Candida albicans nucleoside-diphosphate kinase: Purification and characterization
Soluble nucleoside-diphosphate kinase (NDP kinase) fromCandida albicanswas purified to electrophoretic homogeneity and a partial sequence was determined. The enzyme was kinetically and physically characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed...
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1995
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paper:paper_00039861_v323_n1_p187_Biondi2023-06-08T14:25:02Z Candida albicans nucleoside-diphosphate kinase: Purification and characterization Biondi, Ricardo M. Walz, Katherina adenosine triphosphate antibody cytidine triphosphate guanosine diphosphate guanosine triphosphate nucleoside diphosphate kinase phosphate phosphoprotein polypeptide thymidine triphosphate uridine triphosphate article autoradiography candida albicans catalysis enzyme activity enzyme purification isoelectric focusing nonhuman polyacrylamide gel electrophoresis priority journal thin layer chromatography Amino Acid Sequence Candida albicans Kinetics Molecular Sequence Data Nucleoside-Diphosphate Kinase Phosphorylation Sequence Alignment Support, Non-U.S. Gov't Candida albicans Soluble nucleoside-diphosphate kinase (NDP kinase) fromCandida albicanswas purified to electrophoretic homogeneity and a partial sequence was determined. The enzyme was kinetically and physically characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide of 17 kDa upon staining, by immunodetection with heterologous and homologous antibodies, and by autoradiography of the phosphorylated enzyme. Furthermore, isoelectric focusing of the purified native enzyme showed a single acidic band (pI4.5). These results together with a native molecular mass of 98 kDa suggest a hexameric native enzyme composed of identical subunits. Like NDP kinases from other sources, the catalysis involves a phosphoenzyme intermediate that is rapidly formed upon incubation of the enzyme with ATP. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. Kinetic experiments indicated that GTP and ATP had the lowestKmcompared to UTP, dTTP, and CTP. GDP acted as a preferred acceptor as assessed by kinetic measurements as well as by competition experiments. Experimental data are presented indicating the existence of a membrane-associated NDP kinase. Preliminary characterization of this enzyme suggests that cytosolic and membrane-associated NDP kinases are similar proteins. © 1995 Academic Press, Inc. Fil:Biondi, R.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Walz, K. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 1995 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00039861_v323_n1_p187_Biondi http://hdl.handle.net/20.500.12110/paper_00039861_v323_n1_p187_Biondi |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
adenosine triphosphate antibody cytidine triphosphate guanosine diphosphate guanosine triphosphate nucleoside diphosphate kinase phosphate phosphoprotein polypeptide thymidine triphosphate uridine triphosphate article autoradiography candida albicans catalysis enzyme activity enzyme purification isoelectric focusing nonhuman polyacrylamide gel electrophoresis priority journal thin layer chromatography Amino Acid Sequence Candida albicans Kinetics Molecular Sequence Data Nucleoside-Diphosphate Kinase Phosphorylation Sequence Alignment Support, Non-U.S. Gov't Candida albicans |
| spellingShingle |
adenosine triphosphate antibody cytidine triphosphate guanosine diphosphate guanosine triphosphate nucleoside diphosphate kinase phosphate phosphoprotein polypeptide thymidine triphosphate uridine triphosphate article autoradiography candida albicans catalysis enzyme activity enzyme purification isoelectric focusing nonhuman polyacrylamide gel electrophoresis priority journal thin layer chromatography Amino Acid Sequence Candida albicans Kinetics Molecular Sequence Data Nucleoside-Diphosphate Kinase Phosphorylation Sequence Alignment Support, Non-U.S. Gov't Candida albicans Biondi, Ricardo M. Walz, Katherina Candida albicans nucleoside-diphosphate kinase: Purification and characterization |
| topic_facet |
adenosine triphosphate antibody cytidine triphosphate guanosine diphosphate guanosine triphosphate nucleoside diphosphate kinase phosphate phosphoprotein polypeptide thymidine triphosphate uridine triphosphate article autoradiography candida albicans catalysis enzyme activity enzyme purification isoelectric focusing nonhuman polyacrylamide gel electrophoresis priority journal thin layer chromatography Amino Acid Sequence Candida albicans Kinetics Molecular Sequence Data Nucleoside-Diphosphate Kinase Phosphorylation Sequence Alignment Support, Non-U.S. Gov't Candida albicans |
| description |
Soluble nucleoside-diphosphate kinase (NDP kinase) fromCandida albicanswas purified to electrophoretic homogeneity and a partial sequence was determined. The enzyme was kinetically and physically characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide of 17 kDa upon staining, by immunodetection with heterologous and homologous antibodies, and by autoradiography of the phosphorylated enzyme. Furthermore, isoelectric focusing of the purified native enzyme showed a single acidic band (pI4.5). These results together with a native molecular mass of 98 kDa suggest a hexameric native enzyme composed of identical subunits. Like NDP kinases from other sources, the catalysis involves a phosphoenzyme intermediate that is rapidly formed upon incubation of the enzyme with ATP. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. Kinetic experiments indicated that GTP and ATP had the lowestKmcompared to UTP, dTTP, and CTP. GDP acted as a preferred acceptor as assessed by kinetic measurements as well as by competition experiments. Experimental data are presented indicating the existence of a membrane-associated NDP kinase. Preliminary characterization of this enzyme suggests that cytosolic and membrane-associated NDP kinases are similar proteins. © 1995 Academic Press, Inc. |
| author |
Biondi, Ricardo M. Walz, Katherina |
| author_facet |
Biondi, Ricardo M. Walz, Katherina |
| author_sort |
Biondi, Ricardo M. |
| title |
Candida albicans nucleoside-diphosphate kinase: Purification and characterization |
| title_short |
Candida albicans nucleoside-diphosphate kinase: Purification and characterization |
| title_full |
Candida albicans nucleoside-diphosphate kinase: Purification and characterization |
| title_fullStr |
Candida albicans nucleoside-diphosphate kinase: Purification and characterization |
| title_full_unstemmed |
Candida albicans nucleoside-diphosphate kinase: Purification and characterization |
| title_sort |
candida albicans nucleoside-diphosphate kinase: purification and characterization |
| publishDate |
1995 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00039861_v323_n1_p187_Biondi http://hdl.handle.net/20.500.12110/paper_00039861_v323_n1_p187_Biondi |
| work_keys_str_mv |
AT biondiricardom candidaalbicansnucleosidediphosphatekinasepurificationandcharacterization AT walzkatherina candidaalbicansnucleosidediphosphatekinasepurificationandcharacterization |
| _version_ |
1768544065019707392 |