Expressão heteróloga, purificação e busca de inibidores da NTPDase-1 de Trypanosoma cruzi

• Autor(a) principal: Moura, Christiane Mariotini
• Data de Publicação: 2010
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2327
• topic: Expressão heteróloga; Purificação; Busca de inibidores da NTPDase-1 de Trypanosoma cruzi; Heterologous expression; Purification; Search for inhibitors of the NTPDase-1 of Trypanosoma cruzi; CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

The Chagas’ Disease is an antropozoonose caused by Trypanosoma cruzi, a flagellate protozoan of order Kinetoplastida. T. cruzi presents an ectonucleotidase on its surface, named NTPDase-1. The ectonucleotidases of the E-NTPDase family are enzymes capable of hydrolyse nucleotides tri and/or diphosphates that can be degraded to nucleosides by other ectonucleotidases. Extracellular nucleotides act as signaling molecules in several celullar processes, such as modulation of immune response of mammalian hosts. The ATPe hydrolysis plays a role on T. cruzi infectivity and virulence, as in other parasites such as, Leishmania, Trichomonas, Toxoplasma and Entamoeba. Therefore, NTPDases are taken as potencial targets for the rational design of new drugs for the treatment of Chagas’ disease. In this work we performed the parcial purification of TcNTPDase-1 and produced polyclonal antibodies against it. Futhermore, we evaluated the stability of its activity with respect to time and temperature of storage, standardized an inhibition assay and performed a screening of synthetic adenine analogues as potential inhibitors of NTPDase-1. It was observed that the storage temperature influenced the enzyme activity over time, and temperature of 4 º C was the most efficient to preserve this activity. The purified protein sample is heterogeneous, with inactive oligomers and active monomers and/or dimers. The ATPase activity can be inhibited by adenine and analogues and this inhibition seems to be competitive.