A scintillation proximity assay for studying inhibitors of human tau protein kinase II/cdk5 using a 96-well format.
Dysregulation of the brain-specific tau protein kinase II (TPK II)/cdk5 is reported to play an important role in the pathogenesis of Alzheimer's disease. We report here a quantitative scintillation proximity assay (SPA), which is suitable for determining TPK II/cdk5 activity and its inhibition. It depends upon the phosphorylation of a synthetic histone-based peptide substrate (PKTPKKAKKL), which has been biotinylated at its C-terminus. When this biotinylated peptide is incubated with [gamma-33P] ATP and TPK II/cdk5 under defined assay conditions, product formation is linear with respect to time and enzyme concentration. The production of [33P] phosphorylated peptide is inhibited in the presence of a known TPK II/cdk5 inhibitor but is unaffected in the presence of 1% DMSO. A signal-to-noise ratio of 16:1 was obtained in a 60-min assay with an intra-assay variability of <10% in the 96-well microtiter format. The TPK II/cdk5 SPA is very robust, sensitive and simple to perform.
Evans
David B
DB
Protein Science, Pharmacia Corporation, 7000 Portage Road, 7240-267-117, Kalamazoo, MI 49007, USA.
Rank
Kenneth B
KB
Sharma
Satish K
SK
D000544:Alzheimer Disease / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000032:analysis / Q000037:antagonists & inhibitors*; D006657:Histones / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors*; D012588:Scintillation Counting / Q000379:methods*; D013379:Substrate Specificity
D006657:Histones; D009842:Oligopeptides; D010761:Phosphorus Radioisotopes; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; C495905:CDK5 protein, human; D018844:Cyclin-Dependent Kinases; C079134:tau protein kinase II