Extraction, purification and chemical characterisation of xylogalacturonans from pea hulls

Pea hulls contained 925 mg/g sugar including 659 mg/g cellulosic glucose and 90 mg/g uronic acid. They were de-esterified by NaOH (pH>13 at 4°C, 2 h) and treated with HCl (0.1 mol/l, 80°C, 24 h). The HCl-soluble fraction represented 95 mg/g initial pea hulls. It was rich in galacturonic acid (259 mg/g), xylose (93 mg/g) and rhamnose (91 mg/g), which co-eluted in anion-exchange chromatography. The HCl-soluble fraction was degraded by a rhamnogalacturonan-hydrolase and the reaction products were fractionated by size-exclusion chromatography. Two fractions, representing together 18 mg/g initial pea hulls, were composed almost exclusively of galacturonic acid and xylose and could be defined as xylogalacturonans. The first fraction exhibited a high molar mass, a molar ratio Xyl/GalA of 1 and contained almost 5% of rhamnose. The molar mass of the second fraction was much lower and the molar ratio Xyl/GalA was 0.6. Methylation analysis showed the presence in both fractions of a (1→4) galacturonan backbone highly substituted on O-3 either by terminal xylosyl residues or by short side-chains of (1→2) linked xylosyl residues.     

猴头菌水溶性多糖的分离纯化与结构表征

从猴头菌子实体中分离得到一种新型的水溶性杂多糖HEPF2,分子量大小为1.66′104Da,该多糖由岩藻糖、半乳糖和葡萄糖以1.00:3.69:5.42比例构成,同时也含有微量的3-O-甲基鼠李糖。进一步利用傅立叶变换红外光谱法、糖组成分析、甲基化分析、部分酸水解法和核磁共振法等方法进行结构鉴定,检测结果表明,该杂多糖中包含1→4、1→6结合的葡萄糖和1→6结合的半乳糖残基,连接于主链的侧链残基,包括岩藻糖残基、少数的端基葡萄糖和半乳糖残基。核磁共振法检测结果还表明,1→4结合葡萄糖为β构型,(1→6)结合半乳糖、(1→2,6)结合半乳糖和端基葡萄糖均为α构型。     

Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis

Two types of fucan sulfate were isolated from chloroform/methanol extract of the body wall of the sea cucumber Stichopus japonicus. One type (type A) contained 3.41 mmol fucose/g and 2.35 mmol sulfate/g, and the molecular mass was determined to be 9 kDa by gel permeation chromatography (GPC). Structural analysis suggested that type A consists of a backbone of (1-->3)-linked fucosyl residues that are substituted at C-4 with fucosyl residues, and that fucosyl residues are sulfated at C-2 and/or C-4. Another type (type B) contained 3.90 mmol fucose/g and 3.07 mmol sulfate/g, and the molecular mass was determined to be 32kDa by GPC. Structural analysis showed that type B is largely composed of unbranched (1-->3)-linked fucosyl residues, and that sulfate substitution(s) occur at C-2 and/or C-4. The potential of both types to inhibit osteoclastogenesis was examined by an in vitro assay system, showing that both types of fucan sulfate inhibit osteoclastogenesis more than 95% at 50 microg/mL concentration. These results suggest that types A and B fucan sulfate from sea cucumber are potent inhibitors of osteoclastogenesis.     

Isolation and characterization of a novel polysaccharide as a possible allergen occurring in wheat flour

A new polysaccharide with a molecular weight of 5.0 x 10(4) was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with beta-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.     

Intracellular Localization of GDP-Fucose: Polysaccharide Fucosyl Transferase in Corn Roots (Zea mays L.)

The intracellular site of synthesis of the fucose-rich polysaccharide slime secreted by corn roots was localized by monitoring the distribution of GDP-fucose:polysaccharide fucosyl transferase activity in subcellular fractions of corn roots. Root tip sections were chopped in the presence of 0.56 molar sucrose and 100 millimolar Tris (pH 7.0). After a brief centrifugation, the homogenate was applied to a Sepharose 4B column (1.5 × 30 cm). The turbid, particulate portion of the supernatant fraction eluted at the void volume. Ninety per cent of the enzyme activity was found in the pooled particulate fractions. The particulate fraction was purified on linear sucrose gradients. Gradient fractions were characterized by buoyant density, 280 nanometer absorbance, electron microscope observation, and distributions of NADH-cytochrome c oxidoreductase and fucosyl transferase activities.     

Purification and partial characterization of a cobalamin-binding protein from chicken egg yolk

Cobalamin-binding protein has been purified from chicken egg yolk by using DEAE-cellulose with a NaCl gradient. The resultant protein fraction was subjected to bioaffinity chromatography. The Mr was 38,000 by SDS-PAGE and 39,000 by gel filtration, and indicated that it was a glycoprotein. The Stokes radius was 4.3 nm and the pI 4.1. The protein bound 57CO.B12 with a molar ratio of 1:1 and a Kd of 0.41 microM. The CBP composed 296 amino acids residues. The protein-ligand interaction was inhibited by Cbl analogues.     

Structures of sulfated oligosaccharides in human trachea mucin glycoproteins

The structures of high molecular weight sulfated oligosaccharide chains in mucins purified from the sputum of a patient with cystic fibrosis and blood group H determinant were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by ion exchange chromatography on DEAE-Agarose and a fraction containing multisulfated chains was further purified by lectin affinity chromatography to completely remove small amounts of sialylated chains. A major sulfated oligosaccharide fraction containing chains with an average of 160 to 200 sugar residues was isolated by gel filtration on BioGel P-10 columns and individual subfractions were characterized by methylation analysis, periodate oxidation and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GldNAc in a ratio of 1:2:2.1 and only one galactosaminitol residue for every 160-to 200 sugar residues. The average molecular weight of oligosaccharide chains in these fractions was between 27,000 and 40,000 daltons. Structural analysis showed that these high molecular weight chains contained varying amounts of the repeating unit shown in the following oligosaccharide. Only one in about every 10 repeating units contained sulfate esters.Several shorter chains which contain 2 to 3 sulfate esters were also isolated from this multisulfated oligosaccharide fraction. The structures proposed for these oligosaccharides indicate that they are lower molecular weight chains with the same general structure as those found in the high molecular weight sulfated oligosaccharides. Taken collectively, the results of these studies show that a major sulfated oligosaccharide fraction in resporatory mucin purified from the mucus of patients with cystic fibrosis contains high molecular weight branched chains that consist of a repeating oligosaccharide sequence with sulfate linked to the 6 positions of galactose and possibly GlcNAc residues in the side chains.     

Mannose in the keratan sulfate from bovine cornea

After exhaustive digestion of bovine corneas with a protease, keratan sulfate fractions of different chain length were obtained by ethanol fractionation on a cellulose column, followed by ion-exchange column chromatography. Compositional analysis of these fractions showed that aspartic acid is the predominant amino acid and that the keratan sulfate in each fraction contains aspartic acid and mannose in the molar ratio of 1:3. Molecular weights of these fractions, estimated by gel chromatography, were close to the calculated values based on the molar ratios of the components to three moles of mannose. The results strongly suggested that the keratan sulfate of bovine cornea contains three mannose residues per chain, as an integral component of the linkage region to protein.     

Purification of a lysinonorleucine cross-linked peptide fraction from porcine aorta elastin.

Highly purified elastin from porcine aorta was submitted to elastase digestion. The enzymic products were subjected to gel filtration on Sephadex G 25. The excluded fraction was then submitted to thermolysin digestion and gel filtration. The excluded fraction was submitted to partial acid hydrolysis and gel filtration. Several sub-fractions were obtained. The F3 subfraction containing cross-linking agents (desmosines and lysinonorleucine) was finally subjected to ion exchange chromatography. A highly enriched peptide fraction containing lysinomorleucine was obtained and then purified by preparative electrophoresis. The ratio of enrichment passed from 2 residues of lysinonorleucine (expressed as lysine) from starting material (elastin) to 178 out of 1,000 residues in the final step. In this peptide fraction, if we express in molar ratio and consider the amount of lysinonorleucine is one residue, the following amino-acid composition is Pro:3, Gly:1, Ala:2, LNL:1, Lys:2. No traces of desmosines are detected. The role of lysinonorleucine in bridging functions in elastin is discussed.     

Enzymic and chemical degradation and the fine structure of pectins from sugar-beet pulp

Pectins sequentially extracted from sugar-beet pulp with water (WSP), oxalate (OXP), hot acid (HP), and cold alkali (OHP) have been degraded variously by base-catalysed β-elimination, de-esterification, endopectin lyase, pectin methyl esterase, endopolygalacturonase, and endopectate lyase. The products were studied mainly by chromatography on Sephadex G-100. The pectins contain various amounts of degradation-resistant (hairy) fragments in which the molar ratios of neutral sugar residues to galacturonic acid residues were 4.8, 4.6, 3.8, and 3.7 for WSP, OXP, HP, and OHP, respectively. The molar ratios of rhamnose residues to galacturonic acid residues in these fragments were 0.15, 0.20, 0.38 and 0.35, respectively. The pectins also contained sequences of galacturonic acid residues with relatively little neutral sugar residues attached (smooth fragments). Methyl ester and acetyl groups were distributed fairly regularly along the smooth fragments. Evidence is presented for an association of oligogalacturonic acids with the hairy fragments under the conditions of gel chromatography. Feruloyl groups are located in the hairy fragments. Other phenolic compounds, associated with the purified pectins, appear not to be covalently linked.     

A fucoidan fraction from Ascophyllum nodosum.

A fucoidan fraction was purified from the brown alga Ascophyllum nodosum. The polysaccharide contained L-fucose and sulfate as the only constituents. Combination of methylation analysis, Smith degradation, FTIR and NMR spectroscopy on the native and the de-sulfated polymers demonstrated that the fucoidan consisted of a highly branched core region with primarily alpha-(1-->3)-linked fucosyl residues and a few alpha-(1-->4) linkages. Branch points were at position 2 of the -->3-linked internal residues. The side chains consisted of single and multi-unit fucosyl residues. The combined analytical data suggested also a complex sulfation pattern with substitution principally at position 2 and/or position 4. Such diversity in the structural features of this fucoidan may be of importance for its various biological properties.     

Isolation and partial characterization of uronic acid-containing glycoproteins from Mucor rouxii

Five different fractions containing uronic acids associated with protein were isolated from the cytoplasm of the filamentous form of Mucor rouxii. A signle fraction was isolated from the cell wall by hot sodium dodecyl sulfate followed by ion exchange column chromatography. Two cytoplasmic entities (peaks I and II) were not adsorbed to DEAE Bio-Gel A. The molecular mass of peaks I to V ranged from 16.5 to 210 kDa. The protein-uronic acid ratios were different for each fraction. The cell wall fraction showed a molecular mass of 16.5 kDa, similar to that of peak II but with differences in chromatographic behavior and protein-uronic acid ratio. The possible role of these molecules as acceptors of sugar residues during polyuronide chain growth is discussed.     

Structures of the sugar chains of mouse immunoglobulin G

The asparagine-linked sugar chains of mouse immunoglobulin G (IgG) were quantitatively liberated as radioactive oligosaccharides from the polypeptide portions by hydrazinolysis followed by N-acetylation, and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin (RCA120) affinity high-performance liquid chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Mouse IgG was shown to contain the biantennary complex type sugar chains. Eight neutral oligosaccharide structures, viz, +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1---- 4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc, were found after the sialidase treatment. The molar ratio of the sugar chains with 2,1, and 0 galactose residues was 2:5:3. The galactose residue in the monogalactosylated sugar chains was distributed on Man alpha 1----3 and Man alpha 1----6 sides in the ratio of 1:3. The oligosaccharides were almost wholly fucosylated and contained no bisecting N-acetylglucosamine which is present in human, rabbit, and bovine IgGs.     

Structural characterization of a new anticoagulant fucan sulfate from the brown seaweed Ecklonia kurome.

Methylation analysis of a fucose-containing, sulfated polysaccharide (C-II), which was isolated from the brown seaweed Ecklonia kurome and has a potent anticoagulant activity, showed the presence of 3-O- and 3,4-O-disubstituted fucopyranosyl residues in addition to small proportions of nonreducing, terminal fucofuranosyl and fucopyranosyl groups, and 2,3-di-O- and 2,3,4-tri-O-substituted fucopyranosyl and galactopyranosyl residues with various glycosidic linkages. Methanolysis of C-II gave several neutral oligosaccharide fractions in small proportions and two high-molecular-weight acidic fractions in large proportions. Methylation analysis of the low-sulfated acidic fraction showed that the proportion of 3-O-linked fucosyl residues increases and that of 3,4-O-disubstituted decreased as compared to C-II. Methylation and g.l.c.-m.s. analysis of the neutral oligosaccharide fractions showed the presence of Fuc-(1----3)-Fuc and a fucosyl trisaccharide, in addition to small proportions of Gal-(1----4)-Fuc, Fuc-(1----2)-Fuc, Fuc-(1----4)-Fuc, Fuc-(1----2)-Gal, and Fuc----Gal----Fuc. Methylated C-II was also desulfated by methanolysis, followed by remethylation with (2H3)methyl iodide, and most of (2H3)methyl groups were linked to O-4 of the 3-O-linked fucosyl residues. These results suggested a highly branched, new type of fucan sulfate containing a backbone of (1----3)-linked L-fucosyl residues having sulfate groups mainly attached to C-4.     

Structural characterization of a tobacco rhamnogalacturonan

A rhamnogalacturonan, extracted with hot water from the aqueous ethanol insoluble residue of flue-cured bright tobacco lamina, was purified by tangential flow ultrafiltration, ion chromatography and gel filtration. It was characterized by chemical and spectroscopic methods. Fractionation revealed that the rhamnogalacturonan consisted of a series of polysaccharides with different amounts of methyl-esterified galactopyranosyluronic acid residues in the backbone and different amounts of neutral sugar residues.The main pectic polysaccharide fraction has a backbone consisting of 4-linked α-d-galactopyranosyluronic acid residues interspersed with 2-linked l-rhamnopyranosyl residues. Approximately 22% of the galactopyranosyluronic acid residues are methylated. The main chain is branched at C-4 of rhamnose with neutral sugar side chains containing terminal and 4-linked β-d-galactopyranosyl and terminal and 5-linked α-l-arabinofuranosyl residues. The average degree of polymerization of this tobacco rahamnogalacturonan was estimated to be 400.     

Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Cellular localization, purification and characterization

Gel-filtration analysis of cytosol fraction obtained from unfertilized sea-urchin (Anthocidaris crassispina) eggs on Sephadex G-75 revealed the presence of two Zn-binding-protein fractions. The major Zn-binding protein fraction had a low molecular weight and a low absorbance at 280 nm, properties similar to those of the metallothionein found in the regenerating rat liver. These fractions were further purified by DEAE-cellulose and Sephadex G-50 chromatography. Homogeneity of the Zn-binding protein was judged by polyacrylamide-disc-gel electrophoresis and gel-permeation chromatography in the presence of 6 M-guanidinium chloride. The molecular weight determined by gel-permeation chromatography was 3900. This value is in good agreement with the minimum molecular weight calculated from the amino acid composition, which was 3655. Zn-binding protein is composed of 36 amino acid residues and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and a complete absence of aromatic amino acids, as well as of methionine, histidine and arginine. Zn-binding protein contained 4.1 g-atoms of zinc per mol and a trace of cadmium, but no copper, iron or calcium. The molar ratio of reactive thiol groups to metal ion was calculated to be 2.73:1. Possible roles of this Zn-binding protein in the homoeostasis of zinc in unfertilized sea-urchin eggs are discussed.     

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

Structural studies of the polysaccharide part of the cell wall lipopolysaccharide from Bacteroides fragilis NCTC 9343

The structure of the polysaccharide part of the lipopolysaccharide from Bacteroides fragilis NCTC 9343 has been determined using sugar and methylation analysis as the principal tools. Phenol--water extraction followed by a phenol--chloroform--light petroleum extraction yielded a lipopolysaccharide suitable for structural analysis. Analysis of sugars using alditol acetates showed that the polysaccharide contained L-rhamnose, D-galactose and D-glucose in the approximate molar ratios of 1:5:1. After weak acid hydrolysis, two polysaccharide fractions were isolated by gel permeation chromatography: PSI and PSII with the sugar molar ratios 1:5:1 and 1:2:1 respectively. Chromium trioxide oxidation revealed that all galactosyl residues have the beta configuration, and that the rhamnosyl and glucosyl residues have the alpha configuration. From methylation analysis of lipopolysaccharide and the PS I and PS II fractions the following structures could be deduced.     

The structures of the asparagine-linked sugar chains of human apolipoprotein B-100

The asparagine-linked sugar chains of human apolipoprotein B-100 were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Their structures were elucidated by sequential exoglycosidase digestion in combination with methylation analysis after fractionation by paper electrophoresis and gel permeation chromatography. One neutral and two acidic fractions were obtained by paper electrophoresis in a molar ratio of 7:8:5. The neutral fraction contained high-mannose type oligosaccharides consisting of Man5GlcNAc2 to Man9GlcNAc2. The acidic fractions contained monosialylated and disialylated biantennary complex type oligosaccharides. As minor components in the monosialylated fraction, biantennary complex-type oligosaccharides which were absent one terminal galactose residue, monoantennary complex type, and hybrid type oligosaccharides were detected. Apolipoprotein B-100 was calculated to contain 5-6 mol of high-mannose type and 8-10 mol of complex type oligosaccharides per mole protein.     

Improved Synthesis of 3-14C-3-Hydroxy-3-methylglutaryl Coenzyme A

A new polysaccharide with a molecular weight of 5.0×10⁴ was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with β-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.