Name: spectrophotometric methods for the study of eukaryotic glycogen metabolism
Uploaded: 2021-08-19T15:00:00
Description: Watch this Scientific Journal Video about Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism at JoVE.com

The author would like to thank Karoline Dittmer and Andrew Brittingham for their insights and many helpful discussions. This work was supported in part by grants from the Iowa Osteopathic Education and Research Fund (IOER 03-17-05 and 03-20-04).

1. Determination of glycogen synthase activity
<ol>
	<li>Prepare stock solutions of required reagents as indicated in Table 1 (prior to the experimental day).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td colspan="2">Directions</td>
		</tr>
		<tr>
			<td>50 mM Tris pH 8.0</td>
			<td colspan="2">Dissolve 0.61 g of Tris base in ~ 80 mL of water.&#160; Chill to 4 &#176;C....

<ol>
	<li>Wilson, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glycogen+metabolism+in+yeast+and+bacteria.">Regulation of glycogen metabolism in yeast and bacteria.</a> FEMS Microbiology Reviews. 34 (6), 952-985 (2010).</li><li>Ralton, J. E., Sernee, M. F., McConville, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+function+of+carbohydrate+reserve+biosynthesis+in+parasitic+protists.">Evolution and function of carbohydrate reserve biosynthesis in parasitic protists.</a> Trends in Parasitology. 1471 (21), 00144-00146 (2021).</li><li>Roach, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+and+its+metabolism.">Glycogen and its metabolism.</a> Current Molecular Medicine. 2 (2), 101-120 (2002).</li><li>Thomas, J. A., Schlender, K. K., Larner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+filter+paper+assay+for+UDPglucose-glycogen+glucosyltransferase,+including+an+improved+biosynthesis+of+UDP-14C-glucose.">A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose.</a> Analytical Biochemistry. 25 (1), 486-499 (1968).</li><li>Fox, J., Kawaguchi, K., Greenberg, E., Preiss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+bacterial+glycogen.+Purification+and+properties+of+the+Escherichia+coli+B+ADPglucose:1,4-alpha-D-glucan+4-alpha-glucosyltransferase.">Biosynthesis of bacterial glycogen. Purification and properties of the Escherichia coli B ADPglucose:1,4-alpha-D-glucan 4-alpha-glucosyltransferase.</a> Biochemistry. 15 (4), 849-857 (1976).</li><li>Gilboe, D. P., Larson, K. L., Nuttall, F. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioactive+method+for+the+assay+of+glycogen+phosphorylases.">Radioactive method for the assay of glycogen phosphorylases.</a> Analytical Biochemistry. 47 (1), 20-27 (1972).</li><li>Brown, D. H., Illingworth, B., Cori, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+de+novo+synthesis+of+polysaccharide+by+phosphorylase.">The mechanism of the de novo synthesis of polysaccharide by phosphorylase.</a> Proceedings of the National Academy of Sciences of the United States of America. 47 (4), 479-485 (1961).</li><li>Nelson, T. E., Larner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+micro+assay+method+for+amylo-1,6-glucosidase.">A rapid micro assay method for amylo-1,6-glucosidase.</a> Analytical Biochemistry. 33 (1), 87-101 (1970).</li><li>Leloir, L. F., Olavarria, J. M., Goldemberg, S. H., Carminatti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+glycogen+from+uridine+diphosphate+glucose.">Biosynthesis of glycogen from uridine diphosphate glucose.</a> Archives of Biochemistry and Biophysics. 81 (2), 508-520 (1959).</li><li>Danforth, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+synthetase+activity+in+skeletal+muscle.+Interconversion+of+two+forms+and+control+of+glycogen+synthesis.">Glycogen synthetase activity in skeletal muscle. Interconversion of two forms and control of glycogen synthesis.</a> Journal of Biological Chemistry. 240, 588-593 (1965).</li><li>Wayllace, N. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-coupled+continuous+spectrophotometric+assay+for+glycogen+synthases.">An enzyme-coupled continuous spectrophotometric assay for glycogen synthases.</a> Molecular Biology Reports. 39 (1), 585-591 (2012).</li><li>Shapiro, B., Wertheimer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorolysis+and+synthesis+of+glycogen+in+animal+tissues.">Phosphorolysis and synthesis of glycogen in animal tissues.</a> Biochemical Journal. 37 (3), 397-403 (1943).</li><li>Mezl, V. A., Knox, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+methods+for+the+assay+of+glycogen+phosphorylase+in+tissue+homogenates.">Comparison of two methods for the assay of glycogen phosphorylase in tissue homogenates.</a> Enzyme. 13 (4), 197-202 (1972).</li><li>Mendicino, J., Afou-Issa, H., Medicus, R., Kratowich, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fructose-1,+6-diphosphatase,+phosphofructokinase,+glycogen+synthetase,+phosphorylase,+and+protein+kinase+from+swine+kidney.">Fructose-1, 6-diphosphatase, phosphofructokinase, glycogen synthetase, phosphorylase, and protein kinase from swine kidney.</a> Methods in Enzymology. 42, 375-397 (1975).</li><li>Schreiber, W. E., Bowling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+assay+of+glycogen+phosphorylase+in+the+direction+of+phosphorolysis.">An automated assay of glycogen phosphorylase in the direction of phosphorolysis.</a> Annals of Clinical Biochemistry. 27, 129-132 (1990).</li><li>Nelson, T. E., Kolb, E., Larner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+rabbit+muscle+amylo-1,6-glucosidase-oligo-1,4-1,4-transferase.">Purification and properties of rabbit muscle amylo-1,6-glucosidase-oligo-1,4-1,4-transferase.</a> Biochemistry. 8 (4), 1419-1428 (1969).</li><li>Taylor, C., Cox, A. J., Kernohan, J. C., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Debranching+enzyme+from+rabbit+skeletal+muscle.+Purification,+properties+and+physiological+role.">Debranching enzyme from rabbit skeletal muscle. Purification, properties and physiological role.</a> European Journal of Biochemistry. 51 (1), 105-115 (1975).</li><li>Makino, Y., Omichi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+glycogen+debranching+enzyme+from+porcine+brain:+evidence+for+glycogen+catabolism+in+the+brain.">Purification of glycogen debranching enzyme from porcine brain: evidence for glycogen catabolism in the brain.</a> Bioscience, Biotechnology, and Biochemistry. 70 (4), 907-915 (2006).</li><li>Lee, E. Y. C., Whelan, W. J., Boyer, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Enzymes Vol. 5. , 191-234 (1971).</li><li>Yu, X., Houtman, C., Atalla, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+of+amylose+and+iodine.">The complex of amylose and iodine.</a> Carbohydrate Research. 292, 129-141 (1996).</li><li>Boyer, C., Preiss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+bacterial+glycogen.+Purification+and+properties+of+the+Escherichia+coli+b+alpha-1,4,-glucan:+alpha-1,4-glucan+6-glycosyltansferase.">Biosynthesis of bacterial glycogen. Purification and properties of the Escherichia coli b alpha-1,4,-glucan: alpha-1,4-glucan 6-glycosyltansferase.</a> Biochemistry. 16 (16), 3693-3699 (1977).</li><li>Krisman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+colorimetric+estimation+of+glycogen+with+iodine.">A method for the colorimetric estimation of glycogen with iodine.</a> Analytical Biochemistry. 4, 17-23 (1962).</li><li>Friedman, D. L., Larner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+UDPG-alpha-glucan+transglucosylase.+iii.+Interconversion+of+two+forms+of+muscle+UDPG-alpha-glucan+transglucosylase+by+a+phosphorylation-dephosphorylation+reaction+sequence.">Studies on UDPG-alpha-glucan transglucosylase. iii. Interconversion of two forms of muscle UDPG-alpha-glucan transglucosylase by a phosphorylation-dephosphorylation reaction sequence.</a> Biochemistry. 2, 669-675 (1963).</li><li>Hanashiro, I., Roach, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+of+muscle+glycogen+synthase+that+disable+activation+by+glucose+6-phosphate.">Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate.</a> Archives of Biochemistry and Biophysics. 397 (2), 286-292 (2002).</li><li>Huang, K. P., Cabib, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+glycogen+synthetase+in+the+glucose+6-phosphate-dependent+form.+I.+Purification+and+properties.">Yeast glycogen synthetase in the glucose 6-phosphate-dependent form. I. Purification and properties.</a> Journal of Biological Chemistry. 249 (12), 3851-3857 (1974).</li><li>Pederson, B. A., Cheng, C., Wilson, W. A., Roach, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glycogen+synthase.+Identification+of+residues+involved+in+regulation+by+the+allosteric+ligand+glucose-6-P+and+by+phosphorylation.">Regulation of glycogen synthase. Identification of residues involved in regulation by the allosteric ligand glucose-6-P and by phosphorylation.</a> Journal of Biological Chemistry. 275 (36), 27753-27761 (2000).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D., Tagliabracci, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+and+its+metabolism:+some+new+developments+and+old+themes.">Glycogen and its metabolism: some new developments and old themes.</a> Biochemical Journal. 441 (3), 763-787 (2012).</li><li>Gosselin, S., Alhussaini, M., Streiff, M. B., Takabayashi, K., Palcic, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+spectrophotometric+assay+for+glycosyltransferases.">A continuous spectrophotometric assay for glycosyltransferases.</a> Analytical Biochemistry. 220 (1), 92-97 (1994).</li><li>Wilson, W. A., Pradhan, P., Madhan, N., Gist, G. C., Brittingham, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+synthase+from+the+parabasalian+parasite+Trichomonas+vaginalis:+An+unusual+member+of+the+starch/glycogen+synthase+family.">Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.</a> Biochimie. 138, 90-101 (2017).</li><li>Krisman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-1,4-glucan:+alpha-1,4-glucan+6-glycosyltransferase+from+liver.">alpha-1,4-glucan: alpha-1,4-glucan 6-glycosyltransferase from liver.</a> Biochimica et Biophysica Acta. 65, 307-315 (1962).</li><li>Sandhya Rani, M. R., Shibanuma, K., Hizukuri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+oyster+glucogen.">The fine structure of oyster glucogen.</a> Carbohydrate Research. 227, 183-194 (1992).</li><li>Dittmer, K. E., Pradhan, P., Tompkins, Q. C., Brittingham, A., Wilson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+glycogen+branching+and+debranching+enzymes+from+the+parasitic+protist+Trichomonas+vaginalis.">Cloning and characterization of glycogen branching and debranching enzymes from the parasitic protist Trichomonas vaginalis.</a> Biochimie. 186, 59-72 (2021).</li></ol>

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 &#181;M changes in concentration can be readi...

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 levels1,2,3. 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 glycogen4,5. 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 &#945;1,4-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 chain9,10. 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 &#8594; (Glucose)n+1 + UDP
UDP + ATP &#8594; ADP + UTP
ADP + phosphoenolpyruvate &#8594; pyruvate + ATP
Pyruvate + NADH + H+ &#8594; 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 &#8594; (Glucose)n+1 + Pi
At timed intervals, aliquots of the reaction mixture are removed, and the amount of phosphate liberated is quantified12,13. 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 + Pi &#8594; (Glucose)n-1 + Glucose-1-phosphate
Glucose-1-phosphate &#8594; Glucose-6-phosphate
Glucose-6-phosphate + NADP+ &#8594; 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 &#38; 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 &#945;1,6-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 &#38; Omichi18.
Debranching is a two-step process. The 4-&#945;-glucanotransferase activity of the bifunctional debranching enzyme first transfers three glucose residues from the branch point to the nonreducing end of a nearby &#945;1,4-linked glucose chain. The single, &#945;1,6-linked glucose residue remaining at the branch point is then hydrolyzed by the &#945;1,6-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 &#8594; (Glucose)n-1 + Glucose
Glucose + ATP &#8594; Glucose-6-phosphate + ADP
Glucose-6-phosphate + NADP+ &#8594; 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 &#945;1,4-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 &#945;1,4-linked chains. Thus, amylose, which consists of long, largely unbranched chains of &#945;1,4-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 &#945;1,4-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 &#38; 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 &#945;1,4-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amylopectin (amylose free) from waxy corn</td>
			<td>Fisher Scientific</td>
			<td>A0456</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose</td>
			<td>Biosynth Carbosynth</td>
			<td>YA10257</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP, disodium salt</td>
			<td>MilliporeSigma</td>
			<td>A3377</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose-1,6-bisphosphate, potassium salt</td>
			<td>MilliporeSigma</td>
			<td>G6893</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose-6-phosphate, sodium salt</td>
			<td>MilliporeSigma</td>
			<td>G7879</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-6-phosphate dehydrogenase, Grade I, from yeast</td>
			<td>MilliporeSigma</td>
			<td>10127655001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen, Type II from oyster</td>
			<td>MilliporeSigma</td>
			<td>G8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexokinase</td>
			<td>MilliporeSigma</td>
			<td>11426362001</td>
			<td></td>
		</tr>
		<tr>
			<td>Methacrylate cuvettes, 1.5 mL</td>
			<td>Fisher Scientific</td>
			<td>14-955-128</td>
			<td>Methacrylate is required since some procedures are conducted at 340 nm or below</td>
		</tr>
		<tr>
			<td>&#946;-Nicotinamide adenine dinucleotide phosphate sodium salt</td>
			<td>MilliporeSigma</td>
			<td>N0505</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Nicotinamide adenine dinucleotide, reduced disodium salt</td>
			<td>MilliporeSigma</td>
			<td>43420</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleoside 5&#39;-diphosphate kinase</td>
			<td>MilliporeSigma</td>
			<td>N0379</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoenolpyruvate, monopotassium salt</td>
			<td>MilliporeSigma</td>
			<td>P7127</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoglucomutase from rabbit muscle</td>
			<td>MilliporeSigma</td>
			<td>P3397</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorylase A from rabbit muscle</td>
			<td>MilliporeSigma</td>
			<td>P1261</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle</td>
			<td>MilliporeSigma</td>
			<td>P0294</td>
			<td></td>
		</tr>
		<tr>
			<td>UDP-glucose, disodium salt</td>
			<td>MilliporeSigma</td>
			<td>U4625</td>
			<td></td>
		</tr>
	</tbody>
</table>

spectrophotometric methods for the study of eukaryotic glycogen metabolism

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 &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-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.

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 ...

Watch this Scientific Journal Video about Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism at JoVE.com

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

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

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 ...

The protocols are significant, because they avoid the use of radioactive isotopes and remove a barrier that may prevent many workers from studying enzymes of glycogen metabolism. The procedures described are inexpensive, requiring only access to a simple spectrophotometer. To start with the determination of glycogen synthase activity, thaw the previously prepared stock solutions of UDP glucose, ATP, phosphoenolpyruvate, and NDP kinase on ice.Pre-heat a water bath to 30 degrees celsius. When the stock solutions are ready, prepare sufficient assay mixture according to the number of glycogen synthase assays by adding the reagents to a 15 milliliter tube, as described in the text protocol. Prepare a blank reaction by replacing the NADH from the reaction mixture with the water, and transfer the reaction to a disposable methacrylate cuvette.Use the blank reaction to set zero on the spectrophotometer at the wavelength of 340 nanometers. Take one 770 microliters aliquot of the reaction mixture in a 1.5 milliliter tube, and sequentially add two microliters each of NDP kinase and pyruvate kinase lactate dehydrogenase mixture to the tube. After mixing, gently incubate the tube at 30 degrees celsius in the water bath for three minutes to prewarm the reaction mixture.Then add 30 microliters of the sample containing glycogen synthase in 20 millimolar Tris buffer at pH 7.8, and mix gently before transferring the reaction mixture to a disposal methacrylate cuvette. Place the cuvette into the spectrophotometer, and record the absorbance at 340 nanometers at timed intervals for 10 to 20 minutes. Then, plot the absorbances obtained against the time.To measure the released glucose, transfer 40 microliters of the supernatant from the heated glycogen samples to the disposable methacrylate cuvettes. And add the measured volumes of triethanolamine, hydrochloride magnesium sulfate buffer, NADP ATP mixture and water as described in the manuscript. Mix the mixture by pipetting up and down gently without creating air bubbles.Add 0.5 microliters of glucose-6-phosphate dehydrogenase to each cuvette. After mixing by pipetting, incubate the mixture at room temperature for 10 minutes, and then record the absorbance at 340 nanometers. Next, mix 0.5 microliters of hexokinase to each cuvette as described earlier, and incubate for 15 minutes before recording the absorbent set 340 nanometers.Then continue the incubation at room temperature for five minutes, followed by recording the absorbance. If the absorption has increased from that recorded at 15 minutes, continue incubation for five minutes before recording the final absorption at 340 nanometers. To determine the glycogen branching, combine 650 microliters of iodine calcium chloride color reagent stock with 100 microliters of water in a 1.5 milliliter tube, and mix the solution thoroughly before transferring to a disposable methacrylate cuvette.Place the cuvette in the spectrophotometer to conduct a run in a wavelength scanning mode for collecting the background spectrum from 330 to 800 nanometers. In a separate 1.5 milliliter tube combine 650 microliters of the working iodine calcium chloride color reagent with 50 micrograms of the oyster glycogen, and make up the final volume to 750 microliters with water. After blending the mixture thoroughly, transfer the solution to a disposable methacrylate cuvette to collect an absorption spectrum from 330 to 800 nanometers.Similarly, obtain an absorption spectrum with 50 micrograms of amylopectin and 30 micrograms of amylose as described before. To obtain an indication of the branched structure of an uncharacterized glycogen sample, combine 25 to 50 micrograms of the glycogen with 650 microliters of the working iodine calcium chloride color reagent, and proceed as explained earlier to acquire the absorption spectrum. In the glycogen synthase assays, a linear decrease in the absorption at 340 nanometers over time was observed.Adding too much glycogen synthase to the assay caused the reaction to reach completion within the first two minutes. The control reaction, which contained no glycogen synthase, show no measurable decrease in absorbance. The data from a glycogen phosphorylase assay using a purified enzyme had a linear phase lasting for three minutes.A rate of absorbance increase of around 0.01 to 0.04 units per minute is optimal. In the glycogen de-branching enzyme assay, the reaction showed a linear phase persisting for at least 10 minutes. The representative data from the glycogen branching enzyme assays showed the difference in the absorbance between the control samples that lacked branching enzyme, and the reactions that contained branching enzyme.The narrow dynamic range of the assay is illustrated. The maximum change in the absorbance that can be produced is 0.4 absorbance units, and linearity is lost when the change in the absorption is approximately 0.2 absorbance units. The masses of glycogen, amylopectin, and amylose used in the glycogen branching assessment displayed a maximum absorbance reading of around 0.7 to 0.8, each at different absorbance maxima.The glycogen sample produced two peaks at approximately 400 nanometers and 460 nanometers. The iodine calcium chloride color reagent is very dense. It's important to ensure that the glycogen samples are fully mixed with the reagent prior to collecting an absorption spectrum.

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