Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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.

Protokół

1. Determination of glycogen synthase activity

1. 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.  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 1: Stock solutions required for the assay of glycogen synthase activity.

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.

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

Table 2: Composition reaction mixture for assay of glycogen synthase activity.

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. See Discussion for details.
10. Determine the reaction rate (see Results for details).

2. Determination of glycogen phosphorylase activity

1. Prepare stock solutions as indicated in Table 3 (prior to experimental day).

Component	Directions
125 mM PIPES pH 6.8	Dissolve 3.78 g of PIPES in water.  Adjust the pH to 6.8 with NaOH and make up the volume to 100 mL with water.
8% w/v oyster glycogen	Weigh out 0.8 g of oyster glycogen and add to water.  Make the final volume up to 10 mL with water and warm gently/mix to dissolve glycogen.  Store frozen at -20 °C.
200 mM Na phosphate pH 6.8	Dissolve 2.63 g of Na2HPO4.7H2O and 1.41 g of NaH2PO4.H2O in water.  Bring the volume up to 100 mL with water.
1 mM glucose-1,6-bisphosphate	Dissolve 2 mg of glucose-1,6-bisphosphate in 4 mL of water.  Aliquot and store frozen at -20 °C.  Stable for at least several months.
10 mM NADP	Dissolve 23 mg of NADP in 3 mL of water.  Aliquot and store frozen at -20 °C.  Stable for at least several months.

Table 3: Stock solutions required for the assay of glycogen phosphorylase activity.

2. Pre-heat a water bath to 30 °C
3. Set up each glycogen phosphorylase assay in a 1.5 mL tube by adding the reaction mixture reagents listed below (Table 4).

Component	Volume (µl)
125 mM PIPES buffer pH 6.8	160
Water	70
8% w/v oyster glycogen	100
200 mM Na phosphate 6.8	400
1 mM glucose-1,6-bisphosphate	20
10 mM NADP	20
Final volume	770

Table 4: Composition reaction mixture for assay of glycogen phosphorylase activity.

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.

5. Prepare a blank reaction containing the components listed in Table 4 but add an additional 30 µL of 25 mM PIPES buffer, pH 6.8 (prepared by diluting 125 mM PIPES buffer 1/5 with water). Transfer to a disposable methacrylate cuvette and use to set the zero on the spectrophotometer at 340 nm.
6. Take one 770 µL aliquot of reaction mixture in a 1.5 mL tube. Add 1 µL of glucose-6-phosphate dehydrogenase and 1 µL of phosphoglucomutase, mix gently, and incubate at 30 °C for 3 min to pre-warm the reaction mixture.
7. Add 30 µL of the sample containing glycogen phosphorylase in 25 mM PIPES buffer, pH 6.8. Mix and transfer the reaction mixture to a disposable methacrylate cuvette.
8. 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 glycogen phosphorylase is replaced with 25 mM PIPES buffer should be included. Depending upon the purity of the glycogen phosphorylase sample, other controls may also be needed (see Discussion for details).
9. Determine the reaction rate (see Representative Results for details).

3. Determination of glycogen debranching enzyme activity

1. Prepare stock solutions as indicated in Table 5 (prior to the experimental day).

Component	Directions
100 mM maleate buffer	Dissolve 1.61 g of maleic acid in ~ 80 mL of water.  Adjust the pH to 6.6 with NaOH and make the final volume up to 100 mL with water.
300 mM triethanolamine hydrochloride/ 3 mM MgSO4 pH 7.5	Dissolve 27.85 g of triethanolamine hydrochloride and 0.370 g of MgSO4.7H2O in ~ 400 mL of water.  Adjust the pH to 7.5 with NaOH and make up to a final volume of 500 mL with water.
150 mM ATP/12 mM NADP	Dissolve 1.24 g of ATP in ~ 10 mL of water.  Monitor the pH and add NaOH to maintain a pH of ~ 7.5 as the ATP dissolves.  Add 0.138 g of NADP.  Adjust the pH to ~ 7.5 with NaOH and make up to a final volume of 15 mL with water. Store in aliquots at – 20 °C. Stable for several months.
50 mM Na phosphate buffer pH 6.8	Dissolve 32.81 g of Na2HPO4.7H2O and 17.61 g of NaH2PO4.H2O in water.  Bring the volume up to a final volume of 5 L with water.

Table 5: Stock solutions required for the assay of glycogen debranching enzyme activity.

2. Prepare phosphorylase limit dextrin
   1. Dissolve 0.3 g of oyster glycogen in 10 mL of 50 mM sodium phosphate buffer, pH 6.8.
   2. Dissolve sufficient lyophilized phosphorylase A powder to yield 60 U of activity in 50 mM phosphate buffer, pH 6.8.
      NOTE: Depending upon the lot of phosphorylase A purchased, the mass needed will vary but is generally between 5 and 10 mg of powder.
3. Add 60 U of phosphorylase A to the glycogen solution and transfer to a dialysis bag. Dialyze at room temperature against 1 L of 50 mM sodium phosphate buffer, pH 6.8 for 8 h. Change to the fresh dialysis buffer and continue the incubation overnight.
4. Add another 10 U of phosphorylase A and change to fresh dialysis buffer. After 8 h, again change to fresh dialysis buffer and continue the incubation overnight.
5. Transfer the contents of the dialysis bag to a centrifuge tube and boil for 10 min. Chill on ice, and then centrifuge at 10,000 x g for 15 min.
6. Transfer the supernatant to a dialysis bag and dialyze for 8 h against three changes of 2 L of distilled water.
7. Transfer the contents of the dialysis bag to a 50 mL centrifuge tube. Measure the volume and add two volumes of ice-cold absolute ethanol to precipitate the limit dextrin. Let the tube stand on ice for 30 min.
   NOTE: A white precipitate should begin to form immediately upon the addition of ethanol but, if it does not, add a drop of 3 M NaCl.
8. Centrifuge at 15,000 x g for 15 min and discard the supernatant. Rinse the white pellet of limit dextrin twice with 66% v/v ethanol, using ~30 mL for each rinse.
9. Transfer the limit dextrin to a mortar and allow to air dry completely. When the limit dextrin is dry, grind to a powder with a pestle and transfer to a suitable vessel for storage; dry at 4 °C.
10. For use as debranching enzyme substrate, prepare a 1% w/v solution in water.
11. Pre-heat a water bath to 30 °C.
12. Pre-heat a heating block or water bath to 95 °C.
13. Prepare four 1.5 mL tubes each containing 100 µL of maleate buffer, 80 µL of phosphorylase limit dextrin, and 10 µL of water. These tubes will be used to conduct the debranching enzyme assay. Label two of the tubes Reaction and the other two tubes Control.
14. At timed intervals, add 10 µL of the debranching enzyme sample to the Reaction tubes and 10 µL of buffer which was used to prepare the branching enzyme sample to the Control tubes. Incubate at 30 °C.
15. At defined time points (e.g., 5-, 10-, and 20-min incubation), withdraw 50 µL from each Reaction and Control tube and immediately place into the heating block or water bath at 95 °C. Heat for 3 min.
16. Centrifuge at 15,000 x g for 2 min in order to remove precipitated protein.
    NOTE: At this point, the procedure can be halted if needed. The heated samples can be stored frozen at -20 °C until proceeding to the measurement of released glucose (step 17, below).
17. Measurement of released glucose
    1. Transfer 40 µL of the supernatant from the heated samples to disposable methacrylate cuvettes and add 833 µL of triethanolamine hydrochloride/magnesium sulfate buffer, 67 µL of NADP/ATP mix, and 60 µL of water. Mix by pipetting up and down gently, being careful not to introduce air bubbles.
       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.
    2. Prepare a blank reaction by combining 100 µL of maleate buffer, 80 µL of phosphorylase limit dextrin, and 20 µL of water. Mix well, and transfer 40 µL to a disposable methacrylate cuvette. Add 833 µL of triethanolamine hydrochloride/magnesium sulfate, etc. as described in step 3.17.1.
    3. Set the zero on the spectrophotometer at 340 nm using the blank reaction.
    4. Add 0.5 µL of glucose-6-phosphate dehydrogenase to each cuvette. Mix gently by pipetting up and down slowly. Incubate at room temperature for 10 min, and then record the absorbance at 340 nm.
       NOTE: The absorption values should be low, signifying little contamination of the samples with glucose-6-phosphate.
    5. Add 0.5 µL of hexokinase to each cuvette. Mix gently by pipetting up and down slowly. Incubate at room temperature for 15 min, and then record the absorbance at 340 nm.
    6. Continue the incubation at room temperature for an additional 5 min. Record the absorbance at 340 nm once again. If the absorption has increased from that recorded at 15 min, continue incubation for a further 5 min and again check the absorption. Record the final absorption at 340 nm obtained.
    7. For each sample, subtract the absorbance at 340 nm recorded after the addition of glucose-6-phosphate dehydrogenase from the final absorbance obtained after addition of hexokinase. Plot the values obtained against the length of time that the corresponding sample had been incubated with debranching enzyme.

4. Determination of glycogen branching enzyme activity

1. Prior to the experimental day, prepare iodine/KI solution by first dissolving 2.6 g of KI in 10 mL of water. In a fume hood, weigh out 0.26 g of iodine and add to the KI solution.
   CAUTION: Iodine is harmful when in contact with the skin or if inhaled. Mix to dissolve the iodine and store at 4 °C, protected from the light. Also prepare 125 mM PIPES buffer, pH 6.8 (see Table 3).
2. When beginning experiments, make a working stock of acidified iodine reagent.
   1. Take 45.7 mL of water in a 50 mL tube and add 150 µL of iodine/KI solution followed by 150 µL of 1 M HCl.
   2. Mix well and store at 4 °C, protected from the light. The solution is stable for at least 3 days under these conditions.
3. On the day of the experiment, make a fresh 10 mg/mL solution of amylose.
   1. Weigh out 50 mg of amylose and transfer to a 15 mL tube.
   2. Add 200 µL of absolute ethanol and shake gently.
   3. Add 500 µL of 2 M KOH and shake gently.
      CAUTION: KOH causes severe skin burns and eye damage. Use appropriate personal protective equipment.
   4. Add 0.5 mL of water while shaking gently. If the amylose does not dissolve completely, add an additional 0.5 mL of water.
   5. Adjust the pH to ~6.5 to 7.0 with 1 M HCl.
      CAUTION: HCl may cause eye, skin, and respiratory tract irritation. Use appropriate personal protective equipment.
   6. Add water to reach a final volume of 5 mL.
   7. Sterilize by passage through a 0.2 µm syringe end filter and store at room temperature. Do not chill or freeze.
4. Pre-heat a water bath to 30 °C.
5. Prepare twelve 1.5 mL tubes, each containing 1 mL of acidified iodine reagent. Set aside on the bench. These will be used to stop the branching enzyme reaction.
6. Prepare four 1.5 mL tubes each containing 150 µL of amylose, 150 µL of PIPES buffer, and 45 µL of water. These tubes will be used to conduct the branching enzyme assay. Label two of the tubes Reaction and the other two tubes Control.
7. At timed intervals, add 5 µL of branching enzyme sample to the Reaction tubes and 5 µL of buffer which was used to prepare the branching enzyme sample to the Control tubes. Incubate at 30 °C.
8. At defined time points (e.g., 5, 10, and 15 min incubation), withdraw 10 µL from each Reaction and Control tube and add to the 1.5 mL tubes that contain 1 mL of acidified iodine reagent. Add an additional 140 µL of water and mix well. Transfer to disposable cuvettes.
   NOTE: The samples should be blue in color and the solution should be free of any precipitate. The color formed is stable for at least 2 h at room temperature.
9. Prepare a sample that contains 1 mL of acidified iodine reagent and 150 µL of water. Mix well and transfer to a cuvette. Use this cuvette to set the zero on the spectrophotometer at 660 nm.
10. Read the absorbance of each of the twelve samples at 660 nm. Determine the rate of the branching enzyme reaction by subtracting the absorbance obtained in the presence of branching enzyme (Reaction) from the absorbance when no branching enzyme is present (Control) at each time point. See Representative Results for details.

5. Qualitative assessment of glycogen branching

1. Prepare saturated calcium chloride solution by adding 74.5 g of anhydrous calcium chloride to ~40 mL of water and stirring. Add a little more water and continue to stir. Make the volume up to 100 mL with water and continue stirring until the CaCl₂ is fully dissolved.
2. Prepare a working stock of iodine/CaCl₂ color reagent by mixing 50 µL of KI/iodine stock solution (see step 4.1, above) with 13 mL of saturated CaCl₂ solution in a 15 mL tube. Mix well and store at 4 °C, protected from the light. The solution is stable for at least 1 week under these conditions.
3. Determination of branching
   1. In a 1.5 mL tube, combine 650 µL of iodine/CaCl₂ color reagent stock with 100 µL of water and mix thoroughly. Transfer the solution to a disposable methacrylate cuvette.
      NOTE: The solution in the cuvette should be clear and pale yellow in color.
   2. Place in the spectrophotometer and, running in a wavelength scanning mode, collect a background spectrum from 330 nm to 800 nm.
   3. In a 1.5 mL tube, combine 650 µL of working iodine/CaCl₂ color reagent with 50 µg of oyster glycogen. Bring the final volume to 750 µL with water and mix thoroughly. Transfer the solution to a disposable methacrylate cuvette.
      NOTE: The solution in the cuvette should be clear and a deep orange/brown color.
   4. Place in the spectrophotometer and collect an absorption spectrum from 330 nm to 800 nm.
   5. Repeat steps 4.4.3 through 4.4.4 with 50 µg of amylopectin and 30 µg of amylose.
      NOTE: The amylopectin sample should be yellow/green and the amylose sample should be green/blue. Both samples should be clear. The colored complexes formed are stable, with no change in absorption spectrum, for at least 1 h at room temperature.
   6. To obtain an indication of the branched structure of an uncharacterized glycogen sample, combine 25 µg to 50 µg of glycogen with 650 µL of working iodine/CaCl₂ color reagent. Proceed as above, bringing the volume to 750 µL with water, mixing thoroughly, and transferring to a methacrylate cuvette.
      NOTE: The glycogen sample should yield a yellow/orange to orange/brown color depending upon the degree of branching (length of outer chains) of the glycogen present. Again, the sample should be clear. See Representative Results for details.
   7. Collect the absorption spectrum.

Wyniki

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 ...

Dyskusje

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...

Materiały

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