The Structure of the Aerobacter aerogenes A3(S1) Polysaccharide. I. A Reexamination Using Improved Procedures for Methylation Analysis<sup>*</sup>

Sandford, Paul A.; Conrad, H. Edward

doi:10.1021/bi00869a009

Cited by 458 publications

(120 citation statements)

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“…The purified a-I was methylated by the method of Hakomori (14). The uronic acid residue in the sugar, esterified in the methylation reaction, was reduced with lithium aluminum hydride (28) and the primary hydroxyl group formed from the uronic acid residue was methylated by the method of Hakomori. The fully methylated neutral sugar thus prepared was hydrolyzed and converted into corresponding alditol acetates.…”

Section: Methodsmentioning

confidence: 99%

Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides

Kato

Nevins²

1984

Plant Physiol.

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The structure of a glucuronoarabinoxylan in Zea mays L. (hybrid B73 x Mol7) shoot cell walls has been studied. The water-insoluble fraction of Zea shoot cell walls, pretreated with purified Bacillus subtilis (1 -_ 3),(I -4)-ft-D-glucan 4-glucanohydrolase, was treated with purified B. subtilis endo-(1 -_ 4)-#-xylanase. Carbohydrates (2.6% of the waterinsoluble fraction of Zea shoot cells walls) derived from the enzyme treatment consisted of glucuronoarabinoxylan fragments with molecular weights which varied from a few hundred to over 2.0 x 10' daltons. Structural analyses of the fragments suggested that the glucuronoarabinoxylan had a xylan backbone which contained (1 -_ 4)-j%D-xylopyranosyl residues, with about 60 to 70% substitution at the C-2 or C-3 position with arabinose, glucuronic acid, and other substituents. Furthermore, the glucuronoarabinoxylan contained a phenolic component which appeared to be primarily ferulic acid bonded to carbohydrate, probably by an ester linkage. Sugars on the chromatogram were detected with alkaline silver nitrate (27). Total carbohydrate was determined by the phenol-H2SO4 method (9). Reducing power was measured by the NelsonSomogyi method (25,30). GLC was performed with a Varian model 3700 gas chromatograph.Analysis of Neutral Sugars in Oligo-or Polysaccharide. Oligosaccharide (20 ug as xylose equivalent) was hydrolyzed with 1 M HCI for 3 to 4 h at 100°C. Polysaccharide (100 ,ug as xylose equivalent) was hydrolyzed with 2 M HCI for 5 h at 100°C. After hydrolysis, the acid was removed with a stream of air. Sugars were converted into their corresponding alditol acetates followed by analysis by GLC on a glass column (0.2 x 190 cm) packed 3% SP-2340 (Supelco) at 2 10°C at a helium-flow rate of 35 ml/ min.Methylation Analysis of Poly-or Oligosaccharide. Polysaccharide (1 mg as xylose equivalent) or oligosaccharide (20-100 Og as xylose equivalent) in DMSO (0.2 ml) was methylated with methylsulfinyl carbanion (0.1 ml) and methyl iodide (0.1 ml) by the method of Hakomori (14). The methylated poly-or oligosaccharide, extracted into chloroform followed by evaporation of the extract, was hydrolyzed with 90% HCOOH at 100°C for 1 h. The HCOOH was removed with a stream of air, and the residue treated with 0.5 M HCI at 1O0C for 4 h. The acid was removed with a stream of air. The methylated sugars were converted into their corresponding alditol acetates (21) followed by GLC analysis on a 30-m DB-1 fused silica capillary column or a 10-m SP 2330 glass WCOT capillary column at 150 to 230°C, 4°C min-', with a split ratio of 50:1. The helium carrier flow was 1 ml min-'. For GLC-MS, a combined gas chromatograph-mass spectrometer, Finnegan 4000 with INCOS data system, fitted with a DB-1 fused silica capillary column (25 m) was used.

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Section: Methodsmentioning

confidence: 99%

Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides

Kato

Nevins²

1984

Plant Physiol.

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“…Unfertilized ovules were taken on the day of anthesis and cultured as described by Beasley and Ting (9,10 (43). Following methylation, 1 ml of chloroform-methanol (1:1, v/v) was added and this solution dialyzed for 2 days against four changes of 2 liters each of distilled H20.…”

Section: Methodsmentioning

confidence: 99%

Changes in Biochemical Composition of the Cell Wall of the Cotton Fiber During Development

Meinert¹,

Delmer²

1977

Plant Physiol.

301

199

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The composition of the cell wall of the cotton fiber (Gossypium hirsutum L. Acala SJ-1) has been studied from the early stages of elongation (5 days postaathesis) through the period of secondary wall formation, using cell wals derived both from fibers developing on the plant and from fibers obtained from excised, cultured ovules. The cell wall of the elongating cotton fiber was shown to be a dynamic structure.

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“…Therefore, the acidic polysaccharide (60 mg) was subjected to two subsequent methylations with sodium hydride/dimethyl sulfoxide/methyl iodide, as described by Sandford and Conrad [12]. The reaction product (25 mg), which was readily soluble in chloroform, was then subjected to methanolysis (1 ml of 1.5 N HC1 in absolute methanol/l6 hours/lOO"-in a sealed tube).…”

Section: Oligosaccharide Analysesmentioning

confidence: 99%

Immunochemistry of K Antigens of Escherichia coli. 5. The K Antigen of E. coli 08:K 27 (A):H-

Jann

et al. 1968

Eur J Biochem

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An acidic polysaccharide which consists of D-glUCUroniC acid, D-glUCOSe, D-galaCtoSe and L-fucose in a molar ratio of 1 : 1 : 1 : 1 and which contains 5.2 o/o of 0-acetyl groups was isolated from Escherichia coli 08 : K 27 (A) : H-cells.About go0/, of the total polysaccharide present on the bacterial cell could not be extracted with saline at 60". This polysaccharide fraction was solubilized with 4501, aqueous phenol a t 65". Purification of the acidic polysaccharide was achieved with cetavlon precipitation. It was shown to be homogeneous by ultracentrifugation analysis. The acidic polysaccharide has a high molecular weight (mean value 3 x lo6), gives an aqueous solution with high viscosity ([q] = 5.0 dl/g) and possesses a very large form factor (/ifo = 6.5).Treatment of the acidic polysaccharide with dilute alkali produced smaller molecules of a molecular weight of about 100,000. Alkali treatment did not alter the sugar composition of the acidic polysaccharide nor did it impair its serological properties.The acidic polysaccharide was oxidized by sodium periodate with concomitant destruction of the galactose moiety. Glucuronic acid, glucose and fucose in the polysaccharide resisted periodate oxidation. When the periodate oxidized and sodium borohydride reduced polysaccharide was hydrolyzed, 0.8 moles of glycerol per mole of the oxidized galactose was detected in the hydrolyzate. The methylated acidic polysaccharide was subjected to methanolysis and the methyl glycosides of the methylated sugar constituents were investigated with the aid of gas liquid chromatography. Methyl-tetra-0-methyl-D-galactoside was found to be present in the methanolyzate.Acid hydrolysis of the acidic polysaccharide gave rise to the following charged oligosaccharides : (i) glucuronosyl-(1 : 3)-fucose, (ii) glucosyl-( 1 : 3)-glucuronosyl-( 1 : 3)-fucose and (iii) galactosyl-( 1 : 3)-glucosyl-( 1 : 3)-glucuronosyl-( 1 : 3)-fucose. No neutral oligosaccharides were detected in hydrolyzates of the acidic polysaccharide.It is concluded that the acidic polysaccharide of E. coli 08:K27(A):H-consists of subunits of a molecular weight of about 100,000. These subunits are probably linked through ester bonds between carboxyl groups of glucuronic acid and hydroxyl groups of sugar constituents. It is suggested that the polysaccharide chains of the subunits consist of glucose, glucuronic acid and fucose in repeating order to which galactose is bound in I : 3-linkage, with glucose as the branch The acidic polysaccharide absorbes directly onto erythrocytes. In passive haemagglutination tests the polysaccharide-sensitized red cells reacted strongly with E. coli 08:K27 anti OK and anti K sera but very weakly with anti 0 serum. After absorption of anti OK serum with the purified acidic polysaccharide the antiserum no longer agglutinated the encapsulated strain. The acidic polysaccharide was precipitated by 08:K27 antiserum and by K27 antiserum. This precipitation was inhibited by the tetrasaccharide (iii) and to a lesser extent by the trisaccharide (ii), but...

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The Structure of the Aerobacter aerogenes A3(S1) Polysaccharide. I. A Reexamination Using Improved Procedures for Methylation Analysis^*

Cited by 458 publications

References 18 publications

Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides

Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides

Changes in Biochemical Composition of the Cell Wall of the Cotton Fiber During Development

Immunochemistry of K Antigens of Escherichia coli. 5. The K Antigen of E. coli 08:K 27 (A):H-

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The Structure of the Aerobacter aerogenes A3(S1) Polysaccharide. I. A Reexamination Using Improved Procedures for Methylation Analysis*

Cited by 458 publications

References 18 publications

Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides

Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides

Changes in Biochemical Composition of the Cell Wall of the Cotton Fiber During Development

Immunochemistry of K Antigens of Escherichia coli. 5. The K Antigen of E. coli 08:K 27 (A):H-

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The Structure of the Aerobacter aerogenes A3(S1) Polysaccharide. I. A Reexamination Using Improved Procedures for Methylation Analysis^*