title: A CE-based assay for human protein kinase CK2 activity measurement and inhibitor screening.
abstract: A new assay for protein kinase CK2 activity determination based on the quantification of a phosphorylated substrate was developed. The common CK2 substrate peptide RRRDDDSDDD, conjugated with the fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid at the C-terminus served as the analyte. By means of CZE using 2 mol/L acetic acid as electrolyte and UV detection at 214 nm, the non-phosphorylated and the phosphorylated peptide variants could be resolved within 6 min from a complex assay mixture. By this means, activity of human CK2 could be monitored by a kinetic, as well as an endpoint, method. Inhibition of human recombinant CK2 holoenzyme by 6-methyl-1,3,8-trihydroxyanthraquinone and 4,5,6,7-tetrabromobenzotriazole resulted in IC(50) values of 1.33 and 0.27 microM, respectively, which were similar to those obtained with the standard radiometric assay. These results suggest that the CE/UV strategy described here is a straightforward assay for CK2 inhibitor testing.
authors.affiliation: Bioanalytics, Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gratz
authors.forename: Claudia
authors.initials: C
authors.lastname: Götz
authors.forename: Joachim
authors.initials: J
authors.lastname: Jose
mesh_terms: D047390:Casein Kinase II / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D006801:Humans; D007700:Kinetics; D009282:Naphthalenesulfonates / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: D009282:Naphthalenesulfonates; D010455:Peptides; D010748:Phosphopeptides; D047428:Protein Kinase Inhibitors; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; D047390:Casein Kinase II