Once released outside the cell, adenosine, a naturally occurring ribonucleoside, reveals strong neuroprotective and anti-inflammatory properties. One of the strategies to increase extracellular adenosine consists in inhibiting adenosine kinase (ADK), a ubiquitous enzyme that catalyzes the transfer of _-phosphate from ATP to the 5'-hydroxyl of adenosine generating AMP and ADP.
ADK Assay Kit is a non isotopic assay that allows a continuous monitoring of ADK activity at 340nm through the coupling of ADK-catalyzed phosphorylation of inosine to the oxidation of IMP by a recombinant IMP-dehydrogenase (IMPDH). The assay developed in microplate format allows a HTS search of novel ADK inhibitors.
ADK Assay Kit was validated with a known ADK inhibitor (A-134974, IC50=20nM) and meets the requirements of a convenient and reliable HTS assay (microplate format, add-and measure, spectrophotometric continuous readout, Z-factor = 0.68).
ADK Assay Kit can be also used to evaluate the phosphorylation of novel nucleoside analogues. In the absence of nucleoside
competitor, adenosine kinase phosphorylates inosine resulting in the formation of IMP directly monitored by the formation kinetics of NADH2 catalyzed by IMP-dehydrogenase. In the presence of nucleoside competitor, the phosphorylation of inosine, poor ADK substrate, is inhibited and detected as a decrease in NADH2 formation.