This article details the enzymatic activity of glycogen synthase, an essential enzyme in glycogen synthesis. The process involves the conversion of UDP-glucose into UDP and glycosyl residues, which are then incorporated into glycogen chains.
Glycogen synthase ― a crucial enzyme in glycogen synthesis ― cleaves uridine diphosphate glucose, or UDP-glucose ― a nucleotide sugar ― into UDP and glycosyl residue and adds the released glucose monomer to the growing glycogen chain.
To estimate glycogen synthase activity, begin by taking a reaction mixture containing glycogen, UDP-glucose, ATP, phosphoenolpyruvate, and nicotinamide adenine dinucleotide, or NADH, in a tube. Add nucleoside diphosphate, or NDP, kinase enzyme, and, a pyruvate kinase and lactate dehydrogenase enzyme cocktail. Mix the contents, and incubate.
Add glycogen synthase-containing sample to the tube to initiate the enzymatic reaction.
Glycogen synthase acts on UDP-glucose, generating UDP and a glycosyl residue. The enzyme takes up the free glucose monomer and incorporates it into the reaction mixture's glycogen chain. In the presence of ATP, the reaction mixture's NDP kinase phosphorylates the unused UDP, converting it into UTP and generating ADP.
The enzyme pyruvate kinase acts on ADP and adds a phosphate donated by the reaction mixture's phosphoenolpyruvate to produce ATP and pyruvate. Next, lactate dehydrogenase converts the pyruvate to lactate and oxidizes NADH during the reaction.
Measure the decrease in NADH absorbance at 340 nm to quantify the amount of NADH consumed, which indicates successful enzyme activity.
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.
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