RESUMO
A cDNA with 403 nucleotides encoding the precursor of the toxin PnTx2-6 was cloned and sequenced. Subsequent analysis revealed that the precursor begins with a signal peptide and a glutamate-rich propeptide. The succeeding peptide confirmed the reported sequence of PnTx2-6. The purified toxin exerted complex effects on Na(+) current of frog skeletal muscle. There was a marked decrease of the inactivation kinetics, and a shift to hyperpolarizing potentials of both the Na(+) conductance and the steady-state inactivation voltage dependences, along with a reduction of the current amplitude. The concentration dependence of the modified current suggests a K(D) of 0.8 microM for the toxin-channel complex.
Assuntos
Músculo Esquelético/efeitos dos fármacos , Neuropeptídeos/farmacologia , Canais de Sódio/fisiologia , Aranhas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/análise , Condutividade Elétrica , Eletrofisiologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Cinética , Dados de Sequência Molecular , Músculo Esquelético/fisiologia , Neuropeptídeos/genética , Peptídeos/genética , Peptídeos/farmacologia , Rana catesbeiana , Canais de Sódio/efeitos dos fármacos , Venenos de Aranha/genética , Venenos de Aranha/farmacologia , Aranhas/genéticaRESUMO
The present experiments investigated the effect of a neurotoxin purified from the venom of the spider Phoneutria nigriventer. This toxic component, P. nigriventer toxin 3-6 (PnTx3-6), abolished Ca(2+)-dependent glutamate release with an IC(50) of 74.4nM but did not alter Ca(2+)-independent secretion of glutamate when brain cortical synaptosomes were depolarized by KCl (33mM). This effect was most likely due to interference with the entry of calcium through voltage activated calcium channels (VACC), reducing the increase in the intrasynaptosomal free calcium induced by membrane depolarization with an IC(50) of 9.5nM. We compared the alterations induced by PnTx3-6 with the actions of toxins known to block calcium channels coupled to exocytosis. Our results indicate that PnTx3-6 inhibition of glutamate release and intrasynaptosomal calcium involves P/Q type calcium channels and this toxin can be a valuable tool in the investigation of calcium channels.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Neuropeptídeos/farmacologia , Potássio/farmacologia , Sinaptossomos/metabolismo , Animais , Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Eletrofisiologia , Feminino , Técnicas In Vitro , Masculino , Neuropeptídeos/isolamento & purificação , Potássio/antagonistas & inibidores , Ratos , Ratos Wistar , Sinaptossomos/efeitos dos fármacosRESUMO
Tx1 from the venom of the Brazilian spider, Phoneutria nigriventer, is a lethal neurotoxic polypeptide of M(r) 8600 Da with 14 cysteine residues. It is a novel sodium channel blocker which reversibly inhibits sodium currents in CHO cells expressing recombinant sodium (Nav1.2) channels. We cloned and expressed the Tx1 toxin as a thioredoxin fusion product in the cytoplasm of Escherichia coli. After semipurification by immobilized Ni-ion affinity chromatography, the recombinant Tx1 was purified by reverse phase chromatography and characterized. It displayed similar biochemical and pharmacological properties to the native toxin, and it should be useful for further investigation of structure-function relationship of Na channels.
Assuntos
Neuropeptídeos/isolamento & purificação , Bloqueadores dos Canais de Sódio/isolamento & purificação , Canais de Sódio/efeitos dos fármacos , Venenos de Aranha/genética , Aranhas/genética , Sequência de Aminoácidos , Animais , Ligação Competitiva/efeitos dos fármacos , Brasil , Células CHO , Clonagem Molecular , Cricetinae , Injeções Intraventriculares , Camundongos , Dados de Sequência Molecular , Neuropeptídeos/biossíntese , Neuropeptídeos/toxicidade , Ligação Proteica , Ratos , Ratos Wistar , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/toxicidade , Sódio/metabolismo , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/toxicidade , Canais de Sódio/metabolismo , Especificidade da Espécie , Venenos de Aranha/química , Relação Estrutura-AtividadeRESUMO
A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (0.8 microg/g) was shown to lower blood pressure in experimental rats. In vitro, the isolated proteinase was shown to have neither fibrin(ogeno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity=13.0 and 31.5 U/mg, respectively; crude venom=0.25 and 6.0 U/mg) but had no proteolytic effect on dimethylcasein and insulin B chain. Its enzymatic activity was inhibited by NPGB and PMSF, indicating that the enzyme is a serine proteinase. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen was one of the activities exhibited by LV-Ka.