ADK Phosphorylation Assay Kit was specially designed to evaluate the phosphorylation of novel nucleoside analogues. The assay
allows the test of 12 analogues at 7 different concentrations at the same time (or 6 analogues in duplicata).
This assay is based on competitive inhibition of inosine (IR) phosphorylation by ADK enzyme. In the absence of nucleoside competitor, adenosine kinase phosphorylates inosine resulting in the formation of IMP. IMP is further oxidized by IMPDH to XMP and NADH2.This coupling reaction is immediate when IMPDH activity is much higher than ADK activity in the assay. The enzymatic activity of ADK, which corresponds to the formation kinetics of IMP, is then stoichiometrically and directly monitored by the formation kinetics of NADH2. In the presence of nucleoside competitor, the phosphorylation of inosine, poor ADK substrate, is inhibited detected as a decrease in NADH2 formation.
This assay was validated with antiviral nucleoside analogues such as mizoribine, tubercidine, ribavirin.