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 identif...
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todo:paper_01666851_v70_n1-2_p119_Ulloa2023-10-03T15:04:23Z Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi Ulloa, R.M. Muschietti, J.P. Veron, M. Torres, H.N. Tellez-Iñón, M.T. Nucleoside diphosphate kinase Phosphoenzyme intermediate Trypanosoma cruzi nucleoside diphosphate kinase article controlled study enzyme analysis enzyme purification immunoblotting nonhuman priority journal trypanosoma cruzi Adenosine Triphosphate Animal Cross Reactions Guanosine Diphosphate Kinetics Nucleoside-Diphosphate Kinase Phosphorylation Protein Conformation Protein Denaturation Protozoan Proteins Solubility Species Specificity Substrate Specificity Support, Non-U.S. Gov't Temperature Thymine Nucleotides Trypanosoma cruzi Candida albicans Dictyostelium discoideum Trypanosoma 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°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. © 1995. Fil:Muschietti, J.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Torres, H.N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_01666851_v70_n1-2_p119_Ulloa |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Nucleoside diphosphate kinase Phosphoenzyme intermediate Trypanosoma cruzi nucleoside diphosphate kinase article controlled study enzyme analysis enzyme purification immunoblotting nonhuman priority journal trypanosoma cruzi Adenosine Triphosphate Animal Cross Reactions Guanosine Diphosphate Kinetics Nucleoside-Diphosphate Kinase Phosphorylation Protein Conformation Protein Denaturation Protozoan Proteins Solubility Species Specificity Substrate Specificity Support, Non-U.S. Gov't Temperature Thymine Nucleotides Trypanosoma cruzi Candida albicans Dictyostelium discoideum Trypanosoma Trypanosoma cruzi |
| spellingShingle |
Nucleoside diphosphate kinase Phosphoenzyme intermediate Trypanosoma cruzi nucleoside diphosphate kinase article controlled study enzyme analysis enzyme purification immunoblotting nonhuman priority journal trypanosoma cruzi Adenosine Triphosphate Animal Cross Reactions Guanosine Diphosphate Kinetics Nucleoside-Diphosphate Kinase Phosphorylation Protein Conformation Protein Denaturation Protozoan Proteins Solubility Species Specificity Substrate Specificity Support, Non-U.S. Gov't Temperature Thymine Nucleotides Trypanosoma cruzi Candida albicans Dictyostelium discoideum Trypanosoma Trypanosoma cruzi Ulloa, R.M. Muschietti, J.P. Veron, M. Torres, H.N. Tellez-Iñón, M.T. Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi |
| topic_facet |
Nucleoside diphosphate kinase Phosphoenzyme intermediate Trypanosoma cruzi nucleoside diphosphate kinase article controlled study enzyme analysis enzyme purification immunoblotting nonhuman priority journal trypanosoma cruzi Adenosine Triphosphate Animal Cross Reactions Guanosine Diphosphate Kinetics Nucleoside-Diphosphate Kinase Phosphorylation Protein Conformation Protein Denaturation Protozoan Proteins Solubility Species Specificity Substrate Specificity Support, Non-U.S. Gov't Temperature Thymine Nucleotides Trypanosoma cruzi Candida albicans Dictyostelium discoideum Trypanosoma Trypanosoma cruzi |
| description |
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. © 1995. |
| format |
JOUR |
| author |
Ulloa, R.M. Muschietti, J.P. Veron, M. Torres, H.N. Tellez-Iñón, M.T. |
| author_facet |
Ulloa, R.M. Muschietti, J.P. Veron, M. Torres, H.N. Tellez-Iñón, M.T. |
| author_sort |
Ulloa, R.M. |
| title |
Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi |
| title_short |
Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi |
| title_full |
Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi |
| title_fullStr |
Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi |
| title_full_unstemmed |
Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi |
| title_sort |
purification and characterization of a soluble nucleoside diphosphate kinase in trypanosoma cruzi |
| url |
http://hdl.handle.net/20.500.12110/paper_01666851_v70_n1-2_p119_Ulloa |
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