Overview
This video describes a spectrophotometric assay for the estimation of glycogen synthase activity. The method relies on the reduction in the absorbance of NADH with time and indicates glycogen synthase activity.
Protocol
1. Determination of glycogen synthase activity
- Prepare stock solutions of required reagents as indicated in Table 1 (prior to the experimental day).
- On the day of the assay, prepare a fresh working solution of 4 mM NADH by dissolving 4.5 mg of NADH in 1.5 mL of 50 mM Tris-HCl, pH 8.0. Store on ice, protected from the light.
- Thaw stock solutions of UDP-glucose, ATP, phosphoenolpyruvate, and NDP kinase on ice.
- Pre-heat a water bath to 30 °C.
- Set up each glycogen synthase assay in a 1.5 mL tube by adding the reaction mixture reagents listed in Table 2.
NOTE: To facilitate the set-up, a master mix can be made containing enough of each of the above-listed reagents to complete the number of assays planned. - Prepare a blank reaction, where the NADH in the above mixture is replaced with water. Transfer to a disposable methacrylate cuvette and use this to set the zero on the spectrophotometer at 340 nm.
- Take one 770 µL of the aliquot of reaction mixture in a 1.5 mL tube. Add 2 µL of NDP kinase and 2 µL of pyruvate kinase/lactate dehydrogenase mixture, mix gently, and incubate at 30 °C for 3 min to pre-warm the reaction mixture.
- Add 30 µL of the sample containing glycogen synthase in 20 mM Tris buffer, pH 7.8; mix, and transfer the reaction mixture to a disposable methacrylate cuvette.
- Place the cuvette into the spectrophotometer and record the absorbance at 340 nm at timed intervals for 10 to 20 min. Plot the absorbances obtained against time.
NOTE: A reaction in which the glycogen synthase sample is replaced with 20 mM Tris buffer should be conducted to control for non-enzymatic oxidation of NADH. Depending upon the purity of the sample, other control reactions may be required. - Determine the reaction rate.
Table 1: Stock solutions required for the assay of glycogen synthase activity.
| Component | Directions | ||
| 50 mM Tris pH 8.0 | Dissolve 0.61 g of Tris base in ~ 80 mL of water. Chill to 4 °C. Adjust the pH to 8.0 with HCl and make the volume up to 100 mL with water. | ||
| 20 mM HEPES buffer | Dissolve 0.477 g of HEPES in ~ 80 mL of water. Adjust the pH to 7.0 with NaOH and make up the volume to 100 mL with water. | ||
| 132 mM Tris/32 mM KCl buffer pH 7.8 | Dissolve 1.94 g of Tris base and 0.239 g of KCl in ~90 mL of water. Adjust pH to 7.8 with HCl and make up the volume to 100 mL with water. | ||
| 0.8% w/v Oyster glycogen | Weigh out 80 mg of oyster glycogen and add to water. Make the final volume up to 10 mL with water and warm gently/mix to fully dissolve glycogen. | ||
| 100 mM UDP-glucose | Dissolve 0.31 g of UDP-glucose in water and make the final volume up to 1 mL. Store in aliquots, frozen at -20 °C. Stable for several months. | ||
| 50 mM ATP | Dissolve 0.414 g of ATP in ~ 13 mL of water. Adjust the pH to 7.5 with NaOH and make up the volume to 15 mL with water. Store in aliquots frozen at -20 °C. Stable for several months. | ||
| 100 mM Glucose-6-phosphate pH 7.8 | Dissolve 0.282 g of glucose-6-phosphate in ~ 7 to 8 mL of water. Adjust the pH to 7.8 with NaOH. Make the volume up to 10 mL with water. Store frozen in aliquots at -20 °C. Stable for at least six months. | ||
| 40 mM Phosphoenolpyruvate | Dissolve 4 mg of phosphoenolpyruvate in 0.5 mL of 20 mM HEPES buffer pH 7.0. Store at -20 °C. Stable for at least 1 week. | ||
| 0.5 M MnCl2 | Dissolve 9.90 g of MnCl2 in a final volume of 100 mL water. | ||
| NDP kinase | Reconstitute lyophilized powder with sufficient water to give 1 U/µl solution. Prepare aliquots, freeze in liquid nitrogen, and store at -80 °C. Stable for at least 1 year. | ||
Table 2: Composition reaction mixture for assay of glycogen synthase activity.
| Component | Volume (µl) |
| 160 mM Tris/32 mM KCl buffer pH 7.8 | 250 |
| Water | 179 |
| 100 mM glucose-6-phosphate, pH 7.8 | 58 |
| 0.8 % w/v oyster glycogen | 67 |
| 50 mM ATP | 80 |
| 4 mM NADH | 80 |
| 100 mM UDP-glucose | 28 |
| 40 mM phosphoenolpyruvate | 20 |
| 0.5 M MnCl2 | 8 |
| Final volume | 770 |
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Materials
| Name | Company | Catalog Number | Comments |
| Amylopectin (amylose free) from waxy corn | Fisher Scientific | A0456 | |
| Amylose | Biosynth Carbosynth | YA10257 | |
| ATP, disodium salt | MilliporeSigma | A3377 | |
| D-Glucose-1,6-bisphosphate, potassium salt | MilliporeSigma | G6893 | |
| D-glucose-6-phosphate, sodium salt | MilliporeSigma | G7879 | |
| Glucose-6-phosphate dehydrogenase, Grade I, from yeast | MilliporeSigma | 10127655001 | |
| Glycogen, Type II from oyster | MilliporeSigma | G8751 | |
| Hexokinase | MilliporeSigma | 11426362001 | |
| Methacrylate cuvettes, 1.5 mL | Fisher Scientific | 14-955-128 | Methacrylate is required since some procedures are conducted at 340 nm or below |
| β-Nicotinamide adenine dinucleotide phosphate sodium salt | MilliporeSigma | N0505 | |
| β-Nicotinamide adenine dinucleotide, reduced disodium salt | MilliporeSigma | 43420 | |
| Nucleoside 5'-diphosphate kinase | MilliporeSigma | N0379 | |
| Phosphoenolpyruvate, monopotassium salt | MilliporeSigma | P7127 | |
| Phosphoglucomutase from rabbit muscle | MilliporeSigma | P3397 | |
| Phosphorylase A from rabbit muscle | MilliporeSigma | P1261 | |
| Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle | MilliporeSigma | P0294 | |
| UDP-glucose, disodium salt | MilliporeSigma | U4625 |
