Detection of Glycogen-Debranching System in Trophozoites of Entamoeba histolytica

ABSTRACT. Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4- α -glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl- α -glucoside yielding successively 4-nitrophenyl- α -maltoside and 4-nitrophenyl- α -maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of M_r= 180,000 ± 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

Detection of glycogen-debranching system in trophozoites of Entamoeba histolytica

Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4-alpha-glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl-alpha-glucoside yielding successively 4-nitrophenyl-alpha-maltoside and 4-nitrophenyl-alpha-maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of Mr = 180,000 +/- 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

Glycogen metabolism and cyclic AMP levels in isolated islets of lean and genetically obese mice.

The levels of glycogen and cyclic AMP, incorporation of glucose into glycogen and activities of glycogen synthetase and phosphorylase were determined in pancreatic islets isolated from genetically obese mice and their lean litter-mates. Islets from obese mice had elevated glycogen levels, increased phosphorylase activity and an increased amount of glycogen synthetase in the physiologically more effective I-form, indicating an increased turnover of glycogen. There was no significant difference in cyclic AMP levels between islets of lean and obese mice, but inhibition of phosphodiesterase or stimulation of adenyl cyclase increased cyclic AMP levels more in obese than in lean mouse islets, indicating a more rapid turnover of cyclic AMP in the former. It is suggested that cyclic AMP stimulated phosphorolytic breakdown of glycogen may be one of the mechanisms responsible for the increased insulin secretory response to glucose observed in islets from genetically obese mice.     

Time course for cryoprotectant synthesis in the freeze-tolerant chorus frog, Pseudacris triseriata

Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5-12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.     

Control of glycogen levels in brain

Abstract— Prolonged (6 hr) anaesthesia with phenobarbital in mice or rats results in a doubling or tripling of brain glycogen. Increases were also observed if high levels of plasma glucose were maintained for 6 hr. In alloxan diabetes brain glycogen was not elevated in spite of the high plasma glucose concentrations. However, administration of insulin to such diabetic animals, together with enough glucose to maintain high plasma levels, resulted in at least a doubling of brain glycogen in 6 hr. Phenobarbital can still increase brain glycogen in diabetic animals. In all of the conditions associated with increased glycogen deposition, increases were found in the ratio of brain glucose to plasma glucose. Cerebral glucose-6-P levels were also increased whereas there were no substantial changes in levels of UDP-glucose or glucose-1,6-diphosphate.     

Glycogen debranching enzyme in bovine brain

Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-1,6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5,000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-exchange HPLC, and gel-permeation HPLC. The enzyme preparation had no alpha-glucosidase or alpha-amylase activities and degraded phosphorylase limit dextrin of glycogen with phosphorylase. The molecular weight of the enzyme was 190,000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an alpha-1,6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase.     

Metabolism of the reserve polysaccharide of Streptococcus mitis. Some properties of a pullulanase

1. A pullulanase has been separated from cell extracts of Streptococcus mitis. The enzyme was freed from transglucosylase by fractionation with ammonium sulphate. 2. Pullulanase was produced in the absence of inducers, and addition of glucose or maltose to the broth did not increase the yield of enzyme. 3. The pullulanase acted rapidly on alpha-(1-->6)-bonds in substrates having the structure alpha-maltodextrinyl-(1-->6)-maltodextrin, but had no action on isomaltose, 6-alpha-glucosylmaltodextrins or 6-alpha-maltodextrinylglucoses. 4. 6-alpha-Maltotriosylmaltodextrins were hydrolysed over 10 times faster than 6-alpha-maltosylmaltodextrins. 5. The branch linkages of amylopectin phosphorylase limit dextrin, glycogen phosphorylase limit dextrin and glycogen beta-amylase limit dextrin were hydrolysed. The action of pullulanase on amylopectin and glycogen was accompanied by a rise in the iodine stain of 50% and 30% respectively. 6. A reversal of pullulanase action occurred on incubation with high concentrations of maltotriose. Condensation of maltosyl units to form a branched tetrasaccharide occurred less readily. 7. S. mitis pullulanase was rapidly inactivated at temperatures higher than 40 degrees , and the enzyme did not recover activity on storage at room temperature.     

The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system.

The effects in kidney of streptozotocin-induced diabetes and of insulin supplementation to diabetic animals on glycogen-metabolizing enzymes were determined. Kidney glycogen levels were approximately 30-fold higher in diabetic animals than in control or insulintreated diabetic animals. The activities of glycogenolytic enzymes i.e., phosphorylase (both a and b), phosphorylase kinase, and protein kinase were not significantly altered in the diabetic animals. Glycogen synthase (I form) activity decreased in the diabetic animals whereas total glycogen synthase (I + D) activity significantly increased in these animals. The activities were restored to control values after insulin therapy. Diabetic animals also showed a 3-fold increase in glucose 6-phosphate levels. These data suggest that higher accumulation of glycogen in kidneys of diabetic animals is due to increased amounts of total glycogen synthase and its activator glucose 6-phosphate.     

Histochemical studies on the enzymes of glycogen metabolism in hamster epididymis

Summary Active and total (active + inactive) phosphorylase and glycogen synthetase (I- and D-form) were studied in hamster epididymis in relation to glycogen. Immature and adult, sexually active and regressed animals were examined.Epididymis in adult animals, based on their phosphorylase activity, may be divided into 5 zones. The zone 1 epithelium contains particulate glycogen, rich in phosphorylase and glycogen synthetase. The epithelial cytoplasm also contains moderate phosphorylase activity. The zone 2 epithelium is almost devoid of phosphorylase. The zone 3 epithelium shows considerable phosphorylase activity both in principal and holocrine cells. The epithelium of the zone 4 contains the highest total phosphorylase activity. In the zone 5 epithelium phosphorylase and glycogen are absent, but glycogen synthetase is often observed.Holocrine cells, particularly in zones 3 and 4, contain predominating active phosphorylase, some glycogen, but no synthetase activity. The lumen in the zone 4 often shows a faint staining for glycogen.In immature animals, low phosphorylase activity is always present in the epithelial cells. Holocrine cells are detectable, by their phosphorylase activity, in 4 week animals. The division of zones is usually established slightly before sexual maturation.During the period of sexual regression, phosphorylase diminishes considerably. Glycogen, phosphorylase and glycogen synthetase are, however, detectable in the zone 1 of these animals.     

Regulation of glycogen synthetase and phosphorylase phosphatase activities in rat adipose tissue.

Incubation of a rat adipose tissue homogenate causes a time and temperature dependent activation of glycogen synthetase (UDP glucose:glycogen 4-alpha-glucosyltransferase) and simultaneous inactivation of phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Activation of glycogen synthetase at 15 and 23 degrees C was preceded by a lag period. The duration of the lag period could not be correlated with significant changes in phosphorylase activity. Addition of glucose and methylxanthines caused an increase in the rates of glycogen synthetase activation and phosphorylase inactivation. The effect on glycogen synthetase activation was mainly on the linear phase. Addition of AMP inhibited phosphorylase inactivation and accelerated glycogen synthetase activation. Addition of muscle phosphorylase alpha caused a prolongation of the lag period which lasted until phosphorylase alpha activity had decreased to the level originally present in the preparation. It is concluded that in adipose tissue activation of glycogen synthetase is not dependent on prior inactivation of phosphorylase and that other factors should be looked for to explain the lag period preceding glycogen synthetase activation.     

THE INCORPORATION OF GLUCOSE INTO BRAIN GLYCOGEN AND THE ACTIVITIES OF CEREBRAL GLYCOGEN PHOSPHORYLASE AND SYNTHETASE: SOME EFFECTS OF AMPHETAMINE

Abstract— The effects of amphetamine sulphate (5 mg/kg intraperitoneally) on the incorporation of radioactive carbon from [U-¹⁴C]glucose into the glycogen of mouse cerebral cortex, midbrain and hind-brain have been investigated. In all brain regions studied amphetamine induced a rapid decrease in glycogen followed by a slower return to control values. No significant alterations were observed in the steady state concentration of cerebral glucose. The initial fall in glycogen was associated with a fall in its specific radioactivity relative to that of cerebral glucose, whereas the resynthesis of the polysaccharide was associated with a marked increase in the relative specific radioactivity of glycogen. Other experiments demonstrated that amphetamine initially stimulates the breakdown of prelabelled glycogen and that the resulting molecule has fewer 1,4 linked glucose side chains.
Studies of the relative forms of the enzymes glycogen phosphorylase and glycogen synthetase suggested that rapid post mortem changes were less likely to occur if cerebral tissue was fixed by means of a freeze-blowing technique. Amphetamine administration resulted in a rapid though transient elevation of phosphorylase a activity in mouse forebrain. The level of glycogen synthetase I activity was unchanged initially but was markedly elevated during the period when there was a large increase in the rate of incorporation of glucose into glycogen. It is suggested that cerebral glycogen metabolism is controlled, at least in part, by the interconversion of the 'active' and 'inactive' forms of glycogen phosphorylase and synthetase.     

Fourth expansion and glucose perturbation of the Dictyostelium kinetic model

The kinetic model of carbohydrate metabolism has been expanded to include: (a) the accumulation of alpha and beta-cellulose, insoluble cell-wall glycogen and mucopolysaccharide; (b) the role of RNA turnover as a source of carbon for end-product synthesis and as a buffer regulating the level of uridine nucleotides in this metabolic network; and (c) the role of purine-nucleoside phosphorylase, 5'-AMP nucleotidase, nucleosidediphosphate kinase and polynucleotide phosphorylase. One of many predictions based on this model is that cells differentiating in the presence of glucose will produce sorocarps with an abnormally high trehalose to cellulose ratio. External perturbation of either the model or of developing cells by glucose increases the levels of sorocarp trehalose and glycogen, 5-fold and 6-fold respectively. Evaluation of the experimental data and the simulation analyses have allowed several predictions to be made concerning the compartmentation of metabolites and the permeability of cells to glucose during differentiation.     

The effects of glucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations.

In the isolated perfused rat liver, increasing glucose concentration from 5.5 to 55 mm in the perfusion medium caused a sequential inactivation of glycogen phosphorylase and activation of glycogen synthetase. The latter change was preceded by a lag period which corresponded to the time required to inactivate the major part of the phosphorylase. 2. The same sequence of events was observed in isolated rat hepatocytes incubated at 37C. In this preparation, the rate of phosphorylase inactivation was greatly increased by increasing the concentration of glucose and/or of K+ ions in the external medium. The same agents also caused the activation of glycogen synthetase, but this effect was secondary to the inactivation of phosphorylase. 3. In both types of preparations, the rate of synthetase activation was modulated by the residual amount of phosphorylase a that remained after the initial phase of rapid inactivation and was independent of glucose concentration. 4. In isolated hepatocytes, the rate of conversion of glucose into glycogen was propotional to the activity of synthetase a in the preparation. This conversion was preceded by a lag period which could be shortened by increasing either glucose or K+ concentration in the medium. The incorporation of labelled glucose into glycogen was simultaneous with a glycogenolytic process which could not be attributed to the activity of phosphorylase a.     

INCREASE OF GLUCOSE AND HIGH ENERGY PHOSPHATE RESERVE IN THE BRAIN AFTER HYDROCORTISONE1

Abstract— Young mice treated with hydrocortisone (50 mg/kg) subcutaneously for 10 days showed a doubling of brain glucose. Brain phospho-creatine, glucose-6-phosphate, and ATP increased slightly. Brain glycogen and lactate were unchanged. Total energy reserve of the brain was 23 per cent higher than the control value. Liver glycogen was increased 47 per cent; liver and blood glucose levels were 11 per cent lower than in control animals. Since the animals showed no evidence of sedation, these findings suggest a facilitated transport of glucose from blood into the brain under the influence of hydrocortisone. Other possible explanations include an inhibition of the hexose monophosphate shunt and a proportionate decrease in both the oxidative and glycolytic pathways of the brain, but it was concluded that these explanations are less likely.     

Action of degradative enzymes on the light fraction of bovine septa protein polysaccharide

1. The administration of cortisol and of other glucocorticoid steroids to starved mice produced an increase in liver glycogen content, an elevation of glycogen-synthetase activity and a predominantly particulate localization of both phosphorylase and glycogen-synthetase enzymes. 2. Three daily doses of actinomycin D caused a marked glycogen depletion, a significant decrease in glycogen-synthetase activity, the solubilization of phosphorylase and glycogen synthetase and the following effects on the activities of various other enzymes: a decrease in UDP-glucose pyrophosphorylase and phosphoglucomutase, an increase in glucose 6-phosphate dehydrogenase and no change in glucose 6-phosphatase, 6-phosphogluconate dehydrogenase, pyruvate kinase and UDP-glucose dehydrogenase. 3. Glucose ingestion, but not cortisol administration, reversed the effects of actinomycin D on liver glycogen content and on the activities of phosphorylase and glycogen synthetase.     

Glycogen and glycogen enzymes in the liver and striated muscle of rats under altered thyroid states

Changes induced in liver and striated muscle glycogen and glycogen enzymes (glycogen synthetase, glycogen phosphorylase and alpha-amylase) by hypothyroidism and hyperthyroidism in rats have been determined. There were no changes in liver glycogen synthetase, phosphorylase and amylase activities in the hypothyroid group. Hyperthyroid rats showed lower liver glycogen synthetase, phosphorylase a and amylase activities. In muscle, hypothyroid rats had lower phosphorylase activity. In the hyperthyroid group glycogen synthetase was increased.--The results presented do not completely agree with the glycogen levels found in both tissues studied, and they are obviously more related to other factors such as glucose availability. It can be concluded that under the conditions studied, the glycogen enzyme levels could not alone explain the variations of glycogen levels.     

Effect of hydrocortisone and glucagon on glycogen metabolism in the fetal rat liver.

1. Hydrocortisone increases in vivo incorporation of [14C] glucose into fetal liver glycogen in the last days of gestation, whereas in glucagon-treated fetuses, a slight decrease in the incorporation rate was found. 2. Hydrocortisone increases total synthetase activity as that of synthetase a but was without effect on fetal liver glycogen phosphorylase. 3. Glucagon causes a slight increase in phosphorylase a activity on days 19-21, and was without effect on the activities of synthetase a and total synthetase. 4. Dibutyryl cyclic AMP had no effect on the key enzymes of glycogen metabolism 1 h after injection in utero, whereas after 6 h an increase in phosphorylase a activity was found without any change in synthetase a activity.     

Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the UDPglucose:glycogen 4-alpha-glucosyltransferase.

The Neurospora crassa glycogen synthase (UDPglucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) was purified to electrophoretic homogeneity by a procedure involving ultracentrifugation, DEAE-cellulose column chromatography, (NH4)2SO4 fractionation and 3-aminopropyl-Sepharose column chromatography. The final purified enzyme preparation was almost entirely dependent on glucose-6-P and had a specific activity of 6.9 units per mg of protein. The subunit molecular weight of the glycogen synthase was determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel to be 88 000--90 000. The native enzyme was shown to have a molecular weight of 270 000 as determined by sucrose density gradient centrifugation. Thus, the glucose-6-P-dependent form of the N. crassa glycogen synthase can exist as trimer of the subunit. Limited proteolysis with trypsin or chymotrypsin converted the glucose-6-P-dependent form of the enzyme into an apparent glucose-6-P-independent form. The enzyme was shown to catalyze transfer of glucose from UDPglucose to glycogen as well as to its phosphorylase limit dextrin, but not to its beta-amylase limit dextrin. Moreover, glucose, maltose and maltotriose were not active as acceptors.     

Purification of glycogen debranching enzyme from porcine brain: evidence for glycogen catabolism in the brain

Amylo-1,6-glucosidase from porcine brain was purified to homogeneity by ammonium sulfate fractionation, followed by sequential steps of liquid chromatography on DEAE-Sephacel, Sephacryl S-300, and Super Q. The purified enzyme had both maltooligosaccharide transferase and amylo-1,6-glucosidase activities within a single polypeptide chain, and the combination of these two activities removed the branches of phosphorylase limit dextrin. Based on these results, the purified enzyme was identified as a glycogen debranching enzyme (GDE). The molecular weight of the brain GDE was 170,000 by gel-filtration and 165,000 by reducing SDS-PAGE. The pH profile of maltooligosaccharide transferase activity coincided with that of the amylo-1,6-glucosidase activity (pH optimum at 6.0). The existence of GDE as well as glycogen phosphorylase in the brain explains brain glycogenolysis fully and supports the hypothesis that glycogen is a significant source of energy in this organ.     

Structural rationale for the short branched substrate specificity of the glycogen debranching enzyme GlgX

Glycogen serves as major energy storage in most living organisms. GlgX, with its gene in the glycogen degradation operon, functions in glycogen catabolism by selectively catalyzing the debranching of polysaccharide outer chains in bacterial glycosynthesis. GlgX hydrolyzes α‐1,6‐glycosidic linkages of phosphorylase‐limit dextrin containing only three or four glucose subunits produced by glycogen phosphorylase. To understand its mechanism and unique substrate specificity toward short branched α‐polyglucans, we determined the structure of GlgX from Escherichia Coli K12 at 2.25 Å resolution. The structure reveals a monomer consisting of three major domains with high structural similarity to the subunit of TreX, the oligomeric bifunctional glycogen debranching enzyme (GDE) from Sulfolobus. In the overlapping substrate binding groove, conserved residues Leu270, Asp271, and Pro208 block the cleft, yielding a shorter narrow GlgX cleft compared to that of TreX. Residues 207–213 form a unique helical conformation that is observed in both GlgX and TreX, possibly distinguishing GDEs from isoamylases and pullulanases. The structural feature observed at the substrate binding groove provides a molecular explanation for the unique substrate specificity of GlgX for G4 phosphorylase‐limit dextrin and the discriminative activity of TreX and GlgX toward substrates of varying lengths. Proteins 2010. © 2010 Wiley‐Liss, Inc.