Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Summary

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

Abstract

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of α1,4-linked glucose residues with branches introduced through the addition of α1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Introduction

Glycogen is widely distributed in nature, with the compound being found in bacteria, many protists, fungi, and animals. In microorganisms, glycogen is important for cell survival when nutrients are limiting and, in higher organisms such as mammals, synthesis and degradation of glycogen serve to buffer blood glucose levels^1,2,3. The study of glycogen metabolism is, therefore, of importance to such diverse fields as microbiology and mammalian physiology. Understanding glycogen metabolism requires studying the key enzymes of glycogen synthesis (glycogen synthase and the branching enzyme) and glycogen degradation (glycogen phosphorylase and debranching enzyme). The gold standard assays of glycogen synthase, phosphorylase, branching, and debranching enzyme activities employ radioactive isotopes. For example, glycogen synthase is generally measured in a stopped radiochemical assay by following the incorporation of glucose from UDP-[¹⁴C]glucose (in the case of animal and fungal enzymes) or ADP-[¹⁴C]glucose (in the case of bacterial enzymes) into glycogen^4,5. Similarly, glycogen phosphorylase is measured in the direction of glycogen synthesis, following the incorporation of glucose from [¹⁴C]glucose-1-phosphate into glycogen⁶. The branching enzyme is assayed by measuring the ability of this enzyme to stimulate the incorporation of [¹⁴C]glucose from glucose-1-phosphate into α1,4-linked chains by glycogen phosphorylase⁷, and debranching enzyme activity is determined by following the ability of the enzyme to incorporate [¹⁴C]glucose into glycogen⁸. While very sensitive, allowing their use in crude cell extracts with low levels of enzyme activity, the radioactive substrates are expensive and subject to the regulatory requirements attendant to radioisotope use. These barriers place the use of certain assays out of the reach of many workers. However, over the course of many years, an impressive variety of spectrophotometric approaches to the measurement of these enzymes have been described. In general, these approaches ultimately rely upon measuring the production or consumption of NADH/NADPH, or the generation of colored complexes between glycogen and iodine. Thus, they are straightforward and can be carried out using simple spectrophotometers equipped with only tungsten or xenon flash lamps.

Spectrophotometric assays of glycogen synthase rely upon measuring the nucleoside diphosphate released from the sugar nucleotide donor as glucose is added to the growing glycogen chain^9,10. The procedure for measuring glycogen synthase activity described in section 1 of the protocol, below, is a modification of that outlined by Wayllace et al.¹¹, and the coupling scheme is shown below:

(Glucose)_n + UPD-glucose → (Glucose)_n+1 + UDP

UDP + ATP → ADP + UTP

ADP + phosphoenolpyruvate → pyruvate + ATP

Pyruvate + NADH + H⁺ → Lactate + NAD⁺

Glycogen synthase adds glucose from UDP-glucose onto glycogen. The UDP generated in this process is converted to UTP by nucleoside diphosphate kinase (NDP kinase), in a reaction that generates ADP. The ADP, in turn, then serves as a substrate for pyruvate kinase, which phosphorylates the ADP using phosphoenolpyruvate as a phosphate donor. The resulting pyruvate is converted to lactate by the enzyme lactate dehydrogenase in a reaction that consumes NADH. The assay can, therefore, be performed in a continuous fashion, monitoring the decrease in absorbance at 340 nm as NADH is consumed. It is readily adapted for use with enzymes that require ADP-glucose as a glucose donor. Here, the coupling steps are simpler since the ADP released by the action of glycogen synthase is directly acted upon by pyruvate kinase.

There are a variety of spectrophotometric assays available for the determination of glycogen phosphorylase activity. In the classical version, the enzyme is driven backward, in the direction of glycogen synthesis, as shown below:

(Glucose)_n + Glucose-1-phosphate → (Glucose)_n+1 + P_i

At timed intervals, aliquots of the reaction mixture are removed, and the amount of phosphate liberated is quantified^12,13. In our hands, this assay has been of limited use due to the presence of readily measurable free phosphate in many commercial preparations of glucose-1-phosphate, combined with the high concentrations of glucose-1-phosphate required for phosphorylase action. Rather, we have routinely employed an alternative assay that measures the glucose-1-phosphate released as glycogen is degraded by phosphorylase¹³. A coupled reaction scheme, illustrated below, is employed.

(Glucose)_n + P_i → (Glucose)_n-1 + Glucose-1-phosphate

Glucose-1-phosphate → Glucose-6-phosphate

Glucose-6-phosphate + NADP⁺ → 6-phosphogluconolactone + NADPH + H⁺

The glucose-1-phosphate is converted into glucose-6-phosphate by phosphoglucomutase, and the glucose-6-phosphate is then oxidized to 6-phosphogluconolactone, with the concomitant reduction of NADP⁺ to NADPH. The procedure detailed in section 2 of the protocol, below, is derived from methods described by Mendicino et al.¹⁴ and Schreiber & Bowling¹⁵. The assay can be readily performed in a continuous fashion, with the increase in absorbance at 340 nm over time, allowing the determination of the reaction rate.

Spectrophotometric determination of debranching enzyme activity relies upon the measurement of the glucose released by the action of the enzyme on phosphorylase limit dextrin¹⁶. This compound is made by treating glycogen exhaustively with glycogen phosphorylase. Since glycogen phosphorylase action stops 4 glucose residues away from an α1,6-branch point, the limit dextrin contains glycogen, the outer chains of which have been shortened to ~4 glucose residues. Preparation of phosphorylase limit dextrin is described here, using a procedure derived from those developed by Taylor et al.¹⁷ and Makino & Omichi¹⁸.

Debranching is a two-step process. The 4-α-glucanotransferase activity of the bifunctional debranching enzyme first transfers three glucose residues from the branch point to the nonreducing end of a nearby α1,4-linked glucose chain. The single, α1,6-linked glucose residue remaining at the branch point is then hydrolyzed by the α1,6-glucosidase activity¹⁹. The assay is typically performed in a stopped fashion, the glucose released after a given time (or series of times) being measured in a coupled enzyme assay as shown below:

(Glucose)_n → (Glucose)_n-1 + Glucose

Glucose + ATP → Glucose-6-phosphate + ADP

Glucose-6-phosphate + NADP⁺ → 6-phosphogluconolactone + NADPH + H⁺

The determination of NADPH produced gives a measure of glucose production. The procedure outlined in section 3 of the protocol, below, is based upon one described by Nelson et al.¹⁶. Like the other methods that rely upon the consumption or generation of NADH/NADPH, the assay is quite sensitive. However, the presence of amylases or other glucosidases, which can also liberate free glucose from phosphorylase limit dextrin, will cause significant interference (see Discussion).

The colorimetric determination of branching enzyme activity relies upon the fact that α1,4-linked chains of glucose adopt helical structures that bind to iodine, forming colored complexes²⁰. The color of the complex formed depends upon the length of the α1,4-linked chains. Thus, amylose, which consists of long, largely unbranched chains of α1,4-linked glucose forms a deep blue complex with iodine. In contrast, glycogens, the outer chains of which are generally in the order of only 6 to 8 glucose residues long, form orange-red complexes. If a solution of amylose is incubated with branching enzyme, the introduction of branches into the amylose results in the generation of shorter α1,4-linked glucose chains. Thus, the absorption maximum of the amylose/iodine complexes shifts toward shorter wavelengths. The procedure discussed here is derived from that detailed by Boyer & Preiss²¹ and branching enzyme activity is quantified as a reduction in absorption of the amylose/iodine complex at 660 nm over time.

As should be readily apparent from the discussion above, the fact that the colors of the complexes formed between iodine and α1,4-glucose chains vary with the chain length means that the absorbance spectra of glycogen/iodine complexes should vary with the degree of glycogen branching. This is indeed the case, and less-branched glycogens/glycogens with longer outer chains absorb light at a longer wavelength than glycogens that are more branched/have shorter outer chains. The iodine staining reaction can therefore be used to gain rapid, qualitative data on the degree of glycogen branching²². The orange-brown color forms when glycogen complexes with iodine is not particularly intense. However, color development can be enhanced by the inclusion of saturated calcium chloride solution²². This boosts the sensitivity of the method some 10-fold and allows ready analysis of microgram quantities of glycogen. The assay for the determination of branching described in section 4 of the protocol, below, is adapted from a procedure developed by Krisman²². It is conducted simply by combining the glycogen sample with iodine solution and calcium chloride in a cuvette and collecting the absorption spectrum from 330 nm to 800 nm. The absorbance maximum shifts toward longer wavelengths as the degree of branching decreases.

Collectively, the methods described here provide simple, reliable means of assessing the activities of the key enzymes of glycogen metabolism, and for obtaining qualitative data on the extent of glycogen branching.

Protocol

1. Determination of glycogen synthase activity Prepare stock solutions of required reagents as indicated in Table 1 (prior to the experimental day). Component Directions 50 mM Tris pH 8.0 Dissolve 0.61 g of Tris base in ~ 80 mL of water.  Chill to 4 °C….

Representative Results

Determination of glycogen synthase activity Figure 1 shows representative results from glycogen synthase assays using purified enzymes. In panel A, following a slight lag, there was a linear decrease in the absorption at 340 nm over time for a period of around 12 min. The rate of change in absorption in Figure 1A was ~0.12 absorbance units/min. A rate of change in absorbance between ~0.010 and ~0.20 absorbance units/min is optimal and the …

Discussion

In general, the key advantages of all of the methods presented are their low cost, ease, speed, and lack of reliance upon specialized equipment. The major disadvantage that they all share is sensitivity compared to other available methods. The sensitivity of the procedures that involve production or consumption of NADH/NADPH are easy to estimate. Given that the extinction coefficient of NADH/NADPH is 6.22 M^-1 cm^-1, simple arithmetic indicates that ~10-20 µM changes in concentration can be readi…

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