Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity

A real-time, simple and sensitive method for detection of nucleoside diphos phate (NDP) kinase activity has been developed. The assay is based on detec tion of ATP generated in the NDP kinase reaction between a nucleoside triph osphate and adenosine diphosphate (ADP), by the firefly luciferase system I n the presence of 0.3 mM dGTP, the K-m, for ADP was found to be approximate ly 30 mu M for the NDP kinase from Baker's yeast. In the presence of 250 mu M ADP: the K-m, for dATP alpha S dTTP alpha S, dGTP, dTTP, dCTP and GTP wa s found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respec tively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method w as used to detect NDP kinase contamination in commercial enzyme preparation s.