Spectrophotometric Assay to Determine Glycogen Synthase Activity

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

1. Prepare stock solutions of required reagents as indicated in Table 1 (prior to the experimental day).
2. 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.
3. Thaw stock solutions of UDP-glucose, ATP, phosphoenolpyruvate, and NDP kinase on ice.
4. Pre-heat a water bath to 30 °C.
5. Set up each glycogen synthase assay in a 1.5 mL tube by adding the reaction mixture reagents listed in Table 2.
   NOTE:....

Materials

List of materials used in this article

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