Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi.
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 degrees C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [gamma-32P]ATP or thiophosphorylated with [35S]GTP gamma S. 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.
Ulloa
R M
RM
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Buenos Aires, Argentina.
Muschietti
J P
JP
Veron
M
M
Torres
H N
HN
Tellez-Iñón
M T
MT
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003429:Cross Reactions; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D009701:Nucleoside-Diphosphate Kinase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D015800:Protozoan Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature; D013942:Thymine Nucleotides / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000254:growth & development / Q000276:immunology
D015800:Protozoan Proteins; D013942:Thymine Nucleotides; D006153:Guanosine Diphosphate; C019111:thymidine 3',5'-diphosphate; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase