The protocols are significant, because they avoid the use of radioactive isotopes and remove a barrier that may prevent many workers from studying enzymes of glycogen metabolism. The procedures described are inexpensive, requiring only access to a simple spectrophotometer. 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.
Pre-heat 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 two 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 three 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 disposal 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.
To measure the released glucose, transfer 40 microliters of the supernatant from the heated glycogen samples to the disposable methacrylate cuvettes. And add the measured volumes of triethanolamine, hydrochloride magnesium sulfate buffer, NADP ATP mixture and water as described in the manuscript. Mix the mixture by pipetting up and down gently without creating air bubbles.
Add 0.5 microliters of glucose-6-phosphate dehydrogenase to each cuvette. After mixing by pipetting, incubate the mixture at room temperature for 10 minutes, and then record the absorbance at 340 nanometers. Next, mix 0.5 microliters of hexokinase to each cuvette as described earlier, and incubate for 15 minutes before recording the absorbent set 340 nanometers.
Then continue the incubation at room temperature for five minutes, followed by recording the absorbance. If the absorption has increased from that recorded at 15 minutes, continue incubation for five minutes before recording the final absorption at 340 nanometers. To determine the glycogen branching, combine 650 microliters of iodine calcium chloride color reagent stock with 100 microliters of water in a 1.5 milliliter tube, and mix the solution thoroughly before transferring to a disposable methacrylate cuvette.
Place the cuvette in the spectrophotometer to conduct a run in a wavelength scanning mode for collecting the background spectrum from 330 to 800 nanometers. In a separate 1.5 milliliter tube combine 650 microliters of the working iodine calcium chloride color reagent with 50 micrograms of the oyster glycogen, and make up the final volume to 750 microliters with water. After blending the mixture thoroughly, transfer the solution to a disposable methacrylate cuvette to collect an absorption spectrum from 330 to 800 nanometers.
Similarly, obtain an absorption spectrum with 50 micrograms of amylopectin and 30 micrograms of amylose as described before. To obtain an indication of the branched structure of an uncharacterized glycogen sample, combine 25 to 50 micrograms of the glycogen with 650 microliters of the working iodine calcium chloride color reagent, and proceed as explained earlier to acquire the absorption spectrum. In the glycogen synthase assays, a linear decrease in the absorption at 340 nanometers over time was observed.
Adding too much glycogen synthase to the assay caused the reaction to reach completion within the first two minutes. The control reaction, which contained no glycogen synthase, show no measurable decrease in absorbance. The data from a glycogen phosphorylase assay using a purified enzyme had a linear phase lasting for three minutes.
A rate of absorbance increase of around 0.01 to 0.04 units per minute is optimal. In the glycogen de-branching enzyme assay, the reaction showed a linear phase persisting for at least 10 minutes. The representative data from the glycogen branching enzyme assays showed the difference in the absorbance between the control samples that lacked branching enzyme, and the reactions that contained branching enzyme.
The narrow dynamic range of the assay is illustrated. The maximum change in the absorbance that can be produced is 0.4 absorbance units, and linearity is lost when the change in the absorption is approximately 0.2 absorbance units. The masses of glycogen, amylopectin, and amylose used in the glycogen branching assessment displayed a maximum absorbance reading of around 0.7 to 0.8, each at different absorbance maxima.
The glycogen sample produced two peaks at approximately 400 nanometers and 460 nanometers. The iodine calcium chloride color reagent is very dense. It's important to ensure that the glycogen samples are fully mixed with the reagent prior to collecting an absorption spectrum.
