The Use of β,γ-Methyleneadenosine 5′-Triphosphate to Determine ATP Competition in a Scintillation Proximity Kinase Assay
| Autor: | Tate Bonnie Frances, Robin W. Spencer, William H. Martin, Marcia F. Peterson |
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| Rok vydání: | 1999 |
| Předmět: |
Indoles
Biophysics Binding Competitive Biochemistry Glycogen Synthase Kinase 3 Adenosine Triphosphate GSK-3 medicine Staurosporine Enzyme Inhibitors Protein kinase A Glycogen synthase Phosphorylase kinase Protein Kinase Inhibitors Molecular Biology IC50 biology Chemistry Kinase Glycogen Synthase Kinases Cell Biology Kinetin Kinetics Scintillation proximity assay Purines Calcium-Calmodulin-Dependent Protein Kinases biology.protein Scintillation Counting medicine.drug |
| Zdroj: | Analytical Biochemistry. 271:131-136 |
| ISSN: | 0003-2697 |
| Popis: | A novel method for characterizing the kinetics of protein kinase inhibitors is described. This method uses glycogen synthase kinase beta as the model protein kinase and looks at the shift in IC50 of inhibitors using the nonhydrolyzable ATP analog, beta, gamma-methyleneadenosine 5'-triphosphate, also known as AMP-PCP. Due to its inability to be hydrolyzed, AMP-PCP is being used to characterize known glycogen synthase kinase inhibitors by determining the shift in IC50 at concentrations above its calculated Ki of 490 microM. The assay format for the detection of inhibition is a scintillation proximity assay which is robust and reproducible at very low levels of [gamma-33P]ATP. The use of AMP-PCP coupled with the use of the scintillation proximity assay allows this characterization of inhibition without increasing [gamma-33P]ATP and without significantly diluting the overall assay signal. We have used this method in kinetic analyses to demonstrate that we can detect a significant shift in IC50 with the known ATP competitive inhibitors, staurosporine, Ro 31-8220, and olomoucine. The IC50 for glycogen synthase peptide and lithium chloride, which has been reported to be uncompetitive, remains unchanged. |
| Databáze: | OpenAIRE |
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