polygalacturonic-acid and arabinogalactan

Purification, characterization and biological activities of polysaccharides from Lycium barbarum pedicel were investigated in this study. Two polysaccharides, PLBP-I-I and PLBP-II-I, were obtained from water extracts by anion exchange chromatography and gel filtration. Structural elucidation based on IR,

Topics: Antioxidants; Galactans; Hexuronic Acids; Immunologic Factors; Lycium; Magnetic Resonance Spectroscopy; Pectins; Plant Extracts; Polysaccharides

Two pectic polysaccharides, 50 WCP-II-I and 100 WCP-II-I, were obtained from 50 and 100 °C water extracts of Codonopsis pilosula roots by ion exchange chromatography and gel filtration. The study of the sub-fractions obtained after pectinase degradation showed that the complement fixation activities of these pectins are expressed mainly by their ramified regions. The structure studies of native and sub-fractions showed the 50 WCP-II-I is a pectic polysaccharide, with long homogalacturonan regions (some of the galacturonic acid units were methyl esterified), interrupted by one short rhamnogalacturonan I (RG-I) region. The side chains of the RG-I region are arabinogalactan type I (AG-I) and type II (AG-II) attached on position 4 of rhamnose. The 100 WCP-II-I has two main ramified regions, one is galacturonan region with AG-I side chain on position 2 of GalA, and the other one is RG-I region with AG-II side chain on position 4 of Rha.

Topics: Chromatography, Gel; Chromatography, Ion Exchange; Codonopsis; Galactans; Hexuronic Acids; Pectins; Plant Roots; Polysaccharides; Water

To enable structural characteristics of individual cell wall polysaccharides from rapeseed (Brassica napus) meal (RSM) to be studied, polysaccharide fractions were sequentially extracted. Fractions were analysed for their carbohydrate (linkage) composition and polysaccharide structures were also studied by enzymatic fingerprinting. The RSM fractions analysed contained pectic polysaccharides: homogalacturonan in which 60% of the galacturonic acid residues are methyl-esterified, arabinan branched at the O-2 position and arabinogalactan mainly type II. This differs from characteristics previously reported for Brassica campestris meal, another rapeseed cultivar. Also, in the alkali extracts hemicelluloses were analysed as xyloglucan both of the XXGG- and XXXG-type decorated with galactosyl, fucosyl and arabinosyl residues, and as xylan with O-methyl-uronic acid attached. The final residue after extraction still contained xyloglucan and remaining (pectic) polysaccharides next to cellulose, showing that the cell wall matrix of RSM is very strongly interconnected.

Topics: Brassica napus; Carbohydrate Sequence; Cell Wall; Chromatography, Ion Exchange; Enzyme Assays; Galactans; Glucans; Hexuronic Acids; Molecular Sequence Data; Pectins; Polysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Xylans

Arabinogalactan glycoproteins (AGPs) are implicated in virtually all aspects of plant growth and development, yet their precise role remains unknown. Classical AGPs cover the plasma membrane and are highly glycosylated by numerous acidic arabinogalactan polysaccharides O-linked to hydroxyproline. Their heterogeneity and complexity hindered a structural approach until the recent determination of a highly conserved repetitive consensus structure for a 15-sugar residue arabinogalactan subunit with paired glucuronic carboxyls. Based on NMR data and molecular dynamics simulations, we identify these carboxyls as potential intramolecular Ca(2+)-binding sites. Using rapid ultrafiltration assays and mass spectrometry, we verified that AGPs bind Ca(2+) tightly (K(d) ~ 6.5 μM) and stoichiometrically at pH 5. Ca(2+) binding is reversible in a pH-dependent manner. As typical AGPs contain c. 30 Ca(2+)-binding subunits and are bulk components of the periplasm, they represent a Ca(2+) capacitor discharged at low pH by stretch-activated plasma membrane H(+)-ATPases, hence a substantial source of cytosolic Ca(2+). We propose that these Ca(2+) waves prime the 'calcium oscillator', a signal generator essential to the global Ca(2+) signalling pathway of green plants.

Topics: Binding Sites; Calcium; Calcium Signaling; Cell Membrane; Cytosol; Galactans; Glucuronic Acid; Gum Arabic; Hexuronic Acids; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation; Nicotiana; Periplasm; Plant Cells; Plant Development; Plant Proteins; Protein Binding; Proton-Translocating ATPases; Solanum lycopersicum; Ultrafiltration

Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-alpha-l-arabinan and (1,4)-beta-d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.

Topics: Antibodies, Monoclonal; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Enzyme-Linked Immunosorbent Assay; Galactans; Hexuronic Acids; Pectins; Rhamnose

Polysaccharides (pectin and intracellular and extracellular arabinogalactans) were isolated from campion callus culture cultivated on medium with varied concentrations of pectinase and beta-galactosidase. A decrease in contents of arabinose residues in pectin and arabinogalactans and of galactose residues in arabinogalactans was associated with an increase in the activities of alpha-L-arabinofuranosidase and beta-galactosidase upon addition of pectinase into the medium. Pectinase destroyed the high-molecular-weight (more than 300 kD) fraction of pectin and decreased the content of galacturonic acid residues. alpha-L-Arabinofuranosidase transformed arabinogalactan into galactan, and galactan was destroyed under the influence of galactosidase. The contents of arabinogalactan and/or galactan in the cells were decreased, and it was released into the culture medium. Pectin samples with low contents of arabinose and galactose in the side chains and galactan samples were obtained from the callus grown on the medium with beta-galactosidase. Cultivation of the plant cells on medium containing carbohydrases resulted in modification of pectin and arabinogalactan of the cell walls.

Topics: Arabinose; beta-Galactosidase; Galactans; Glycoside Hydrolases; Hexuronic Acids; Pectins; Polygalacturonase; Polysaccharides; Silene

The Aspergillus nigerbeta-1,4-endogalactanase encoding gene (galA) was cloned and characterized. The expression of galA in A. niger was only detected in the presence of sugar beet pectin, d-galacturonic acid and l-arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well as the arabinanolytic genes. The corresponding enzyme, endogalactanase A (GALA), contains both active site residues identified previously for the Pseudomonas fluorescensbeta-1,4-endogalactanase. The galA gene was overexpressed to facilitate purification of GALA. The enzyme has a molecular mass of 48.5 kDa and a pH optimum between 4 and 4.5. Incubations of arabinogalactans of potato, onion and soy with GALA resulted initially in the release of d-galactotriose and d-galactotetraose, whereas prolonged incubation resulted in d-galactose and d-galactobiose, predominantly. MALDI-TOF analysis revealed the release of l-arabinose substituted d-galacto-oligosaccharides from soy arabinogalactan. This is the first report of the ability of a beta-1,4-endogalactanase to release substituted d-galacto-oligosaccharides. GALA was not active towards d-galacto-oligosaccharides that were substituted with d-glucose at the reducing end.

Topics: Amino Acid Sequence; Arabinose; Aspergillus niger; beta-Galactosidase; Carbohydrate Sequence; Cloning, Molecular; Enzyme Activation; Fungal Proteins; Galactans; Gene Expression Regulation, Developmental; Glycoside Hydrolases; Hexuronic Acids; Hydrolysis; Molecular Sequence Data; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization