RESUMEN
The soluble venom from the scorpion Tityus metuendus was characterized by various methods. In vivo experiments with mice showed that it is lethal. Extended electrophysiological recordings using seven sub-types of human voltage gated sodium channels (hNav1.1 to 1.7) showed that it contains both α- and ß-scorpion toxin types. Fingerprint analysis by mass spectrometry identified over 200 distinct molecular mass components. At least 60 sub-fractions were recovered from HPLC separation. Five purified peptides were sequenced by Edman degradation, and their complete primary structures were determined. Additionally, three other peptides have had their N-terminal amino acid sequences determined by Edman degradation and reported. Mass spectrometry analysis of tryptic digestion of the soluble venom permitted the identification of the amino acid sequence of 111 different peptides. Search for similarities of the sequences found indicated that they probably are: sodium and potassium channel toxins, metalloproteinases, hyaluronidases, endothelin and angiotensin-converting enzymes, bradykinin-potentiating peptide, hypothetical proteins, allergens, other enzymes, other proteins and peptides.
Asunto(s)
Venenos de Escorpión/química , Venenos de Escorpión/toxicidad , Escorpiones , Secuencia de Aminoácidos , Animales , Células CHO , Cricetulus , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Péptidos/química , Proteoma , Bloqueadores de los Canales de Sodio , Canales de Sodio/efectos de los fármacosRESUMEN
The venom from the Brazilian scorpion Tityus stigmurus was fractionated by high performance liquid chromatography (HPLC) and the corresponding components were used for molecular mass determination using electrospray ion trap mass spectrometry. One hundred distinct components were clearly assigned showing molecular masses from 216.5 to 44,800.0 Da. Fifteen new components were isolated and sequenced, four of them to completion: Tst-3 (similar to Na(+) channel specific scorpion toxins), Tst-17 (a K(+) channel blocking peptide similar to Tc1), Tst beta KTx (a peptide with identical sequence as that of TsTX-K beta toxin earlier described to exist in T. serrulatus venom) and finally a novel proline-rich peptide of unknown function. Among the eleven components partially sequenced were two enzymes: hyaluronidase and lysozyme. The first enzyme has a molecular mass of 44,800.0 Da. This enzyme showed high activity against the substrate hyaluronan in vitro. Amino acid sequence of the second enzyme showed that it is similar to other known lysozymes, with similar molecular mass and sequence to that of bona fide lysozymes reported in public protein data banks. Finally, this communication reports a correlation among HPLC retention times and molecular masses of folded scorpion toxins as well as a comparative structural and physiological analysis of components from the venom of several species of the genus Tityus.
Asunto(s)
Proteínas de Insectos/química , Bloqueadores de los Canales de Potasio/química , Proteómica , Venenos de Escorpión/química , Escorpiones , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Electrofisiología , Hialuronoglucosaminidasa/análisis , Proteínas de Insectos/farmacología , Datos de Secuencia Molecular , Peso Molecular , Muramidasa/análisis , Técnicas de Placa-Clamp , Mapeo Peptídico , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/farmacología , Canales de Potasio de la Superfamilia Shaker/efectos de los fármacos , Canales de Potasio de la Superfamilia Shaker/metabolismo , Especificidad de la Especie , Espectrometría de Masa por Ionización de Electrospray , Spodoptera/citología , Spodoptera/efectos de los fármacosRESUMEN
A new arthropod selective toxin was purified from the venom of the Venezuelan scorpion Tityus discrepans, and its amino acid sequence, cDNA clone and biological activity are reported here. The amino acid sequence of this peptide, named ardiscretin (from arthropod toxin of T. discrepans) was completed by Edman degradation and mass spectrometry. It is a single polypeptide composed by 61 amino acids with an amidated cysteine residue at the C-terminal end, closely packed by four disulfide bridges. The atomic mass unit (a.m.u.) experimentally determined was 7103.8 a.m.u. This peptide was shown to be specific for invertebrates (crickets, triatomides, crabs and squids), but non-toxic to mice, at the dose assayed. Ardiscretin inhibits the Na(+)-currents of squid giant axons in an apparent irreversible manner, whose inhibitory effect is reached at 30 microM toxin concentration. Sequence comparison showed that it is phylogenetically closely related to insect-specific scorpion toxins. Ardiscretin produced a small depolarization and induced repetitive firing in squid axons resembling those of DDT [1,1'(p-chlorobenzyl)2-tricloretane] in its ability to slow down action potential, to induce repetitive firing, and in that the concentration required for any effect in squid axon is rather high.
Asunto(s)
Neurotoxinas/química , Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Artrópodos/efectos de los fármacos , Secuencia de Bases , Cartilla de ADN , Masculino , Ratones , Datos de Secuencia Molecular , Neurotoxinas/genética , Neurotoxinas/farmacología , Filogenia , Reacción en Cadena de la Polimerasa , Venenos de Escorpión/genética , Venenos de Escorpión/farmacología , Alineación de Secuencia , Canales de Sodio/efectos de los fármacosRESUMEN
Two almost identical proteins with 70 amino acid residues each, closely packed by four disufide bridges, and molecular masses of 7899.5 and 7884.7 were isolated and sequenced from the venom of the scorpion Isometrus vittatus from Pakistan. They differ by an acidic amino acid residue (glutamic or aspartic) at the same position 55 of the peptide chain, however, they exhibit the same length, the same charge and are undistinguishable when separated by C(18) reverse phase HPLC. The mixture of the two proteins called IsomTx1 depolarizes the cockroach isolated axon; artificial repolarization is followed by sustained repetitive activity, artificial hyperpolarization facilitates bursting activity observed as an answer to rapid depolarization to -60 mV. The depolarization is antagonized by TTX. In voltage-clamp experiments IsomTx1 increases axonal sodium permeability which has a particular importance between resting and threshold potentials and moderately slows down the fast inactivation. These characteristics closely resemble those of other anti-insect scorpion toxins classified as contractive toxins from Androctonus and Buthotus venoms.