Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi

ULLOA RM; MUSCHIETTI JP; VERON M; TORRES HN; TÉLLEZ-IÑÓN MT

MOLECULAR AND BIOCHEMICAL PARASITOLOGY

ELSEVIER

Año: 1995 vol. 70 p. 119 - 129

0166-6851

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms ofsoluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypan~~~ma 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 16.5kDa 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 K, values of the enzyme for ATP, GDP and dTDP were 0.2 f 0.008 mM, 0.125 f 0.012 mM and 0.4 k 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 [ Y-~~P]ATP or thiophosphorylated with [35S]GTpyS. 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.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 16.5kDa 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 K, values of the enzyme for ATP, GDP and dTDP were 0.2 f 0.008 mM, 0.125 f 0.012 mM and 0.4 k 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 [ Y-~~P]ATP or thiophosphorylated with [35S]GTpyS. 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.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 K, values of the enzyme for ATP, GDP and dTDP were 0.2 f 0.008 mM, 0.125 f 0.012 mM and 0.4 k 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 [ Y-~~P]ATP or thiophosphorylated with [35S]GTpyS. 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.K, values of the enzyme for ATP, GDP and dTDP were 0.2 f 0.008 mM, 0.125 f 0.012 mM and 0.4 k 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 [ Y-~~P]ATP or thiophosphorylated with [35S]GTpyS. 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.[ Y-~~P]ATP or thiophosphorylated with [35S]GTpyS. 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.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.