RESUMEN
The pathological alterations induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi, were studied in mice. Neuwiedase was devoid of hemorrhagic activity when tested in the skin up to a dose of 200 microgram, and also after intramuscular injection in the gastrocnemius. However, it induced bleeding when applied onto the mouse cremaster muscle in intravital microscopy experiments, and caused pulmonary hemorrhage when injected intravenously at doses higher than 5 microgram/g. Median lethal dose (LD(50)) by the intravenous route was 5 microgram/g, whereas LD(50) of crude venom was 0.47 microgram/g. After intramuscular injection, neuwiedase induced a mild myotoxic effect, evidenced histologically and by the increment in plasma creatine kinase activity, but it was devoid of hemorrhagic and thrombotic effects. In contrast, crude B. neuwiedi venom induced prominent hemorrhage and myonecrosis in gastrocnemius muscle. Both venom and neuwiedase induced an inflammatory reaction in muscle tissue characterized by abundant polymorphonuclear leukocytes. Moreover, a conspicuous edema developed in the foot pad after subcutaneous injection of neuwiedase. Anti-neuwiedase antibodies produced in rabbits were effective in the neutralization of hemorrhagic activity of crude venom, evidencing immunological cross-reactivity between neuwiedase and other hemorrhagic metalloproteinases present in the venom, and suggesting that metalloproteinases devoid of, or having low, hemorrhagic activity could be good immunogens to generate antibodies effective against high molecular mass metalloproteinasas having potent hemorrhagic activity. It is concluded that neuwiedase, despite its lack of hemorrhagic effect when injected in the gastrocnemius muscle, contributes to local tissue damage by inducing edema, inflammatory infiltrate and mild myotoxicity, and by degrading extracellular matrix components. In addition, large doses of neuwiedase may contribute to pulmonary bleeding
Asunto(s)
Bothrops , Venenos de Crotálidos/enzimología , Metaloendopeptidasas/toxicidad , Venenos de Víboras/toxicidad , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Creatina Quinasa/metabolismo , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/inmunología , Venenos de Crotálidos/toxicidad , Edema/inducido químicamente , Edema/tratamiento farmacológico , Hemorragia/inducido químicamente , Hemorragia/tratamiento farmacológico , Técnicas In Vitro , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Dosificación Letal Mediana , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/inmunología , Ratones , Músculos/efectos de los fármacos , Músculos/patología , Pruebas de Neutralización , Factores de Tiempo , Venenos de Víboras/antagonistas & inhibidores , Venenos de Víboras/inmunologíaRESUMEN
The hydrolysis of phospholipids by class II phospholipase A2 (PLA2) involves a Ca2+ ion cofactor bound to the Asp49 residue in the active site region. In the lysine 49 phospholipase A2 homologues (Lys49-PLA2), the Asp49 residue is substituted by Lys, and consequently the Lys49-PLA2s show no Ca2+ binding and no detectable phospholipid hydrolysis. Nevertheless, the Lys49-PLA2s demonstrate membrane damaging activity by an incompletely understood Ca2+-independent mechanism of action. Using a combination of steady-state and time-resolved fluorescence techniques, we have examined the effect of pH on the monomer-dimer equilibrium of bothropstoxin I (BthTX-I), a Lys49-PLA2 from the venom of Bothrops jararacussu which contains a single Trp77 residue located at the dimer interface. At pH 5.0, we observe a decreased quantum yield, a decreased rotational correlation time, and an increased bimolecular quenching rate constant with iodide. These results are consistent with a pH-induced dissociation of the BthTX-I dimer, with the consequent exposure of the Trp77 residue to aqueous solvent. In the presence of liposomes, membrane damaging activity is observed only under conditions in which the dimeric form of the BthTX-I is favored. These results demonstrate that the dimeric form of the protein is essential for the initiation of the Ca2+-independent membrane damaging activity.
Asunto(s)
Calcio/metabolismo , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/metabolismo , Liposomas/metabolismo , Lisina/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Animales , Venenos de Crotálidos/toxicidad , Dimerización , Fluoresceínas/metabolismo , Polarización de Fluorescencia , Concentración de Iones de Hidrógeno , Fosfolipasas A/toxicidad , Fosfolipasas A2 , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Soluciones , Espectrometría de Fluorescencia , Triptófano/metabolismoRESUMEN
Bothopstoxin-I (BthTX-I) is a homodimeric Lys49-PLA2 homologue from the venom of Bothrops jararacussu in which a single Trp77 residue is located at the dimer interface. Intrinsic tryptophan fluorescence emission (ITFE) quenching by iodide and acrylamide has confirmed that a dimer to monomer transition occurs on reducing the pH from 7.0 to 5.0. Both the monomer and the dimer showed an excitation wavelength-dependent increase in the fluorescence emission maximum, however the excitation curve of the dimer was blue-shifted with respect to the monomeric form. No differences in the absorption or circular dichroism spectra between pH 5.0 and 7.0 were observed, suggesting that this curve shift is due neither to altered electronic ground states nor to exciton coupling of the Trp residues. We suggest that fluorescence resonance energy homotransfer between Trp77 residues at the BthTX-I dimer interface results in excitation of an acceptor Trp population which demonstrates a red-shifted fluorescence emission.
Asunto(s)
Venenos de Crotálidos/química , Animales , Bothrops , Dimerización , Transferencia de Energía , Concentración de Iones de Hidrógeno , Yoduros , Modelos Moleculares , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia/métodos , TriptófanoRESUMEN
Piratoxins (PrTX) I and III are phospholipases A2 (PLA2s) or PLA2 homologue myotoxins isolated from Bothrops pirajai snake venom, which also induce myonecrosis, bactericidal activity against Escherichia coli, disruption of artificial membranes, and edema. PrTX-III is a catalytically active hemolytic and anticoagulant Asp49 PLA2, while PrTX-I is a Lys49 PLA2 homologue, which is catalytically inactive on artificial substrates, but promotes blockade of neuromuscular transmission. Chemical modifications of His, Lys, Tyr, and Trp residues of PrTX-I and PrTX-III were performed, together with cleavage of the N-terminal octapeptide by CNBr and inhibition by heparin and EDTA. The lethality, bactericidal activity, myotoxicity, neuromuscular effect, edema inducing effect, catalytic and anticoagulant activities, and the liposome-disruptive activity of the modified toxins were evaluated. A complex pattern of functional differences between the modified and native toxins was observed. However, in general, chemical modifications that significantly affected the diverse pharmacological effects of the toxins did not influence catalytic or membrane disrupting activities. Analysis of structural changes by circular dichroism spectroscopy demonstrated significant changes in the secondary structure only in the case of N-terminal octapeptide cleavage. These data indicate that PrTX-I and PrTX-III possess regions other than the catalytic site, which determine their toxic and pharmacological activities.
Asunto(s)
Músculos/efectos de los fármacos , Fosfolipasas A/metabolismo , Fosfolipasas A/farmacología , Acetofenonas/química , Animales , Bioensayo , Bothrops , Dicroismo Circular , Venenos de Crotálidos/química , Venenos de Crotálidos/metabolismo , Venenos de Crotálidos/farmacología , Cristalografía por Rayos X , Bromuro de Cianógeno/química , Diafragma/efectos de los fármacos , Diafragma/inervación , Diafragma/fisiología , Edema/inducido químicamente , Inhibidores Enzimáticos/química , Fosfolipasas A2 Grupo II , Técnicas In Vitro , Liposomas/química , Liposomas/metabolismo , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Nitrobencenos/química , Peroxidasa/análisis , Fosfolipasas A/química , Fosfolipasas A2 , Estructura Secundaria de Proteína , Proteínas de Reptiles , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA(2) homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA(2) homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins.
Asunto(s)
Venenos de Crotálidos/toxicidad , Músculo Esquelético/efectos de los fármacos , Fosfolipasas A/toxicidad , Animales , Bothrops , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía por Intercambio Iónico , Venenos de Crotálidos/química , Venenos de Crotálidos/aislamiento & purificación , Edema/inducido químicamente , Dosificación Letal Mediana , Liposomas , Masculino , Ratones , Músculo Esquelético/patología , Necrosis , Fosfolipasas A/aislamiento & purificaciónRESUMEN
Myotoxin-I (MjTX-I) was purified to homogeneity from the venom of Bothrops moojeni by ion-exchange chromatography on CM-Sepharose. Its molecular weight, estimated by SDS-PAGE, was 13,400 (reduced) or 26, 000 (unreduced). The extinction coefficient (E(1.0 mg/ml)(1.0 cm)) of MjTX-I was 1.145 at lambda = 278 nm, pH 7.0, and its isoelectric point was 8.2 at ionic strength mu = 0.1. When lyophilized and stored at 4 degrees C, dimeric, trimeric, and pentameric forms of the protein were identified by SDS-PAGE. This "heterogeneous" sample could be separated into three fractions by gel filtration on Sephadex G-50. The fractions were analyzed by isoelectric focusing, immunoelectrophoresis, and amino acid composition, which indicated that heterogeneity was the result of different levels of self-association. Protein sequencing indicated that MjTX-I is a Lys49 myotoxin and consists of 121 amino acids (M(r) = 13,669), containing a high proportion of basic and hydrophobic residues. It shares a high degree of sequence identity with other Lys49 PLA(2)-like myotoxins, but shows a significantly lower identity with catalytically active Asp49 PLA(2)s. The three-dimensional structure of MjTX-I was modeled based on the crystal structures of three highly homologous Lys49 PLA(2)-like myotoxins. This model showed that the amino acid substitutions are conservative, and mainly limited to three structural regions: the N-terminal helix, the beta-wing region, and the C-terminal extended random coil. MjTX-I displays local myotoxic and edema-inducing activities in mice, and is lethal by intraperitoneal injection, with an LD(50) value of 8.5 +/- 0.8 mg/kg. In addition, it is cytotoxic to myoblasts/myotubes in culture, and disrupts negatively charged liposomes. In comparison with the freshly prepared dimeric sample, the more aggregated forms showed significantly reduced myotoxic activity. However, the edema-inducing activity of MjTX-I was independent of molecular association. Phospholipase A(2) activity on egg yolk, as well as anticoagulant activity, were undetectable both in the native and in the more associated forms. His, Tyr, and Trp residues of the toxin were chemically modified by specific reagents. Although the myotoxic and lethal activities of the modified toxins were reduced by these treatments, neither its edema-inducing or liposome-disrupting activities were significantly altered. Rabbit antibodies to native MjTX-I cross-reacted with the chemically modified forms, and both the native and modified MjTX-I preparations were recognized by antibodies against the C-terminal region 115-129 of myotoxin II from B. asper, a highly Lys49 PLA(2)-homologue with high sequencial similarity.
Asunto(s)
Bothrops , Venenos de Crotálidos/química , Venenos de Crotálidos/toxicidad , Neurotoxinas/química , Neurotoxinas/toxicidad , Fosfolipasas A/química , Fosfolipasas A/toxicidad , Secuencia de Aminoácidos , Animales , Bothrops/genética , Venenos de Crotálidos/genética , Edema/inducido químicamente , Fosfolipasas A2 Grupo II , Inmunoquímica , Punto Isoeléctrico , Dosificación Letal Mediana , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Neurotoxinas/genética , Fosfolipasas A/genética , Conformación Proteica , Estructura Cuaternaria de Proteína , Conejos , Proteínas de Reptiles , Homología de Secuencia de AminoácidoRESUMEN
The crude aqueous extract from the leaves of Casearia sylvestris, a plant found in Brazilian open pastures, was assayed for its ability to inhibit phospholipase A2 (PLA2) activity and some biological activities of bee and several snake venoms, and of a number of isolated PLA2s. The extract induced partial inhibition of the PLA2 activity of venoms containing class I, II and III PLA2s. When tested against the purified toxins, it showed the highest efficacy against class II PLA2s from viperid venoms, being relatively ineffective against the class I PLA2 pseudexin. In addition, C. sylvestris extract significantly inhibited the myotoxic activity of four Bothrops crude venoms and nine purified myotoxic PLA2s, including Lys-49 and Asp-49 variants. The extract was able to inhibit the anticoagulant activity of several isolated PLA2s, with the exception of pseudexin. Moreover, it partially reduced the edema-inducing activity of B. moojeni and B. jararacussu venoms, as well as of myotoxins MjTX-II and BthTX-I. The extract also prolonged the survival time of mice injected with lethal doses of several snake venoms and neutralized the lethal effect induced by several purified PLA2 myotoxins. It is concluded that C. sylvestris constitutes a rich source of PLA2 inhibitors.
Asunto(s)
Antitoxinas/farmacología , Venenos de Abeja/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Extractos Vegetales/farmacología , Rosales/química , Venenos de Serpiente/metabolismo , Animales , Anticoagulantes/farmacología , Venenos de Abeja/antagonistas & inhibidores , Venenos de Crotálidos/metabolismo , Relación Dosis-Respuesta a Droga , Edema/tratamiento farmacológico , Electroforesis en Gel de Poliacrilamida , Masculino , Ratones , Fosfolipasas A2 , Venenos de Serpiente/antagonistas & inhibidores , Factores de TiempoRESUMEN
The isolation and partial characterization of a serine protease with arginine ester hydrolase activity from Bothrops jararacussu snake venom are described. The purification procedure consisted of a gel filtration of the crude venom on Sephadex G-75 followed by an ion-exchange chromatography of the active fraction on DEAE-cellulose and a rechromatography on Bio-Rex 70 resin. The esterase fraction (DI-III), M(r) = 25,000 by SDS-PAGE, showed proteolytic activity on fibrinogen and casein. After 2 hr incubation, the A alpha and B beta chains of fibrinogen were intensely hydrolysed, while the gamma chain kept apparently intact, even after 20 hr of incubation. In spite of that, DI-III did not clot fibrinogen. DI-III induced edema in the rat paw. Although unable to release bradykinin, it induced contractions of the isolated rat uterus. DI-III did not catalyse the hydrolysis of bradykinin. Its arginine ester hydrolase activity was completely inhibited by diisopropyl fluorophosphate after 1 hr incubation, but not by phenylmethylsulfonyl fluoride under the same conditions.
Asunto(s)
Bothrops , Hidrolasas de Éster Carboxílico/metabolismo , Venenos de Crotálidos/enzimología , Miometrio/efectos de los fármacos , Animales , Bradiquinina/metabolismo , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Hidrolasas de Éster Carboxílico/farmacología , Venenos de Crotálidos/farmacología , Femenino , Fibrinógeno/metabolismo , Humanos , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Miometrio/fisiología , Ratas , Ratas WistarRESUMEN
Crotamine, a 4.88 kDa neurotoxic protein, has been purified to apparent homogeneity from Crotalus durissus venom by gel filtration on Sephadex G-75. When injected (i.p. or s.c.) in adult male Swiss mice (20-25 g), it induced a time-dose dependent analgesic effect which was inhibited by naloxone, thus suggesting an opioid action mechanism. When compared with morphine (4 mg/kg), crotamine, even in extremely low doses (133.4 microg/kg, i.p., about 0.4% of a LD50 is approximately 30-fold more potent than morphine (w/w) as an analgesic. On a molar basis it is more than 500-fold more potent than morphine. It is also much more potent than the lower molecular weight crude fractions of the same venom. The antinociceptive effects of crotamine and morphine were assayed by the hot plate test and by the acetic acid-induced writhing method. Therefore, both central and peripheral mechanisms should be involved. Histopathological analysis of the brain, liver, skeletal muscles, stomach, lungs, spleen, heart, kidneys and small intestine of the crotamine injected mice did not show any visible lesion in any of these organs by light microscopy. Since crotamine accounted for 22% (w/w) of the desiccated venom, it was identified as its major antinociceptive low molecular weight peptide component.
Asunto(s)
Analgésicos/farmacología , Venenos de Crotálidos/farmacología , Crotalus/fisiología , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Masculino , Ratones , Naloxona/farmacología , Naloxona/toxicidad , Neurotoxinas/farmacología , Dimensión del DolorRESUMEN
Crotamine, a basic, myonecrotic, histamine-releasing neurotoxin, was isolated from Crotalus durissus terrificus venom. Carboxypeptidase A was shown to be activated by crotamine when acting upon N-carbobenzoxyglycil-L-phenylalanine. However the activity of carboxypeptidase B upon the substrate hippuryl-L-arginine was not enhanced by this toxin. Teh basic histamine releasers protamine and compound 48/80 also activated carboxypeptidase A. These three agents activated both alpha-chymotrypsin when acting upon acetyl-L-tyrosine ethyl ester and also five snake venom phospholipase-like myotoxins acting upon egg yolk phosphatidylcholine. These findings suggest that the action of these agents during histamine release may involve the participation of specific intermediary hydrolases which, upon activation, would enhance their cytolytic effects on the sequence of events which lead to granule extrusion and histamine release from mast cells.
Asunto(s)
Venenos de Crotálidos/farmacología , Liberación de Histamina/efectos de los fármacos , Fosfolipasas A/metabolismo , Protaminas/farmacología , p-Metoxi-N-metilfenetilamina/farmacología , Carboxipeptidasas/efectos de los fármacos , Carboxipeptidasas/metabolismo , Quimotripsina/efectos de los fármacos , Quimotripsina/metabolismo , Fosfolipasas A/efectos de los fármacos , Venenos de Serpiente/farmacologíaRESUMEN
A detailed procedure for the isolation of a new clotting enzyme from the venom of Bothrops jararacussu (common name jararacuçu) is described. The estimated mol. wt of the native protein was 30,100 but 37,500 after reduction by dithiothreitol. Two major close bands corresponding to pI 5.18 and 5.20 were detected by electrofocusing but, after methanolysis, a single band focused at pI 8.20. The mol. wt of the protein moiety of this glycoprotein was 28,500, showing V-V-G-A-D-N-C-N-F-N... as N-terminal sequence. The content of neutral sugar was 4.8% and that of total sugars 5.3%. This clotting factor degraded only the A alpha-chain of the fibrinogen molecule. The stability of the clot, when produced in the presence of aprotinin opens new uses for snake clotting enzymes in the production of fibrin glue.
Asunto(s)
Factores de Coagulación Sanguínea/aislamiento & purificación , Bothrops , Venenos de Crotálidos/química , Adhesivo de Tejido de Fibrina/metabolismo , Trombina/aislamiento & purificación , Aminoácidos/análisis , Animales , Factores de Coagulación Sanguínea/química , Cromatografía en GelRESUMEN
The antibothropic complex (ABC) from opossum (species Didelphis albiventris) serum was purified by chromatography on DEAE-Sephacel. It showed an acidic character and two polypeptide chains of ca. 45 kDa and 48 kDa, respectively. Lyophilized opossum serum or the ABC (100 micrograms), as well as ethylenediamine tetraacetate (0.25 mumoles) were able to completely neutralise the hemorrhagic effect of 50 micrograms of the desiccated venoms of Bothrops moojeni, Bothrops pirajai and Bothrops jararacussu. The myotoxic (100 micrograms venom in mice) and edematogenic (90 micrograms venom in rats) activities of Bothrops moojeni and Bothrops jararacussu venoms, as well as of the major myonecrotic protein (myotoxin-I) isolated from Bothrops moojeni venom, were also totally inhibited by the ABC (200 micrograms and 270 micrograms, respectively). The lyophilized opossum serum (30 micrograms) and the ABC (30 micrograms) reduced to 50% the phospholipase A2 activity of Bothrops moojeni venom (10 micrograms). The clotting activity of Bothrops alternatus and Bothrops moojeni (20 micrograms) on bovine plasma was also significantly inhibited by the ABC (60 micrograms).