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At least 937 records · Page 52Linked to original sources
PubMed1988

Chromosomal localization of the gene for human glucosamine-6-sulphatase to 12q14.

Glucosamine-6-sulphatase (G6S), a lysosomal enzyme found in all cells, is involved in the catabolism of heparin, heparan sulphate, and keratan sulphate. Deficiency of G6S results in the accumulation of undegraded substrate and the lysosomal storage disorder mucopolysaccharidosis type IIID (Sanfilippo D syndrome). Regional mapping by in situ hybridization of a 3H-labelled human G6S cDNA probe to human metaphase chromosomes indicated that the G6S gene is localized to chromosome 12 at q14. The localization of the G6S gene to chromosome 12 was confirmed using the G6S cDNA clone in Southern blot hybridization analysis of DNA from human x mouse hybrid cell lines.

Animals↗
PubMed2005

Production of N-acetyl-beta-D-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme.

A bacterium, GJ-18, having strong chitinolytic activity was isolated from coastal soil. The isolated strain was identified as Aeromonas sp. by morphological and biochemical properties along with 16S rRNA gene sequence. The crude chitinolytic activity of culture supernatants was maximal on the 5th day of culture. Below 45 degrees C, chitin was effectively hydrolyzed to N-acetyl-beta-D-glucosamine (GlcNAc) by Aeromonas sp. GJ-18 crude enzymes, but hydrolysis decreased markedly above 50 degrees C. The optimum pH for enzyme activity was 5.0. TLC and HPLC analysis revealed that, below 45 degrees C, the major reaction product was GlcNAc with a small amount of (GlcNAc)(2) and (GlcNAc)(3), whereas above 50 degrees C the major product was (GlcNAc)(2). When swollen chitin (100 mg) was incubated with crude enzyme preparations (10 U) at 40 degrees C, chitin was hydrolyzed to 83.0 and 94.9% yield of GlcNAc within 5 and 9 days, respectively.

Acetylglucosamine↗
PubMed2005

Selective preparation of N-acetyl-D-glucosamine and N,N'-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp.

A bacterium, Aeromonas sp. GJ-18, having strong chitinolytic activity was isolated from coastal soil and used for crude enzyme preparations. This enzyme preparation contained N-acetyl-D-glucosaminidase and N,N'-diacetylchitobiohydrolase. N-Acetyl-D-glucosaminidase was inactive above 50 degrees C, but N,N'-diacetylchitobiohydrolase was stable at this temperature. Utilizing the temperature sensitivities of the chitin degradation enzymes in crude enzyme preparation, N-acetyl-D-glucosamine (GlcNAc) and N,N'-diacetylchitobiose [(GlcNAc)(2)] were selectively produced from chitin. At 45 degrees C, GlcNAc was produced as a major hydrolytic product (94% composition) with a yield of 74% in 5 d, meanwhile at 55 degrees C (GlcNAc)(2) was the major product (86%) with a yield of 35% within 5 d.

Acetylglucosamine↗
PubMed2006

Chemoenzymatic synthesis of stable isotope labeled UDP-N-[2H]-acetyl-glucosamine and [2H]-acetyl-chitooligosaccharides.

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5'-diphospho-N-[(2)H]-acetyl-glucosamine and the chemical preparation of N-[(2)H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.

Acetylation↗
PubMed1983

Demonstration of the enzymatic mechanisms of alpha-N-acetyl-D-glucosamine-1-phosphodiester N-acetylglucosaminidase (formerly called alpha-N-acetylglucosaminylphosphodiesterase) and lysosomal alpha-N-acetylglucosaminidase.

An enzyme that is capable of removing the outer N-acetylglucosamine residues from phosphodiesters present on the high-mannose-type oligosaccharides of newly synthesized lysosomal enzymes has been described. This enzyme has been called an alpha-N-acetylglucosaminylphosphodiesterase, based upon its substrate specificity and on inhibitor studies. In this study it is demonstrated by the 18O enrichment method that the enzyme cleaves the C-O bond rather than the O-P bond, and therefore acts by a glycosidase type of mechanism. In addition, the enzyme has no significant activity toward alpha-N-acetylglucosamine 1-phosphate, and therefore requires an underlying phosphodiester for activity. In accordance with the IUB recommendations for enzyme nomenclature, it is therefore suggested that the enzyme be renamed alpha-N-acetyl-D-glucosamine-1-phosphodiester N-acetylglucosaminidase (systematic name, 2-acet-amido-2-deoxy-alpha-D-glucose 1-phosphodiester acetamidodeoxyglucohydrolase). For convenience, the trivial name phosphodiester glycosidase is proposed. Lysosomal alpha-N-acetylglucosaminidase also has a glycosidase type of mechanism but it is active toward alpha-N-acetylglucosamine 1-phosphate as well as phosphodiesters with outer N-acetylglucosamine residues.

Acetylglucosaminidase↗
PubMed1985

Characteristic [3H]glucosamine-labeled glycoproteins in two-dimensional electrophoretograms of human renal cancer cells: identification as cathepsin D.

Two-dimensional electrophoretograms of extracts of [3H]glucosamine-labeled human renal cancer cells demonstrated a series of components (Mr 48,000 and 30,000) that are only poorly expressed in similarly labeled normal kidney epithelial cell cultures [S. Ogata, R. Ueda, and K. O. Lloyd (1981) Proc. Natl. Acad Sci. USA 78, 770-774]. These characteristics are also exhibited by [3H]Man-labeled samples and by concanavalin A-binding glycoproteins from [35S]Met-labeled cells. It is now shown that these species are the precursor chain (Mr 48,000) and native heavy chain (Mr 30,000) forms of the lysosomal enzyme, cathepsin D. These results were obtained by precipitation with a specific anti-cathepsin D serum and by binding of the components to pepstatin-Sepharose. Cathepsin D heavy chain is heterogeneous, having three major species with pI's of 5.7, 5.3, and 4.9; all forms are glycosylated with high mannose-type chains [approximate size: Man5(GlcNAc)2] and are partially phosphorylated. Despite these indications of dissimilarities in cathepsin D levels, the actual levels of total acid protease activity were not significantly higher in renal cancer cells than in normal kidney epithelial cells.

Aspartic Acid Endopeptidases↗
PubMed1986

Transbilayer migration (flip-flop) of 12-(4-azido-2-nitrophenoxy)stearoyl glucosamine in large unilamellar phospholipid vesicles.

The ability of the glycolipid photoprobe, 12-(4-azido-2-nitrophenoxy)-stearoyl[1-14C]glucosamine (12-APS-GlcN), to undergo transbilayer flip-flop and intermembrane transfer between liposomes was examined. It was found that probe which was incorporated into membranes during the preparation of large unilamellar vesicles (LUVs) could be rapidly and completely extracted by incubation of these donor vesicles (in the liquid-crystalline state) with probe-free acceptor vesicles.

Azides↗
PubMed1992

Improved affinity chromatographic purification of D-mannose-N-acetyl-D-glucosamine-specific lectin from the bark of Sophora japonica eliminating the loss by sugar specific self-aggregation.

A novel D-mannose-N-acetyl-D-glucosamine-specific lectin of Sophora japonica bark, B-SJA-II, which showed self-aggregation based on sugar specificity, was purified by affinity chromatography on maltamyl-Sepharose subsequent to chromatographic separation on lactamyl-Sepharose to remove a major D-galactose-N-acetyl-D-galactosamine specific lectin, B-SJA-I. However, the yield of this method was low as a result of the sugar-specific precipitation and binding to other glycoproteins. A modified method was developed to circumvent this problem. All the purification procedures, except for the final chromatographic separation, were carried out in the presence of the haptenic sugar and the sugar-specific adsorption of B-SJA-II onto the adsorbent was carried out in a dialysis bag by gradually removing the sugar. This method gave a yield eight times higher than the original method.

Acetylation↗
PubMed1994

A novel assay for typing Rh antigens in blood-stains using a lectin specific to the bisecting N-acetyl-D-glucosamine side chain of glycoprotein.

A unique sandwich enzyme-linked immunosorbent assay for the determination of Rh antigens in blood stains has been developed using Rh antisera and phaseolus vulgaris E4/peroxidase conjugate (PHAE4/PO). The appropriate antiserum for detecting Rh C, c, D, E or e was coated on the inner surface of microplate wells, and the sample antigens from blood stains, solubilized with n-octyl-beta-D-glucopyranoside, were then placed in the wells. After washing the wells repeatedly, PHAE4/PO was added. Bound PHAE4/PO was detected by the development of colors using o-phenylenediamine/H2O2. All Rh antigens corresponding to the antisera were clearly detected using this technique. The detection limit expressed by sample dilution was more than 2 x 10(5) times (volume/dried blood weight) for the various antigens from the fresh 5 x 5 mm2 blood stain. Even when the blood stain samples were left beside a sunny window at room temperature for 2 months, Rh antigens were still detected. When the ABH, MN, P1, Kidd, Duffy and Lewis blood grouping systems were tested with similar ELISA procedures PHAE4 did not recognize any antigen. Since PHAE4 specifically recognizes and combines with the bisecting N-acetyl-D-glucosamine side chain, it was concluded that the glycoprotein was a component of all Rh antigens immune complexes.

Acetylglucosamine↗
PubMed1992

Crystallization and preliminary crystallographic studies of glucosamine-6-phosphate deaminase from Escherichia coli K12.

Hexameric glucosamine-6-phosphate deaminase from Escherichia coli has been crystallized isomorphously with both phosphate and ammonium sulphate as precipitants, over a wide pH range (6.0 to 9.0). The crystals belong to space group R32 and the cell parameters in the hexagonal setting are a = b = 125.9 A and c = 223.2 A. A complete native data set was collected to 2.1 A resolution. Self-rotation function studies suggest that the hexamers sit on the 3-fold axis and have point group symmetry 32, with a non-crystallographic dyad relating two monomers linked by an interchain disulfide bridge. A possible packing for the unit cell is proposed.

Aldose-Ketose Isomerases↗
PubMed1990

N-acetyl-D-glucosamine is present in cysts and trophozoites of Giardia lamblia and serves as receptor for wheatgerm agglutinin.

Previously, on the basis of lectin binding and glycosidase digestion assays, we have suggested that N-acetyl-D-glucosamine residues (GlcNAc) are major structural components of both trophozoites and in vivo cysts of the intestinal parasite Giardia lamblia. In this report we confirm that GlcNAc is present both in trophozoites and in vitro cysts as assessed by lectin binding and glycosidase digestion assays, galactosyltransferase labeling, immunochemical analysis using antibodies specific for GlcNAc and its beta 1-4 oligomers, and by gas chromatography/mass spectrometry (GC/MS). The results show that wheatgerm agglutinin (WGA) binds specifically to intact trophozoites and in vitro cysts as well as to SDS-PAGE separated proteins. WGA binding to the separated proteins was markedly reduced after their digestion with N-acetyl-beta-D-glucosaminidase, supporting the conclusion that WGA is reacting with terminal beta-linked GlcNAc residues. Labeling of trophozoites and cysts by 3H-exogalactosylation with galactosyltransferase further confirmed the presence of terminal GlcNAc in both surface and intracellular glycoproteins. The presence of GlcNAc is also supported by microfluorometric analysis using antibodies to (GlcNAc)1, (GlcNAc)2, and (GlcNAc)3, which revealed a sugar-inhibitable binding of the antibody to live trophozoites. Finally, the presence of GlcNAc in both cysts and trophozoites was unequivocally confirmed by GC/MS analysis of detergent-extracted membranes and of glycoproteins isolated by affinity chromatography on WGA-agarose. GC/MS analysis also revealed mannose (Man), N-acetyl-D-galactosamine (GalNAc), fucose (Fuc), galactose (Gal), glucose (Glc) and N-acetylneuraminic acid (NANA) to be present in cysts. All these sugars were also present in trophozoites, except for GalNAc. The glycoproteins isolated by WGA affinity chromatography were 5- to 40-fold enriched in GlcNAc, further supporting the conclusion that WGA reacts with GlcNAc in Giardia. In summary, the data presented here provide biological and chemical evidence for GlcNAc in both cysts and trophozoites of G. lamblia and are consistent with previously published results from this and other laboratories.

Acetylglucosamine↗
PubMed1984

The inactivation of glucosamine synthetase from bacteria by anticapsin, the C-terminal epoxyamino acid of the antibiotic tetaine.

Incubation of anticapsin with the purified glucosamine synthetase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase, amino transferring, EC 5.3.1.19) from Escherichia coli, Pseudomonas aeruginosa, Arthrobacter aurescens and Bacillus thuringiensis led to the formation of an inactive enzyme irreversibly modified. The inactivation reaction followed pseudo-first-order kinetics. The rate of the inactivation reaction at various concentrations of anticapsin exhibited saturation kinetics, implying that anticapsin binds reversibly to the enzyme prior to inactivation. The determined Kinact is in the range of 10(-5) M (B. thuringiensis) and 10(-6) M (E. coli, P. aeruginosa, A. aurescens ). The addition of glutamine protected the amidotransferase from inactivation by anticapsin . The anticapsin was demonstrated to be a mixed type or competitive inhibitor with respect to glutamine with a Ki value of 10(-6) to 10(-7) M. Reaction of anticapsin with the enzyme exhibits the characteristics of affinity labelling of the glutamine binding site. Chemical modification of the enzyme thiol group with various reagents, 5,5'-dithiobis-(2-nitrobenzoic) acid, 6,6'- dithiodinicotinic acid, 1,1'- dithiodiformamidine , N-ethylmaleimide and iodoacetamide, resulted in an inactive enzyme.

Alanine↗
PubMed1990

Zinc binding and its trapping by allosteric transition in glucosamine-6-phosphate deaminase from Escherichia coli.

Glucosamine-6-phosphate isomerase deaminase from Escherichia coli, a typical allosteric enzyme, becomes less cooperative and 50% inhibited when treated with zinc. This metal cation behaving as a tight-bound and slow partial inhibitor. Modification of a pair of vicinal reactive thiols with some sulfhydryl reagents mimics this effect. On the other hand, sulfhydryl reactivity disappears in the presence of saturating concentrations of Zn2+, which does not modify the kinetics of S-methylated enzyme, a finding that indicates that vicinal thiols are an essential part of the zinc-binding site. Allosteric activation of the deaminase causes trapping of the metal, which cannot be released by dialysis against a buffer containing EDTA. Cadmium and nickel(II) cations also produce a similar effect.

Aldose-Ketose Isomerases↗
PubMed1991

Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy.

The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated. A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic. Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.

Aldose-Ketose Isomerases↗
PubMed1993

Chemical modification studies of the active site of glucosamine-6-phosphate synthase from baker's yeast.

Glucosamine-6-phosphate synthase from baker's yeast has been purified 100-fold with a final recovery of 70%. The purification procedure involved thiol-affinity chromatography. Chemical modification studies of the enzyme revealed the presence of cysteine, Glu/Asp-carboxyl and probably histidine at the glutamine binding site and, on the other hand, arginine and probably another histidine at the D-fructose 6-phosphate binding site. A few glutamine analogs, including 6-diazo-5-oxo-L-norleucine (DON), anticapsin and N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP), were shown to inactivate the enzyme in a time- and concentration-dependent manner. Anticapsin, the most active in the series, exhibited an inactivation constant, Kinact, of 9.5.10(-6) M.

Binding Sites↗
PubMed1990

Enhancement of nonspecific resistance to microbial infections of synthetic lipid A-subunit analogues of GLA-27 modified at the C1 position of the glucosamine backbone.

The C1 position of lipid A-subunit analogue GLA-27, 4-O-phosphono-D-glucosamine carrying N-3-tetradecanoyloxytetradecanoyl(C14-O-(C14)) and 3-O-tetradecanoyl (C14) groups, was S-acetylated, thiolated or phosphorylated. Enhancement of nonspecific resistance to Pseudomonas aeruginosa and vaccinia virus infections of these chemically modified compounds were investigated. Thiolation augmented the nonspecific resistance to P. aeruginosa infection. Protective activity against vaccinia virus infection was reduced by all the chemical modifications. NK cell activity was found not to be effected by S-acetylation, but to be decreased slightly by thiolation or phosphorylation. IFN-inducing activity was reduced remarkably by thiolation or S-acetylation, or completely diminished by phosphorylation, compared with that of GLA-27.

Animals↗
PubMed1988

Molecular cloning and overexpression of the glucosamine synthetase gene from Escherichia coli.

A recombinant plasmid carrying a 4.6 kg restriction endonuclease NcoI-ClaI fragment of genomic DNA from Escherichia coli K12 was constructed. This plasmid complements the glmS mutation. Subcloning into pUC18 gave plasmid pGM10 encoding the structural gene of glucosamine synthetase, as judged by overexpression of enzyme activity and the isolation in high yield of the pure protein.

Cloning, Molecular↗