The fine structure of the branched α-D-glucan from the blue-green alga Anacystis nidulans: comparison with other bacterial glycogens and phytoglycogen

The fine structure of the glycogen from the blue-green alga Anacystis nidulans has been examined. After selective hydrolysis of all (1→6)-α-D linkages by a bacterial isoamylase, the resulting mixture of linear chains was subjected to gel-permeation chromatography. For purposes of comparison, the glycogens from Escherichia coli and Arthrobacter sp., amylopectin, phytoglycogen from sweet corn, and shell-fish glycogen were treated similarly. The profiles of the unit chains of A. nidulans glycogen and phytoglycogen were closely similar. There was no close resemblance in the size distribution of unit chains for A. nidulans glycogen, other bacterial glycogens, and amylopectin.     

Glucosamine is a normal component of liver glycogen

There have been several reports of the incorporation of glucosamine into liver glycogen by an intraperitoneal injection of galactosamine, but it has not previously been considered that glucosamine is a normal component of liver glycogen. We now report that glucosamine occurs endogenously in rabbit- and pig-liver glycogens in the amount of about 1 nmol per 10 mg glycogen. Like the glucosamine incorporated by exogenous administration of galactosamine, the endogenous glucosamine takes the place of 1,4-linked alpha-glucose residues. It is found in both the outer and inner chains of the glycogen molecule.     

Comparison of the Unit-chain Distributions of Glycogens from Different Biological Sources,Revealed by Anion Exchange Chromatography

The precise distributions of α-1,4-unit-chains of several kinds of glycogens, average chain length 7–13, from different sources, e.g., mammals, shellfish, mushrooms, and microorganisms, were compared using high performance anion exchange chromatography (HPAEC). These glycogens were completely debranched, and a series of maltosaccharides derived from individual unit-chains were analyzed. Most glycogens had similar chromatographic profiles, with degrees of polymerization (DPs), from 4–5 to 35; oyster glycogen contained shorter chains of DP 2–3. On the other hand, molar-based distribution patterns of these glycogens appeared to be specific to their origins. Ratios of their A- to B-chains varied from 0.6: 1 to 1.2: 1, as estimated by quantitative HPAEC of maltosaccharides by debranching of the β-limit dextrins. Possible chain lengths of the exterior A-chains were also discussed.     

Relationship between structure and immunostimulating activity of enzymatically synthesized glycogen

Glycogen acts as energy and carbon reserves in animal cells and in microorganisms. Although anti-tumor activity has recently been reported for shellfish glycogen and enzymatically synthesized glycogen, the activity of glycogen has not yet been fully clarified. We enzymatically prepared various sizes of glycogens with controlled structures to investigate the relationship between the structure and immunostimulating activity of glycogen. The results revealed that glycogens with a weight-average molecular weight (M(w)) of more than 10,000K hardly activated RAW264.7, a murine macrophage cell line, whereas glycogens of M(w) 5000K and 6500K strongly stimulated RAW264.7 in the presence of interferon-gamma (IFN-gamma), leading to augmented production of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). Comparing the fine structure of the glycogens, the average-number of chain length, as well as the exterior and the interior chain lengths of the glycogens, had minor correlation between active and less-active glycogen derivatives. The available evidence suggests that the macrophage-stimulating activity of glycogen is strictly related to its molecular weight rather than to any fine structural property.     

Multiple branching in glycogen and amylopectin

The β-amylase limit dextrins of glycogen and amylopectin are completely debranched by joint action of isoamylase and pullulanase. Action of isoamylase alone results in incomplete debranching as a consequence of the inability of this enzyme to hydrolyze those A-chains that are two glucose units in length (half the total number of A-chains). From the reducing powers released by isoamylase acting (a) alone and (b) in conjunction with pullulanase, the relative numbers of A- (unsubstituted) and B- (substituted) chains in the β-dextrins, and therefore in the native polysaccharides themselves, can be calculated. Examination of a series of glycogens and amylopectins in this way showed that the ratio of A-chains: B-chains is markedly higher in amylopectins (1.5–2.6:1) than in glycogens (0.6–1.2:1). Glycogen typically contains A-chains and B-chains in approximately equal numbers; amylopectin typically contains approximately twice as many A-chains as B-chains. These polysaccharides therefore differ in degree of multiple branching as well as in average chain length. A consequence of these findings is that amylopectin cannot be formed in vivo by debranching of a glycogen precursor, as proposed by Erlander, since it is impossible to increase the A:B chain ratio by action of a debranching enzyme.     

Stimulation of macrophage by enzymatically synthesized glycogen: the relationship between structure and biological activity

Glycogen is exclusively known as an energy and carbon reserve in animal cells and micro-organisms. We synthesized glycogens of varying molecular weight by using three enzymes, and investigated the relationship between the structure and immunostimulating activity of glycogen. These results indicated that glycogens with a molecular weight of more than 1.0×10⁷ hardly activated RAW264.7, a murine macrophage cell line, whereas glycogens of 5.0–6.5×10⁶ strongly stimulated RAW264.7 in the presence of interferon-γ, leading to augmented production of nitric oxide, tumour necrosis factor-α and interleukin-6. Additionally, the number-average unit chain length and the exterior and interior chain lengths of the glycogens showed a minor correlation between active and less-active glycogen derivatives. On the other hand, the binding activity of glycogen toward RAW264.7 did not depend on the molecular weight of glycogen. The available evidence suggests that the macrophage-stimulating activity of glycogen is strictly related to its molecular weight rather than to fine structural properties.     

Demonstration of intracytoplasmic glycogen of megakaryocytes and blood platelets by means of the periodic acid-thiocarbohydrazide-silver proteinate method

Summary The glycogen of megakaryocytes and blood platelets has been investigated in glutaraldehyde and osmium tetroxide fixed tissues by the periodic acid-thiocarbohydrazide-silver proteinate method (PA-TCH-SP). The PA-TCH-SP method involves the staining of intracytoplasmic glycogens more densely than the routine lead citrate method. Glycogen having a mean particle diameter of 21.1 nm has been shown localizing in the matrix of mature megakaryocytes, while that of glycogen in the platelets was 26.2 nm. The staining pattern of the glycogen in blood platelets was classified into three groups according to staining intensity.It is found that the PA-TCH-SP method is a very suitable one for the demonstration of intracytoplasmic glycogen from the viewpoints of reaction specificity, reproducibility, fineness of reaction products, sufficiency of electron density, and experimental cost. This method is also a very useful one for differentiating intracytoplasmic glycogens and ribosomes.     

Demonstration of intracytoplasmic glycogen of megakaryocytes and blood platelets by means of the periodic acid-thiocarbohydrazide-silver proteinate method.

The glycogen of megakaryocytes and blood platelets has been investigated in glutaraldehyde and osmium tetroxide fixed tissues by the periodic acid-thiocarbohydrozide-silver proteinate method (PA-TCH-SP). The PA-TCH-SP method involves the staining of intracytoplasmic glycogens more densely than the routine lead citrate method. Glycogen having a mean particle diameter of 21.1 nm has been shown localizing in the matrix of mature megakaryocytes, while that of glycogen in the platelets was 26.2 nm. The staining pattern of the glycogen in blood platelets was classified into three groups according to staining intensity. It is found that the PA-TCH-SP method is a very suitable one for the demonstration of intracytoplasmic glycogen from the viewpoints of reaction specificity, reproducibility, fineness of reaction products, sufficiency of electron density, and experimental cost. This method is also a very useful one for differentiating intracytoplasmic glycogens and ribosomes.     

The structure and deposition of human-liver glycogens

1. Glycogen was extracted from and the amount determined in human foetal livers ranging in age from 13(1/2) to 26 weeks. 2. The detailed structures of human foetal- and child-liver glycogens were examined and shown to be essentially the same. 3. The deposition of glycogen in different mammalian species is discussed.     

Quantitative estimation of the pathways followed in the conversion to glycogen of glucose administered to the fasted rat

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.     

Molecular and metabolic heterogeneity of liver glycogen

On refeeding after starvation, the resynthesis of rabbit-liver glycogen proceeds inhomogeneously and over-produces material of low molecular weight. The fate of radioactivity incorporated into glycogen from d-glucose-¹⁴C can be explained if glycogen of high molecular weight is synthesised on a protein backbone. Confirmation of this view is given by the effect upon glycogen of reagents that break disulphide bonds; these cause loss of the polysaccharide of high molecular weight. Buoyant densities of glycogens are found to be independent of molecular weight and even of extensive degradation. It is concluded that glycogen synthesis proceeds by two routes; one results in the production of polysaccharide of high molecular weight which has a protein backbone capable of forming disulphide bonds, and another results in the production of polysaccharide of low molecular weight which has either no protein backbone or a protein backbone that is incapable of forming disulphide bridges. Apart from size, the two species are physicochemically indistinguishable.     

The Structure of Neurospora crassa Glycogen

The mycelia of a wild type strain of Neurospora crassa (6068, IFO) contain a polysaccharide which is stained reddish brown by iodine. The polysaccharide purified by repeated precipitation with ethanol is made up of d-glucose and has a molecular weight of about (more than) 2 × 10⁷, 101 S on ultracentrifugation analysis, an average chain length of 10, β-amylolysis limit of 33.6%, and α-amylolysis limit of 58.3%. The highly branched structure, therefore, resembles to that of a typical glycogen. The properties of the glycogen from N. crassa are discussed in comparison with the commercial glycogens from shellfish and rabbit liver.     

The nature of glycogen complexing with iodine in the presence of CaCl2

The absorption and dichroism of muscle glycogen--iodine complexes depending on CaCl2 concentration were studied. It was shown that besides intensification of glycogen staining with iodine, high concentrations of CaCl2 cause destabilization of the alpha-glucan helix as well as disturbances in the formation of a specific chromophore of the iodine-glycogen complex which manifest themselves as a loss of dichroism. The stained chromophore formed by a simultaneous decrease in the dichroic absorption seems to be generated in the non-helical regions of the glycogen molecule and is thus nonspecific. This nonspecific chromophore is a potential source of errors in spetrophotometrical assays of glycogen structure. Study of rabbit skeletal muscle and liver glycogens by the Krisman method based on the use of concentrated solutions of CaCl2 failed to reveal any differences in glycogen structure that are normally detectable at low concentrations of CaCl2. The unfavourable effect of high concentrations of CaCl2 on helix formation should be taken into consideration when studying the stoichiometry of iodine interaction with alpha-glucan.     

The distribution of glucosamine in mammalian glycogen from different species, organs and tissues

The reasons for the occurrence of trace amounts of glucosamine in animal liver glycogens have been explored. Human liver glycogen is now shown to contain this amino sugar. Galactosamine, known to be the source of the incorporated glucosamine, is found to give rise to glucosamine in glycogen when administered orally, or as the N-acetyl derivative. The rabbit can also incorporate glucosamine into kidney glycogen but not into glycogen in heart or skeletal muscle. These experiments led to the discovery that glucosamine is incorporated into rabbit liver glycogen in such a way that there is intermolecular heterogeneity in the content of glucosamine, suggesting that there exists more than one pool of liver glycogen.     

Regulation of lysosomal glycogen metabolism: studies of the actions of mammalian acid alpha-glucosidases

1. Acid alpha-glucosidases were purified to homogeneity from rat liver, rat skeletal muscle and human placenta. The properties of these enzymes were investigated. 2. Their pH optima for activity toward various substrates were in the range 4-5. 3. Time course and pH dependence experiments revealed that all glycogen substrates were not hydrolysed at the same rate; the rate of hydrolysis was inversely related to the molecular size of the substrate. The most rapidly hydrolysed glycogen substrate was the smallest (commercial oyster) while the least rapidly hydrolysed was the largest (native rat or rabbit liver). Intermediate sized glycogens were hydrolysed at intermediate rates. 4. Glycogen hydrolysis was stimulated by added sodium ions; this stimulation was pH dependent. 5. It is suggested that lysosomal glycogen metabolism may be controlled by pH, salt concentration and the size of the glycogen substrate. 6. Since the high molecular weight glycogen associated with lysosomes is formed by disulphide bridges between lower molecular weight material it is proposed that an important step of lysosomal glycogen degradation is disulphide bond reduction.     

Isolation of a polydisperse high-molecular-weight glycogen from rat liver

A new method is described for the isolation of glycogen from rat liver using centrifugation, gentle heating, and gel chromatography. The prepared polysaccharide was judged by both sucrose density gradient centrifugation and the absorbance spectrum of an I₂-glycogen complex to be highly branched, polydisperse, and of an unusually high molecular weight upon comparison to other glycogens. Using adult fasted rats, this glycogen was shown to be better than high-molecular-weight cold water-ethanol extracted glycogen for the binding of glycogen metabolizing enzymes. Further, the addition of 0.5% ($\text{w}\text{v}$) of the glycogen to a crude liver extract from newborn rats facilitated the isolation of an almost 700-fold purified glycogen synthase with 40% recovery. It is suggested that this glycogen could also be used to study the role of enzyme binding in the regulation of carbohydrate metabolism.     

Glycogen from Klebsiella pneumoniae M 5 al and Escherichia coli K 12

Summary 1. A continuous two stage cultivation method for two strains of Klebsiella pneumoniae and Escherichia coli yielding high cell mass and relatively high glycogen contents is described. The stage 1 cells (carbon-limited) were fed with the nitrogen source ammonia (which also neutralized simultaneously) soly via the pH-stat. In stage 2, the cells grew nitrogen-limited, a small excess of the carbon source was maintained by continuous addition of a glucose solution.2. Through the action of lysozyme, the glycogen could be quantitatively solubilized from the alkali-insoluble cell material obtained through alkaline hydrolysis of the cells in dimethylsulfoxide-3M aqueous potassium hydroxide. The possibility that the glycogen is in part covalently linked to the peptidoglycan and localized in the periplasm is discussed. 3. Analytical data for the glycogens isolated from the two bacterial strains is given.     

The effect of fixation on the chemical extraction of glycogen from rat liver

Synopsis Small samples of rat liver, weighing 15 mg or less, were either (a) frozen in liquid nitrogen or (b) fixed at 4°C for 5 min to 2 hr in absolute alcohol, alcoholic picric acid (Rossman's fluid), or aqueous picric acid (Bouin's fluid). The tissue samples were analysed for total glycogen content by a modification of the procedure described by Goodet al. (1933).Comparable yields of glycogen were extracted from freshly frozen and fixed tissue samples. The time of fixation had no apparent effect on the amount of glycogen that could be extracted chemically. Dissolved glycogen was not detectable in the fixatives.It is concluded that (a) the fixatives used in this study do not significantly affect the yield of chemically extractable glycogen from liver; (b) fixation is extremely rapid; and (c) alcoholic fixatives are not significantly superior to aqueous picric acid fixatives for preservation of chemically extractable glycogen in very small samples of tissue.     

Comparative structural analyses of purified glycogen particles from rat liver,human skeletal muscle and commercial preparations

Glycogen is a cellular energy store that is crucial for whole body energy metabolism, metabolic regulation and exercise performance. To understand glycogen structure we have purified glycogen particles from rat liver and human skeletal muscle tissues and compared their biophysical properties with those found in commercial glycogen preparations. Ultrastructural analysis of commercial liver glycogens fails to reveal the classical α-rosette structure but small irregularly shaped particles. In contrast, commercial slipper limpet glycogen consists of β-particles with similar branching and chain lengths to purified rat liver glycogen together with a tendency to form small α-particles, and suggest it should be used as a source of glycogen for all future studies requiring a substitute for mammalian liver glycogen.     

Crystallisation of malto-oligosaccharides as models of the crystalline forms of starch: minimum chain-length requirement for the formation of double helices

The multigram preparation of malto-oligosaccharides of average d.p. ∼11, by the debranching of glycogen using Cytophaga isoamylase is described. Debranched glycogen and fractions derived therefrom readily crystallise from hot, concentrated aqueous solution to give 40–70% of crystalline materials having sharp X-ray diffraction patterns characteristic of A-, B-, and C-type (intermediate) starch polymorphs. The polymorphic form obtained is dependent on chain length, concentration, and temperature, the A-type being favoured by shorter chain-length, higher concentration, and higher crystallisation temperature. For pure oligomers, the minimum chain-length required for crystallisation (formation of double helices) is 10. In the presence of longer chains, oligomers as short as maltohexaose can co-crystallise. These results explain the known differences in aggregation properties of glycogens and amylopectins.