Studies on a novel polysaccharide gel from the fruit of Thaumatococcus daniellii (Benth)

T. daniellii gel contains residues of L-arabinose, D-xylose, D-glucuronic acid, and 4-O-methyl-D-glucuronic acid in the ratios 1.00:7.20:1.91:0.66, together with nitrogen (1?%) and ash (3.1 %). The ash-free gel contains 76% of pentose and 24% of uronic acid; 25% of the uronic acid occurs as the 4-O-methyl derivative. All of the uronic acid residues in the polysaccharide are susceptible to periodate oxidation. Methylation studies suggest that the uronic acids occur as terminal side-substituents to a xylan back-bone and that the polysaccharide is highly branched. Enzymolysis with β-D-glucuronidase liberates a substantial part of the uronic acid, but does not completely depolymerise the gel.     

Automated analysis of uronic acids by high-performance liquid chromatography with photometric and fluorimetric postcolumn labelling using 2-cyanoacetamide

A convenient method for the rapid and sensitive automated analysis of uronic acids, based on high-performance anion-exchange chromatography on a Hitachi 2633 column and photometric as well as fluorimetric postcolumn labeling with 2-cyanoacetamide, has been developed. This method allows the simultaneous determination of 1-1000 nmol of D-mannuronic and D-galacturonic acids and 5-1000 nmol of L-iduronic and D-glucuronic acids in approx 70 min with high precision by photometric monitoring. In fluorimetric monitoring the linearity range was 1-1000 nmol for all these uronic acids, but reproducibility was rather low at the lowest limit of linearity. Application of this method to the analysis of component uronic acids in some polyuronides has suggested the inadequacy of generally accepted conditions for hydrolysis.     

羊栖菜褐藻糖胶抗凝血活性的研究

本文研究了羊栖菜褐藻糖胶的化学组成和抗凝血活性之间的关系。采用热水提取得羊栖菜粗多糖,CaCl2纯化得褐藻糖胶,DEAE Sepharose CL-6B柱层析与Sepharose CL-6B柱层析对褐藻糖胶进行分级,得到F1、F2、F31、F32和F33五个级分,均为岩藻糖、半乳糖和甘露糖等糖基组成的杂多糖,并含有硫酸酯和糖醛酸以及少量的蛋白质,相对分子质量范围2．5万～95万。采用活化部分凝血活酶时间(APTT)和凝血酶时间(TT)检测了这5个级分的抗凝血活性,结果显示,羊栖菜褐藻糖胶能显著延长APTT的凝血时间,而对TT的影响不明显。F1、F31和F32对APTT的影响比较显著,而F2、F33和羊栖菜粗多糖的影响较小。研究表明,羊栖菜褐藻糖胶主要是通过抑制内源凝血途径而达到抗凝血的效果,其抗凝血活性与褐藻糖胶的硫酸基含量成正相关,而与相对分子质量和糖醛酸含量无关。     

Isolation of a polysaccharide from the inner cell wall, intine, of pollen of Cryptomeria japonica

Isolation and solubilization of the intine of pollen grainsof Cryptomeria japonica were attempted. The intine was separatedeffectively from other cell fragments by passing it througha column of glass beads and dissolving it in an EDTA solution.The extract was fractionated by column chromatography on DEAE-celluloseand gelfiltration through Sephadex G-200, and a polysaccharidecomponent was obtained. This was found to consist of galactose,rhamnose, arabinose, xylose and uronic acid. (Received October 13, 1976; )     

Improved chromatographic separations on anion-exchange resins. II. Separation of uronic acids in acetate medium and detection with a noncorrosive reagent

An ion-exchange chromatographic system is described which is capable of separating complex uronic acid mixtures in about 2.5 hr. The system has the following advantages: (i) The resin is commercially available; (ii) no column temperature or buffer changes are needed during a run; (iii) the detection reagent is highly sensitive and also noncorrosive; and (iv) organic contaminants such as amino acids and reducing monosaccharides do not interfere with the separations. Several hundred samples can be run without column regeneration or equilibration. Furthermore, the system can be easily built by modification of existing conventional amino acid or sugar analyzers.     

Cell wall carbohydrates in Phycomyces blakesleeanus burgeff

The carbohydrate composition of the cell walls from spores, mycelium and sporangiophores of Phycomyces blakesleeanus was analyzed. Spore wall polysaccharides contained over 50% glucose, about 20% uronic acids, 10% mannose and 10% amino-sugars. During the growth of the hyphae amino-sugars became the main carbohydrate (45%); uronic acids contributed some 25%, glucose and fucose 10% and galactose nearly 6%. Sporangiophores contained almost 90% aminosugars and some 6% uronic acids. Traces of rhamnose were found in all wall preparations. A similar picture emerged from studies on the incorporation of [U-¹⁴C]-glucose into wall materials.Furthermore we looked for a GDP-fucose synthesizing system and found an increasing activity during early germination. This rise in activity was inhibited by cycloheximide but not by 5-fluorouracil.     

絮凝剂BP25的化学组成及结构研究

从活性污泥里筛选到一株巨大芽孢杆菌 (Bacillusmegaterium)A2 5,它能分泌胞外絮凝剂。用乙醇沉淀及SephadexS 50 0分子筛层析得到絮凝剂纯品BP2 5。通过Bradford反应、琼脂糖凝胶电泳及硫酸 酚法测定糖 ,证明BP2 5是一类多糖类物质。应用核磁共振技术证明其不含有糖醛酸。经三甲基硅醚和甲基化的气相色谱 质谱分析 ,证明多糖BP2 5含有葡萄糖和甘露糖两种单糖 ,其摩尔比为 4∶1。其连接键型包括α 1 ,6糖苷键和α 1 ,3糖苷键。由此推导出BP2 5可能的单元结构。     

Glycosaminoglycans from earthworms (<Emphasis Type="Italic">Eisenia andrei</Emphasis>)

The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M and 2.0 M NaCl, showed the presence of acidic polysaccharides on agarose gel electrophoresis. Monosaccharide compositional analysis showed that galactose and glucose were most abundant monosaccharides in both fractions. Depolymerization of the polysaccharide mixture with glycosaminoglycan-degrading enzymes confirmed the presence of chondroitin sulfate/dermatan sulfate and heparan sulfate in the 2.0 M NaCl fraction. The content of GAGs (uronic acid containing polysaccharide) in the 2.0 M NaCl fraction determined by carbazole assay was 2%. Disaccharide compositional analysis using liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) analysis after chondroitinase digestion (ABC and ACII), showed that the chondroitin sulfate/dermatan sulfate contained a 4-O-sulfo (76%), 2,4-di-O-sulfo (15%), 6-O-sulfo (6%), and unsulfated (4%) uronic acid linked N-acetylgalactosamine residues. LC–ESI–MS analysis of heparin lyase I/II/III digests demonstrated the presence of N-sulfo (69%), N-sulfo-6-O-sulfo (25%) and 2-O-sulfo-N-sulfo-6-O-sulfo (5%) uronic acid linked N-acetylglucosamine residues.     

Further biochemical investigations on glycosaminoglycans extracted from Euglena gracilis]

Polyanionic glycans extracted from Euglena gracilis have been studied by biochemical, chromatographic and electrophoretic analysis. Our results show the presence of a fraction which precipitate with CPC and another one which not precipitate with CPC. The CPC precipitable material fractionated on CPC-Cellulose column shows the presence of 5 Glycosaminoglycans; the not CPC precipitable material contains uronic acid, galactose, sulfate, galactosamine and cannot be related to Keratan sulfate.     

裙带菜褐藻糖胶的分离纯化及其结构分析

裙带菜经水提法提取得到褐藻糖胶粗多糖,经DEAE-Sepharose FF离子交换层析和 Sepharose 4B层析后,得到Sl、S2两个单一组分,相对分子质量分别为550 808、38 335.基本结构及单糖组成分析表明,二者均含有岩藻糖、糖醛酸、硫酸基、半乳糖、甘露糖、葡萄糖、鼠李糖、木糖、阿拉伯糖,但含量差别较大,推测与二者的生理活性的差异有关.     

Uronic acid content in polysaccharides: Estimation by decarboxylation involving use of gas chromatography

A quantitative method for determining uronic acid content in different polysaccharides has been described. The decarboxylation reaction was run with 57% hydriodic acid (HI) in a small flask attached directly to the gas circuit of a chromatograph. After the reaction was completed, the resultant carbon dioxide was supplied to the chromatographic column. A sample uronic acid (0.5–10.0 μmol) yielded quantitative results with a relative standard deviation of 2%. The contribution of nonuronic materials to the error did not exceed 0.6%. The total analysis time amounted to 50–60 min.     

Isolation and characterization of disaggregatase from Methanosarcina mazei LYC

At certain stages in its growth cycle, Methanosarcina mazei produces an enzyme (disaggregatase) that causes aggregates to separate into single cells. M. mazei S-6 and LYC both produce this enzymatic activity, although the specificities of activities differ. The disaggregatase of M. mazei S-6 had little effect on strain LYC cells, but the disaggregatase of M. mazei LYC disaggregated both strain LYC and strain S-6 cells. The disaggregatase of M. mazei LYC was purified by column chromatography, and it apparently consisted of two similar subunits with a combined molecular size of about 180,000 Da. Strain S-6 culture supernatants contained 14 U of activity per liter when activity was measured as uronic acids released from purified cell wall material. When the activity was quantified as the release of uronic acids from boiled M. mazei S-6 cells, the highest activity was found at pH 4.7 and at 35 degrees C.     

Isolation and characterization of disaggregatase from Methanosarcina mazei LYC.

At certain stages in its growth cycle, Methanosarcina mazei produces an enzyme (disaggregatase) that causes aggregates to separate into single cells. M. mazei S-6 and LYC both produce this enzymatic activity, although the specificities of activities differ. The disaggregatase of M. mazei S-6 had little effect on strain LYC cells, but the disaggregatase of M. mazei LYC disaggregated both strain LYC and strain S-6 cells. The disaggregatase of M. mazei LYC was purified by column chromatography, and it apparently consisted of two similar subunits with a combined molecular size of about 180,000 Da. Strain S-6 culture supernatants contained 14 U of activity per liter when activity was measured as uronic acids released from purified cell wall material. When the activity was quantified as the release of uronic acids from boiled M. mazei S-6 cells, the highest activity was found at pH 4.7 and at 35 degrees C.     

Presence of heparan sulfate proteoglycan in thyroid tissue

Glycosaminoglycan (GAG) was extracted from the porcine thyroid gland with a buffer containing 5.3 M guanidine-HCl and proteolytic enzyme inhibitors and was fractionated by subsequent isodensity CsCl centrifugation. 60% of uronic acid positive materials was accumulated in the bottom one-fourth fraction with high buoyant density. More than 90% of this uronic acid positive material in the thyroid tissue was heparin or heparan sulfate (sensitive to nitrous acid treatment) and the rest was chondroitin sulfate or dermatan sulfate (sensitive to chondroitinase ABC treatment). When the accumulated high buoyant density GAG was analyzed on a Sepharose CL-6-B column, approximately 14% of the heparin sulfate were in the macromolecular portion as a form of proteoglycan because it was destroyed by the papain digestion or alkaline borohydride treatment which extensively digests protein or releases GAG from protein by the elimination reaction, respectively. This study demonstrates the existence of heparin sulfate proteoglycan in thyroid tissue for the first time.     

Determination of hexuronic acids in glycosaminoglycans by automated ion-exchange chromatography.

This report describes a procedure for analyzing glucuronic and iduronic acids using the Technicon automated sugar chromatography system. Glueronic and iduronic acids of standard samples of glycosaminoglycans have been analyzed after hydrolysis by formic acid. The method has been applied to quantitate uronic acids in chondroitin sulfates and dermatan sulfate mixtures obtained by Dowex 1 Cl^? column fractionation of glycosaminoglycans from aortas of different animal species. The results are in good agreement with those obtained by the gas-liquid chromatographic technique.     

Ascorbic acid and development of xylem and phloem cells in the pine trunk

Changes in the levels of ascorbic acid (AA), its oxidized form, dehydroascorbic acid (DHA), and uronic acids as initial precursors for the AA synthesis were studied as related to the degree of xylem and phloem cell development in the course of early and late wood formation in the trunks of Scots pine (Pinus sylvestris L.). The cells of mature and conducting phloem, cambial zone, differently developed cells in the zones of cell enlargement and maturation were obtained by successive scraping tissue layers from trunk segments of 20–25-year-old trees; tissue identification was checked anatomically and histochemically. The contents of compounds tested were calculated per dry weight and per cell basis. We found great differences in the contents of AA and DHA and also in their ratio in dependence of the wood type developing in the pine trunks during growth period and on the stage of differentiation of xylem and phloem cells. Changes in the AA content during xylem cell differentiation were accompanied by changes in the content of uronic acids. The amounts of AA, DHA, and uronic acids were the highest at the stage of early lignification and reduced with tracheid maturation. The AA to DHA ratio changed differently in the course of early and late xylem lignification. It reduced from the start of lignification to the formation of early mature xylem and, in contrast, increased in mature late wood; this indicates a difference in the level of redox processes in these tissues.     

Re-examination of the products of the action of galactose oxidase. Evidence for the conversion of raffinose to 6'-carboxyraffinose

Galactose oxidase is a fungal enzyme which is known to oxidize the C-6 hydroxymethyl of galactose to an aldehyde group. When the products of a galactose oxidase-catalase treatment of raffinose were examined by gel filtration and ion exchange chromatography, we found that, in addition to the expected 6'-aldehydoraffinose, two other components were present. Of these two components, the major one was retained on a column of AG 1-X8 (formate), gave a positive carbazole reaction for uronic acid, and on paper chromatograms had a mobility identical with that of 6'-carboxyraffinose. The infrared spectrum of the compound showed a carbonyl absorbance at 1725 cm-1 and was distinguishable from the spectra of raffinose and 6'-aldehydoraffinose. These data showed that raffinose was partly converted to 6'-carboxyraffinose when treated with galactose oxidase and catalase. The conversion of [3H]raffinose to [3H]6'-carboxyraffinose increased gradually with time of oxidation from 22% at 6 h to 68% at 96 h. Results of other experiments provided evidence that this was an enzymic conversion and depended on the presence of galactose oxidase. The activities responsible for the formation of aldehyde and uronic acid could not be separated by affinity chromatography, gel electrophoresis, or ion exchange chromatography, indicating that the same enzyme is responsible for both activities. Treatment of galactose, melibiose, and stachyose with galactose oxidase and catalase also resulted in the formation of the corresponding uronic acids. These studies indicate that galactose oxidase not only converts the C-6 hydroxymethyl group of galactose to an aldehyde group, but also catalyzes further oxidation to the carboxyl group.     

Chemical characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test

The lipopolysaccharides (LPS) extracted from Proteus strains OX2, OX19, and OXK used as antigens in the Weil-Felix test, were characterized by chemical analysis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To separate the O-polysaccharide, core-oligosaccharide, and lipid A moieties, each LPS was treated with 2% acetic acid, centrifuged, and applied to Sephadex G-50 column. The core-oligosaccharides contained L-glycero-D-mannoheptose, D-glycero-D-mannoheptose, glucose (Glc), galactose, 3-deoxy-D-mannooctulosonic acid, uronic acid, phosphate, glucosamine (GlcN), and galactosamine (GalN). The lipid A preparations contained GlcN, GlcN-phosphate, and three fatty acids (myristic, plamitic, and beta-hydroxymyristic acids). However, the O-polysaccharides of OX2- and OXK-LPS had different chemical compositions which consisted of Glc, GlcN, and quinovosamine, and Glc, uronic acid, and GalN, respectively, while OX19-LPS seemed to lack O-polysaccharide.     

Extracellular agglutination factor of myxamoebae produced by Dictyostelium discoideum NC-4

A non-dialyzable specific agglutination factor of myxamoebae obtained from culture broth during the growth phase of Dictyostelium discoideum NC-4 was thermostable but the agglutination activity disappeared below pH 5.0. In the case of formalinized myxamoebae, digestion of the factor with Pronase decreased the activity, but periodate treatment of the factor did not affect the activity. Myxamoebal agglutination by this factor was inhibited by the addition of uronic acid, polyuronide (protuberic acid), and cell-surface polysaccharide prepared from the myxamoebae, but the agglutination was not affected by citric acid or glycine.The factor was purified by ethanol precipitation, column chromatography using DEAE-cellulose and Sepharose-2B, and zone electrophoresis. Chemical analysis of the purified factor gave 61.0% carbohydrate and 26.1% protein, and glucose, mannose, xylose and rhamnose (molar ratios of 9.3 : 3.2 : 2.1 : 1.0) were detected as the component sugars. The content of uronic acid was 12.9%.When the myxamoebae of the growth phase were starved in Millipore-supporting medium, the agglutination activity was detected in the supernatant of the medium.     

Is uronic acid oxidase involved in the hormonal regulation of abscission in explants of citrus leaves and fruits?

The role of uronic acid oxidase in abscission was studied in explants of citrus ( Citrus sinensis L. Osbeck; var. Shamouti) leaves and fruits. In leaf explants, activity of uronic acid oxidase prior to onset of abscission and the rate of abscission were markedly accelerated by ethylene and delayed by 2,4-dichlorophenoxyacetie acid. Similar results were obtained for uronic acid oxidase activity in the exocellular fraction of young fruit explants. In mature fruit explants, treated with ethylene, an immediate increase in activity was evidenty in the non-active shoot/peduncle abscission zone, whereas in the calyx abscission zone the rise in activity occurred after a prolonged exposure to ethylene, when most of the fruits had already abscised. Whenever ethylene enhanced uronic acid oxidase activity, 2,4-dichlorophenoxyacetic acid delayed it. A gradient of decreasing activity or uronic acid oxidase was recorded from both sides of the abscission zone in leaves and fruits toward the separation line, where activity was the lowest as compared with the activity found in adjacent tissues. It is suggested that uronic acid oxidase is involved in senescence and cell wall degradation. However, it is yet questionable whether this enzyme is directly related to the control mechanism of abscission.