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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. Glycogen Metabolism By Shimon Evidence for active ism in normal in the Normal W. Moses, glycogen mature Nava Bashan, metabol- red blood related to RBC HE MATURE glucose (RBC) has meet no synthesis contrast, glycogen tients type affected with VI).9’#{176}Sidbury cells is is not broken possible metabolism to glycogen study the mature blood tion under of radioactivity active glucose Prom Beer the Hadassa, and Submitted accepted 10, 10, by W. Moses, Research 1972; USPHS first Grant M.D.: Lecturer, revision investigator, Am Head and the either and in degrada- distribution incorporation Hospital is also metabolism synthesis University, the of Negev Hadassa radio- University, Medical School, May 19, 1972; second revision July 7, 1972; 12672-03. of Negev Pediatric Alisa Gutman, M.D.: Senior Hebrew University, Hadassa 836 Central pa- glycogen enzyme unless glycogen after It active of normal determining molecule Hebrew cell.9 an accumulates and In of development of glycogen metabolism, mature of glycogen Negev Biochemistry, present.48 7972. Shimon Senior glycogen be Israel. March July rates Laboratory, of the is missing. investigate conditions cell catalyzing erythrocytes maintains in the presence glycogen to the active in blood disease (type III and in affected red blood in an degree whereas measuring to in of environmental red enzymes shown stage of significant Research Supported Center; cell, Department Jerusalem, early mature erythrocyte steady state favors, within the into glycogen. Pediatric Sheva, although been absence incubation on normal glycogen-storage that the glycogen or phosphorylase was undertaken varying 14C-Uglycogen substantial deposition be observed. demonstrated an the breakdown, amylo-1,6-glucosidase The present red of any the normal in which the of from view down that activities, in been types assumed remnant that, may depends have has pH for into accumulation of glycoin erythrocytes with affecting glycogen The stores,3 breakdown certain et al. vestigial erythrocyte breakdown, glycogen glycogen and optimum any significant gen, whereas enzyme defects requirements.’2 deposition Gutman incorporation ERYTHROCYTE energy significant glycogen a its Cell 7.6. Replacing the radioactive glucose employed for incorporation after 1 hr of incubation with non labeled glucose resulted in a gradual loss of radioactivity from erythrocyte glycogen. In normal cells, glycogen synthesis and breakdown do not result in concentration HUMAN to The glucose was pH in the medium. The major part of the incorporated radioactivity resided in the outer branches of the glycogen T Blood and Alisa molecule. cells (RBC) is presented. Initial rates of ‘4C-U-glucose incorporation into erythrocyte glycogen were found to be independent of substrate concentration over a range of 3.3-16.6 mM. Incorporation of label into glycogen was initially linear but reached a plateau after a variable period of time that was Inversely Red Pediatrics University, Research, Lecturer, Medical Clinical School, “B” and Pediatric heva, Beer-S Soroka Research, israel. Medical Biochemistry, Jerusalem, Blood, Nava Soroka Bashan, Center, Beer-Sheva, Department of Medical M.Sc.: Israel. Biochemistry, Israel. Vol. 40, No. 6 (December), 1972 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. GLYCOGEN METABOLISM IN RED BLOOD MATERIALS The various enzymes, Boehringer, Mannheim, Diazyme reagent was glucose was obtained Preparation Fresh drawn fugation After pressed the red 0.15 M originally The before CELL AND 837 METHODS coenzymes, and glycolytic Germany or Sigma Chemicals, obtained from Miles Chemicals, from the Radiochemical intermediates St. Louis, Elkhart, Center, were Mo. Ind. Amersham, obtained from Radioactive 14C-U- England. of Eryfhrocytes blood was obtained from healthy laboratory personnel. The blood samples were into heparinized tubes, and the erythrocytes (RBC) were sedimented by centriin the cold. aspiration of the supernatant and the buffy coat, the remaining blood was through a cotton-wool sieve as described by Busch and Pelz.11 Two passages of blood cells suspension through the syringe containing the cotton wool soaked in NaCl solution were found to remove 99% of leukocytes and 98% of thrombocytes present. erythrocytes subsequently were washed three times with 10 vol of cold 0.15 M NaCl their use in the various studies. Analytical Methods Glucose was determined by the glucose oxidase method.12.13 Maltose was measured by the Somogy and Nelson method,14 and glycogen was measured with diazyme reagent.’5 ‘4C-U-Clucose incorporation into glycogen was measured in an incubation mixture contaming 30% RBC suspension, 6.6 mM glucose, 2.5 Ci 14C-U-glucose, 15 mM NaHPO4, 120 mM glycylglycine buffer, in a shaking acetic acid described.’8 and scintillation spectrometer. Analysis water bath carrier the 7.8, in glycogen, Radioactivity of pH at 37#{176}C. The and a final volume reaction was the measurements distribution of of 6 ml. stopped polysaccharide were 14C-U-glycosyl made units This by mixture the was isolated a Packard with between was addition the incubated of as trichloropreviously Tricarb outer liquid branches and the limit dextrin fraction of the glycogen was carried out after p-amylolysis.17 Maltose, liberated by fl-amylase, was separated from other compounds by descending paper chromatography utilizing Whatman No. 1 paper. The solvent used was butanolpyridine-water (3:2,5:1,5 by v/v). Separation of glycogen from other polysaccharides was performed as follows: after 14C-U-glucose was incorporated into intact RBC as described above, an equal volume of 6% HgC12 was added to precipitate the protein. Nonlabeled glycogen was added to the supernatant. The total glycogen was subsequently extracted by repeated alternate precipitations with alcohol and resuspension in water as previously described.7 The polysaccharide suspension was subsequently run through a Biogel-200 column. Eluting phase was water. Glycogen and radioactivity were determined in each of the 2-ml fractions collected by a fraction collector. RESULTS Incorporation of Glucose Incubation of normal of a radioactive appearance of material. radioactivity concentration. This Biogel-200 column polysaccharide of the with This in material extracted could Glycogen RBC (Fig. nonlabeled incorporated Into 1), be recovered material a fraction was which from glycogen ‘4C-U-glucose further showed erythrocytes added. In as maltose resulted was identified that sedimented in as the appearance glycogen at 60% by the ethanol characterized by separation on a that the peaks of the radioactive were addition, identical most on f3-amylolysis with of the (Fig. the peaks radioactivity 2). From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 838 MOSES, The rate of incorporation cyte glycogen was 0.04 ± This rate could not have of radioactivity of moles glucose 0.01 been due to BASHAN, ‘4C-U-glucose incorporated/g contamination of AND GUTMAN into erythroHb per hr. the RBC by leuko- cytes or thrombocytes. The maximum rate of glucose incorporation attributable to the number of leukocytes and thrombocytes remaining in the cell suspension after the removal procedure would have been 0.05 m&moles’8 and 0.03 m&moles, the respectively.’9 incorporation The reaction. initial As linear slope shown represents in Fig. the the 2, curve net rate of approaches a plateau after 2 hr. indicating a steady state that is the result of an equilibrium state between the rates of incorporation into and loss of radioactivity from glycogen. On examining the distribution of radioactivity within the glycogen molecule, an Increase of radioactivity is observed mainly in the outer branches and to a small extent (about 10%) in the limit dextrin fraction (core). To exclude the from physical possibility trapping chromatographic with cold similar gen separating glycogen, and glycogen was observed 16.6 mM (Fig. figure), glucose and noted. Further No for the resulted during the first carried mixed through was found in the spot. of ‘4C-U-glucose into from nature of erythrocyte glycogen glycogen metabolism in blood cells was obtained in experiments that showed that replacement radioactive glucose by nonlabeled glucose after 1 hr of incubation resulted a gradual decrease of radioactivity from RBC glycogen (Fig. 4). The incorporation maximum maximum of glucose activity being for glycolytic into reached activity. glycogen at pH was 7.6, shown which is to be pH very near red of in dependent, to the pH Fig. 1. Comparison of peaks of added cold glycogen with radioactive erythrocyte glycogen after passage through Biogel-200 column. Incubation of ABC ‘4C-U-glucose was performed as described. After denaturation with cold TCA, 20 mg of glycogen were added to supernatant. Glycogen 0. U was separated by cose alcohol. resuspended plied a glyco- concentration was varied from in the low glucose medium shown in the lower part of the in radioactivity dynamic core was was in the maltose incorporation medium glucose 3). After 2-hr incubation had almost disappeared (as decrease maltose radioactivity of in the molecule mixture when a progressive evidence the was detected initial rate detected glycogen the radioactive subsequently procedure. all radioactivity difference in the 3.3 to (3.3 mM), radioactivity within procedure, and chromatographic No was that the of maltose to collected a Biogel-200 in which column radioactivity 60 X 3 elution and glycogen phase-water. were from radioactive alu- repeated precipitations with Precipitated glycogen was in 5 ml of water and ap- Samples determined. of 2 ml were From www.bloodjournal.org by guest on June 18, 2017. For personal use only. GLYCOGEN METABOLISM It is apparent tion medium from the IN RED BLOOD Fig. 5 that incorporation at low rate with ing fresh cells been incubated a fresh one had 839 concentrations of ‘4C-U-glucose with time up to 2 hr. However, when the rate of incorporation tended to phenomenon could not be ascribed to in the composition of the incubation mixture CELL no effect of RBC into in the glycogen the concentration of cells decrease as a function depletion of substrate or medium, since replacing on in an incubation medium resulted in a normal rate this phenomenon, in which other of incorporation. Fig. 2. Distribution of incorporated ‘4C-U-gtucose between outer tiers and limit dextrin fraction. Aliquots of glycogen were exposed to /3-amylolysis. Subsequently, separation of outer branches from the limit dextrin fractions (core) was performed by paper chromotography, and radioactivity of the various fractions was determined. incuba- was linear was increased, of time. This other changes the incubation whereas incubat- had previously cells 2 0 3 Hours 20 U 15 E 10 . 5 Gkicose- Cncorporohon ___________________________ 2 Glucose utihsotion 0 IS 0 E ---- 0 Hours Fig. 3. ‘4C-U-Glucose incorporation into glycogen and glucose utilization with two substrate concentrations. One milliliter of ABC was suspended in ml of incubation medium containing 3.3 (open circles) or 16.6 (closed cirdes) mM glucose. Samples of medium were removed at specified intervals and were analyzed for the radioactivity glycogen. for glucose incorporated and into 2 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 840 MOSES, BASHAN, AND GUTMAN Fig. 4. Pulse studies of glucose incorporation into glycogen in normal erythrocytes. Initial incubation condition as in Methods. After 1 hr of incubation, red blood cells were washed in 0.15 M NaCI and reincubated in an incubation mixture as before, but without added radioactivity. Open circles represent cells reincubated in nonradioactive incubation mixture. Black dots represent controls. 25 Fig. 5. Effect of ABC concentration on rate of glucose incorporation into glycogen. RBC suspensions of different concentrations were incubated with ‘4C-U-glucose. After 2 hr, cells from tube containing highest ABC concentration were separated from medium and washed with cold 0.9#{176}/o NaCI solution. (A) Cells were subseE quently reincubated in fresh incuba. tion medium. (B) Separated incubation medium was used for incubation with fresh ABC. Black circles, 0.5 ml ABC; open circles, 1.0 ml ABC; black squares; 2.0 ml ABC. (A), (B) Final volume of incubation mixture was 4 ml. Composition of incubation mixture as described ---‘-#{149}-. in methods. flme (hours) From www.bloodjournal.org by guest on June 18, 2017. For personal use only. GLYCOGEN METABOLISM To establish of the mature blood cell suspension the younger older cell than that the glucose erythrocyte and suspension, cell counts IN RED BLOOD the was exposed were observed higher density last fraction ing that that but of mature this conclusion the blood was stored for 24 stored red blood hr the cells, as well by ACD in suspension into did show is not only (Table 1). no the : red order to blood separate density, from high glycogen cells. incorporation which had in in which older of reticulocytes presented in cell cells a function in the red performed hr 1 ‘4C-U-glucose of incorporation is also present to be of lower of younger comprising devoid for are known incorporated was were centrifugation fraction RBCs capacity red to into glycogen of reticulocytes experiments which The 841 incorporation not only following erythrocytes, population. CELL more However, readily even incorporation, a function Further evidence performed reticulocytes were capacity to incorporate this indicat- of studies the reticulocyte reticulocytes to support on found; blood yet glucose this into glycogen. DISCUSSION This report tains an formed active Most the radioactivity. metabolism present limit The glycogen experiment in the steady that blood. Both to the can mature not column erythrocyte be main- attributed separation to state, reached into glycogen, and indicate that the incorporation of radioactive and not into other components of the cell. in the outer tiers of the glycogen molecule, the rate after of contains only small amounts of of radioactivity from erythro- incorporation, mm 30 units of initial as 12.5% for 2 hr. from shown increase equilibrium erythrocyte in RBC The linear an incubation with time an represents glycosyl whereas by the the glycogen. presence concentration factors of pulse ‘4C-U-glucose between entry The of 50% incorporation responsible for this and plateau is RBC conis a function of Table of Incorporation of 14C-U-Glucose Into Glycogen in Red Blood Separated According to Their Relative Densities or Stored 1. Rates other experiments 4). exit of radioactive approached after centration, that dextrin fraction (core) initial rate of disappearance is similar (Fig. incorporation as indicating breakdown studies is indeed into glycogen of the label is found whereas The evidence glycogen elements enzymatic glucose cyte presents linear phenomenon Rates Cells of Incorporation Retlculocytes (#{176}/o) ‘Centrif by careful obtained Activity (mmole/mI RBC/hr) Unspun RBC Upper layer of spun RBC’ Lower layer of spun ABC’ 1.3 3.9 0 20.1 42 12.5 RBC 0 12.1 stored for24 hr in ACD ugatlon was performed at 4#{176}C, 25,000 g for 60 nihi. Upper layer was obtained aspiration of approximately 10#{176}/o of top fraction of spun ABC. Lower layer was by free flow of lowest fraction from bottom of punctured centrifugation tube. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 842 MOSES, are not immediately reach a plateau was BASHAN, AND GUTMAN The observation that the time required to of RBC concentration does not support the possibility that a time-dependent running down of the synthetic mechanism of the cells was responsible for the observed phenomenon. The fact that most of the radioactivity resided in the outer tiers of the glycogen molecule suggests that under the conditions of the experiment very little branching of the newly formed chains took place. The limitation imposed on de novo contribute to the glycogen slowing of this phenomenon The accumulation not be implicated by suspension. environment increasing cell RBC hemoglobin important factor of a function special of interest metabolism by the rate of branching but can not of synthesis It has, therefore, takes place concentration. could possibly regulating the glycogen metabolism. The separation and only synthesis down may explain the possibly dependence on the concentration of RBC in the incubation mixture. of an inhibitory factor in the incubation medium could in the observed decrease in the rate of incorporation in concentrated cell in the intracellular rated apparent. a function aging in view the of in that mature that that oxygen glycogen presence be an an involved as of of might metabolism erythrocytes, well-known a change is accele- saturation pH and of enzymes intracellular activities establish but of be assumed incubation Differences change relative studies reticulocytes to during is not well. active in This is glycogen in young nucleated red cell precursors that seems to be retained form in the mature red blood cell. The steady state present in the normal red blood cell does not lead to glycogen accumulation. This may be related to the minimal functional activity of glycogen synthetase,2#{176} in contrast to markedly higher activities of the enzymes catalyzing glycogen breakdown.8 In abnormal conditions, namely in the absence of erythrocyte amylo-1,6-glucosidase and of phosphorylase, in a vestigial yet the normal resulting in steady glycogen the normal functional state of glycogen accumulation that synthesis may and breakdown is reach several hundred upset, times level. REFERENCES 1. Murphy, J. R.: Erythrocyte metabolism. H. Glucose metabolism and pathways. J. Lab. Clin. Med. 55:286, 1960. 2. Bishop, C.: Overall red cell metabolism. In Bishop, C. and Surgenor, D. M. (Eds.): The Red Blood Cell. New York, Academic, 1964, p. 148. 3. Bartels, H.: Untersuchungen zur Frage des Glycogengehaltes von Erythrocyten. In Deutsch, E., Gerlach, E., and Moser, K. (Eds.): Metabolism and Membrane Permeability of Erythrocytes. Stuttgart, Thieme, 1968, p. 132. 4. Steinitz, K.: Amylo-1,6-glucosidase branching enzyme) In erythrocytes. fuah 62:275, 1962. (deHare- 5. Spencer-Peet, J.: Erythrocyte synthetase in glycogen storage resulting from the absence of from liver. Clin. Chim. Acta 6. Cornblath, M., Steiner, D. and King, J.: Uridine-diphosphoglucose cosyltransferase in human Clin. Chim. Acta 12:270, glycogen deficiency this enzyme 10:481, 1964. F., Bryan, P., glu- erythrocytes. 1965. Chayoth, R.: Determination of amylo1,6-glucosidase (debrancher) activity in patients with type III glycogenosis and In their relatives. M.S. Thesis. Tel-Aviv University, Tel-Aviv, Israel, 1965. 8. Moses, S. W., Chayoth, R., Leving, S., Lazarovitz, E., and Rubinstein, D.: Glucose and glycogen metabolism in erythrocytes of 7. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. GLYCOGEN METABOLISM IN RED BLOOD glycogen storage disease type III. J. Clin. Invest. 47:1343, 1968. 9. Sidbury, J. B., Cornblath, M., Fisher, J., and House, E.: Glycogen in erythrocytes of patients with glycogen storage disease. Pediatrics 27:103, 1961. 10. -, Gitzelman, R., and Fisher, J.: The glycogenosis: Further observation on glycogen in erythrocytes of patients with glycogenosis. Helv. Paediat. Acta 516:506, 1961. 11. Busch, D. K., and Pelz, K.: Erythrocyten Isolierung aus Blut mit Baumwolle. Klin. Wschr. 44:983, 1960. 12. Somogyi, M.: The determination of blood sugar. J. Biol. Chem. 160:69, 1945. 13. Kingsley, C. R., and Getchele, G.: Direct ultramicro glucose oxidase method for the determination of glucose in biological fluids. Clin. Chem. 6:466, 1960. 14. Nelson, N.: A photometric adaptation of the Somogy method for the determination of glucose. J. Biol. Chem. 153:375, 1944. 15. Johnson, J. A., and Fusaro, R. M.: An CELL 843 method for the quantitative determination of glycogen. Anal. Biochem. 5:370, 1963. 16. Steinitz, K.: Laboratory diagnosis of glycogen disease. In Sobota, H., and Stewart, C. P. (Eds.): Advances in Clinical Chemistry, Vol. IX. New York, Academic, 1966, p. 254. 17. French, D.: Structure of glycogen and its amylolytic degradation. In Whelan, W. J., and Cameron, M. P. (Eds.): Ciba Foundation Symposium: Control of Glycogen Metabolism. London, Churchill, 1964, p. 7. 18. Karpatkin, S., and Charmatz, A.: Heterogeneity of human platelets. III. Glycogen metabolism in platelets of different sizes. Brit. J. Haemat. 19:135, 1970. 19. Agam, G., and Gutman, A.: The synthesis of glycogen in leucocyte from various precursors. Europ. Clin. Biol. Res. In press. 20. Moses, S. W., and Bashan, N.: Properties of glycogen synthetase in normal red blood cell. Israel J. Med. Sci. 7:1213, 1971. enzyme From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1972 40: 836-843 Glycogen Metabolism in the Normal Red Blood Cell Shimon W. 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