Spectrophotometric Assay to Determine Glycogen Synthase Activity
Published: April 30, 2023
Abstract
Source: Wilson W.A., Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism. J. Vis. Exp. (2021).
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 e…
Declarações
The authors have nothing to disclose.
Materials
| 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 |
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Citar este artigo
Spectrophotometric Assay to Determine Glycogen Synthase Activity. J. Vis. Exp. (Pending Publication), e21321, doi: (2023).