PURIFICATION AND CHARACTERIZATION OF A SOLUBLE NUCLEOSIDE DIPHOSPHATEKINASE IN TRYPANOSOMA-CRUZI

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme w as purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated ND P kinase was identified which accounts for 30% of total enzymatic acti vity. Western blot analysis of the soluble NDP kinase revealed a 16.5- kDa monomer recognized by polyclonal antibodies to NDP kinase from Dic tyostelium 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 mon omers. The K-m 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 fo und to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphoryla ted with [gamma-P-32]ATP or thiophosphorylated with [S-35]GTP gamma S. The incubation of the P-32-labelled phosphoenzyme with unlabelled nuc leoside 5'-diphosphates resulted in the formation of P-32-labelled nuc leoside 5'-triphosphates without strict base specificity, indicating t hat the reaction mechanism of the T. cruzi enzyme is the same as repor ted for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside S-diphosphates, GTP was preferentially formed.