Activation of 4-alpha-glucanotransferase activity of porcine liver glycogen debranching enzyme with cyclodextrins

Glycogen debranching enzyme (GDE) is a single polypeptide chain containing distinct active sites for 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Debranching of phosphorylase limit dextrin from glycogen is carried out by cooperation of the two activities. We examined the effects of cyclodextrins (CDs) on debranching activity of porcine liver GDE using a fluorogenic branched dextrin, Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/84), as a substrate. B5/84 was hydrolyzed by the hydrolytic action of 4-alpha-glucanotransferase to B5/81 and maltotriose. The fluorogenic product was further hydrolyzed by the amylo-alpha-1,6-glucosidase activity to the debranched product, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (G8PA), and glucose. alpha-, beta- and gamma-CDs accelerated the liberation of B5/81 from B5/84, indicating that the 4-alpha-glucanotransferase activity was activated by CDs to remove the maltotriosyl residue from the maltotetraosyl branch. This led to acceleration of B5/84 debranching. The extent of 4-alpha-glucanotransferase activation increased with CD concentration before reaching a constant value. This suggests that there is an activator binding site and that the binding of CDs stimulates 4-alpha-glucanotransferase activity. In the porcine liver, glycogen degradation may be partially stimulated by the binding of a glycogen branch to this activator binding site.     

Acceptor specificity of 4-alpha-glucanotransferases of mammalian glycogen debranching enzymes

Glycogen debranching enzyme (GDE) has two distinct active sites for its 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. The GDE 4-alpha-glucanotransferases of mammals show stringent donor specificity; only alpha-glucans with an alpha-1,6-linked maltotetraosyl or maltotriosyl branch function as donors of a maltotriosyl or maltosyl residue. In this study, we investigated the acceptor specificity of the 4-alpha-glucanotransferases using methyl alpha-maltooligosides, p-nitrophenyl alpha-maltooligosides, and pyridylaminated maltooligosaccharides of various sizes as the acceptor substrates, and phosphorylase limit dextrin as the donor substrate. High-performance liquid chromatography analysis of the transfer products indicated that maltotriosyl and maltosyl residues were specifically transferred from phosphorylase limit dextrin to acceptors with a maltopentaosyl residue comprising a nonreducing-end. These results suggest that the acceptor binding sites in the active sites of mammalian GDE 4-alpha-glucanotransferases are composed of tandem subsites that are geometrically complementary to five glucose residues.     

Active site mapping of amylo-alpha-1,6-glucosidase in porcine liver glycogen debranching enzyme using fluorogenic 6-O-alpha-glucosyl-maltooligosaccharides

Glycogen debranching enzyme (GDE) has two enzymatic activities, 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase. Products with 6-O-alpha-glucosyl structures formed from phosphorylase limit dextrin by the 4-alpha-glucanotransferase activity are hydrolyzed to glucose by the amylo-alpha-1,6-glucosidase activity. Here, we probed the active site of amylo-alpha-1,6-glucosidase in porcine liver GDE using various 6-O-alpha-glucosyl-pyridylamino (PA)-maltooligosaccharides, with structures (Glcalpha1-4)(m)(Glcalpha1-6)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol residue). Fluorogenic dextrins were prepared from 6-O-alpha-glucosyl-alpha-, beta-, or gamma-cyclodextrin through partial acid hydrolysis, followed by fluorescent tagging of the reducing-end residues of the hydrolysates and separation by gel filtration and reversed-phase HPLC. Porcine liver GDE hydrolyzed dextrins with the structure Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glc to glucose and the corresponding PA-maltooligosaccharides, whereas other dextrins were not hydrolyzed. Thus, substrates must have two glucosyl residues sandwiching the isomaltosyl moiety to be hydrolyzed. The rate of hydrolysis increased as m increased and reached maximum at m = 4. The rates were the highest when n = 1 but did not vary much with changes in n. Of the dextrins examined, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (6(3)-O-alpha-glucosyl-PA-maltoheptaose) was hydrolyzed most rapidly, suggesting that it fits the best in the amylo-alpha-1,6-glucosidase active site. It is likely that the active site accommodates 6(2)-O-alpha-glucosyl-maltohexaose and that the interactions of seven glucosyl residues with the active site allow the most rapid hydrolysis of the alpha-1,6-glucosidic linkage of the isomaltosyl moiety.     

Fluorogenic substrates of glycogen debranching enzyme for assaying debranching activity

Glycogen debranching enzyme (GDE) degrades glycogen in concert with glycogen phosphorylase. GDE has two distinct active sites for maltooligosaccharide transferase and amylo-1,6-glucosidase activities. Phosphorylase limit dextrin from glycogen is debranched by cooperation of the two activities. Fluorogenic branched dextrins were prepared as substrates of GDE from pyridylaminated maltooctaose (PA-maltooctaose) and maltotetraose, taking advantage of the synthetic action of Klebsiella pneumoniae pullulanase. Their structures were as follows: Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (B3), Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B4), Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5), Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B6), Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B7), and Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B8). These dextrins were incubated with porcine skeletal muscle GDE. No fluorogenic product was found in the digest of B8. The fluorogenic products from B3, B4, and B5 were PA-maltooctaose only. PA-maltooctaose, PA-maltoundecaose, and 6(7)-O-alpha-glucosyl-PA-maltooctaose were from B7. PA-maltooctaose and 6(6)-O-alpha-glucosyl-PA-maltooctaose were from B6. These results indicate that the maltooligosaccharide transferase removed the maltotriosyl residues from the maltotetraosyl branches by hydrolysis or intramolecular transglycosylation to expose 6-O-alpha-glucosyl residues, and then the amylo-1,6-glucosidase hydrolyzed the alpha-1,6-glycosidic linkages of the products rapidly. Probably, 6-O-alpha-glucosyl-PA-maltooctaoses from B7 and B6 were less susceptible to the amylo-1,6-glucosidase than were those from B3, B4, and B5. Taking this into account, B3, B4, and B5 are suitable substrates for GDE assay.     

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.     

Glycogen debranching pathway deduced from substrate specificity of glycogen debranching enzyme

Glycoconjugate Journal - Glycogen debranching enzyme (GDE) is bifunctional in that it exhibits both 4-α-glucanotransferase and amylo-α-1,6-glucosidase activity at two distinct catalytic...     

Association of bi-functional activity in the N-terminal domain of glycogen debranching enzyme

Glycogen debranching enzyme (GDE) in mammals and yeast exhibits α-1,4-transferase and α-1,6-glucosidase activities within a single polypeptide chain and facilitates the breakdown of glycogen by a bi-functional mechanism. Each enzymatic activity of GDE is suggested to be associated with distinct domains; α-1,4-glycosyltransferase activity with the N-terminal domain and α-1,6-glucosidase activity with the C-terminal domain. Here, we present the biochemical features of the GDE from Saccharomyces cerevisiae using the substrate glucose(n)-β-cyclodextrin (Gn-β-CD). The bacterially expressed and purified GDE N-terminal domain (aa 1–644) showed α-1,4-transferase activity on maltotetraose (G4) and G4-β-CD, yielding various lengths of (G)_n. Surprisingly, the N-terminal domain also exhibited α-1,6-glucosidase activity against G1-β-CD and G4-β-CD, producing G1 and β-CD. Mutational analysis showed that residues D535 and E564 in the N-terminal domain are essential for the transferase activity but not for the glucosidase activity. These results indicate that the N-terminal domain (1–644) alone has both α-1,4-transferase and the α-1,6-glucosidase activities and suggest that the bi-functional activity in the N-domain may occur via one active site, as observed in some archaeal debranching enzymes.     

Cyclization reaction catalyzed by glycogen debranching enzyme (EC 2.4.1.25/EC 3.2.1.33) and its potential for cycloamylose production

Glycogen debranching enzyme (GDE) has 4-alpha-glucanotransferase and amylo-1,6-glucosidase activities in the single polypeptide chain. We analyzed the detailed action profile of GDE from Saccharomyces cerevisiae on amylose and tested whether GDE catalyzes cyclization of amylose. GDE treatment resulted in a rapid reduction of absorbance of iodine-amylose complex and the accumulation of a product that was resistant to an exo-amylase (glucoamylase [GA]) but was degraded by an endo-type alpha-amylase to glucose and maltose. These results indicated that GDE catalyzed cyclization of amylose to produce cyclic alpha-1,4 glucan (cycloamylose). The formation of cycloamylose was confirmed by high-performance anion-exchange chromatography, and the size was shown to range from a degree of polymerization of 11 to a degree of polymerization around 50. The minimum size and the size distribution of cycloamylose were different from those of cycloamylose produced by other 4-alpha-glucanotransferases. GDE also efficiently produced cycloamylose even from the branched glucan substrate, starch, demonstrating its potential for industrial production of cycloamylose.     

Reassessment of the catalytic mechanism of glycogen debranching enzyme

The amylo-1,6-glucosidase catalytic activity of glycogen debranching enzyme allows it to hydrolyze alpha-D-glucosyl fluoride, in the absence or presence of glycogen or oligosaccharides, releasing equal amounts of fluoride and glucose at rates comparable to those seen with the natural substrates. 2-Deoxy-2-fluoro-alpha-D-glucosyl fluoride is found to be a poor substrate, rather than the covalent inhibitor that would be expected for a glucosidase which catalyzes hydrolysis of the glycosidic linkage with retention of anomeric configuration. In fact, analysis of the glucosidase reaction by NMR reveals that the debranching enzyme hydrolyzes the glycosidic linkage with inversion of configuration, releasing beta-D-glucose from both alpha-glucosyl fluoride and its natural substrate, the phosphorylase limit dextrin. In contrast, its transferase activity necessarily proceeds with retention of configuration. As has been seen with other "inverting" glycosidases, the debranching enzyme releases beta-D-glucose from beta-D-glucosyl fluoride in the presence of oligosaccharides such as maltohexaose and cyclomaltoheptaose but, unlike the others, not in their absence. An intermediate glucosyl-alpha-(1,6)-cyclomaltoheptaose has been detected by NMR analysis. In the presence of a water-soluble carbodiimide, a single mole of glycine ethyl ester is incorporated into each mole of the debranching enzyme, resulting in its inactivation when measured by the combined assay for both transferase and glucosidase activities. Measurement of the latter two activities independently indicates that it is the transferase activity which is inactivated, while the glucosidase activity, measured with alpha-D-glucosyl fluoride as substrate, is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Identification of the catalytic residues of bifunctional glycogen debranching enzyme

Eukaryotic glycogen debranching enzyme (GDE) possesses two different catalytic activities (oligo-1,4-->1,4-glucantransferase/amylo-1,6-glucosidase) on a single polypeptide chain. To elucidate the structure-function relationship of GDE, the catalytic residues of yeast GDE were determined by site-directed mutagenesis. Asp-535, Glu-564, and Asp-670 on the N-terminal half and Asp-1086 and Asp-1147 on the C-terminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes. The five mutant enzymes, D535N, E564Q, D670N, D1086N, and D1147N were constructed. The mutant enzymes showed the same purification profiles as that of wild-type enzyme on beta-CD-Sepharose-6B affinity chromatography. All the mutant enzymes possessed either transferase activity or glucosidase activity. Three mutants, D535N, E564Q, and D670N, lost transferase activity but retained glucosidase activity. In contrast, D1086N and D1147N lost glucosidase activity but retained transferase activity. Furthermore, the kinetic parameters of each mutant enzyme exhibiting either the glucosidase activity or transferase activity did not vary markedly from the activities exhibited by the wild-type enzyme. These results strongly indicate that the two activities of GDE, transferase and glucosidase, are independent and located at different sites on the polypeptide chain.     

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.     

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.     

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.     

Crystal structure of glycogen synthase: homologous enzymes catalyze glycogen synthesis and degradation

Glycogen and starch are the major readily accessible energy storage compounds in nearly all living organisms. Glycogen is a very large branched glucose homopolymer containing about 90% alpha-1,4-glucosidic linkages and 10% alpha-1,6 linkages. Its synthesis and degradation constitute central pathways in the metabolism of living cells regulating a global carbon/energy buffer compartment. Glycogen biosynthesis involves the action of several enzymes among which glycogen synthase catalyzes the synthesis of the alpha-1,4-glucose backbone. We now report the first crystal structure of glycogen synthase in the presence and absence of adenosine diphosphate. The overall fold and the active site architecture of the protein are remarkably similar to those of glycogen phosphorylase, indicating a common catalytic mechanism and comparable substrate-binding properties. In contrast to glycogen phosphorylase, glycogen synthase has a much wider catalytic cleft, which is predicted to undergo an important interdomain 'closure' movement during the catalytic cycle. The structures also provide useful hints to shed light on the allosteric regulation mechanisms of yeast/mammalian glycogen synthases.     

Enzymes of glycogen mobilization in the photosynthetic procaryote,Anacystis nidulans

Glycogen, the principal storage compound of assimilatory products in Anacystis nidulans, is synthesized in the light and degraded in the dark. ¹⁴C-labelled glycogen and its radioactive limit dextrin obtained by phosphorylase action were used as substrates to identify enzymes involved in glycogen mobilization. A crude homogenate of cells kept in the dark contained the following enzymes: glycogen phosphorylase (EC 2.4.1.1.) that is firmly bound to glycogen, a debranching enzyme that hydrolyzes 1,6--glucosidic bonds, and an -glucosidase (EC 3.2.1.20). Other amylolytic enzymes were not detectable Using ion exchange chromatography on DEAE-cellulose, -glucosidase and the debranching enzyme could be partly separated from each other and completely from the phosphorylase-glycogen complex. On the basis of their known substrate specificities, the cooperation of these 3 enzymes is sufficient to account for the complete conversion of glycogen into glucose and glucose 1-phosphate.     

Some regulatory properties of glycogen phosphorylase from Streptococcus salivarius

Purified (200-fold) glycogen phosphorylase (EC 2.4.1.1) of Streptococcus salivarius was activated by AMP and NaF when assayed both in the direction of synthesis and in the direction of phosphorolysis. Activation by NaF + AMP was greater than the sum of their individual effects. In the direction of synthesis, the K_m for AMP was 0.25 mm and was decreased to 0.125 mm in the presence of NaF. The K_m for NaF was 0.49 m and was decreased to 0.40 m in the presence of AMP. Glycogen phosphorolysis was similarly affected by AMP and NaF, except that above a concentration of 2 mm AMP was inhibitory. The effects of AMP and NaF were reversible since preincubation with these compounds, followed by dialysis, restored activity almost to the control values although some inhibition of enzyme activity was noted with the samples preincubated with NaF. The presence of both NaF and AMP had no effect on the K_m values for glucose-1-P and glycogen in the direction of synthesis, but increased the V of the enzyme.When assayed in the absence of AMP and NaF in the direction of synthesis, the enzyme was slightly inhibited by glucose and glucose-6-P, and activated by P-enolpyruvate and ADP-glucose. In the presence of AMP and NaF, the enzyme was inhibited by glucose, glucose-6-P and ADP-glucose, but was activated by P-enolpyruvate. Fructose-1,6-P₂ had no effect on the enzyme. The enzyme was further activated in the absence of AMP and NaF by adenosine, ATP, GMP, cyclic AMP and ADP, and was slightly inhibited by GTP and GDP. In the presence of AMP and NaF, however, these compounds, with the exception of adenosine, either did not show any effect or were slightly inhibitory. Adenosine was slightly stimulatory with NaF + AMP, but not with AMP alone. In the direction of phosphorolysis, the enzyme was inhibited by glucose and ADP-glucose, and activated by P-enolpyruvate, fructose-1,6-P₂ and ATP, both in the presence and absence of AMP + NaF.     

Adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscle with increased glycogen content

The effect of glycogen content on the activation of glycogen phosphorylase during adrenaline stimulation was investigated in soleus muscles from Wistar rats. Furthermore, adrenergic activation of glycogen phosphorylase in the slow-twitch oxidative soleus muscle was compared to the fast-twitch glycolytic epitrochlearis muscle. The glycogen content was 96.4 +/- 4.4 mmol (kg dw)(-1) in soleus muscles. Three hours of incubation with 10 mU/ml of insulin (and 5.5 mM glucose) increased the glycogen content to 182.2+/-5.9 mmol (kg dw)(-1) which is similar to that of epitrochlearis muscles (175.7+/-6.9 mmol (kg dw)(-1)). Total phosphorylase activity in soleus was independent of glycogen content. Adrenaline (10(-6) M) transformed about 20% and 35% (P < 0.01) of glycogen phosphorylase to the a form in soleus with normal and high glycogen content, respectively. In epitrochlearis, adrenaline stimulation transformed about 80% of glycogen phosphorylase to the a form. Glycogen synthase activation was reduced to low level in soleus muscles with both normal and high glycogen content. In conclusion, adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscles with increased glycogen content. Glycogen phosphorylase activation during adrenaline stimulation was much higher in epitrochlearis than in soleus muscles with a similar content of glycogen.     

Metabolic function of glycogen phosphorylase and trehalose phosphorylase in fruit-body formation ofFlammulina velutipes

Glycogen phosphorylase in the vegetative mycelium ofFlammulina velutipes converts glycogen to α-glucose 1-phosphate (G1P) in the colony during fruit-body development. Glycogen may contribute to the synthesis of trehalose as the starting material in the vegetative mycelium during the fruiting process of the colony, and the trehalose produced is translocated into the fruit-bodies as the main carbohydrate substrate for their development. Trehalose phosphorylase activity in the vegetative mycelium was at a relatively high level until fruit-body initiation, suggesting the turnover of this disaccharide during the vegetative stage of the colony development. Trehalose phosphorylase activity in the stipes showed a peak level at the early phase of fruit-body development, suggesting the continuing phosphorolysis of trehalose by this enzyme. The stipes also showed a high specific activity of phosphoglucomutase at a sufficient level to facilitate the conversion of G1P to α-glucose 6-phosphate (G6P). In the pilei a large amount of G1P remained until the growth of the fruit-bodies ceased. Trehalase activities in the stipes and pilei were at a very low level, and this enzyme may not contribute to the catabolism of trehalose in the fruit-body development.     

Hormonal regulation of glycogen metabolism in neonatal rat liver

1. The development of active and inactive phosphorylase was determined in rat liver during the perinatal period. No inactive form could be found in tissues from animals less than 19 days gestation or older than the fifth postnatal day. 2. The regulation of phosphorylase in organ cultures of foetal rat liver was examined. None of the agents examined [glucagon, insulin or dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate)] changed the amount of phosphorylase activity. 3. Glycogen concentration in these explants were nevertheless decreased more than twofold by 4h of incubation with glucagon or dibutyryl cyclic AMP. Incubation with insulin for 4h increased the glycogen content twofold. 4. Glycogen synthetase activity was examined in these explants. I-form activity (without glucose 6-phosphate) was found to decrease by a factor of two after 4h of incubation with dibutyryl cyclic AMP, whereas I+D activity (with glucose 6-phosphate) remained nearly constant. Incubation for 4h with insulin increased I-form activity threefold, with only a slight increase in I+D activity. 5. When explants were incubated with insulin followed by addition of dibutyryl cyclic AMP, the effects of insulin on glycogen concentration and glycogen synthetase activity were reversed. 6. These results indicate that the regulation of glycogen synthesis may be the major factor in the hormonal control of glycogen metabolism in neonatal rat liver.