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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.

The catalytic subunit of cyclic AMP-dependent protein kinase from Trypanosoma cruzi epimastigote forms was purified by ionic-exchange chromatography, affinity chromatography and sucrose gradient centrifugation. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the purified preparations showed a main polypeptide band with a mobility of about 40 kDa. In Western blots this band immunoreacted with a polyclonal antibody specific for the catalytic subunit of bovine heart protein kinase A. Hydrodynamic and molecular parameters of this subunit are as follows: molecular weight, 40,000 +/- 3000; sedimentation constant, 2.8 +/- 0.3 S; Stokes' radius, 2.8 +/- 0.2 nm; frictional ratio, 1.28 +/- 0.05. Purified preparations of T. cruzi catalytic and regulatory subunits reconstitute a holoenzyme with a sedimentation constant. 8.6 +/- 1.17 S. This data together with those previously reported by Ulloa et al. [8] indicate that the T. cruzi cyclic AMP-dependent protein kinase holoenzyme is a tetramer with the structure R2C2 of about 200 kDa. The apparent Km of the catalytic subunit for ATP and histone IIA or kemptide as phosphate donor and acceptor, respectively, were 40 microM, 48.6 microM and 26 microM, respectively.

Calmodulin has been purified from Trypanosoma cruzi epimastigote forms by ion-exchange chromatography, gel filtration and affinity chromatography on 2-chloro-10-(3-aminopropyl)phenotiazine-Sepharose. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the factor showed a polypeptide band with an apparent molecular weight of 16 000. In addition, cyclic AMP phosphodiesterase activity from T. cruzi epimastigote forms was purified by ion-exchange chromatography and affinity chromatography on a brain calmodulin-Sepharose column. The enzyme was activated by homologous calmodulin as well as by bovine brain and Neurospora crassa calmodulins. The activation required micromolar concentrations of Ca2+ and it was blocked by EGTA and by some neuroleptic drugs such as chlorpromazine, fluphenazine and compound 48/80. Activations were observed at micromolar concentrations of cyclic AMP as substrate. In addition, T. cruzi calmodulin was also active in bringing about the stimulation of brain phosphodiesterase.