Moles Of N-acetylglucosamine Research Articles

Structural and functional analysis of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn-superoxide dismutase (Cu/ZnSOD) (HLSOD). To improve its yield, the effect of increased concentration of Cu 2+ (from 1 to 750 μg/ml) on growth and enzyme biosynthesis was studied. The primary structure of this fungal enzyme has been determined by Edman degradation of peptide fragments derived from proteolytic digest. A single chain of the protein, consisting of 152 amino acid residues, reveals a very high degree (74–85%) of structural homology in comparison to the amino acid sequences of other fungal Cu/ZnSODs. The difference of the molecular masses of H. lutea Cu/ZnSOD, measured by MALDI-MS (15,935 Da) and calculated by its amino acid sequence (15,716 Da), is attributed to the carbohydrate chain of one mole of N-acetylglucosamine, attached to the N-glycosylation site Asn 23-Glu-Ser. HLSOD protected mice from mortality after experimental influenza A/Aichi/2/68 (H3N2) virus infection. Using the glycosylated HLSOD, the survival rate is increased by 66% (protective index=86.1%) and the survival time prolonged by 5.2 days, similar to the application of ribavarin, while non-glycosylated bovine SOD conferred lower protection.

Isolation and characterization of a novel Forssman active acidic glycosphingolipid with branched isoglobo-, ganglio-, and neolacto-series hybrid sugar chains.

Equine kidney and spleen contain a Forssman active glycosphingolipid, and the structure of this glycolipid has been reported to be that of a globopentaosylceramide (GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1, 4Galbeta-1,4Glcbeta-1,1'Ceramide). We found that equine kidney contains several other anti-Forssman antibody-reactive glycosphingolipids. One of these acidic Forssman active glycosphingolipids was isolated and characterized by means of NMR, mass spectrometry, permethylation studies, and TLC-immunostaining. This glycolipid contains three moles of galactose, one mole of glucose, three moles of N-acetylgalactosamine, one mole of N-acetylglucosamine, and one mole of N-acetylneuraminic acid, and is stained on TLC with anti-Forssman antibodies and anti-GM2 ganglioside antibodies. HOHAHA and ROESY experiments and permethylation studies showed this glycolipid oligosaccharide to be branched at the innermost galactose; one chain has an isoglobo structure with a terminal Forssman disaccharide and the other chain is branched through the linkage of N-acetylglucosaminebeta-1,6 to the inner galactose. The nonreducing end of the GM2 trisaccharide is linked to this glucosamine. The structure of the oligosaccharide of the glycolipid presented here is a novel type, having branched isoglobo-, ganglio-, and neolacto-series oligosaccharides. Mass spectrometric analyses indicated the ceramide moiety of the glycolipid to be composed predominantly of hydroxy fatty acids (C20:0, C22:0, C23:0, C24:0, and C25:0) and hydroxysphinganine. GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1,3[GalNAcbet a-1, 4(NeuAcalpha-2,3)Galbeta-1,4GlcNAcbeta-1,6]Galbeta+ ++-1,4Glcbeta-1, 1'Ceramide

Rodent kinin-forming enzyme systems—II: Purification and characterization of an acid protease from murphy-sturm lymphosarcoma

An acid protease from the rat Murphy-Sturm lymphosarcoma (MSLS) tumor was purified 640-fold by extraction of the tumor tissue, acid precipitation with glacial acetic acid, ammonium sulfate precipitation, DEAE-Sephadex A-50 batch adsorption, QAE-Sephadex A-50 column chromatography, Sephadex G-200 gel filtration, and CM-32 cellulose chromatogaphy. The protease hydrolyzed bovine hemoglobin and formed vasopeptide kinins when incubated with purified rat plasma kininogen. Two protease fractions obtained by Sephadex G-200 gel filtration had identical molecular weights of 39,500–41,000 and were similar in other physico-chemical and kinetic characteristics. The purified enzyme showed three major isozymic forms (α, β and γ) with isoelectric points (pI) of 5.2, 5.5 and 5.8, respectively, and nearly identical amino acid compositions. The enzyme had a high moles percent of both aspartic and glutamic acids. The carbohydrate moiety of the enzyme contained 2 moles of N-acetylglucosamine and 8 moles of mannose per mole of enzyme. The pH optimum for the digestion of bovine hemoglobin was approximately 3.0 with a sharp decline of activity on either side of the pH optimum. The protease activity was very stable above pH 3.4. The K m values for the purified enzyme fractions A and B were 31.17 and 31.19 μM, respectively, and the corresponding V max values were 6.17 and 5.5 μM tyrosine per mg per min at 37° and pH 3.O. The enzyme was inhibited strongly by pepstatin ( K i = 31 × 10 −9M and α = 0.1). The acid protease released kinin from purified rat plasma kininogen at an initial rapid rate which plateaued at 460 ng bradykinin equivalents/mg substrate after a 2-hr incubation at 37°.

A glycopeptide containing 15 amino acid residues derived from hepatitis B surface antigen particles: Demonstration of immunogenicity to raise anti-HBs in mice

The major polypeptides composing hepatitis B surface antigen (HBsAg) particles are P-I and P-II. P-II shares the same amino acid sequence as P-I and contains an additional carbohydrate moiety of mol. wt approximately 5000. When a purified preparation of P-II was digested with Nagarse and then with Pronase P, it gave rise to a glycopeptide containing 15 amino acid residues and the carbohydrate moiety of P-II. The N-terminal amino acid sequence of the glycopeptide was determined to be Lys-Pro-Thr-Asp-Gly-Asn-. The polysaccharide moiety contained 5 moles of N-acetylglucosamine and was connected with Asn at the sixth position from the N-terminus. When mice were immunized against this HBsAg glycopeptide, they raised humoral antibodies which bound to each of three preparations of P-I derived from HBsAg particles of subtypes adw, adr and ayw, thereby indicating that the sequence of 15 amino acids in the glycopeptide would constitute a common antigenic structure of HBsAg.

Demonstration of a new amidase acting on glycopeptides

An enzyme preparation from almond emulsin cleaved the peptide-carbohydrate linkage of stem bromelain glycopeptide, Asn-Asn (oligosaccharide)-Glu-Ser-Ser. The resulting products were determined to be an intact peptide, Asn- Asp -Glu-Ser-Ser, and an intact oligosaccharide unit with two moles of N-acetylglucosamine. So far as tested the enzyme hydrolyzed glycopeptides with 3 to 10 amino acids, while both asparagine-oligosaccharide from ovalbumin and Asn-GlcNAc were not. Thus, the enzyme is a new amidase capable of hydrolyzing aspartylglycosylamine linkage in glycopeptides with multiple amino acid residues.

Studies on saccharogenic amylase from Rhizopus javanicus. IV. The structure of the carbohydrate moiety of a glycopeptide GP-I-b from Rhizopus saccharogenic amylase.

The structure of the carbohydrate moiety of GP-I-b which is one out of three glycopeptides isolated from a Pronase digest of the saccharogenic amylase of Rhizopus javanicus sp. 3-46, was investigated by enzymatic and chemical techniques. Nine moles of mannose followed by one mole of N-acetylglucosamine were released per mole of GP-I-b when it was treated sequentially with purified jack bean α-mannosidase and β-N-acetylglucosaminidase. Methylation of GP-I-b gave 3, 6-di-O-methyl derivative from the N-acetylglucosamine residues, and 2, 3, 4, 6-tetra-O-methyl, 3, 4, 6-tri-O-methyl and 2, 4-di-O-methyl derivatives from the mannose residues in an approximate ratio of 3:4:2. A smaller glycopeptide (F-1) containing two moles each of mannose and N-acetylgluco-samine per mole of asparagine was obtained when GP-I-b was subjected to one step of the Smith degradation. Exhaustive methylation of F-1 gave 3, 6-di-O-methyl derivative of N-acetylglucosamine, and 2, 3, 4, 6-tetra-O-methyl and 2, 3, 4-tri-O-methyl derivatives of mannose in a ratio of 1.00:0.85. Controlled acetolysis of GP-I-b yielded mannose, O-α-mannosyl-(1→2)-O-α-mannosyl-(1→3)-mannose and a smaller glycopeptide which was resistant to the acetolysis. From these and previous evidences, the following structure was determined for GP-I-b.

Structure of the Carbohydrate Units of IgA1 Immunoglobulin: I. COMPOSITION, GLYCOPEPTIDE ISOLATION, AND STRUCTURE OF THE ASPARAGINE-LINKED OLIGOSACCHARIDE UNITS

Abstract The carbohydrate composition of an IgA (α1 subtype) myeloma protein has been determined. The carbohydrate, present only on the heavy chain, was found to consist of 3 moles of sialic acid, 9 mole of galactose, 5.4 moles of mannose, 0.8 moles of fucose, 8.8 moles of N-acetylglucosamine, and 5 moles of N-acetylgalactosamine per mole of heavy chain. Four major glycopeptide-containing fractions were isolated following pronase degradation of the protein. Glycopeptide I consisted of galactose, N-acetylgalactosamine, threonine, serine, and proline in the molar ratio 4:5:4:5:9. Each of the N-acetylgalactosamine residues was found to be involved in an O-glycosidic linkage, demonstrating that there are five O-glycosidically linked oligosaccharide units per heavy chain. The structure of this glycopeptide is presented in the following paper (Baenziger, J., and Kornfeld, S. (1974) J. Biol. Chem. 249, 7270–7281. The three other glycopeptide fractions contained 1 to 2 residues of sialic acid, 0 to 0.7 residue of fucose, 2 residues of galactose, 3 residues of mannose, 4 to 5 residues of N-acetylglucosamine, and 1 residue of asparagine. Glycopeptide IIA was a homogeneous glycopeptide with the following structure: Glycopeptide fractions IIB and IIC did not appear to be homogeneous, but instead each contained 2 glycopeptides. In both cases, one of the glycopeptides was probably the disialyl form of IIA containing a residue of sialic acid linked α2,6 to the terminal galactose. The other glycopeptide had 2 rather than 3 nonreducing termini arising from the core mannose. Both of these terminated in sialic acid and had the same sequence as that found for the sialic acid-containing terminus of IIA. These termini arose from positions 3 and 6 of the core mannose. The core of this glycopeptide differed from that of IIA in having a residue of fucose which was most likely linked α1,6 to the N-acetylglucosamine involved in the N-glycosidic linkage to the peptide.

The structure of the carbohydrate moiety of a glycopeptide GP-I-a from Rhizopus saccharogenic amylase.

The structure of the carbohydrate moiety of GP–I–b which is one out of three glycopeptides isolated from a Pronase digest of the saccharogenic amylase of Rhizopus javanicus sp. 3–46, was investigated by enzymatic and chemical techniques.Nine moles of mannose followed by one mole of N-acetylglucosamine were released per mole of GP–I–b when it was treated sequentially with purified jack bean α-mannosidase and β-N-acetylglucosaminidase.Methylation of GP–I–b gave 3, 6-di-O-methyl derivative from the N-acetylglucosamine residues, and 2, 3, 4, 6-tetra-O-methyl, 3, 4, 6-tri-O-methyl and 2, 4-di-O-methyl derivatives from the mannose residues in an approximate ratio of 3: 4: 2.A smaller glycopeptide (F–l) containing two moles each of mannose and N-acetylglucosamine per mole of asparagine was obtained when GP–I–b was subjected to one step of the Smith degradation. Exhaustive methylation of F–l gave 3, 6-di-O-methyl derivative of Nacetylglucosamine, and 2, 3, 4, 6-tetra-O-methyl and 2, 3, 4-tri-O-methyl derivatives ...

Isolation of N-Acetyllactosamine and Galactosyl-β-d-(1 → 4)-N-acetyllactosamine from Beef Brain Glycopeptides

Abstract Forty-eight beef brains were dehydrated with acetone, extracted with chloroform-methanol solutions to remove lipid, and digested with Pronase. The glycopeptides were quantitatively recovered after gel filtration on P-4 resin (yield, 98 g with a carbohydrate content of 55%). The glycopeptides were further purified by chromatography on DEAE-cellulose and P-30 resin to give three fractions, obtained in a total yield of 75%, based on the fucose content of the Pronase digest. The three fractions were analyzed for pentoses, hexoses, hexosamines, hexuronic acid, sialic acid, sulfate, phosphorus, nitrogen, fatty acid, and peptide. Two of the fractions, which were free of fatty acid and hexuronic acid, were pooled (total weight, 5.6 g), desialized, and subjected to partial acid hydrolysis (pH 1.1, 100°, 90 min). The partial hydrolysate was fractionated by gel filtration on P-2 resin to give four peaks. Peak 3 was further purified by DEAE-cellulose and charcoal-Celite chromatography, and yielded 26 mg of a substance that was identified as N-acetyllactosamine by its composition, reduction with NaBH4 to give galactose and N-acetylglucosaminitol, complete cleavage with β-galactosidase, optical rotation, resistance to heating under alkaline conditions, and cochromatography with authentic N-acetyllactosamine in two solvent systems and with borate electrophoresis. Its identity was confirmed by mass spectroscopy of the per(trimethylsilyl)ated derivative. Peak 2 of the partial acid hydrolysate was further purified by deionization and preparative paper chromatography (yield, 3 mg). The substance contained 2 moles of galactose and 1 mole of N-acetylglucosamine. Reduction with NaBH4 established that the N-acetylglucosamine occupied the reducing end. Extended hydrolysis with β-galactosidase quantitatively cleaved the trisaccharide to monosaccharides. Partial hydrolysis with β-galactosidase yielded galactose and N-acetyllactosamine. Smith degradation gave erythritol in an 85% yield. These results indicated that the trisaccharide was galactosyl-β-d-(1 → 4)-N-acetyllactosamine.

Porcine Pancreatic Lipase: A GLYCOPROTEIN

Porcine pancreatic lipase has been purified to homogeneity as determined with analytical polyacrylamide gels. Both isoenzymes of lipase were shown to be glycoproteins containing 3.8 moles of mannose and 2.9 moles of N-acetylglucosamine per mole of enzyme.

The enzymic degradation of ovalbumin and its glycopeptides.

1. Ovalbumin glycopeptides, freed from all amino acids other than aspartic acid and a small proportion of leucine by repeated digestion with Pronase, were hydrolysed by 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (glycoaspartamidase) to the corresponding oligosaccharides. The glycoaspartamidase did not attack ovalbumin itself. 2. Ovalbumin, with mannose/hexosamine ratio 5:4, lost 1.5moles of N-acetylglucosamine and more than 2moles of mannose after incubation with alpha-mannosidase and beta-N-acetylglucosaminidase respectively. 3. In ovalbumin glycopeptides with approximate mannose/hexosamine ratios 5:3 and 5:4, one and two N-acetylglucosamine residues respectively were accessible to the action of beta-N-acetylglucosaminidase. 4. A mixture of alpha-mannosidase and beta-N-acetylglucosaminidase, acting on an ovalbumin glycopeptide with mannose/hexosamine ratio 5:3.7, removed nearly 4moles of mannose and 1.5moles of N-acetylglucosamine. 5. alpha-Mannosidase removed about 1.5moles of mannose from the ovalbumin oligosaccharide with mannose/hexosamine ratio approx. 5:3. The subsequent action of beta-N-acetylglucosaminidase liberated less than 1mole of N-acetylglucosamine and made at least 1mole further of mannose accessible to alpha-mannosidase action. 6. It is concluded that the carbohydrate moiety of ovalbumin is linked through a glycosyl group to asparagine. In a molecule with mannose/hexosamine ratio 5:4, there are two beta-N-acetylglucosamine residues linked together in a terminal position, followed by alpha-mannose. There is also present a side chain containing two alpha-mannose units.

Chemical characterization of the polysaccharide of the hydatid membrane of Echinococcus granulosus

An acetone powder of the hydatid membrane has been prepared, and the proteins were extracted with 0.5 N NaOH. Galactose, N-acetylgalactosamine, and N-acetylglucosamine were detected in the deproteinized acetone powder. The molar ratio of galactose to hexosamines was 1:1, and the ratio between both amino sugars was found to be 9–10 moles of N-acetylgalactosamine to 1 mole of N-acetylglucosamine. No sugars were found in the water and NaOH extracts.