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

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Nucleoside diphosphate (NDP) kinases are crucial enzymes involved in nucleotide metabolism.
• Accurate detection of NDP kinase activity is essential for biochemical research and quality control of enzyme preparations.
• Existing methods may lack sensitivity, real-time capability, or simplicity.

Purpose of the Study:

• To develop a novel, real-time, simple, and sensitive assay for quantifying NDP kinase activity.
• To characterize the kinetic parameters of NDP kinase using the developed assay.
• To demonstrate the utility of the assay for detecting enzyme contamination.

Main Methods:

• Development of a real-time assay utilizing the firefly luciferase system to detect ATP produced during NDP kinase reactions.
• Enzymatic assays were performed using various nucleoside triphosphates and adenosine diphosphate (ADP).
• Kinetic parameters (Km) were determined for ADP and different nucleoside triphosphates.

Main Results:

• The assay demonstrated high sensitivity with a detection limit of 0.05 mU of NDP kinase.
• Linear responses were observed for NDP kinase activity between 0.05-50 mU.
• Kinetic parameters were determined, showing varying affinities for different substrates, e.g., Km for ADP was ~30 microM for yeast NDP kinase.

Conclusions:

• A robust and sensitive real-time assay for NDP kinase activity has been successfully developed.
• The assay provides valuable kinetic data and is suitable for routine use in research and quality control.
• The method effectively detected NDP kinase contamination in commercial enzyme preparations, highlighting its practical application.