title: 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 glucokinase, P. chrysogenum APS kinase and human ERK1, kinases specific for sugar, nucleotide and protein respectively, are assayed. To assess the compatibility of the method for high-throughput assays, Z' factors >0.5 are also obtained for the three kinases.
authors.affiliation: R&D Systems Inc., Minneapolis, Minnesota, United States of America. leon.wu@rndsystems.com
authors.forename: Zhengliang L
authors.initials: ZL
authors.lastname: Wu
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005941:Glucokinase / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D051379:Mice; D010407:Penicillium / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D005941:Glucokinase; C019617:adenylylsulfate kinase; D048049:Extracellular Signal-Regulated MAP Kinases; D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases; C027201:ectoATPase