Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi |
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Authors: | Rita M Ulloa Jorge P Muschietti Michel Veron H ctor N Torres and Marí a T Tellez-I n |
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Institution: | a Instituto de Investigaciones en Ingeniería Genética y Biología Molecular and Facultad de Ciencias Exactas y Naturales. Obligado 2490, 1428, Buenos Aires, Argentina b Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France |
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Abstract: | A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The Km values of the enzyme for ATP, GDP and dTDP were 0.2 ± 0.008 mM, 0.125 ± 0.012 mM and 0.4 ± 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65°C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with γ-32P]ATP or thiophosphorylated with 35S]GTPγS. The incubation of the 32P-labelled phosphoenzyme with unlabelled nucleoside 5′-diphosphates resulted in the formation of 32P-labelled nucleoside 5′-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5′-diphosphates, GTP was preferentially formed. |
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Keywords: | Nucleoside diphosphate kinase Trypanosoma cruzi Phosphoenzyme intermediate |
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