RESUMO
A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M(r)=61,000, pI approximately 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans to improve thrombin-like activity of BjussuSP-I toxin.
Assuntos
Fatores de Coagulação Sanguínea/química , Bothrops , Venenos de Crotalídeos/enzimologia , Serina Endopeptidases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Coagulação Sanguínea/isolamento & purificação , Fatores de Coagulação Sanguínea/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Calicreínas/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Trombina/química , Tempo de TrombinaAssuntos
Fatores de Coagulação Sanguínea/efeitos adversos , Trombose , Animais , Argentina , Fatores de Coagulação Sanguínea/administração & dosagem , Fatores de Coagulação Sanguínea/química , Modelos Animais de Doenças , Combinação de Medicamentos , Desenho de Fármacos , Feminino , Humanos , Ratos , Trombose/induzido quimicamente , Trombose/prevenção & controleRESUMO
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.