Enzymatic synthesis of low molecular weight amyloses with modified terminal groups.

Low molecular weight amyloses with modified terminal groups were synthesized by cyclomaltohexaose (alpha-cyclodextrin) transfer using (1----4)-alpha-D-glucan: 4-alpha-D-(1----4)- alpha-D-glucopyranosyltransferase (cyclising) (EC 2.4.1.19) from Bacillus macerans. 4-Nitrophenyl alpha-malto-oligosaccharides d.p. 2-7 served as acceptors, and cyclomaltohexaose served as the donor. The reaction was optimized to obtain a majority of species of definite chain lengths in a range of d.p. 10-20, depending upon the chain length of the acceptor. The course of the coupling reactions, as well as the action of the enzyme in disproportionation, cyclisation, and hydrolysis of the products, were observed by h.p.l.c. analysis of the oligomer distributions. Using a 15-fold molar excess of cyclomaltohexaose and 0.5 units enzyme per mumol of acceptor at pH 5.2, the chromatograms revealed that the products of the coupling reaction were predominant during the first reaction period. By incubating the acceptors with the enzyme, but without the donor, the mechanism of disproportionation was elucidated as a transfer of malto-oligosaccharyl residues dependent upon the substrate chain length. The minimum chain length required for a direct cyclisation reaction was d.p. 7. The results were confirmed by separation and investigation of the products of hydrolysis and cyclisation, which were nonmodified alpha-malto-oligosaccharides and cyclomalto-oligosaccharides.     

A comparative study of the main components of the nucleolus in different populations of rat trophoblast cells during differentiation. II. The nucleoli of the trophospongium cells, the glycogen cells and the secondary giant cells

A comparative study was performed of the arrangement of different nucleolar components during differentiation of trophoblast cell populations in the junctional zone of placenta (glycogen cells and trophospongium) and in the secondary giant cells. Each cell type is characterized by specific interrelation of nucleolar components. Some glycogen cells show signs of segregation of nucleolar components: strands of nucleolar components with fibrillar centers (FCs) are displaced to the periphery of the nucleolus and contact with the perinucleolar chromatin. Large reticular nucleoli in trophospongium cells contain many FCs which are gathered into several "chains" by strands of dense fibrillar component. Such a "chain" has also been found in nucleoli of secondary giant cells, with greater number of FCs in each "chain". Relationship between the arrangement of nucleolar components and the level of cell differentiation is discussed.     

Glycogen synthase phosphatase of rat liver. Its separation from phosphorylase phosphatase on DE-52 columns.

Glycogen synthase D was prepared from rat liver by chromatographing the glycogen pellet on DE-52 columns. It was free of glycogen and phosphorylase and converted readily into synthase I upon incubation with glycogen synthase phosphatase. With this synthase D as substrate, the identity of rat liver glycogen synthase phosphatase was studied by means of DE-52 column chromatography. Under the conditions developed, synthase phosphatase emerged from the columns as a sharp, single peak, and phosphorylase phosphatase came off later. The two phosphatases were also different from each other in stability, synthase phosphatase being less stable than phosphorylase phosphatase.     

Low-energy conformers of pamidronate and their intramolecular hydrogen bonds: a DFT and QTAIM study

Extensive DFT and ab initio calculations were performed to characterize the conformational space of pamidronate, a typical pharmaceutical for bone diseases. Mono-, di- and tri-protic states of molecule, relevant for physiological pH range, were investigated for both canonical and zwitterionic tautomers. Semiempirical PM6 method were used for prescreening of the single bond rotamers followed by geometry optimizations at the B3LYP/6-31++G(d,p) and B3LYP/6-311++G(d,p) levels. For numerous identified low energy conformers the final electronic energies were determined at the MP2/6-311++G(2df,2p) level and corrected for thermal effects at B3LYP level. Solvation effects were also considered via the COSMO and C-PCM implicit models. Reasonable agreement was found between bond lengths and angle values in comparison with X-ray crystal structures. Relative equilibrium populations of different conformers were determined from molecular partition functions and the role of electronic, vibrational and rotational degrees of freedom on the stability of conformers were analyzed. For no level of theory is a zwitterionic structure stable in the gas-phase while solvation makes them available depending on the protonation state. Geometrically identified intramolecular hydrogen bonds were analyzed by QTAIM approach. All conformers exhibit strong inter-phosphonate hydrogen bonds and in most of them the alkyl-amine side chain is folded on the P-C-P backbone for further hydrogen bond formation.

Optimized synthesis of specific sizes of maltodextrin glycosides by the coupling reactions of Bacillus macerans cyclomaltodextrin glucanyltransferase

Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase, EC 2.4.1.19), in reaction with cyclomaltohexaose and methyl alpha-D-glucopyranoside, methyl beta-D-glucopyranoside, phenyl alpha-D-glucopyranoside, and phenyl beta-D-glucopyranoside gave four kinds of maltodextrin glycosides. The reactions were optimized by using different ratios of the individual d-glucopyranosides to cyclomaltohexaose, from 0.5 to 5.0, to obtain the maximum molar percent yields of products, which were from 68.3% to 78.6%, depending on the particular D-glucopyranoside, and also to obtain different maltodextrin chain lengths. The lower ratios of 0.5-1.0 gave a wide range of sizes from d.p. 2-17 and higher. As the molar ratio was increased from 1.0 to 3.0, the larger sizes, d.p. 9-17, decreased, and the small and intermediate sizes, d.p. 2-8, increased; as the molar ratios were increased further from 3.0 to 5.0, the large sizes completely disappeared, the intermediate sizes, d.p. 4-8, decreased, and the small sizes, d.p. 2 and 3 became predominant. A comparison is made with the synthesis of maltodextrins by the reaction of CGTase with different molar ratios of d-glucose to cyclomaltohexaose.     

Affinity glycogen synthetase to crosslinked glycogen.

The affinity of yeast glycogen synthetase to glycogen modified by crosslinking has been studied under various experimental conditions. It was found that the higher the degree of crosslinking, the lower the affinity of glycogen synthetase to glycogen. The amount of glycogen synthetase adsorbed from the solution depends on the amount of crosslinked glycogen added and is inversely proportional to the concentration of the soluble glycogen. The stability of the complex formed between yeast glycogen synthetase and the crosslinked glycogen was found to be maximal at neutral pH range. The presence of glucose 6-phosphate, uridine 5′-di-phosphate, and uridine 5′-diphosphate glucose enhanced the stability of the complex.     

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.     

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.     

Analysis of amino acids as DABS-derivatives with a sensitivity to the femtomole level using RP-HPLC narrow-bore columns

Summary In this paper we report the complete separation of amino acids as DABS-derivatives using a 3µm Supelcosil LC-18 (25 cm × 2.1 mm I.D.) narrowbore column. The system described makes it possible to perform the analysis of DABS-amino acids with a sensitivity to the femtomole level. We have also studied the conditions necessary for using the narrow-bore columns for routine analysis, paying particular attention to the problem of providing adequate protection for the analytical column. We have found it very suitable to use a (2 cm × 2.1 mm I.D.) guard column filled with a 40µm Pelliguard LC-18, pellicular packing resin, without affecting the complete resolution of the DABS-amino acids. Comparing the results obtained using conventional HPLC columns (3–5µm Supelcosil LC-18) of different lengths (15 and 25 cm × 4.6 mm I.D.) with those obtainable with the narrow-bore columns used in this work, it is possible to achieve a much greater sensitivity using the narrow-bore columns. In short, using the appropriate guard column and the standard HPLC apparatus used, the narrow-bore columns are very useful for routine analyses of DABS-amino acids with a sensitivity at the femtomole level.     

Triiodo-L-thyronine stimulates glycogen synthesis in rat hepatocyte cultures

The hyperthyroid state is associated with low hepatic glycogen levels, but paradoxically with a high activity of glycogen synthase and low activity of glycogen phosphorylase. We determined the effects of triiodo-L-thyronine (T₃) on glycogen synthesis and glycogen synthase activity in rat hepatocytesin vitro. Culture of rat hepatocytes with T₃ (100 nM–1 M) for 16 h–40 h increases glycogen synthesis from glucose and gluconeogenic precursors. The stimulation of glycogen synthesis by T₃ was associated with an increase in the activity of glycogen synthase and was additive with the long-term effects of insulin but not with the short-term stimulation of glycogen synthesis by insulin. Culture of hepatocytes with T₃ (at concentrations up to 1 M) did not affect the responsiveness of glycogen synthesis to short-term stimulation by insulin but culture with 10 M-T₃ decreased the responsiveness to insulin without affecting the basal rate. It is suggested that the high activity of glycogen synthase in the hyperthyroid state is due to a direct effect of T₃ on the hepatocyte, but the low hepatic glycogen content is probably due to either secondary metabolite and/or endocrine changes or to impaired responsiveness to insulin. T₃ may have an anabolic role in the control of hepatic glycogen storage in the euthyroid postprandial state. (Mol Cell Biol120: 151–158, 1993)Abbreviations T₃ triiodo-L-thyronine     

Structure and solution properties of enzymatically synthesized glycogen

Recently, a new enzymatic process for glycogen production was developed. In this process, short-chain amylose is used as a substrate for branching enzymes (BE, EC 2.4.1.18). The molecular weight of the enzymatically synthesized glycogen (ESG) depends on the size and concentration of the substrate. Structural and physicochemical properties of ESG were compared to those of natural source glycogen (NSG). The average chain length, interior chain length, and exterior chain length of ESG were 8.2-11.6, 2.0-3.3, and 4.2-7.6, respectively. These values were within the range of variation of NSG. The appearances of both ESG and NSG in solution were opalescent (milky white and slightly bluish). Furthermore, transmission electron microscopy and atomic force microscopy showed that ESG molecules formed spherical particles, and that there were no differences between ESG and NSG. Viscometric analyses also showed the spherical nature of both glycogens. When ESG and NSG were treated with pullulanase, a glucan-hydrolyzing enzyme known to degrade glycogen only on its surface portion, both glycogens were similarly degraded. These analyses revealed that ESG shares similar molecular shapes and surface properties with NSG.     

Glycosphingolipid acyl chain orientational order in unsaturated phosphatidylcholine bilayers.

The glycosphingolipid, galactosyl ceramide (GalCer), was studied by 2H nuclear magnetic resonance (NMR) in fluid phospholipid bilayer membranes, with regard to arrangement of its acyl chain. For this purpose, species with perdeuterated 18-carbon fatty acid (18:0[d35]GalCer) or with perdeuterated 24-carbon fatty acid (24:0[d47] GalCer) were dispersed in bilayers of the 18-carbon phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). For 18:0[d35] GalCer, smoothed profiles of the order parameter, SCD, were found to be very similar to one another over the range of glycolipid concentration, 5-40 mol%. In addition, they were very similar to orientational order parameter profiles well known from the literature on phospholipid and glycolipid acyl chains (which deals in general with membranes of homogeneous chain length in the range 14-18 carbons). Corresponding order parameter profiles for the long-chain species, 24:0[d47] GalCer, were also similar to one another for glycolipid concentrations between 5 and 40 mol%. Their shapes, however, were distinctly different from those of the shorter chain analogues. SCD profiles for the two species were quantitatively similar to a membrane depth of C15. SCD values at C16 and C17 were approximately 20 and 30%, respectively, higher for the long-chain glycosphingolipid than for its short-chain analogue in SOPC. Nitroxide spin labels attached rigidly to C16 of the long-chain glycolipid in SOPC gave electron paramagnetic resonance (EPR) order parameters that were twice as high as for a spin label at C16 on the shorter chain glycolipid. Comparison was made between spectra of 24:0[d47] GalCer in SOPC and fully hydrated bilayers of the pure 24:0[d47] GalCer, a system that is considered to be partially interdigitated in fluid and gel phases. The resultant 2H NMR order parameter profiles displayed similar features, indicating that related organizational properties exist in these fluid systems. Effective chain length of 24:0[d47] GalCer within the SOPC membrane was calculated using the method of Schindler and Seelig (1975. Biochemistry, 14:2283-2287). The result suggested that the long-chain fatty acid should protrude roughly one third of the host matrix chain length across the bilayer midplane. However, a treatment of the same order parameters making very few assumptions about chain conformation indicated a high degree of orientational flexibility for the "extra" length of the long chain fatty acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effects of fatty acids on glycolysis and glycogen metabolism in vascular smooth muscle

The effects of fatty acids of different chain lengths on aerobic glycolysis, lactic acid production, glycogen metabolism and contractile function of vascular smooth muscle were investigated. Porcine carotid artery segments were treated with 50 microM iodoacetate and perchloric acid tissue extracts were then analyzed by 31P-NMR spectroscopy to observe the accumulation of phosphorylated glycolytic intermediates so that the activity of the Embden-Myerhof pathway could be tracked under various experimental paradigms. Aerobic glycolysis and lactate production in resting arteries were almost completely inhibited with 0.5 mM octanoate, partially inhibited with 0.5 mM acetate and unaffected by 0.5 mM palmitate. Inhibition of glycolysis by octanoate was not attributable to inhibition of glucose uptake or glucose phosphorylation. Basal glycogen synthesis was unchanged with palmitate and acetate, but was inhibited by 52% with octanoate incubation. The characteristic glycogenolysis which occurs upon isometric contraction with 80 mM KCl in the absence of fatty acid in the medium was not demonstrable in the presence of any of the fatty acids tested. Glycogen sparing was also demonstrable in norepinephrine contractions with octanoate and acetate, but not with palmitate. Additionally, norepinephrine-stimulated isometric contraction was associated with enhanced synthesis of glycogen amounting to 6-times the basal rate in medium containing octanoate. Contractile responses to norepinephrine were attenuated by 20% in media containing fatty acids. Thus, fatty acids significantly alter metabolism and contractility of vascular smooth muscle. Fatty acids of different chain lengths affect smooth muscle differentially; the pattern of substrate utilization during contraction depends on the contractile agonist and the fatty acid present in the medium.     

Insulin resistance and the mitochondrial link. Lessons from cultured human myotubes

In order to better understand the impact of reduced mitochondrial function for the development of insulin resistance and cellular metabolism, human myotubes were established from lean, obese, and T2D subjects and exposed to mitochondrial inhibitors, either affecting the electron transport chain (Antimycin A), the ATP synthase (oligomycin) or respiratory uncoupling (2,4-dinitrophenol). Direct inhibition of the electron transport chain or the ATP synthase was followed by increased glucose uptake and lactate production, reduced glycogen synthesis, reduced lipid and glucose oxidation and unchanged lipid uptake. The metabolic phenotype during respiratory uncoupling resembled the above picture, except for an increase in glucose and palmitate oxidation. Antimycin A and oligomycin treatment induced insulin resistance at the level of glucose and palmitate uptake in all three study groups while, at the level of glycogen synthesis, insulin resistance was only seen in lean myotubes. Primary insulin resistance in diabetic myotubes was significantly worsened at the level of glucose and lipid uptake. The present study is the first convincing data linking functional mitochondrial impairment per se and insulin resistance. Taken together functional mitochondrial impairment could be part of the pathophysiology of insulin resistance in vivo.     

Scanning tunneling microscopy of mercapto-hexyl-oligonucleotides attached to gold.

6-mercapto hexyl-oligonucleotides bind to a gold surface strongly enough to permit imaging by a scanning tunneling microscope (STM). STM images showed worm-like chains that were approximately 12-(A-wide for single-stranded DNA and 20-(A-wide for double-stranded DNA. The chain lengths corresponded to 3.4 +/- 0.4 A per basepair for double-stranded DNA and 2.2 +/- 0.4 A per base for single-stranded DNA. This unexpectedly short length for single-stranded DNA was confirmed using oligomers with both single- and double-stranded regions. When the attachment of the samples was weakened (by imaging in water or scraping with the STM tip) the images changed to pairs of "blobs," apparently reflecting the attachment points of the molecules to the gold surface. Given this interpretation, images of DNA containing a five-base bulge imply that the bulge bends the oligomer by 90 degrees +/- 20 degrees.     

Synergism of glucose and fructose in net glycogen synthesis in perfused rat livers

Synergism of glucose and fructose in net glycogen synthesis was studied in perfused livers from 24-h fasted rats. With either glucose or fructose alone, net glycogen deposition did not occur (p greater than 0.10 for each), whereas the addition of both together resulted in significant glycogen accumulation (net glycogen accumulation was 0.21 +/- 0.03 mumol of glucose/g of liver/min at 2 mM fructose and 30 mM glucose, p less than 0.001). To better understand this synergism, intermediary substrate levels were compared at steady state with various glucose levels in the absence and in the presence of 2 mM fructose. Independent of fructose, hepatic glucose and glucose 6-phosphate increased proportionally when glucose level in the medium was raised (r = 0.86, p less than 0.001). Unlike glucose 6-phosphate, UDP-glucose did not consistently increase with glucose (p greater than 0.10); in fact, there was a small decrease at a very high glucose level (30 mM), a result consistent with the well-established activation of glycogen synthase by glucose. With elevated glucose, the level of glucose 6-phosphate was strongly correlated with glycogen content (r = 0.71, p less than 0.01, slope = 32). Adding fructose increased the "efficiency" of glucose 6-phosphate to glycogen conversion: the effect of a given increment in glucose 6-phosphate upon glycogen accumulation was increased 2.6-fold (r = 0.73, p less than 0.01, slope = 86). A kinetic modeling approach was used to investigate the mechanisms by which fructose synergized glycogen accumulation when glucose was elevated. Based on steady-state hepatic substrate levels, net hepatic glucose output, and net glycogen synthesis rate, the model estimated the rate constants of major enzymes and individual fluxes in the glycogen metabolic pathway. Modeling analysis is consistent with the following scenario: glycogen synthase is activated by glucose, whereas glucose-6-phosphatase was inhibited. In addition, the model supports the hypothesis that fructose synergizes net glycogen accumulation due to suppression of phosphorylase. Overall, our analysis suggests that glucose enhances the metabolic flux to glycogen by inducing a build up of glucose 6-phosphate via combined effects of mass action and glucose-6-phosphatase inhibition and activating glycogen synthase and that fructose enhances glycogen accumulation by retaining glycogen via phosphorylase inhibition.     

The intracellular polysaccharide of the rumen bacterium Eadie's Oval

Summary The intracellular storage polysaccharide of the rumen organism Eadie's Oval has been purified and found to be a glucan of the glycogen type consisting solely of -1,4- and -1,6-linked glucose units. It is highly branched with mean exterior and interior chain lengths of 7 and 3 respectively, polydisperse with a mean molecular weight of approximately 4.3×10⁶.     

Chain-length specificities of maize starch synthase I enzyme: studies of glucan affinity and catalytic properties

It is widely known that some of the starch synthases and starch-branching enzymes are trapped inside the starch granule matrix during the course of starch deposition in amyloplasts. The objective of this study was to use maize SSI to further our understanding of the protein domains involved in starch granule entrapment and identify the chain-length specificities of the enzyme. Using affinity gel electrophoresis, we measured the dissociation constants of maize SSI and its truncated forms using various glucans. The enzyme has a high degree of specificity in terms of its substrate-enzyme dissociation constant, but has a greatly elevated affinity for increasing chain lengths of alpha-1, 4 glucans. Deletion of the N-terminal arm of SSI did not affect the Kd value. Further small deletions of either N- or C-terminal domains resulted in a complete loss of any measurable affinity for its substrate, suggesting that the starch-affinity domain of SSI is not discrete from the catalytic domain. Greater affinity was displayed for the amylopectin fraction of starch as compared to amylose, whereas glycogen revealed the lowest affinity. However, when the outer chain lengths (OCL) of glycogen were extended using the phosphorylase enzyme, we found an increase in affinity for SSI between an average OCL of 7 and 14, and then an apparently exponential increase to an average OCL of 21. On the other hand, the catalytic ability of SSI was reduced several-fold using these glucans with extended chain lengths as substrates, and most of the label from [14C]ADPG was incorporated into shorter chains of dp < 10. We conclude that the rate of catalysis of SSI enzyme decreases with the OCL of its glucan substrate, and it has a very high affinity for the longer glucan chains of dp approximately 20, rendering the enzyme catalytically incapable at longer chain lengths. Based on the observations in this study, we propose that during amylopectin synthesis shorter A and B1 chains are extended by SSI up to a critical chain length that soon becomes unsuitable for catalysis by SSI and hence cannot be elongated further by this enzyme. Instead, SSI is likely to become entrapped as a relatively inactive protein within the starch granule. Further glucan extension for continuation of amylopectin synthesis must require a handover to other SS enzymes which can extend the glucan chains further or for branching by branching enzymes. If this is correct, this proposal provides a biochemical basis to explain how the specificities of various SS enzymes determine and set the limitations on the length of A, B, C chains in the starch granule.     

Polyglucosan neurotoxicity caused by glycogen branching enzyme deficiency can be reversed by inhibition of glycogen synthase

Uncontrolled elongation of glycogen chains, not adequately balanced by their branching, leads to the formation of an insoluble, presumably neurotoxic, form of glycogen called polyglucosan. To test the suspected pathogenicity of polyglucosans in neurological glycogenoses, we have modeled the typical glycogenosis Adult Polyglucosan Body Disease (APBD) by suppressing glycogen branching enzyme 1 (GBE1, EC 2.4.1.18) expression using lentiviruses harboring short hairpin RNA (shRNA). GBE1 suppression in embryonic cortical neurons led to polyglucosan accumulation and associated apoptosis, which were reversible by rapamycin or starvation treatments. Further analysis revealed that rapamycin and starvation led to phosphorylation and inactivation of glycogen synthase (GS, EC 2.4.1.11), dephosphorylated and activated in the GBE1‐suppressed neurons. These protective effects of rapamycin and starvation were reversed by overexpression of phosphorylation site mutant GS only if its glycogen binding site was intact. While rapamycin and starvation induce autophagy, autophagic maturation was not required for their corrective effects, which prevailed even if autophagic flux was inhibited by vinblastine. Furthermore, polyglucosans were not observed in any compartment along the autophagic pathway. Our data suggest that glycogen branching enzyme repression in glycogenoses can cause pathogenic polyglucosan buildup, which might be corrected by GS inhibition.