Spectrophotometric Assay to Determine Glycogen Synthase Activity

Protokol

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: 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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

Malzemeler

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