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Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives
- Source :
- Analytical and Bioanalytical Chemistry. 401:1881-1888
- Publication Year :
- 2011
- Publisher :
- Springer Science and Business Media LLC, 2011.
-
Abstract
- Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP(2) derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.
- Subjects :
- Phosphatidylinositol 4,5-Diphosphate
chemistry.chemical_classification
Cell type
Chemistry
Kinetic analysis
Electrophoresis, Capillary
Sensitivity and Specificity
Biochemistry
Fluorescence
Article
Thin-layer chromatography
Analytical Chemistry
Kinetics
chemistry.chemical_compound
Capillary electrophoresis
Enzyme
Chromatography, Thin Layer
Phosphatidylinositol
Enzyme Inhibitors
Phosphatidylinositol 3-Kinase
PI3K/AKT/mTOR pathway
Fluorescent Dyes
Phosphoinositide-3 Kinase Inhibitors
Subjects
Details
- ISSN :
- 16182650 and 16182642
- Volume :
- 401
- Database :
- OpenAIRE
- Journal :
- Analytical and Bioanalytical Chemistry
- Accession number :
- edsair.doi.dedup.....4c1d4656d40361e8530b4f3ddb3f9fb7