Measurement of nucleoside diphosphate kinase-Nm23 activity by anion-exchange high-performance liquid chromatography.
A first-order assay to detect the activity of nucleoside diphosphate kinase (NDP-kinase; EC 2.7.4.6) was developed. In this assay, the activity of NDP-kinase is measured using various deoxy- and ribonucleotide triphosphates as phosphate donors and dADP as phosphate acceptor. The enzyme activity is determined by quantifying, after anion-exchange HPLC, the amount of newly synthesized dATP. Contrary to the most common coupled enzymic assays or isotopic assays the use of different donor-acceptor pairs is not restricted. The resolution of the procedure described is limited only by the chromatographic separation of substrate and product pairs participating in the reaction.
National Laboratory for Viral Oncology, LCDC, Health Canada, Ottawa, Ontario.
G
G
Pulido-Cejudo
J
J
Gagnon
J M
JM
Leclerc
K
K
Jamison
J
J
Gordon
J
J
Campione-Piccardo
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009701:Nucleoside-Diphosphate Kinase / Q000032:analysis*; D010710:Phosphates / Q000737:chemistry; D012265:Ribonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D010710:Phosphates; D012265:Ribonucleotides; D006160:Guanosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase