Conversion of N-acetylglucosamine to CMP-N-acetylneuraminic acid in a cell-free system of rat liver

A soluble fraction of rat liver converts glucosamine and N-acetylglucosamine in the presence of ATP and UTP to N-acetylneuraminic acid. This system, when supplemented with CTP, forms CMP-N-acetylneuraminic acid in high yield. Nicotinamide was found to enhance the synthesis of UDP-N-acetylglucosamine and N-acetylneuraminic acid. Kinetic analysis reveals N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, N-acetylmannosamine, N-acetylmannosamine 6-phosphate and N-acetylneuraminic acid 9-phosphate as intermediates. Under certain experimental conditions, however, an epimerisation of N-acetylglucosamine to N-acetylmannosamine was seen.     

Regulation of n-acetylglucosamine uptake in yeast

Various yeasts have been investigated for their ability to grow on N-acetylglucosamine as the sole carbon source and only those which are associated with the disease, candidiasis, gave positive results. The yeasts unable to grow on N-acetylglucosamine lacked the capacity to transport the aminosugar across the cell membrane. In pathogenic yeasts, two systems of different affinity for substrate were found to operate in the uptake of N-acetylglucosamine. In glucose-grown cells a constitutive, low affinity uptake system was present, but upon addition of inducer, a specific high affinity uptake system was synthesized. Experiments with the inhibitors of macromolecule synthesis suggested that the synthesis of RNA and protein is necessary for induction whereas the synthesis of DNA is not.In glucose-grown Candida albicans cells which are devoid of N-acetylglucosamine enters into the cells as phosphorylated form using a constitutive uptake system. Uranyl acetate (0.01 mM) which binds to cell membrane-associated polyphosphates, inhibited completely the inducible uptake of N-acetylglucosamine. Labelling experiments, designed to determine the temporal sequence of appearance of N-acetylglucosamine in intracellular free sugar and sugar-phosphate pools, indicated that N-acetylglucosamine first appeared in the cells as phosphorylated form. Similar results were obtained with Saccharomyces cerevisiae 3059 and some other yeasts which are devoid of N-acetylglucosamine kinase in both uninduced and induced conditions. These results are consistent with the model of van Steveninck that involves phosphorylation during transport. Furthermore, inhibitors of energy metabolism (arsenate, azide and cyanide), proton conductor (m-chlorocarbonylcyanide phenylhydrazine) and dibenzyl diammonium ion (membrane permeable cation) inhibited the inducible N-acetylglucosamine uptake in C. albicans.     

Studies on hog spleen N-acetylglucosamine kinase. II. Allosteric regulation of the activity of N-acetylglucosamine kinase

Kinetic studies of hog spleen N-acetylglucosamine kinase indicate that N-acetylglucosamine-6-phosphate (GlcNAc-6-p), the product of the reaction and UDP-N-acetylglycosamine (UDP-GlcNAc), the end product of the pathway of N-acetylglucosamine (GlcNAc) metabolism, are noncompetitive inhibitors. Maximum inhibitory effect of these metabolites occurs around pH 7.5, whereas the kinase reaction is optimal within a pH range of 8.6–9.4. Binding of these inhibitors with the enzyme shows cooperative homotropic interactions. Hill plot of GlcNAc saturation kinetics of the enzyme which yielded an interaction coefficient of n = 1.66 suggests the presence of at least two binding sites for the substrate on the enzyme molecule.     

Preparation and properties of biologically active fluorescent heparins

Hog mucosal heparin (N-sulfate, 0.84 mol; O-sulfate, 1.55 mol; N-acetyl, 0.12 mol; anticoagulant activity assayed by the method of U.S. Pharmacopeia, 161 USP units/mg) or its partially N-desulfated heparin (N-sulfate, 0.71 mol; O-sulfate, 1.47 mol; N-acetyl 0.12 mol; anticoagulant activity, 117 USP units/ mg) was reacted with 5-isothiocyanatofluorescein in 0.5M carbonate buffer (pH 8.5) at 35°C for 6 h to yield the corresponding N-fluoresceinylthiocarbamoyl heparins (λ_em 516 nm, λ_ex 491 nm; degree of substitution 0.006 and 0.013, respectively, anticoagulant activity, 174 and 140 USP units/mg, respectively).The fluorescent heparin (degree of substitution, 0.006; 174 USP units/mg) was injected into rabbits intravenously. The half-life of the fluorescent heparin determined by fluorometry was 24 min, that determined by the clotting time assay was 39 min. The time-course of concentration and the half-life of the fluorescent heparin and of the starting heparin obtained by the clotting the assay were virtually identical.     

The metabolism of d-galactosamine and N-acetyl-d-galactosamine in rat liver

d-[1-¹⁴C]Galactosamine appears to be utilized mainly by the pathway of galactose metabolism in rat liver, as evidenced by the products isolated from the acid-soluble fraction of perfused rat liver. These products were eluted in the following order from a Dowex 1 (formate form) column and were characterized as galactosamine 1-phosphate, sialic acid, UDP-glucosamine, UDP-galactosamine, N-acetylgalactosamine 1-phosphate, N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and an unidentified galactosamine-containing compound. In addition, [1-¹⁴C]glucosamine was found in the glycogen, an incorporation previously shown to result from the substitution of UDP-glucosamine for UDP-glucose in the glycogen synthetase reaction. Analysis of the [1-¹⁴C]glucosamine-containing disaccharides released from glycogen by β-amylase provided additional evidence that they consist of a mixture of glucose and glucosamine in a 1:1 ratio, but with glucose predominating on the reducing end. UDP-N-acetylgalactosamine was shown to result from the reaction of UTP with N-acetylgalactosamine 1-phosphate in the presence of a rat liver extract.     

The enzymic synthesis of β-[32P]UDP-N-acetylglucosamine

Cells of Micrococcus sp. 2102 incorporate inorganic [³²P]phosphate from the medium into the sugar-phosphate polymer of the wall. Controlled acid hydrolysis of sodium dodecyl sulphate-extracted cells gives N-acetylglucosamine 6-[³²P]phosphate which can be purified by ion-exchange chromatography and incubated with UTP in the presence of crude preparations of phosphoacetylglucosamine mutase from Neurospora crassa and UTP: N-acetylglucosamine 1-phosphate phosphotransferase from Bacillus licheniformis which act in concert to synthesise β-[³²P]UDP-N-acetylglucosamine.     

Determination of galactosamine impurities in heparin sodium using fluorescent labeling and conventional high-performance liquid chromatography

Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia.     

Effects of enzymatic deglycosylation on the biological activities of human thrombin and antithrombin

Sequential digestion of human thrombin and antithrombin with neuraminidase, βgalactosidase, β-N-acetylglucosaminidase, and endo-β-N-acetylglucosaminidase D resulted in the successive removal of sialic acid, galactose, N-acetylglucosamine, and mannose and more N-acetylglucosamine residues. The products obtained after each stage of deglycosylation had electrophoretic mobilites that were consistent with the calculated change in mass expected from the cleavage of the sugar moieties. The modified thrombins did not lose fibrinogen-clotting activity, amidolytic activity, nor the ability to form complexes with antithrombin. In addition, asialothrombin and asialoagalactothrombin caused the same extent of platelet release as did control thrombin. The products obtained after removal of sugars from antithrombin retained thrombin-neutralizing activity. In the presence of heparin the inhibition of thrombin as well as factor Xa was enhanced. Thus, the sugar residues of thrombin and antithrombin are not required for the formation of enzyme-inhibitor complexes or for the other activities that were measured.     

A procedure for the synthesis of uridine-5'-diphospho-N-acetylglucosamine-14C

A complete procedure for the synthesis of 1-¹⁴C-glucosamine-labeled UDP-N-acetylglucosamine is described. Glucosamine is first phosphorylated with ATP and hexokinase to form glucosamine 6-phosphate. This is N-acetylated with acetic anhydride, and the product is converted to UDP-N-acetylglucosamine by incubation with a crude yeast extract. The sugar nucleotide is isolated from the incubation mixture by paper electrophoresis, and purified by paper chromatography.     

Heparan Sulfate 2-O-Sulfotransferase Is Required for Triglyceride-rich Lipoprotein Clearance

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153–164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st^f/f) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1^f/f) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st^f/fAlbCre⁺ mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1^f/fAlbCre⁺ mice. In contrast, Hs6st1^f/fAlbCre⁺ mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.     

Synthesis of Fucosyl-N-Acetylglucosamine Disaccharides by Transfucosylation Using α-l-Fucosidases from Lactobacillus casei

AlfB and AlfC α-l-fucosidases from Lactobacillus casei were used in transglycosylation reactions, and they showed high efficiency in synthesizing fucosyldisaccharides. AlfB and AlfC activities exclusively produced fucosyl-α-1,3-N-acetylglucosamine and fucosyl-α-1,6-N-acetylglucosamine, respectively. The reaction kinetics showed that AlfB can convert 23% p-nitrophenyl-α-l-fucopyranoside into fucosyl-α-1,3-N-acetylglucosamine and AlfC at up to 56% into fucosyl-α-1,6-N-acetylglucosamine.     

Isolation and purification of Lea blood-group active and related glycolipids from human plasma of blood-group A Lea individuals

From 8 1 of human plasma of blood-group A Le^a nonsecretors three different Le^a blood-group active ceramide pentasaccharides (a total of 4.65 mg) have been isolated, all revealing glucose, galactose, N-acetylglucosamine and fucose in molar ratios of 1 : 2 : 1 : 1 as determined by gas liquid chromatography. A fourth blood-group active fraction (0.72 mg) represents a mixture of a Le^a active ceramide pentasaccharide and an A active ceramide hexasaccharide (molar ratio 7.7 : 2.3 as calculated from the content of different aminosugars). Additionally, two different globosides, two different hematosides and a new N-acetylglucosamine containing ceramide tetrasaccharide were obtained. All 9 glycolipid fractions demonstrated homogeneity in analytical high performance thin layer chromatography (HPTLC) using 4 different solvent systems. 0.2 μg of each Le^a active glycolipid completely inhibited the agglutination of O Le(a + b ?) erythrocytes by 50 μl of 4 hemagglutinating units of caprine anti Le^a serum. At least 0.04 μg of each Le^a antigen are sufficient for incubation to convert 9 × 10⁷ O Le(a?b?) erythrocytes into Le^a-positive cells. Mainly due to the relatively low content of the blood-group A glycolipid in plasma (0.17 mg/8 1), previously negative erythrocytes readily become agglutinable by anti Le^a sera and not by anti A sera after incubation with appropriate plasma.     

Presence of two neutral disaccharides containing N-acetylhexosamine in bovine colostrum as free forms

Two neutral disaccharides which comprise 74.0% of the neutral oligosaccharides other than lactose were isolated from bovine colostrum taken 6 h after parturition. The chemical structures were revealed to be galactosyl-β-1,4-N-acetylglucosamine (N-acetyllactosamine, 70.3%) and N-acetylgalactosaminyl-β-1,4-glucose (3.7%). The two carbohydrates were the newly found oligosaccharides from mammalian milk in the free forms. 7 days after parturition, they had completely disappeared from bovine milk.     

Formation of polyprenol-linked mono- and oligosaccharides in Phaseolus aureus

A crude membrane preparation from Phaseolus aureus hypocotyls catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into a acid labile glycolipid and a methanol insoluble fraction. Addition of dolichyl monophosphate to the incubation mixture stimulated the formation of both the mannolipid and the methanol insoluble endproduct. Thin-layer chromatography of endogenous lipid and of the stimulated lipid fraction revealed that both compounds run identical. Ficaprenyl monophosphate also stimulates the incorporation of mannose; however, the ficaprenyl monophosphate mannose formed is not identical to the endogenous mannolipid. This suggests that the endogenous acceptor has the properties of an α-saturated polyprenyl monophosphate rather than those of the ficaprenyl phosphate type. The same membrane preparation also incorporates N-acetylglucosamine into an acid labile glyolipid as well as into a polymer fraction. Evidence is presented that the N-acetylglucosamine containing lipid consists of a mixture of dolichyl pyrophosphate N-acetylglucosamine and dolichyl pyrophosphate di-N-acetylchitobiose. It seems likely that the two compounds have a precursor-product relationship. Incubation of dolichyl pyrophosphate di-N-acetylchitobiose together with GDP-mannose gives rise to lipid-bound mannosyl-di-N-acetylchitobiose. Radioactivity from either the [¹⁴C]mannolipid or the N-acetyl[¹⁴C]glucosamine containing lipid is incorporated into a methanol insoluble product to 3.4 and 6.3%, respectively; it seems, at least in part, to be a glycoprotein.     

Hyoscyamine N-oxide in Atropa belladonna

Separated organs of Atropa belladonna have been examined for their total alkaloid, hyoscyamine and hyoscyamine N-oxide contents during ontogenesis. Marked fluctuations in N-oxide content were observed, the highest being found in the ripe fruit. [G-³H]-atropine was fed to A. belladonna fruits and radioactively labelled hyoscyamine N-oxide isolated.     

Inhibitory effect of glucose and adenosine 3′,5′-monophosphate on the synthesis of inducible N-acetylglucosamine catabolic enzymes in yeast

Glucose can block the utilization of N-acetylglucosamine in Saccharomyces cerevisiae, a facultative aerobe, but not in Candida albicans, an obligatory aerobe. Furthermore, glucose represses the synthesis of the enzymes of the N-acetylglucosamine catabolic pathway in S. cerevisiae, but not in C. albicans. The results suggest that catabolite repression is present in S. cerevisiae, but not in C. albicans. Cyclic AMP added to S. cerevisiae cells maintained in a glucose medium cannot bring about their release from catabolite repression. On the contrary, the synthesis of inducible enzymes of N-acetylglucosamine pathway was inhibited by cyclic AMP in both the yeasts. This seems to indicate that cyclic AMP can penetrate into the yeast cells. Furthermore, cyclic AMP inhibits protein synthesis, suggesting that protein synthesis in yeast is under cyclic AMP control.     

Interaction of lipoprotein lipase with native and modified heparin-like polysaccharides

1. Lipoprotein lipase (EC 3.1.1.34), which was previously shown to bind to immobilized heparin, was now found to bind also to heparan sulphate and dermatan sulphate and to some extent to chondroitin sulphate. 2. The relative binding affinities were compared by determining (a) the concentration of NaCl required to release the enzyme from polysaccharide-substituted Sepharose; (b) the concentration of free polysaccharides required to displace the enzyme from immobilized polysaccharides; and (c) the total amounts of enzyme bound after saturation of immobilized polysaccharides. By each of these criteria heparin bound the enzyme most efficiently, followed by heparan sulphate and dermatan sulphate, which were more efficient than chondroitin sulphate. 3. Heparin fractions with high and low affinity for antithrombin, respectively, did not differ with regard to affinity for lipoprotein lipase. 4. Partially N-desulphated heparin (40–50% of N-unsubstituted glucosamine residues) was unable to displace lipoprotein lipase from immobilized heparin. This ability was restored by re-N-sulphation or by N-acetylation; the N-acetylated product was essentially devoid of anticoagulant activity. 5. Partial depolymerization of heparin led to a decrease in ability to displace lipoprotein lipase from heparin–Sepharose; however, even fragments of less than decasaccharide size showed definite enzyme-releasing activity. 6. Studies with hepatic lipase (purified from rat post-heparin plasma) gave results similar to those obtained with milk lipoprotein lipase. However, the interaction between the hepatic lipase and the glycosaminoglycans was weaker and was abolished at lower concentrations of NaCl. 7. The ability of the polysaccharides to release lipoprotein lipase to the circulating blood after intravenous injection into rats essentially conformed to their affinity for the enzyme as evaluated by the experiments in vitro.     

Structures of triclinic mono- and di-N-acetylglucosamine: lysozyme complexes--a crystallographic study.

N-acetylglucosamine and di-N-acetylglucosamine bind to lysozyme in the triclinic crystal form. Attempts to bind tri-N-acetylglucosamine were unsuccessful. Difference syntheses showed both GalNAc³ and di-GalNAc to be bound as the β-anomers, and revealed shifts in the positions of some of the lysozyme atoms. As was found in the binding studies with tetragonal lysozyme, the side chain of Trp62 moved toward the bound inhibitor. The carbonyl oxygen atom of Ala107 appeared to shift slightly, but there was no suggestion of movement in the lobes of the molecules as was evident in the tetragonal crystals.     

Characterization of a glycoprotein isolated from a continuous tumor-cell line of human lung carcinoma

A glycoprotein with a molecular weight of 62 000 has been isolated from a tumor-cell line, A549, and purified to homogeneity by gel chromatography. The glycoprotein contained sialic acid, galactose, mannose, N-acetylglucosamine and a relatively high amount of glutamic acid and proline. The data indicated that the overall composition of this glycoprotein was different from that of the glycoprotein of M_r 62 000 isolated from lung lavage of patients with alveolar proteinosis. The glycoprotein did not react with the antiserum from lung lavage of patients with alveolar proteinosis. The glycoprotein did not react with the antiserum raised against glycoprotein of M_r 62 000 isolated from lung lavage of patients with alveolar proteinosis.     

Competitive inhibition of Neurospora crassa chitin synthetase activity by tunicamycin.

Chitin synthetase from Neurospora crassa was inhibited in vitro by tunicamycin. The drug was found to be kinetically a linear competitive inhibitor (K_i ～ 480 μm) with respect to the substrate, UDP-N-acetylglucosamine. Since tunicamycin and UDP-N-acetylglucosamine are structurally similar and there exists linear competitive inhibition, it is likely that tunicamycin inhibits enzyme activity by directly competing with the substrate for access to the enzyme.