1. A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases
- Author
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Tamas Balla, Andrew W. Tai, and Naveen Bojjireddy
- Subjects
Adenosine ,Biophysics ,Biology ,Biochemistry ,Chromatography, Affinity ,Article ,Cell Line ,Wortmannin ,chemistry.chemical_compound ,Adenosine Triphosphate ,High-Throughput Screening Assays ,Animals ,Humans ,Dimethyl Sulfoxide ,Phosphatidylinositol ,Radiometry ,1-Phosphatidylinositol 4-Kinase ,Protein Kinase Inhibitors ,Molecular Biology ,Enzyme Assays ,Kinase ,RNA ,Cell Biology ,Molecular biology ,PI4K2A ,Adenosine Diphosphate ,Androstadienes ,Kinetics ,chemistry ,Isotope Labeling ,Cattle ,PI4KA ,PI4KB - Abstract
Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.
- Published
- 2011