RESUMEN
Two new toxins were purified from Leiurus quinquestriatus hebraeus (Lqh) scorpion venom, Lqh II and Lqh III. Lqh II sequence reveals only two substitutions, as compared to AaH II, the most active scorpion alpha-toxin on mammals from Androctounus australis Hector. Lqh III shares 80% sequence identity with the alpha-like toxin Bom III from Buthus occitanus mardochei. Using bioassays on mice and cockroach coupled with competitive binding studies with 125I-labeled scorpion alpha-toxins on rat brain and cockroach synaptosomes, the animal selectivity was examined. Lqh II has comparable activity to mammals as AaH II, but reveals significantly higher activity to insects attributed to its C-terminal substitution, and competes at low concentration for binding on both mammalian and cockroach sodium channels. Lqh II thus binds to receptor site 3 on sodium channels. Lqh III is active on both insects and mammals but competes for binding only on cockroach. The latter indicates that Lqh III binds to a distinct receptor site. Thus, Lqh II and Lqh III represent two different scorpion toxin groups, the alpha- and alpha-like toxins, respectively, according to the structural and pharmacological criteria. These new toxins may serve as a lead for clarification of the structural basis for insect vs mammal selectivity of scorpion toxins.
Asunto(s)
Neurotoxinas/toxicidad , Venenos de Escorpión/química , Canales de Sodio/efectos de los fármacos , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Cucarachas , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neurotoxinas/química , Ratas , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Sinaptosomas/efectos de los fármacosRESUMEN
The solution structure of the anti-mammal and anti-insect LqqIII toxin from the scorpion Leiurus quinquestriatus quinquestriatus was refined and compared with other long-chain scorpion toxins. This structure, determined by 1H-NMR and molecular modeling, involves an alpha-helix (18-29) linked to a three-stranded beta-sheet (2-6, 33-39, and 43-51) by two disulfide bridges. The average RMSD between the 15 best structures and the mean structure is 0.71 A for C alpha atoms. Comparison between LqqIII, the potent anti-mammal AaHII, and the weakly active variant-3 toxins revealed that the LqqIII three-dimensional structure is closer to that of AaHII than to the variant-3 structure. Moreover, striking analogies were observed between the electrostatic and hydrophobic potentials of LqqIII and AaHII. Several residues are well conserved in long-chain scorpion toxin sequences and seem to be important in protein structure stability and function. Some of them are involved in the CS alpha beta (Cysteine Stabilized alpha-helix beta-sheet) motif. A comparison between the sequences of the RII rat brain and the Drosophila extracellular loops forming scorpion toxin binding-sites of Na+ channels displays differences in the subsites interacting with anti-mammal or anti-insect toxins. This suggests that hydrophobic as well as electrostatic interactions are essential for the binding and specificity of long-chain scorpion toxins.
Asunto(s)
Péptidos/química , Péptidos/farmacología , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Secuencia de Aminoácidos , Animales , Encéfalo/efectos de los fármacos , Drosophila melanogaster/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/farmacología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , Canales de Sodio/efectos de los fármacos , Soluciones , Relación Estructura-ActividadRESUMEN
Scorpion toxin Lqq III binds to a single class of high affinity (Kd = 72 +/- 19 pM) and low capacity (Bmax = 2.5 +/- 0.2 pmol/mg) binding sites in cockroach neuronal membranes. Its binding was inhibited by Lqh alpha IT (IC50 = 80 +/- 30 pM) and sea-anemone toxin ATX II (IC50 = 2.5 +/- 0.3 nM), suggesting that Lqq III is a specific probe for receptor site 3 on cockroach sodium channels. This was confirmed by competitive binding experiments between 125I-Lqq III and scorpion alpha-toxins which have less toxicity in insects.
Asunto(s)
Péptidos/metabolismo , Venenos de Escorpión/metabolismo , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Cucarachas , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , EscorpionesRESUMEN
One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha insect-selective toxins and shows a strong sequence identity (> 90%) with Lqh alpha IT and LqqIII, two insect alpha-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.
Asunto(s)
Neurotoxinas/toxicidad , Venenos de Escorpión/aislamiento & purificación , Venenos de Escorpión/toxicidad , Potenciales de Acción/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/aislamiento & purificación , Periplaneta/efectos de los fármacos , Venenos de Escorpión/química , Venenos de Escorpión/genética , Escorpiones , Relación Estructura-ActividadRESUMEN
A depressant toxin active on insects, Buthacus arenicola IT2, was isolated from the venom of the North African scorpion B. arenicola and its structural and pharmacological properties were investigated. B. arenicola IT2 is a single polypeptide of 61 amino acid residues, including 8 half-cystines but no methionine and histidine, with a molecular mass of 6835 Da. Its amino acid sequence is 79-95% identical to other depressant toxins from scorpions. When injected into the cockroach Blatella germanica, B. arenicola IT2 induced a slow depressant flaccid paralysis with a LD50 of 175 ng. B. arenicola IT2 has two non-interacting binding sites in cockroach neuronal membranes: one of high affinity (Kd1 = 0.11 +/- 0.04 nM) and low capacity (Bmax1 = 2.2 +/- 0.6 pmol/mg), and one of low affinity (Kd2 = 24 +/- 7 nM) and high capacity (Bmax2 = 226 +/- 92 pmol/mg). Its binding to these two sites was completely inhibited by Leiurus quinquestriatus quinquestriatus IT2, a depressant toxin from L. quinquestriatus quinquestriatus. Reciprocal-binding experiments between B. arenicola IT2 and the excitatory insect-toxin A. australis Hector IT revealed competition between the two toxins for the high-affinity sites of B. arenicola IT2. B. arenicola IT2 has a higher affinity than L. quinquestriatus hebraeus IT2, a depressant toxin from L. quinquestriatus hebraeus. Thus, B. arenicola IT2 represents an interesting tool to study the receptor site for depressant toxins on insect sodium channels.
Asunto(s)
Venenos de Escorpión/química , Toxinas Biológicas/química , Secuencia de Aminoácidos , Animales , Unión Competitiva , Membrana Celular/efectos de los fármacos , Cucarachas , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Parálisis/inducido químicamente , Venenos de Escorpión/farmacología , Venenos de Escorpión/toxicidad , Canales de Sodio/efectos de los fármacos , Especificidad de la Especie , Toxinas Biológicas/farmacología , Toxinas Biológicas/toxicidadRESUMEN
Two neurotoxins, BmK I and BmK II, were purified from the venom of the Chinese scorpion Buthus martensi Karsch. The complete amino acid sequences of both toxins, each containing 64 amino acid residues, were determined by the automatic sequencing of reduced and S-carboxymethylated toxins and their peptides, obtained after cleavage with TPCK-treated trypsin and Staphylococcus aureus V8 protease, respectively. Toxicity as minimum lethal dose tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II. Only two amino acid replacements were found: at position 59 Val in BmK I was replaced by Ile in BmK II, and at position 62 a basic Lys residue in BmK I was substituted by a neutral Asn residue in BmK II. These features suggest that the positively charged residue (Lys or Arg) in the C-terminal position 62 (or 61 or 63) may also play an important role in facilitating the interaction between scorpion neurotoxins and the receptor on sodium channels. The effects of BmK I on nerve excitability were examined with the crayfish axon using intracellular recording and voltage-clamp conditions. The results indicate that BmK I preferentially blocks the sodium channel inactivation process. Thus, functional and structural similarities suggest that BmK I and BmK II belong to group 3 of scorpion alpha-type toxins.
Asunto(s)
Neurotoxinas/química , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Secuencia de Aminoácidos , Animales , Astacoidea/metabolismo , Cromatografía en Capa Delgada , Dípteros , Electroforesis en Gel de Poliacrilamida , Gryllidae , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Ratones , Datos de Secuencia Molecular , Neurotoxinas/administración & dosificación , Neurotoxinas/genética , Neurotoxinas/aislamiento & purificación , Neurotoxinas/toxicidad , Técnicas de Placa-Clamp , Venenos de Escorpión/administración & dosificación , Venenos de Escorpión/genética , Venenos de Escorpión/toxicidad , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Canales de Sodio/efectos de los fármacos , Especificidad de la Especie , Staphylococcus aureus/enzimología , Clorometilcetona de Tosilfenilalanila/farmacología , Tripsina/metabolismoRESUMEN
Sodium channels posses receptor sites for many neurotoxins, of which several groups were shown to inhibit sodium current inactivation. Receptor sites that bind alpha- and alpha-like scorpion toxins are of particular interest since neurotoxin binding at these extracellular regions can affect the inactivation process at intramembranal segments of the channel. We examined, for the first time, the interaction of different scorpion neurotoxins, all affecting sodium current inactivation and toxic to mammals, with alpha-scorpion toxin receptor sites on both mammalian and insect sodium channels. As specific probes for rat and insect sodium channels, we used the radiolabeled alpha-scorpion toxins AaH II and LqhalphaIT, the most active alpha-toxins on mammals and insect, respectively. We demonstrate that the different scorpion toxins may be classified to several groups, according to their in vivo and in vitro activity on mammalian and insect sodium channels. Analysis of competitive binding interaction reveal that each group may occupy a distinct receptor site on sodium channels. The alpha-mammal scorpion toxins and the anti-insect Lqh alphaIT bind to homologous but not identical receptor sites on both rat brain and insect sodium channels. Sea anemone toxin ATX II, previously considered to share receptor site 3 with alpha-scorpion toxins, is suggested to bind to a partially overlapping receptor site with both AaH II and Lqh alphaIT. Competitive binding interactions with other scorpion toxins suggest the presence of a putative additional receptor site on sodium channels, which may bind a unique group of these scorpion toxins (Bom III and IV), active on both mammals and insects. We suggest the presence of a cluster of receptor sites for scorpion toxins that inhibit sodium current inactivation, which is very similar on insect and rat brain sodium channels, in spite of the structural and pharmacological differences between them. The sea anemone toxin ATX II is also suggested to bind within this cluster.
Asunto(s)
Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Oxocinas , Venenos de Escorpión/toxicidad , Canales de Sodio/química , Secuencia de Aminoácidos , Animales , Cucarachas , Saltamontes , Activación del Canal Iónico , Toxinas Marinas/farmacología , Ratones , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Venenos de Escorpión/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Sinaptosomas/metabolismo , Veratridina/farmacologíaRESUMEN
We describe the secondary structure and the overall fold of toxin III from the venom of the scorpion Leiurus quinquestriatus quinquestriatus determined using two-dimensional-1H-NMR spectroscopy. This protein, which contains 64 amino acids and 4 disulfide bridges, belongs to the long-chain toxin category and is highly toxic to both mammals and insects. The overall fold was determined on the basis of 1208 inter-proton-distance restraints derived from NOE measurements and 90 psi, phi dihedral-angle restraints derived from NOE connectivities and 3JNH-alphaH coupling constants using the HABAS program. This fold, which mainly consists of an alpha-helix packed against a small antiparallel three-stranded beta-sheet, and of several turns and loops, is similar to that of other long-chain scorpion toxins. Aromatic and non-polar residues form several patches on the surface of the protein which alternate with patches of charged and polar residues. Such a topology should be important in the interactions of toxin III with sodium channels in membranes. Two weakly constrained loops introduce some flexibility to the structure which could be related to the activity of this toxin. The central core of toxin III is compared with the cysteine-stabilized alpha beta motif (an alpha-helix connected to a beta-sheet through two disulfide bridges) found in insect defensins and plant thionins. Defensins and thionins are small proteins (approximately 40--50 amino acid residues) containing three or four disulfide bridges, respectively. This comparison confirms that the cysteine-stabilized alpha beta motif is a common core to a number of small proteins from different origins and having different activities.
Asunto(s)
Neurotoxinas/química , Péptidos/química , Venenos de Escorpión/química , Secuencia de Aminoácidos , Simulación por Computador , Disulfuros , Péptidos y Proteínas de Señalización Intercelular , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Hyperthermia and profuse perspiration are rarely absent in severe cases of scorpion envenomation. Based on these observations, the aim of this study was to determine the therapeutic effects of dantrolene, on experimental poisoning by the venom of Androctonus australis hector. Dantrolene is a directly acting muscle relaxant which lowers the body temperature in malignant hyperthermia. The results indicate that the early use of this drug raises the LD50 in experimentally poisoned mice. If these results are transposable to humans, dantrolene could be a useful therapeutic adjuvant.
Asunto(s)
Antivenenos/uso terapéutico , Dantroleno/uso terapéutico , Fármacos Neuromusculares/uso terapéutico , Venenos de Escorpión/antagonistas & inhibidores , Picaduras de Arañas/tratamiento farmacológico , Animales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The insect-selective neurotoxin (BmK IT) of scorpion Buthus martensi Karsch was first reduced and S-alkylated, and then digested by TPCK-trypsin and Staphylococcus aureus V-8 Protease. The enzymatic peptides were purified on TLC-plastic sheet and submitted to determine their amino acid compositions and sequences. The sequence of the 70 amino acid residues of BmK IT was established with reference to the primary structure of AaH IT, another excitatory insect-selective toxin from the venom of North African scorpion Androctonus australis Hector. About 75% of the homologous sequence was found in the molecules of BmK IT and AaH IT. It is obvious that the results contribute toward better understanding of the molecular structure characteristics, structure/activity relationship of scorpion insect-selective toxins, and they can serve as the molecular basis for utilizing the toxins as a tool to clarify molecular mechanism involved in channel gating, and to infer the possibility of developing them as new selective bioinsecticides.
Asunto(s)
Neurotoxinas/química , Venenos de Escorpión , Secuencia de Aminoácidos , Animales , Datos de Secuencia MolecularRESUMEN
The primary structure of toxin III of Leiurus quinquestriatus quinquestriatus (Lqq III) was elucidated by automatic Edman degradation of the reduced and S-carboxymethylated protein and derived tryptic peptides. Like other scorpion toxins that are active on sodium channels, Lqq III, consisting of 64 amino acids, is a 7 kDa single-chain polypeptide crosslinked by four disulfide bridges. It belongs to the alpha-toxin group, as judged by competition experiments with 125I AaH II for binding to rat brain synaptosomes (K0.5 = 7 x 10(-7) M). Lqq III is the first alpha-toxin to be characterized that is highly toxic to mice [LD50 = 50 micrograms (7.1 nmol)/kg body wt], by subcutaneous injection, insects Blatella germanica [LD50 = 60 ng (8.5 pmol)/g body wt.] and Musca domestica [LD50 = 120 ng (17 pmol)/g body wt]. When tested via the intracerebroventricular route, the toxicity for mice [55 micrograms (8 nmol)/kg] was of the same order as that found by subcutaneous injection, indicating that Lqq III has a higher affinity for peripheral sodium channels that for those of the central nervous system. There are three differences between the sequences of Lqq III and Lqh alpha IT, an alpha-toxin isolated from the venom of Leiurus quinquestriatus hebraeus. These substitutions are found at positions 20, 24, and 64 (Ser-->Ala,Asp-->Glu and His-->Arg, respectively). Surprisingly Lqh alpha IT is only weakly active in mice [LD50 = 5 mg (0.7 mumol)/kg], while in insects its toxicity is similar to that of Lqq III [140 ng (20 pmol)/g body wt blowfly larvae]. These observations are relevant to the definition of scorpion toxin structure-activity relationships.
Asunto(s)
Neurotoxinas/toxicidad , Péptidos/toxicidad , Venenos de Escorpión/toxicidad , Secuencia de Aminoácidos , Animales , Cucarachas , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/aislamiento & purificación , Péptidos/química , Péptidos/aislamiento & purificación , Venenos de Escorpión/química , Venenos de Escorpión/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
Aside from serotherapy, the treatment of scorpion venom intoxication is symptomatic. The aim of this study was to compare the efficacy of drugs usually used in scorpion venom intoxications (atropine, propranolol) to that of other compounds, chosen in light of the pathophysiology of scorpion venom intoxication: dipyridamole, doxapram, quinine formate, lysine-acetyl-salicylate, valproate and verapamil. Using mice, the parameters evaluated were the preventive and therapeutic effects of drugs during experimental venom intoxication by Androctonus australis Hector and one of its toxins AaH 1, and by Buthus occitanus and Tityus serrulatus tunetanus on the other hand. It was found that although most of the drugs used could prolong the survival of the animals, the administration of verapamil and more so that of aspirin or quinine formate led to a 50 to 100% loss of venom and toxin toxicity, depending on the drug and the origin of the venom. In the case of propranolol, doxapram, atropine, dipyridamole and valproate, no or little protection were observed. If these results are confirmed in humans, the systematic use of these drugs could be a simple means for treating scorpion venom intoxication. The problem of scorpion venom intoxication poses a health problem both in the North African Maghreb and in the Americas. As a result of considerable information campaigns, the number of scorpion venom intoxications in Tunisia has dropped from 3000 in 1967 to 1000 per year in the 1980s. Serotherapy has reduced mortality to 0.35%, most deaths occurring in underweight children. In light of the large number of countries in which there is a risk of scorpion venom intoxication in the summertime, however, its prevention and treatment remain a major problem.
Asunto(s)
Aspirina/uso terapéutico , Quinina/uso terapéutico , Venenos de Escorpión/toxicidad , Verapamilo/uso terapéutico , Animales , Femenino , Dosificación Letal Mediana , Ratones , Ratones Endogámicos C57BL , Intoxicación/tratamiento farmacológico , Intoxicación/prevención & control , Ácido Valproico/uso terapéutico , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
The amino acid sequences of insect-selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S-carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half-cystine from an external position, which is characteristic of anti-mammal toxins, to an internal sequence position. Lqq IT2 (61 residues) displays the half-cystine residue in position 12, common to the sequence of all known anti-mammal toxins; it induces flaccid paralysis on insects but is non-toxic for the mouse. Lqq IT2 structurally defines a new type of anti-insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.
Asunto(s)
Insectos , Venenos de Escorpión/análisis , Venenos de Escorpión/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Cromatografía Líquida de Alta Presión , Datos de Secuencia Molecular , Radioinmunoensayo , Homología de Secuencia de Ácido NucleicoRESUMEN
Protein X (PX) previously isolated from human pancreatic juice is an inactive protein of 14 kDa which has been shown to be a degradation product liberated by proteolysis of 19 kDa precursors. Polyclonal antibodies against P19 and PX were prepared in rabbits by injection of the two proteins purified by SDS polyacrylamide gel electrophoresis. These antibodies reacted with a form of trypsin 1 (DFP-trypsin 1) which was shown to be partly proteolysed. Immunological studies were performed with pancreatic juice proteins and partially purified trypsinogen 1 using antibodies directed against PX, P19 and trypsin 1. The results of immunoprecipitation and immunoadsorbent chromatography show that these different antisera recognized a protein of 25 kDa. Immunoblotting has permitted to characterize this protein as a trypsinogen 1-like molecule which would be a form of inert protein generated by uncontrolled trypsinogen activation.
Asunto(s)
Aminoácidos/análisis , Proteínas de Unión al Calcio/análisis , Proteínas del Tejido Nervioso , Jugo Pancreático/análisis , Tripsinógeno/análisis , Proteínas de Unión al Calcio/inmunología , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Humanos , Inmunodifusión , Técnicas de Inmunoadsorción , Litostatina , Pruebas de Precipitina , Tripsinógeno/inmunologíaRESUMEN
Seven polypeptides highly toxic to mice were isolated from the venom of the scorpion, Centruroides suffusus suffusus (Css), and their chemical and toxic properties were characterized. It was shown that the most active toxins by intracerebroventricular injection are less active when injected subcutaneously. The complete amino acid sequence (66 residues) of toxin II (Css II) has been determined. The C-terminal end is amidated as found for most other scorpion toxins. Css II is a beta-type toxin, previously used to define the binding site for activation of the sodium channel. Using rat brain synaptosomes, we demonstrated that all Css toxins compete with 125I-Css II to bind to site 4 and should be considered as beta-scorpion toxins. Specific binding parameters for Css VI, one of the most active toxins, were determined: KD = 100 pM; capacity in binding sites, 2.2 pmol of toxin/mg of synaptosomal protein. Css VI was shown to inhibit gamma-aminobutyric acid uptake by synaptosomes: K 0.5 = 100 pM, which agrees with its KD. Competition experiments between the seven Css toxins and 125I-Css II for antiserum raised against Css II demonstrated that all these toxins have common antigenic properties.
Asunto(s)
Venenos de Escorpión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Unión Competitiva , Encéfalo/metabolismo , Técnicas In Vitro , Canales Iónicos/metabolismo , Cinética , Ratones , Ratas , Venenos de Escorpión/metabolismo , Venenos de Escorpión/toxicidad , Sodio/metabolismo , Sinaptosomas/metabolismoRESUMEN
The complete amino acid sequence of toxin XI of the North African scorpion Buthus occitanus tunetanus has been elucidated by automatic sequencing of the reduced and alkylated toxin and of the peptides obtained after tryptic cleavage restricted to arginyl bonds. This toxin is structurally homologous to toxin II of Androctonus australis Hector, the most active among the alpha-toxins, but is far less potent, both in vivo and in vitro. This work points out 12 mutations, many of which are conservative. Nevertheless, the most striking difference is the replacement of the lysine residue at position 58, known to be important in the activity of AaH toxin II, by a valine residue. Thus, it seems that the presence of a positive charge at this location facilitates the interactions between the receptor on the sodium channel and the alpha-type toxins.
Asunto(s)
Neurotoxinas , Venenos de Escorpión , Secuencia de Aminoácidos , Animales , Mutación , Fragmentos de Péptidos/análisis , Venenos de Escorpión/genética , Venenos de Escorpión/aislamiento & purificación , Escorpiones , Especificidad de la Especie , TripsinaRESUMEN
A method of rapid and selective detection and purification of contracture-inducing insect toxins from Buthinae scorpion venoms is described in this paper. It consists of two main steps: the first one is specific as it uses immunoaffinity chromatography with antibodies against Androctonus australis Hector IT; the second, reverse phase high pressure liquid chromatography, allows the final separation of the different toxins present in each of the three venoms studied. Two, three and four insect toxins have been purified, respectively, from the venoms of Androctonus australis Hector, Buthus occitanus mardochei and Leiurus quinquestriatus quinquestriatus. This work demonstrates that, in Buthinae venoms, contracture-inducing insect toxins antigenically related to AaH IT, the first one purified, constitute the most important and, in some cases, the only toxins present.
Asunto(s)
Venenos de Escorpión/aislamiento & purificación , Aminoácidos/análisis , Animales , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Contractura/inducido químicamente , Inmunoquímica , Insectos/efectos de los fármacos , Venenos de Escorpión/inmunología , Ponzoñas/inmunología , Ponzoñas/aislamiento & purificaciónRESUMEN
The complete covalent structure of the insect toxin purified from the venom of the North-African scorpion Androctonus australis Hector was described. Its amino acid sequence was established by phenylisothiocyanate degradation of several protein derivatives and proteolytic fragments in a liquid protein sequencer using either a "protein" or a "peptide" program. The position of the four disulfide bridges were deduced by analysis of proteolytic peptides before and after diperformic oxidation, and by partial labeling of the half cystine residues with [14C]-iodoacetic acid and determining the specific radioactivities of the S-[14C]-carboxymethylated phenylthiohydantoin cysteines. The sequences of the insect and mammal toxins from scorpions can be aligned with homology with the positions of seven half-cystine residues as registers. The mammal and insect toxins have three disulfide bridges at homologous positions. The mammal and insect toxins have three disulfide bridges at homologous positions. The fourth bridge is different in that Cys12 in mammal toxin II is replaced by Cys38 in the insect toxin. It is likely that the position of the disulfide bridges is the same for all scorpion neurotoxins active on mammals. We believe that the shift of one half-cystine residue in the insect toxin may induce a conformational change in the structure of the protein, which, in turn, may partially account for the total specificity of this toxin for insect nervous system.
Asunto(s)
Venenos de Escorpión , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Fenómenos Químicos , Química , Quimotripsina , Disulfuros , Isotiocianatos , Venenos de Escorpión/aislamiento & purificación , Tiocianatos , TripsinaRESUMEN
The amino acid sequence of neurotoxin III, purified from the venom of the North African scorpion Androctonus australis Hector, has been determined by Edman degradation using a liquid-phase sequencer. Carboxypeptidase A hydrolyses confirmed not only the sequence of the five last residues but also the presence of a free alpha-carboxylic group at the C-terminus. Edman degradation was conducted on one hand with the Quadrol [N,N,N',N'-tetrakis(2-hydroxypropyl)ethylene diamine] program and S-alkylated protein before or after coupling with sulfophenylisothiocynate (the first 34 residues were thus identified), on the other hand on tryptic and chymotryptic peptides with a dimethylbenzylamine program (residues 1--23 and 31--34 were confirmed, the positions of residues 35-64 were established). Neurotoxin III was found to belong to the same group of scorpion toxins active on mammals as neurotoxin I purified from the same venom (50 homologous positions exist in the two proteins).