Proglycogen: a low-molecular-weight form of muscle glycogen.

We recently reported that muscle contains a trichloroacetic acid-precipitable component having Mr approx. 400 kDa that can be glucosylated by an endogenous enzyme acting on UDPglucose. This component contains within itself the autocatalytic, self-glucosylating protein glycogenin, the primer for glycogen synthesis. We now report that this substance, to which we give the name proglycogen, is a glycogen-like molecule constituting about 15% of total glycogen. It acts as a very efficient acceptor of glucose residues added from UDPglucose. Further, that the endogenous enzyme that adds the glucose to proglycogen is not the autocatalytic protein but a glycogen synthase-like enzyme. Proglycogen may be an intermediate in the synthesis and degradation of macromolecular glycogen and may exist and be metabolized as a separate entity. Consideration should now be given to the revival of the concept that tissue contains two forms of glycogen. One is proglycogen. The other is the 'classical', macromolecular glycogen. Additionally, proglycogen and glycogen may be glucosylated by different forms of synthase.     

Combined action of Escherichia coli glycogen synthase and branching enzyme in the so-called "unprimed" polyglucoside synthesis.

Mutants of Escherichia coli which are unable to synthesize glycogen were used to study the so-called “unprimed” synthesis of glycogen. The glycogen synthase has been partially purified from these mutants. During the purification, attempts were made to separate the activity which requires the addition of an exogenous primer (primed activity) from the activity which does not require a primer but is highly dependent on the presence of some salts such as citrate and EDTA (unprimed activity). No separation between these two activities could be achieved but the results obtained by chromatography on DEAE-Sephadex indicate that there is a single form of glycogen synthase which is responsible for both unprimed and primed activity. The evidence that a single protein was necessary to catalyze these two reactions was given by the findings that mutants defective in glycogen synthase activity were unable to catalyze glucosyl transfer without added primer. At low concentration, the glycogen synthase purified from a branching enzyme negative mutant catalyzed the unprimed reaction at a slow rate even in presence of salts. A protein activator of this reaction was found in mutants lacking glycogen synthase but not in mutants lacking branching enzyme. The hypothesis that this activator is the branching enzyme itself was supported by the observation that it co-purified with the branching enzyme from a E. coli strain defective in glycogen synthase activity. EDTA or Triton X-100 increased the stimulation of the unprimed synthesis by the branching enzyme. The apparent affinity of the glycogen synthase for glycogen was increased twofold in the presence of EDTA but the branching enzyme further increased the effect of EDTA. The combined action of the glycogen synthase and the branching enzyme on the endogenous glucan associated with the synthase may account for the unprimed activity observed in vitro.     

Purification of Glycogen Phosphorylase from Bovine Brain and Immunocytochemical Examination of Rat Glial Primary Cultures Using Monoclonal Antibodies Raised Against This Enzyme

The physiological function in brain of glycogen and the enzyme catalyzing the rate-limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700-fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia-rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron-rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of the brain.     

Appearance of a nonfunctional isozyme of hepatic glycogen synthase in late gestation

Glycogen levels, glycogen synthase activities, and glycogen synthase protein levels were determined in liver tissues obtained from 14- to 19-day-old fetal mice, newborn mice, and adult mice. The results of these experiments demonstrate a significant increase in the quantity of hepatic glycogen synthase beginning at Day 17 of gestation and reaching adult levels at birth. However, during the same time period, there is a dramatic decrease in total glycogen synthase activity suggesting that the accumulating glycogen synthase molecules are unable to transfer UDP-glucose to glycogen. These inversely coordinated changes in the quantity and activity of glycogen synthase are consistent with the suggestion that glycogen synthesis in the near-term fetal mouse is being maintained by preexisting enzyme, while accumulating enzyme molecules may represent a quiescent isozyme.     

Phosphate incorporation during glycogen synthesis and Lafora disease

Glycogen is a branched polymer of glucose that serves as an energy store. Phosphate, a trace constituent of glycogen, has profound effects on glycogen structure, and phosphate hyperaccumulation is linked to Lafora disease, a fatal progressive myoclonus epilepsy that can be caused by mutations of laforin, a glycogen phosphatase. However, little is known about the metabolism of glycogen phosphate. We demonstrate here that the biosynthetic enzyme glycogen synthase, which normally adds glucose residues to glycogen, is capable of incorporating the β-phosphate of its substrate UDP-glucose at a rate of one phosphate per approximately 10,000 glucoses, in what may be considered a catalytic error. We show that the phosphate in glycogen is present as C2 and C3 phosphomonoesters. Since hyperphosphorylation of glycogen causes Lafora disease, phosphate removal by laforin may thus be considered a repair or damage control mechanism.     

The calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Purification, subunit structure and substrate specificity

A calmodulin-dependent glycogen synthase kinase distinct from phosphorylase kinase has been purified approximately equal to 5000-fold from rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate (0-33%), and chromatographies on phosphocellulose, calmodulin-Sepharose and DEAE-Sepharose. 0.75 mg of protein was obtained from 5000 g of muscle within 4 days, corresponding to a yield of approximately equal to 3%. The Km for glycogen synthase was 3.0 microM and the V 1.6-2.0 mumol min-1 mg-1. The purified enzyme showed a major protein staining band (Mr 58 000) and a minor component (Mr 54 000) when examined by dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 696 000 by sedimentation equilibrium centrifugation, indicating a dodecameric structure. Electron microscopy suggested that the 12 subunits were arranged as two hexameric rings stacked one upon the other. Following incubation with Mg-ATP and Ca2+-calmodulin, the purified protein kinase underwent an 'autophosphorylation reaction'. The reaction reached a plateau when approximately equal to 5 mol of phosphate had been incorporated per 58 000-Mr subunit. Both the 58 000-Mr and 54 000-Mr species were phosphorylated to a similar extent. Autophosphorylation did not affect the catalytic activity. The calmodulin-dependent protein kinase initially phosphorylated glycogen synthase at site-2, followed by a slower phosphorylation of site-1 b. The protein kinase also phosphorylated smooth muscle myosin light chains, histone H1, acetyl-CoA carboxylase and ATP-citrate lyase. These findings suggest that the calmodulin-dependent glycogen synthase kinase may be a enzyme of broad specificity in vivo. Glycogen synthase kinase-4 is an enzyme that resembles the calmodulin-dependent glycogen synthase kinase in phosphorylating glycogen synthase (at site-2), but not glycogen phosphorylase. Glycogen synthase kinase-4 was unable to phosphorylate any of the other proteins phosphorylated by the calmodulin-dependent glycogen synthase kinase, nor could it phosphorylate site 1 b of glycogen synthase. The results demonstrate that glycogen synthase kinase-4 is not a proteolytic fragment of the calmodulin-dependent glycogen synthase kinase, that has lost its ability to be regulated by Ca2+-calmodulin.     

Turkey liver xanthine dehydrogenase. Reactivation of the cyanide-inactivated enzyme by sulphide and by selenide

1. The glycogen present in the liver of rat foetuses was labelled by injecting a trace amount of [6-(3)H]glucose into the mother at 19.5 days of gestation. The radioactivity incorporated in the glycogen 4h after the administration of the label was still present 38h later. A large proportion of this radioactivity was on the outer chains of the polysaccharide. These results indicate that there is normally almost no glycogen degradation in the foetal liver. In contrast, glycogen breakdown occurs very rapidly in the livers of foetuses whose mother is anaesthetized. 2. Glycogen synthetase is present in the liver at day 16 of gestation at a concentration as high as 30% of that in the adult, but essentially as an inactive (b) enzyme. The appearance of synthetase phosphatase between days 18 and 19 corresponds to that of synthetase a and to the beginning of glycogen synthesis. From day 19 to 21.5 the amount of synthetase a present in the foetal liver is just sufficient to account for the actual rate of glycogen deposition. 3. The content of total phosphorylase in the foetal liver increases continuously from day 16 to birth. However, a precise measurement of the a and b forms of the enzyme in the liver of non-anaesthetized foetuses is not possible. Taking the rate of glycogenolysis as an appropriate index of phosphorylase activity, we conclude that this enzyme is almost entirely in the inactive form in the foetal liver under normal conditions. 4. The accumulation of glycogen in the liver during late pregnancy may therefore be explained by a relatively slow rate of synthesis and a nearly total absence of degradation.     

‘Insulin-like’ effects of lithium ion on isolated rat adipocytes II. Specific activation of glycogen synthase

Lithium ion, like insulin, activated adipocyte glycogen synthase with or without glucose in the medium. However, the effect of lithium ion was much greater than that of insulin under both conditions. The lithium-activated glycogen synthase was stable to both Sephadex chromatography and ethanol precipitation of the enzyme, indicating that the effect of lithium ion on glycogen synthase was through covalent modification of the enzyme. Glycogen synthase was significantly activated by lithium ion under conditions where concentrations of cellular ATP were unaffected. The effect of lithium ion on glycogen synthase was rapid and observed at concentrations as low as 1 to 3 mM, reaching a maximum at the concentration of 40 mM. It was thus the most sensitive of all the effects studied (see previous paper). Insulin further stimulated glycogen synthase at low concentrations but not at maximal concentration of lithium ion. Lithium-activated glycogen synthase was inhibited by both epinephrine and dibutyryl cyclic AMP, but was not affected by the removal of extracellular Ca++. Interestingly, lithium ion had no detectable effect on basal pyruvate dehydrogenase as well as on epinephrine-stimulated phosphorylase. The failure of lithium ion to thus mimic insulin actions on pyruvate dehydrogenase and on phosphorylase suggests that the action of lithium ion on glycogen synthase is quite specific and may be mediated by stimulating a phosphatase or by inhibiting a protein kinase acting specifically on glycogen synthase.     

Turbidimetric method for determination of glycogen phosphorylase activity and its use for estimation of equilibrium position of enzymic reaction

A turbidimetric method has been developed for the continous monitoring of the enzyme reaction catalyzed by glycogen phosphorylase. This method is based on the registration of the turbidity of glycogen solution at wavelengths above 300 nm. It has been shown that increase in the turbidity is strictly proportional to the quantity of glucose 1-phosphate formed during the enzyme reaction. The method has the advantage of continuity, and it is suitable for determining the initial rate of catalytic synthesis or degradation of glycogen in a relatively simple and fast way. The kinetic experiments may be carried out under various conditions. The method of calculation of the overall equilibrium constant of the enzyme reaction catalyzed by glycogen phosphorylase has been elaborated. This method is based on the analysis of the dependence of the initial rate of the enzyme reaction on the proportiona of the substrate of the forward reaction: [P_i]/([P_i]+[G-1-P]).     

Stimulation of glycogen synthase phosphorylation by calcium-dependent regulator protein.

Phosphorylation of skeletal muscle glycogen synthase catalyzed by a protein kinase is stimulated up to 10-fold by the calcium-dependent regulator (CDR) protein. Half-maximal stimulation requires about 1 microgram of CDR/ml. Phosphorylation by the CDR-dependent synthase kinase is more rapid at pH 8.6 than at pH 6.8 and is blocked by ethylene glycol bis(beta-aminoethyl-ether)N,N'-tetraacetic acid and trifuloperazine. Approximately 60 to 70% of the phosphate is incorporated into the trypsin-insensitive region of glycogen synthase resulting in conversion of the a form to the b form of the enzyme. The CDR-dependent synthase kinase is not myosin light chain kinase, as this enzyme does not phosphorylate glycogen synthase. Furthermore, synthase phosphorylation by the cAMP-dependent protein kinase catalytic subunit is not affected by CDR. The possibility that CDR-dependent synthase kinase may be phosphorylase kinase is being investigated.     

Effects of hormones and serum on glycogen metabolism in adult rat liver parenchymal cell primary cultures.

Parenchymal cells from adult rat liver, isolated by a collagenase perfusion technique, have been maintained in primary culture and a detailed study on carbohydrate metabolism carried out over the initial 48-hour culture period. The glucose concentration of the medium exerts a major influence on glycogen accumulation by the cells. Insulin, particularly at high glucose concentrations, stimulates glycogen biosynthesis, whereas glucagon prevents glycogen accumulation. Dexamethasone was without effect on glycogen metabolism. Glucose appears to stimulate glycogen accumulation by activation of glycogen synthetase enzyme. However, there is a gradual loss of synthetase activity throughout the culture period. Similar decreases in activity were noted for pyruvate kinase, aldolase and hexokinase. Glucose, insulin and dexamethasone were unable to prevent these decreases in enzyme activity. Foetal bovine serum contains fructose and this hexose appears to be the factor in serum which is responsible for the activation of glycogen accumulation in the presence of physiological glucose concentrations. The lactic acid content of the serum may also stimulate glycogen accumulation. In general, there is a gradual loss of the pattern of carbohydrate metabolism typical of differentiated hepatocytes during the culture period.     

Hexose phosphates as regulators of hepatic glycogen synthase phosphatases

The activity of glycogen synthase phosphatase from smooth endoplasmic reticulum of liver was stimulated markedly by galactose-6- and fructose-6-phosphates and to a lesser extent by glucose-1- and 2-deoxyglucose-6-phosphates. The synthase phosphatase of liver cytosol showed strong activation by glucose-1-, glucose-6- and fructose-6-phosphates and smaller activation by galactose-6- and 2-deoxyglucose-6-phosphates. Kinetic analysis showed that the activators did not affect the Km for glycogen synthase D, for either enzyme. The mechanism of activation of the two phosphatases by hexose phosphates appears to be by combination of the activator at a specific activator site on the enzyme rather than by substrate modulation. It is concluded that certain hexose phosphates, particularly fructose-6-phosphate and glucose-1-phosphate, can function as regulators of hepatic synthase phosphatase activity, and that this may explain the ability of elevated blood glucose to increase both glycogen synthase I activity and glycogen synthesis in the liver.     

The hysteretic properties of glycogen synthase I.

Glycogen-free synthase I from human polymorphonuclear leukocytes is activated by its own substrate, glycogen, in a slow, time-dependent process (hysteretic activation). This lag in response to addition of glycogen depends on the concentration of glycogen, pH and temperature. At pH 7.4 and at a temperature of 30 degrees C, the half-time of activation t 1/2 decreases from 89 min at 0.004 mg/ml glycogen to 6 min at 25 mg/ml. The activation is accelerated by increasing temperature and pH, but is not influenced by enzyme concentration, glucose 6-phosphate, UDP, high ionic strength, EDTA, mercaptoethanol, glucose, sucrose or amylase limit dextrin. The Km for UDP-glucose (0.024 mM) and the activity ratio were unchanged during the activation process. The activation can be described by vt = vf + (vo - vf) e-kt where vt, vf and vo are velocities at times t, O and infinity and k is a complex rate constant. Evidence from ultracentrifugation and kinetic studies is presented to substantiate the hypothesis that the underlying mechanism is a simple biolecular process: enzyme + glycogen in equilibrium enzyme-glycogen complex, with the dissociation constant Ks = 0.003 mg/ml. The hysteretic activation may become rate-limiting during experiments in vitro with synthase. The possibility of a physiological role in glycogen metabolism, perhaps in the form of a concerted hysteresis with H+ is discussed.     

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.     

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.     

Specific mixed disulfide formation with purified bovine cardiac glycogen synthase I and glutathione

Bovine cardiac glycogen-free glycogen synthase I reacts with oxidized glutathione at low temperature to partially inactivate the enzyme. Evidence is presented that a mixed disulfide between glutathione and the enzyme is formed in this reaction. A short incubation of the GSSG-treated enzyme with dithiothreitol restores full enzyme activity. The reaction with GSSG is pH dependent and the product is quite stable at neutral pH. Oxidation of one sulfhydryl group in glycogen synthase is associated with a loss of 60-70% of the enzyme activity. Further modification of protein sulfhydryls has less effect on the enzyme activity. Other low molecular weight disulfides also inactivate glycogen synthase and treatment with [35S]cystine to produce a 40% loss of enzyme activity gave rise to a single major radioactive peptide after cyanogen bromide digestion. Thus the GSSG-mediated inactivation of glycogen synthase apparently occurs through a single reactive sulfhydryl group that forms a mixed disulfide with low molecular weight disulfide molecules. Uridine 5'-diphosphate glucose and glycogen prevent the inactivation of glycogen-free glycogen synthase with GSSG, and glucose 6-phosphate retards the rate of inactivation. Reduction and reactivation of the GSSG-oxidized glycogen synthase is not affected by glycogen and it occurs readily at neutral pH with dithiothreitol, mercaptoethanol, or cysteamine. Oxidation of the reactive sulfhydryl group with GSSG has no effect on the rate of glycogen synthase phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.     

Stabilising normal and mis-sense variant alpha-glucosidase

alpha-Glucosidase (EC 3.2.1.3) is a lysosomal enzyme that hydrolyses alpha-1,4- and alpha-1,6-linkages of glycogen to produce free glucose. A deficiency in alpha-glucosidase activity results in glycogen storage disorder type II (GSD II), also called Pompe disease. Here, d-glucose was shown to be a competitive inhibitor of alpha-glucosidase and when added to culture medium at 6.0 g/L increased the production of this protein by CHO-K1 expression cells and stabilised the enzyme activity. D-Glucose also prevented alpha-glucosidase aggregation/precipitation and increased protein yield in a modified purification scheme. In fibroblast cells, from adult-onset GSD II patients, D-glucose increased the residual level of alpha-glucosidase activity, suggesting that a structural analogue of d-glucose may be used for enzyme enhancement therapy.     

Lysosomal glycogen accumulation in rat liver and its in vivo kinetics after a single intraperitoneal injection of acarbose, an alpha-glucosidase inhibitor

A single intraperitoneal injection of acarbose (400 mg/kg) into rats caused lysosomal accumulation of glycogen in the liver, mimicking the cytological characteristics of human glycogen storage disease type II (Pompe's disease). The animal model is therefore useful for studying the pathogenesis of the disease. In the present study, we applied this model to examine the lysosomal hydrolytic pathway of glycogen in vivo. To quantify the lysosomal glycogen, the lysosome-rich fraction was rapidly prepared from liver homogenate by agglutination in the presence of Ca2+. Then the fraction was treated with alpha-amylase in isotonic medium to remove cytosolic glycogen, followed by transfer to hypotonic conditions in the presence of Triton X-100 to destroy total glycogen. The amount of lysosomal glycogen was calculated from the difference between the glycogen levels measured before and after the treatment under hypotonic conditions, and then it was corrected based on measurements of the intactness (%) of lysosomes and the recovery (%) of the lysosomal marker enzyme (beta NAGase). We observed no measurable lysosomal glycogen in normal liver by this method, and this was confirmed by electron microscopy. After administration of acarbose, the lysosomal glycogen level increased to 2.5 mg/g liver within 2 days, and then decreased gradually at a rate of 0.4 mg/day/g. The accumulation of glycogen in the lysosomes at an initial velocity of 1.5 mg/day/g liver may be considered as the amount of glycogen that would normally be degraded by acid alpha-glucosidase. Therefore, assuming that the liver breaks down about 40 mg glycogen/day/g, we estimated that about 3% of the glycogen would be hydrolyzed by the lysosomal pathway.     

Glycogen synthesis in resident and thioglycollate-elicited rat peritoneal exudate cells

The values of the A0.5 for glucose-6-P, apparent Km for UDPglucose and -/+glucose-6-P activity ratio are similar for glycogen synthase derived from rat resident and thioglycollate-elicited peritoneal macrophages; the specific activity is 7-fold higher for the enzyme from thioglycollate-elicited macrophages. The rate of incorporation of [14C]glucose into macrophage glycogen is 7-fold greater in the elicited population that that in the resident one; the values of the S0.5 for glucose are similar. The in vitro activation of glycogen synthase proceeds at a greater rate and extent for the enzyme from elicited macrophages; thus, phosphatase activity may be reduced in resident macrophages relative to that in thioglycollate-elicited ones.