Fluorometric TLC assay for evaluation of protein kinase inhibitors.
A fluorometric assay for measuring protein kinase activity has been developed. The assay is based on the separation of fluorescently marked substrate 5-carboxytetramethylrhodamine-kemptide (5-TAMRA-kemptide) from its phosphorylated counterpart by TLC and quantification of the product ratiometrically by fluorescence imaging. The utility of the assay was demonstrated by measuring the activity of cAMP-dependent protein kinase. 5-TAMRA-kemptide was characterized as a substrate of this kinase by the kinetic parameters K(m)(app) and V(max). The attachment of 5-TAMRA dye to the N terminal of kemptide decreased the K(m)(app) value but did not have a significant effect on the rate and stoichiometry of the phosphorylation reaction. The inhibitory potency of three known inhibitors was evaluated with the new assay. The closeness of the obtained inhibitory activities of the compounds to the activities determined with the phosphocellulose paper-binding assay, as well as the Z' factor value of 0.5, demonstrates the reliability of the new assay for evaluation of inhibitors of protein kinases.
Viht
Kaido
K
Institute of Organic and Bioorganic Chemistry, University of Tartu, 2 Jakobi Street, 51014 Tartu, Estonia.
Vaasa
Angela
A
Raidaru
Gerda
G
Enkvist
Erki
E
Uri
Asko
A
D002855:Chromatography, Thin Layer / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D015203:Reproducibility of Results; D012235:Rhodamines; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D012235:Rhodamines; C037642:kemptide; D011494:Protein Kinases