| Assay Method Information | |
| | Determination of Inhibitory Potencies by Coupled ATPase Activity Assay |
| Description: | Inhibitory potencies of compounds were determined in a coupled ATPase activity assay using SERCA microsomes at 14 different inhibitor concentrations. In a plastic cuvette, the enzyme and the inhibitor were incubated 37 deg C in the assay buffer containing the enzyme pyruvate kinase and lactate dehydrogenase. After checking for potential background rates of NADH oxidation, the reaction was started by adding ATP. Rates of the rate-limiting NADH oxidation were determined spectroscopically for 5 min with a spectrophotometer operating at a wavelength of 340 nm. ATP hydrolysis rates as a function of inhibitor concentration were fit to a three-parameter logistic equation (amplitude, offset, and IC50). The IC50 was the concentration of inhibitor that caused inhibition of half of the ATPase activity observed in the absence of inhibitor. Reported IC50 values were the average of at least three independent trials. |
| Affinity data for this assay | |
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