Radiochemical Assessment of Glycogen Synthase Enzyme Activity in Animal Tissue

Summary

The animal tissue assay utilizes nuclear scintillation detection to determine the enzymatic activity of glycogen synthase by measuring the incorporation of radio-labeled glucose into glycogen.

Abstract

Glycogen is an important metabolite to many eukaryotic cells, integral to energy storage and quick energy release. The building of the molecule is dependent on glycogen synthase, a highly regulated enzyme with the primary function of facilitating the sequential attachment of glucose units in a growing glycogen polymer. This enzyme is allosterically activated by glucose-6-phosphate and inhibited at multiple sites by phosphorylation. Knowledge of glycogen synthase enzymatic activity is a key component of understanding glycogen metabolism, muscle energy levels, and metabolic disorders such as diabetes and glycogen storage diseases. Described here is a highly sensitive and effective method of determining the activity of glycogen synthase in animal muscle tissue by measuring the amount of integrated glucose, from radio-labeled [14C]-UDP-glucose, into glycogen molecules. This procedure utilizes radioactive ¹⁴C and will require the proper authorization and lab space for use and disposal. This procedure will also require access to a liquid scintillation counter.

Introduction

Glycogen, a highly branched polysaccharide composed of glucose units, serves as an important energy reserve in various tissues, including muscles, liver, and brain¹. Its synthesis and degradation are orchestrated by a complex system of specialized enzymes and regulatory proteins. Vital to its biosynthesis is the enzyme glycogen synthase (GYS), of which there are two isoforms (GYS1 and GYS2). GYS1 is a regulatory enzyme directly responsible for the creation of glycogen from glucose in non-liver cells, while GYS2 functions in the liver. This process, known as glycogenesis, is the sequential addition of an individual unit of glucose onto a growing....

Protocol

All procedures were approved by the Ball State University Animal Care and Use Committee. Male and female cohorts of B6;129-Gaa^tm1Rabn/J wildtype mice, aged 3 months, are used as a representative example in this manuscript. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation prior to assay

1. Harvest tissue by cervical dislocation¹⁶ and immediately freeze in liquid N2 to preserve glycogen stores and store in liquid N2 or ultralow freezer until the start of the assay.
2. Prepare the stock solutions prior to the start of the glycogen s....

Results

Following the protocol, a printout created from the scintillation detector will contain the measured amount of ionizing radiation emitted from each chromatography paper containing vial, directly correlated to the activity level of glycogen synthase. The scintillation detector will compute the counts per minute (CPM) of detected radiation for each sample, blank, and total over a set amount of time. First convert CPM to DPM (disintegrations per min), which is based on the efficiency of the scintillation counter used (DPM=C.......

Discussion

Measurement of glycogen synthase enzyme activity is a valuable tool for monitoring glycogen metabolism in various metabolic and disease states. Measurement in the absence and presence of glucose-6-phosphate (G6P) offers insight into the regulatory status of the enzyme and an indirect index of the phosphorylation state. [U-¹⁴C]-UDP-glucose incorporation assay is an efficient and well-documented technique for measuring glycogen synthase enzymatic activity, offering high sensitivity, substrate specificity, and ad.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
[14C] Uridine 5-diphospho-glucose	American Radiolabeled Chemicals	ARC 0154
31 ETCHR chromatography paper	Whatman	3031 915
Benzamidine hydrochloride hydrate	Sigma	B6506
Beta mercaptoethanol	Sigma	M3148
Dry bath	Torey Pines Scientific	SC25
Ethanol (200 proof)	PharmcoAaper	111000200
Ethylene glycol tetraacetic acid (EGTA)	Sigma	E4378
Ethylenediaminetetraacetic acid (EDTA)	Sigma	E5134
Glucose-6-phosphate sodium salt	Sigma	G7879
Glycogen from rabbit liver (Type III)	Sigma	G8876
Microplate, flat-bottom, 96 well	Greiner	655101
Microplate, pcr, 96 well	DOT Scientific	951-PCR
Multi-Purpose Scintillation Counter	Beckman Coulter	LS 6500
Potassium fluoride dihydrate	Sigma	221872
Protease inhibitor cocktail	GoldBio	GB-108-2
Scintillation cocktail	Reasearch Products International	111195
Scintillation vials	Reasearch Products International	125509
Sodium fluoride	Sigma	S7920
Tissue Tearor	Biospec Products	Model 398
Tosyllysine Chloromethyl Ketone (TLCK)	Sigma	T7254
Trizma base (99.9%)	Sigma	T1503
Trizma hydrochloride	Sigma	T3253
Uridine 5-diphospo-glucose	Sigma	U-4625