Characterization-Carbohydrate-Sequence-Scientifique-N-d
Buy now , Université des Sciences et Technologies de Lille, Several O-linked oligosaccharides of the jelly coat surrounding the eggs of Axolotl maculatum were analysed by 1H-NMR spectroscopy. The four major oligosaccharidealditols released by reductive beta-elimination display either the Lewisx (Lex) determinant or the sequence GalNAc(beta 1-4)[Fuc(alpha 1-3)]GlcNAc. This last structure has previously been characterized in allergenically active oligosaccharides isolated from the sea squirt H-antigen, and in the N-linked glycans of Schistosoma mansoni and human urokinase. It represents the major carbohydrate chain found in A. maculatum, the oviduct of which constitutes an excellent source of beta 1-4-acetylgalactosaminyl transferase activity. Moreover, the carbohydrate chains isolated from A.
maculatum are quite different from those found in seven other amphibian species, in which the presence of species-specific material has been characterized. The role of carbohydrates appears more and more apparent during the fertilization process, and the diversity of the O-linked oligosaccharides supports such a Structures of oligosaccharide-bound forms of the endoglucanase V from Humicola Cellulose, a polymer of beta-1,4-linked glucose residues, is the major polysaccharide component of plant cell walls and the most abundant biopolymer. The underlying mechanisms of the enzymatic degradation of cellulose are of increasing commercial and ecological significance. Endoglucanase V, from the cellulolytic soil hyphomycete Humicola insolens, is an endocellulase, the catalytic core of which consists of 2 amino acids and is known to hydrolyze the beta-1,4 links with inversion of configuration at the anomeric carbon. The major products of cellulose hydrolysis are cellobiose and cellotriose. The crystal structures of the endoglucanase V (EGV) from H. insolens, in native, product (cellobiose), inactive mutant (DN), and oligosaccharide-bound [(DN)-cellohexaose] forms, have been determined at resolutions of 1 A or better.
EGV consists of a six-stranded beta-barrel domain with long interconnecting loops. A A groove exists along the surface of the enzyme, and this contains the catalytic residues, Asp and Asp 121. The two catalytic aspartates sit to either side of the substrate binding groove in an ideal conformation for facilitating cleavage by inversion, their carboxyl groups being separated by approximately 8 A. Seebio 2'-Fucose lactose between substrate and inactive mutant reveals excellent density for an oligosaccharide in six of the enzyme's seven substrate binding subsites. No sugar moiety, however, is seen bound to the -1 subsite at the point of cleavage. The geometry of the cleavage site suggests that the enzyme would favor the binding of sugars with an elongated glycosidic bond, as found in the transition state, as opposed to the binding of substrate. The oligosaccharide complexes reveal solvent water suitably placed for participation in a single displacement reaction as first suggested by Koshland in 1953 [Koshland, D.
E. (1953) Biol. Rev. 28, 416-436]. A large conformational change takes place upon substrate binding. This lid flipping has the effect of increasing the hydrophobic environment of the catalytic proton donor, enclosing the active site at the point of cleavage, and bringing a third aspartate (Asp 114) in close proximity to the substrate. Site-directed mutagenesis of the catalytic residues has been used to confirm their significance in catalysis.
Fast sequencing of oligosaccharides the reagent-array analysis method.A method of oligosaccharide analysis involving controlled fragmentation resulting from enzymatic digestion is presented. The principle involves generating a set of fragments from the original oligosaccharides, characterizing them in terms of their hydrodynamic volumes, determining their molar proportions, and identifying the oligosaccharides by comparison with a computer-generated data base. Experimentally, this technique involves incubation of aliquots of a sample with a set of defined mixtures of exoglycosidases followed by pooling of the products and a single analysis on the product pool. This method has several practical advantages over current techniques, including speed and the ability to use smaller amounts of starting material. The detection of the intensity-versus-hydrodynamic volume profile is limited only by the specific activity of the labeling method. The ability to perform the enzyme digestions is limited by the individual Km values of the enzymes.
Comparative composition, diversity, and abundance of oligosaccharides in early lactation milk from commercial dairy and beef cows.Research, Washington State University, Pullman 99164.
