequence shows no similarity with that of other FXa inhibitors (antistasin and ghilianten) isolated from leech saliva. On the other hand, the N-terminal sequence of lefaxin presents significant sequence similarity with nitric oxide carrier proteins myohemerythrin from the annelid Nereis diversicolor and prolixin S from the triatoma Rhodnius prolixus. Interestingly, prolixin S also proved to be an anticoagulant protein acting on FXa.

Allosteric changes of thrombin catalytic site induced by interaction of bothrojaracin with anion-binding exosites I and II.

Bothrojaracin, a 27-kDa C-type lectin from Bothrops jararaca venom, is a selective and potent thrombin inhibitor (K(d) = 0.6 nM) which interacts with the two thrombin anion-binding exosites (I and II) but not with its catalytic site. In the present study, we analyzed the allosteric effects produced in the catalytic site by bothrojaracin binding to thrombin exosites. Opposite effects were observed with alpha-thrombin, which possesses both exosites I and II, and with gamma-thrombin, which lacks exosite I. On the one hand, bothrojaracin altered both kinetic parameters K(m) and k(cat) of alpha-thrombin for small synthetic substrates, resulting in an increased efficiency of alpha-thrombin catalytic activity. This effect was similar to that produced by hirugen, a peptide based on the C-terminal hirudin sequence (residues 54-65) which interacts exclusively with exosite I. On the other hand, bothrojaracin decreased the amidolytic activity of gamma-thrombin toward chromogenic substrates, although this effect was observed with higher concentrations of bothrojaracin than those used with alpha-thrombin. In agreement with these observaions, bothrojaracin produced opposite effects on the fluorescence intensity of alpha- and gamma-thrombin derivatives labeled at the active site with fluorescein-Phe-Pro-Arg-chloromethylketone. These observations support the conclusion that bothrojaracin binding to thrombin produces two different structural changes in its active site, depending on whether it interacts exclusively with exosite II, as seen with gamma-thrombin, or with exosite I (or both I and II) as observed with alpha-thrombin. The ability of bothrojaracin to evoke distinct modifications in the thrombin catalytic site environment when interacting with exosites I and II make this molecule an interesting tool for the study of allosteric changes in the thrombin molecule.

Pharmacokinetics of the toxic fraction of Centruroides limpidus limpidus venom in experimentally envenomed rabbits and effects of immunotherapy with specific F(ab')2.

Envenomations after scorpion stings are a major health problem throughout the world. Their specific treatment is immunotherapy which consists of the injection of specific antibody. In this article, we studied the pharmacokinetics of the toxic fraction of Centruroides limpidus limpidus venom (fraction II) in experimentally envenomed rabbits. After an intravenous injection, fraction II (FII) was rapidly distributed and eliminated from the body (terminal half-life of 1.9 h). When injected subcutaneously, high concentrations of FII were measured in the vascular space rapidly after the injection (Tmax = 1 h) and FII was eliminated with a terminal half-life of 1.8 h, close to that determined after intravenous injection. These observations go along with the rapid onset of clinical symptoms observed after accidental envenomations. To investigate the mechanism of action of antivenom, we examined the effects of the intravenous administration of antivenom (horse F(ab')2 directed against Centruroides venoms) on the pharmacokinetics of FII. Immunotherapy performed 2 h after the experimental envenomation largely increased the area under the concentration time curve of FII compared to that calculated in absence of immunotherapy (13,000 versus 170 ng h ml(-1), respectively). These observations agree with previous findings which showed that specific antibody fragments are able to remove drugs from their site of action and sequester them in the vascular space. These studies provide a powerful tool to determine an excellent procedure for further improvement of immunotherapy.

Bothrops jararaca snakes produce several bothrojaracin isoforms following an individual pattern.

More than one isoform of bothrojaracin (BJC), a potent and specific thrombin inhibitor isolated from Bothrops jararaca venom, has been found in individual venoms collected from adult snakes. Variations in snake venom composition have previously been associated with factors such as age, sex, geographic origin, season of the year and diet. In order to obtain further information concerning individual patterns of expression of BJC isoforms, we have analyzed five individual Bothrops jararaca snake venoms collected at the same time from adult female snakes from the same geographic region. As expected, crude venoms showed a similar migration pattern on SDS-PAGE. BJC was purified using a procedure which includes an affinity chromatography step (PPACK-thrombin Sepharose). A slight variation in the amount of BJC obtained from individual venom samples was noticed. Inhibition of thrombin-induced platelet aggregation as well as migration pattern on SDS-PAGE (under reducing and non-reducing conditions) and isoelectric focusing varied considerably among BJC samples from the five snakes. The amino-terminal sequences (residues 1-34) of individual BJC samples were compared with the sequence deduced from isolated cDNAs encoding alpha and beta chains of BJC. A high degree of homology was detected, although some residues differed from one sample to other. Altogether, data confirmed the heterogeneity found for BJC purified from individual snakes. Thus, the results indicate that: (1) individual specimens of Bothrops jararaca have different patterns of BJC isoform expression; and (2) it seems that genetic factors, at least in part, determine the variability found in BJC production.

Fibrino(geno)lytic properties of purified hementerin, a metalloproteinase from the leech Haementeria depressa.

The fibrino(geno)lytic protein designated hementerin contained in crude extracts of the salivary complex of Haementeria depressa leeches was purified to apparent homogeneity by gel filtration, ion exchange chromatography and preparative SDS-PAGE. It is a single-chain 80 kDa, PhMeSO2F-resistant, calcium-dependent, metalloproteinase, which specifically degrades fibrin(ogen) through a plasminogen-independent pathway. The amino terminal sequence of 8 residues shows 80% similarity with hementin, another fibrino(geno)lytic protein purified from Haementeria ghilianii leeches. However, their activities differ somewhat in terms of kinetics and with regard to the structure of the fibrin(ogen) fragments they may produce. Cleavage by hementerin of fibrinogen Aalpha, gamma and Bbeta chains, in that order, produces 270 kDa to 67 kDa fragments which differ from those produced by plasmin. Hementerin was also able to degrade cross-linked fibrin although at a lower rate as compared to fibrinogen. In conclusion, hementerin is a plasminogen-independent fibrino(geno)lytic metalloproteinase that degrades fibrinogen faster than fibrin, prevents blood coagulation and destroys fibrin clots in vitro.

Purification from Bothrops lanceolatus (fer de lance) venom of a fibrino(geno)lytic enzyme with esterolytic activity.

Bothrops lanceolatus venom has high caseinolytic, phospholipasic, esterolytic and hemorrhagic activities. In spite of having no coagulant effect on plasma, this venom contains a thrombin-like enzyme. Using gel filtration and ion-exchange chromatographies, we have purified an esterolytic fraction (F-II-1a) from this venom with a protein yield of 4% and a 58% recovery in enzyme activity. SDS-PAGE in the presence of beta-mercaptoethanol showed that the enzyme is a single chain polypeptide with a MW=38,100. Immunodiffusion and immunoelectrophoresis of fraction F-II-1a against serum from horses immunized with B. lanceolatus venom and against rabbit antiserum prepared using fraction F-II-1a both showed a single immunoprecipitin line. The Km and Vmax values for TAME hydrolysis were 0.85 mM and 38.6 micromol/min/mg, respectively. The esterolytic activity was completely inhibited by PMSF (10 mM) but not by EDTA (20 mM). Fraction F-II-1a hydrolyzed the alpha and beta chains of fibrinogen. Degradation of the alpha chain occurred within 10 min while that of the beta-chain was slower. The enzyme had no effect on the gamma-chain even after 4 h of hydrolysis.

Variability of bothrojaracin isoforms and other venom principles in individual jararaca (Bothrops jararaca) snakes maintained under seasonally invariant conditions.

Bothrojaracin (BJC) is a potent thrombin inhibitor isolated from the venom of Bothrops jararaca. Venoms from individual snakes have been shown to vary in BJC content, and more than one molecular variant (isoform) has been identified in the same venom. In order to determine whether the production of this protein and its isoforms varies under seasonally invariant conditions, an analysis was made of BJC isolated from venoms collected individually once a month for 10 months from two female B. jararaca snakes kept under conditions of constant temperature and photoperiod. The crude venom from each individual snake exhibited a characteristic pattern of protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with no noticeable variation throughout the collecting period. BJC from individual venoms was purified by gel filtration on Sephacryl S-200 followed by an affinity column (PPACK-thrombin Sepharose). BJC content and other activities such as phospholipase A2, azocaseinolytic activity and inhibition of thrombin-induced platelet aggregation varied considerably among the samples. Purified BJC from both snakes inhibited fibrin coagulation and migrated as a single band of 27,000 mol. wt on SDS-PAGE. However, the BJC pattern on non-denaturing PAGE differed between the two snakes, with four to six bands per sample each month, which were all recognized by polyclonal anti-BJC antibodies. The isoelectric focusing pattern of BJC was also characteristic for each snake, with only minor differences throughout the collecting period. These results indicate that under seasonally invariant conditions: (1) there was a considerable variation over the 10-month period in the production of BJC and other important venom activities such as phospholipase A2 and proteinases; (2) individual B. jararaca snakes produced a distinctive array of BJC isoforms; and (3) despite quantitative differences, there were essentially no qualitative differences in the production of BJC isoforms by individual snakes during the 10-month period.

Distinct bothrojaracin isoforms produced by individual jararaca (Bothrops jararaca) snakes.

Bothrojaracin (apparent mol. wt 27,000) is a potent inhibitor of thrombin previously isolated from the venom of Bothrops jararaca. Several molecular variants (isoforms) have been identified in a pool of venom collected from a large number of animals. In order to determine whether an individual snake produces a single type of bothrojaracin or multiple isoforms, we analyzed the bothrojaracin content of venoms collected individually from six adult B. jararaca snakes. Bothrojaracin was found in all venoms, but its activity was especially high in three of them. After purification on an alpha-thrombin affinity column, followed by gel filtration on Superose 12 HR, proteins from these three venoms migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as single bands of 27,000 and their amino-terminal sequences (residues 1-28) revealed extensive homology with bothrojaracin. In contrast, the material purified from the three venoms with low bothrojaracin activity consisted of bothrojaracin together with inactive proteins. Differences in the sequences obtained for bothrojaracin isolated from individual venoms indicated the existence of more than one isoform in the venom of an individual snake.

Clinical indicators of envenoming and serum levels of venom antigens in patients bitten by Bothrops lanceolatus in Martinique. Research Group on Snake Bites in Martinique.

An enzyme-linked immunosorbent assay was developed to measure venom antigen levels in the serum of 40 patients bitten by Bothrops lanceolatus. The grading system used for the severity of envenomation (grades 1 to 4, minor to major) was predominantly based on the presence of local signs. Serum venom levels increased with the grade of severity (P < 0.001, by Spearman's rank correlation test); they were 6 +/- 6 ng/mL (mean +/- SD) in clinically non-envenomed patients (grade 1, n = 3), 7.6 +/- 11.7 (n = 17), 44.3 +/- 41.8 (n = 17), and 80.3 +/- 34.1 ng/mL (n = 3) in patients diagnosed as grade 2, 3 and 4 respectively. However, venom antigens could not be detected in the serum of 54% of patients who showed clinical signs of envenomation. Most patients diagnosed as grade 2, 3 or 4 were given 20, 40 and 60 mL of a monospecific F(ab')2 antivenom, respectively. Venom concentrations > or = 15 ng/mL were observed in all patients with progressive aggravation of swelling despite the use of early antivenom therapy. No venom was detectable in blood samples taken after completion of serotherapy. All patients recovered. These results confirm the efficacy of both the clinical severity scoring system used and the therapeutic regimen.

Molecular structure and mechanism of action of the crotoxin inhibitor from Crotalus durissus terrificus serum.

An antivenom protein has been identified in the blood of the snake Crotalus durissus terrificus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotalus serum (CICS), and to analyze its mechanism of action. CICS has been purified from blood serum of the Crotalus snake by gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non-covalent association of 23-25-kDa subunits. Two different subunit peptides were identified by SDS/PAGE, however, their N-terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues. The neutralizing effect of purified CICS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex. The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system.

Crotoxin, half-century of investigations on a phospholipase A2 neurotoxin.

Crotoxin, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a neurotoxic phospholipase A2 which exerts its pathophysiological action by blocking the neuromuscular transmission. Crotoxin acts primarily by altering the acetylcholine release from the nerves terminals through a mechanism which has not yet been elucidated. It also acts on postsynaptic membranes by stabilizing the acetylcholine receptor in an inactive conformation very similar to the desensitized state. Crotoxin is made of two dissimilar subunits: a basic and weakly toxic phospholipase A2 component-B, and an acidic and non toxic component-A which does not possess any enzymatic activity. Binding experiments showed that crotoxin subunits dissociate when crotoxin interacts with biological membranes: Component-B binds, whereas component-A appears free in solution. The phospholipase A2 subunit binds in a non saturable, non specific manner, on any kind of biological membranes, whereas in the presence of component-A it interacts only with a limited number of high affinity binding sites present on synaptic membranes but not on erythrocyte membranes. Although the target site (acceptor) of crotoxin has not yet been formally identified, binding experiments carried out with small unilamellar phospholipid vesicles of different compositions indicate that some negatively charged phospholipids like mono and diphosphoinositide phosphates might be an important component of crotoxin acceptor site. Crotoxin is in fact a mixture of several isoforms which have very similar but not identical polypeptide sequences. An individual Crotalus durissus terrificus snake is able to synthesize several crotoxin isoforms which may result of the expression of several isogenes and/or of post-translational events. When compared in quantitative manner, the crotoxin isoforms slightly but significantly differ in their enzymatic and pharmacological properties. Finally, immunochemical investigations carried out with polyclonal antibodies prepared against both crotoxin subunits, showed that non precipitating anti-component-B- antibodies (Fab) inhibit the phospholipase A2 activity of crotoxin and neutralize its lethal potency, suggesting that the catalytic and toxic sites of crotoxin are closely related.

Crotoxin, half-century of investigations on a phospholipase A2 neurotoxin.

Crotoxin, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a neurotoxic phospholipase A2 which exerts its pathophysiological action by blocking the neuromuscular transmission. Crotoxin acts primarily by altering the acetylcholine release from the nerves terminals through a mechanism which has not yet been elucidated. It also acts on postsynaptic membranes by stabilizing the acetylcholine receptor in an inactive conformation very similar to the desensitized state. Crotoxin is made of two dissimilar subunits: a basic and weakly toxic phospholipase A2 component-B, and an acidic and non toxic component-A which does not possess any enzymatic activity. Binding experiments showed that crotoxin subunits dissociate when crotoxin interacts with biological membranes: Component-B binds, whereas component-A appears free in solution. The phospholipase A2 subunit binds in a non saturable, non specific manner, on any kind of biological membranes, whereas in the presence of component-A it interacts only with a limited number of high affinity binding sites present on synaptic membranes but not on erythrocyte membranes. Although the target site (acceptor) of crotoxin has not yet been formally identified, binding experiments carried out with small unilamellar phospholipid vesicles of different compositions indicate that some negatively charged phospholipids like mono and diphosphoinositide phosphates might be an important component of crotoxin acceptor site. Crotoxin is in fact a mixture of several isoforms which have very similar but not identical polypeptide sequences. An individual Crotalus durissus terrificus snake is able to synthesize several crotoxin isoforms which may result of the expression of several isogenes and/or of post-translational events. When compared in quantitative manner, the crotoxin isoforms slightly but significantly differ in their enzymatic and pharmacological properties. Finally, immunochemical investigations carried out with polyclonal antibodies prepared against both crotoxin subunits, showed that non precipitating anti-component-B- antibodies (Fab) inhibit the phospholipase A2 activity of crotoxin and neutralize its lethal potency, suggesting that the catalytic and toxic sites of crotoxin are closely related.