| Assay Method Information | |
| | In Vitro PDHK1 Activity Assay |
| Description: | The inhibitory potency of compounds of PDHK1 activity was determined in an enzymatic assay. Pyruvate dehydrogenase complex (PDHc) is an enzyme that catalyzes the reaction of pyruvate and a lipoamide to give the acetylated dihydrolipoamide and carbon dioxide. PDHc has three subunits E1, E2 and E3. E1 has 3 serine phosphorylation sites S293, S232, S300. If E1 was phosphorylated, PDH will be inactivated. PDHK can phosphorylate PDH protein (also named PDHE1) in PDH complex (PDC) to inactivate it. In the PDHK1 enzymatic assays, the final reaction mixture contains 1 nM of PDHK1 (in house purification from Escherichia coli), 25 nM of PDHE1 (in house purification from Escherichia coli) and 20 μM of ATP, and the experiment was reacted at 30° C. for 45 minutes. After adding detection reagent AlphaScreen Histidine (Nickel Chelate, from Perkin Elmer), and anti-phosphorylation s-293 antibody (from Abcam), Anti-Rabbit IgG Alpha (from Perkin Elmer)), the plate was incubated at 30° C. for 60 min before measuring the light change using a luminometer (Envision). |
| Affinity data for this assay | |
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