Name: spectrophotometric methods for the study of eukaryotic glycogen metabolism
Uploaded: 2021-08-19T15:00:00.000+00:00
Duration: 7 min 59 s
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).

<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>

There are no known conflicts of interest associated with this work and there has been no financial support for this work that could have influenced its outcome.

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><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>&amp;beta;-Nicotinamide adenine dinucleotide phosphate sodium salt</td><td>MilliporeSigma</td><td>N0505</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide, reduced disodium salt</td><td>MilliporeSigma</td><td>43420</td><td></td></tr><tr><td>Nucleoside 5&#039;-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 ...

spectrophotometric-methods-for-study-eukaryotic-glycogen

Universidad de Cartagena UDEC

Education

Research

JoVE Core

JoVE Journal

Biochemistry

Anatomy and Physiology

Analytical Chemistry

Unit 1

Chemical Applications of Statistical Analyses

Absorption of Nutrients

Biochemistry of the Cell

SCIENTISTS IN ACTION

3.6K Views.  Des Moines University. Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

Video: Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Immunology and Infection

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached to each other by &#945;-1,6 glucoside linkages, referred to as branch points. Among the different forms of carbon storage (i.e., starch, &#946;-glucan), glycogen is probably one of the oldest and most successful storage polysaccharides found across the living world. Glucan chains are organized so that a large amount of glucose can quickly be stored or fueled in a cell when needed. Numerous complementary techniques have been developed over the last decades to solve the fine structure of glycogen particles. This article describes Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). This method quantifies the population of glucan chains that compose a glycogen particle. Also known as chain length distribution (CLD), this parameter mirrors the particle size and the percentage of branching. It is also an essential requirement for the mathematical modeling of glycogen biosynthesis.

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis FACE Method

In the present protocol, the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) technique is used to determine the chain length distribution (CLD) and the average chain length (ACL) of glycogen.

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Biology

A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, leading to the biased characterization of glycogen fine molecular structure. To understand the dynamic changes of glycogen structures and the versatile functions of glycogen particles in bacteria, it is essential to isolate glycogen with minimal degradation. In this study, a mild glycogen isolation method is demonstrated by using cold-water (CW) precipitation via sugar density gradient ultra-centrifugation (SDGU-CW). The traditional trichloroacetic acid (TCA) method and potassium hydroxide (KOH) method were also performed for comparison. A commonly used lab strain, Escherichia coli BL21(DE3), was used as a model organism in this study for demonstration purposes. After extracting glycogen particles using different methods, their structures were analyzed and compared through size exclusion chromatography (SEC) for particle size distribution and fluorophore-assisted capillary electrophoresis (FACE) for linear chain length distributions. The analysis confirmed that glycogen extracted via SDGU-CW had minimal degradation.

Bacterial glycogen structure is greatly impacted by extraction methods which may result in molecular degradation and/or biased sampling. It is essential to develop methods to minimize these problems. Here, four extraction methods have been compared using size distribution and chain length distribution as key criteria for minimizing extraction artifacts.

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, ...

Transmission Electron Microscopy to Quantify Glycogen Distribution in Human Skeletal Muscles

Source: Jensen, R., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/63347">Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy.</a> J. Vis. Exp. (2022)
In this video, we demonstrate the sample preparation of human skeletal muscle tissue and its staining for transmission electron microscopy to visualize and quantify subcellular glycogen distribution.

Source: Jensen, R., et al. Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy. J. Vis. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Microscopy Techniques

Spectrophotometric Assay to Determine Glycogen Synthase Activity

Source:&#160; Wilson W.A., <a href="https://appjove.unicartagenaproxy.elogim.com/v/63046">Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism.</a> J. Vis. Exp. (2021).
This video describes a spectrophotometric assay for the estimation of glycogen synthase activity. The method relies on the reduction in the absorbance of NADH with time and indicates glycogen synthase activity.

Source:&#160; Wilson W.A., Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism. J. Vis. Exp. (2021).
This video describes a ...

Assay Techniques

Biochemical assays

Glycogen Branching Assay to Measure the Degree of Glycogen Branching

Source: Wilson, W. A., <a href="https://appjove.unicartagenaproxy.elogim.com/v/63046">Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism.</a> J. Vis. Exp. (2021).
In this video, we describe a qualitative spectrophotometric assay to determine the extent of branching in a glycogen sample with an uncharacterized structure. Comparing the absorbance maxima of the uncharacterized sample with that of polysaccharides with known branched structures indicates the presence of branching in the uncharacterized sample.

Source: Wilson, W. A., Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism. J. Vis. Exp. (2021).
In this video, we describe a ...

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Summary

Explore More Videos

Chapters in this video

Determination of the Glycogen Content in Cyanobacteria

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method

Biochemical Titration of Glycogen In vitro

A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles

Transmission Electron Microscopy to Quantify Glycogen Distribution in Human Skeletal Muscles

Spectrophotometric Assay to Determine Glycogen Synthase Activity

Glycogen Branching Assay to Measure the Degree of Glycogen Branching