Spectrophotometric Assay to Determine Glycogen Synthase Activity
Trascrizione
To start with the determination of glycogen synthase activity, thaw the previously prepared stock solutions of UDP-glucose, ATP, phosphoenolpyruvate, and NDP kinase on ice. Preheat a water bath to 30 degrees Celsius.
When the stock solutions are ready, prepare sufficient assay mixture according to the number of glycogen synthase assays, by adding the reagents to a 15-milliliter tube, as described in the text protocol.
Prepare a blank reaction by replacing the NADH from the reaction mixture with the water, and transfer the reaction to a disposable methacrylate cuvette. Use the blank reaction to set 'Zero' on the spectrophotometer at the wavelength of 340 nanometers.
Take one 770 microliters aliquot of the reaction mixture in a 1.5-milliliter tube, and sequentially add 2 microliters each, of NDP kinase and pyruvate kinase/lactate dehydrogenase mixture to the tube.
After mixing gently, incubate the tube at 30 degrees Celsius in the water bath, for 3 minutes to prewarm the reaction mixture. Then, add 30 microliters of the sample containing glycogen synthase, in 20 millimolar Tris buffer at pH 7.8, and mix gently before transferring the reaction mixture to a disposable methacrylate cuvette.
Place the cuvette into the spectrophotometer, and record the absorbance at 340 nanometers at timed intervals for 10 to 20 minutes. Then, plot the absorbances obtained against the time.
