| Abstract
| - We report a novel, real-time fluorogenic kinase assay. Thepeptide substrates are synthesized with a fluorescent dyeand a hydrocarbon tail. The substrate self-assembles intomicelles, increasing the local concentration of the dye andquenching its fluorescence. Upon phosphorylation, thefluorescence intensity increases 4−6-fold due to micellereorganization. Both dynamic light scattering data andcryoelectron microscope images show that the size andthe shape of the phosphopeptide micelles are significantlydifferent from micelles of substrate peptide. The systemprovides a robust fluorescence increase in a real-timeprotein kinase assay. Unlike other fluorogenic systems,the fluorophore may be distant from the serine, threonine,or tyrosine that is phosphorylated. Assays for severalkinases, including PKA, PKC, p38, MAPKAP K2, akt,Erk1, and src-family kinases, have been developed. IC50values of inhibitors for PKC βII determined with thistechnology are consistent with published values. Theutility of this assay to high-throughput screening wasdemonstrated with Sigma's LOPAC library, a collectionof 640 compounds with known biological activities, andsatisfactory results were obtained.
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