Nouvelles acquisitions structurales sur les substances pectiques de la pulpe de raisin

<p style="text-align: justify;">Après une mise au point rapide sur les connaissances actuelles des polyosides pectiquesdans les végétaux, les résultats acquis sur la structure des substances pectiques de la pulpe deraisin sont exposés, avec un rappel de la méthodologie utilisée.</p><p style="text-align: justify;">Un matériel insoluble à l'alcool (MIA) a été préparé à partir de pulpe de raisin. Quatre fractionspectiques en ont été isolées après traitements successifs par l'eau (25°C; PSE), l'oxalate(25°C; PSOX, l'acide (HCI 0.05M, 80°C; PSH) et la soude (O,05M, 4°C; PSOH).</p><p style="text-align: justify;">Les PSE (35 p.100) et PSH (55 p. 100) représentent l'essentiel du matériel pectique extrait.Les PSE sont séparées par chromatographie d'échange d'ions en fractions neutre (PSEn~ 13 p. 100) et acide (PSEa ~ 87 p. 100). Les PSEa et les PSH sont constituées principalementd'acide galacturonique (PSEa 63 p. 100, PSH 53 p. 100), fortement estérifié par du méthanol(degré d'estérification : PSEa 77 p. 100, PSH 68 p. 100), tandis que des quantités faibles d'acideglucuronique sont détectées dans les PSEn (2 p. 100). Les oses neutres (PSEn 65 p. 100, PSEa28 p. 100, PSH 19 p. 100) sont principalement de l'arabinose et du galactose suivis dans unordre décroissant du rhamnose, glucose, xylose, mannose et fucose. Des protéines sont égaIementdétectées en association avec les polyosides.</p><p style="text-align: justify;">L'action d'une endopolygalacturonase et d'une endopectine-Iyase sur PSEa et PSH met enévidence des zones «lisses» homogalacturoniques dégradées par les enzymes et des zones«hérissées» rhamnogalacturoniques riches en chaînes latérales d'oses neutres, insensibles àl'attaque enzymatique. Le traitement du MIA par une endopectine-Iyase solubilise un matérielpectique (ZH-MIA) riche en oses neutres (56 p. 100) particulièrement en arabinose, et contenantde l'acide galacturonique (23 p. 100) et des protéines (11 p. 100).</p><p style="text-align: justify;">La perméthylation associée à l'hydrolyse spécifique de l'arabinose par une α-L-arabinofuranosidaseet à la RMN du ¹³C a permis d'établir la structure des chaînes latérales. Des (1 → 3) / (1 → 6) arabinogalactanes, où l'arabinose est essentiellement sous forme terminale non réductrice, dominent dans les ZH-PSE, tout comme dans les PSEn confirmant leur caractère d'arabinogalactane-protéine. En revanche ces structures ainsi que les (1 → 4) arabinogalactanes sont minoritaires dans les ZH-PSH et ZH-MIA où les structures de type (1 → 5) arabinanes et rhamnogalacturonanes sont prédominants.</p><p style="text-align: justify;">+++</p><p style="text-align: justify;">After a brief review of available knowledges on plant pectic polysaccharides, structural data on pectic substances from the pulp or grape berries and related analytical techniques are reported.</p><p style="text-align: justify;">An alcohol insoluble residue (MIA) was prepared from pulp of grape berries, which was sequentially extracted with water (25°C), oxalate (25°C), acid (0,05N HCI, 80°C) and sodium hydroxide (0,05N 4°C) yielding four pectic fractions, respectively, PSE, PSOX, PSH and PSOH. PSE (35 p. 100) and PSH (55 p. 100) represented the main part of extracted pectic material.</p><p style="text-align: justify;">PSE were fractionated by ion-exchange chromatography into neutral (PSEn ~ 13 p. 100) and acidic (PSEa ~ 87 p. 100) fractions. PSEa and PSH were constituted mainly of galacturonic acid (PSEa 63 p. 100, PSH 53 p. 100) highly methylesterified (esterification degree : PSEa 77 p. 100; PSH 68 p. 100), whereas PSEn contained minute amounts of glucuronic acid (2 p. 100). Neutral sugars (PSEn 65 p. 100, PSEa 28 p. 100, PSH 19 p. 100) were mainly arabinose and galactose followed by decreasing amounts of rhamnose, xylose, glucose, mannose and fucose. Proteins were also detected along with the polysaccharides.</p><p style="text-align: justify;">Degradation of PSEa and PSH by endopolygalacturonase and endopectin-lyase evidenced «smooth» homogalacturonic areas sensitive to enzymatic degradation and «hairy» rhamnogalacturonic zones highly substituted by neutral sugar side-chains and resistant to enzyme action. Treatment of MIA With endopectinlyase released pectic material (ZH-MIA) rich in neutral sugars (56 p. 100), especially arabinose, and containing galacturonic acid (23 p. 100) and proteins (11 P. 100).</p><p style="text-align: justify;">Structure of neutral sugar side-chains was investigated using methylation analysis associated with specific hydrolysis of arabinose residues with an α-L-arabinofuranosidase, and ¹³C NMR spectroscopy. ZH-PSE exhibited a structure of 3,6 -linked arabinogalactan substitued by monomeric terminal arabinose. Similar structures were detected in PSEn which relates them to arabino-3,6-galactan-proteins. Conversely PSH or ZH-MIA showed mainly arabinan-like and rhamnogalacturonan structures associated with minor proportions of 3,6- and 4-linked arabino-galactans.</p>

Glycoproteins from the cell wall of Phaseolus coccineus

1. The use of a modified sodium chlorite/acetic acid delignification procedure for the solubilization of a hydroxyproline-rich glycoprotein fraction from the depectinated cell walls of Phaseolus coccineus is described. 2. The crude glycoprotein was associated with some pectic material; hydroxyproline and serine were the most abundant amino acids, and arabinose, galactose and galacturonic acid the predominant monosaccharides. 3. The bulk of the hydroxyproline is O-glycosidically substituted with tetra- and tri-arabinofuranosides. From methylation analysis the linkages in these arabinosides could be inferred. 4. Ion-exchange chromatography of the crude glycoprotein gave one major and two minor hydroxyproline-rich fractions, with similar amino acid but different monosaccharide composition. 5. In the major fraction, serine appears to be O-glycosidically substituted with a single galactopyranoside residue that can be removed by the action of alpha-galactosidase but not beta-galactosidase. Removal of arabinofuranoside residues by partial acid hydrolysis greatly enhanced the action of alpha-galactosidase. 6. Methylation followed by carboxy reduction with LiAl2H4 has shown the presence of (1 leads to 4)-linked galacturonic acid in the crude glycoprotein fraction but not in the major fraction from the ion-exchange column. Hence the bulk of the pectic material is not associated with the major glycoprotein component. It is suggested that the glycoprotein is held in the wall by phenolic cross-links. 7. Similarities with the glycopeptide moiety of potato lectin provides further evidence for a class of hydroxyproline-rich glycoproteins with common features.

A STUDY OF THE PECTIN PRESENT IN THE BARK OF AMABILIS FIR (ABIES AMABILIS)

A pectic material has been isolated from the bark of Abies amabilis (Dougl.) Forbes in a yield of 2%. On hydrolysis it yielded D-galacturonic acid, D-galactose, and L-arabinose in a ratio of 85:4:11, and also traces of rhamnose. The product, when submitted to several conventional fractionation methods, appeared homogeneous. Further resolution could be effected by acidification of an aqueous solution of the pectin, followed by ultracentrifugation. The insoluble portion (50%) was an electrophoretically homogeneous galacturonan with [α]D + 246°. The material remaining in solution (30%), here referred to as a pectic acid, had [α]D + 225° and on hydrolysis gave D-galacturonic acid, D-galactose, and L-arbabinose in a ratio of 74:7:19, as well as traces of rhamnose.The structure of the galacturonan was established by partial hydrolysis and methylation. It consisted of α-D-galacturonic acid residues linked together by (1 → 4)-glycosidic bonds to a linear macromolecule. The same techniques were applied to the pectic acid. While a unique structural formula could not be assigned in this case, one probable alternative involved a framework of (1 → 4)-linked α-D-galacturonic acid residues together with a few residues of 1,2,4-linked L-rhamnose. Some of the galacturonic acid units carried at C-2 and C-3 side chains which were terminated by D-galactopyranose and L-arabinofuranose residues. A few of the latter also occurred as inner units, probably in the side chains. This appears to be the first time a pectic material has been resolved into a galacturonan and a pectic acid containing the four sugar residues usually found in pectins. It is probable that the pectin occurring to a limited extent in wood has a similar composition.

STRUCTURE OF A HEMICELLULOSE FROM MAPLE WOOD PREVIOUSLY EXTRACTED BY LIQUID AMMONIA

The extraction of maple wood meal with anhydrous liquid ammonia under pressure near 20° altered the wood in such a way that an additional small amount of lignin could be extracted with ethanol, and 1.9% of crude hemicelluloses with hot water. An additional 0.25% of nitrogen was retained, apparently as insoluble amides, by the residual wood. After elimination of pectic material by acetylation, the three subtractions of the hemicellulose acetate had identical specific rotations of −61 ± 1° in chloroform; all three corresponded in composition to a combination of one methylglucuronic anhydride to six anhydroxylose units. A conventional study by the methylation method suggested that the hemicellulose was a branched-chain structure averaging four anhydroxylose units linked 1—4, with one also substituted in the second position, and another linked 1—3.