Phosphatase-coupled universal kinase assay and kinetics for first-order-rate coupling reaction.

ABSTRACT: Kinases use adenosine-5'-triphosphate (ATP) as the donor substrate and generate adenosine-5'-diphosphate (ADP) as a product. An ADP-based phosphatase-coupled kinase assay is described here. In this assay, CD39L2, a nucleotidase, is added into a kinase reaction to hydrolyze ADP to AMP and phosphate. The phosphate is subsequently detected using malachite green phosphate-detection reagents. As ADP hydrolysis by CD39L2 displays a first-order rate constant, relatively simple equations are derived to calculate the coupling rate and the lagging time of the coupling reaction, allowing one to obtain kinase kinetic parameters without the completion of the coupling reaction. ATP inhibition of CD39L2-catalyzed ADP hydrolysis is also determined for correction of the kinetic data. As examples, human glu

SUBMITTER: Wu ZL 

PROVIDER: S-EPMC3154929 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature