Notes

Separation Oligosaccharides Saccharomyces Cerevisiae Digestion H Chromatography Elution Naoh Gradient Detector Pad
The elution times of homologous oligosaccharides fell on a straight line having a slope characteristic of the structural type. The response of the PAD detector per mole of oligosaccharide increased about 2-fold going from Man3GlcNAc to Man13GlcNAc, and appeared to depend primarily on the oxidation of the reducing-end N-acetylglucosamine unit common to all the oligosaccharides. The digestion of a ManGlcNAc with jack-bean alpha-mannosidase was monitored by injecting portions of the crude reaction mixture, and the intermediates were characterized by their elution positions and n.m.r. spectra in the anomeric proton region.

One commercial jack-bean alpha-mannosidase preparation contained a novel endolytic activity that released N-acetylglucosamine from the reducing ends of the oligosaccharides and was shown to convert P----6 alpha Man----6 alpha Man----6 beta Man----4 alpha beta GlcNAc to P----6 alpha Man----6 alpha Man----6 alpha beta Man plus free N-acetylglucosamine. Another commercial jack-bean alpha-mannosidase converted the ManGlcNAc to a Man3GlcNAc having the structure alpha Man----6 beta Man----4 alpha beta GlcNAc, [formula see text] whereas the Oerskovia sp. alpha-mannosidase converted the same oligosaccharide to a Man4GlcNAc having the structure alpha Man----6 alpha Man----6 beta Man----4 Analysis of glycoprotein-derived oligosaccharides by high-pH anion-exchange The technique of high-pH anion-exchange chromatography with pulsed amperometric detection has recently been shown to be a powerful method for resolving closely related oligosaccharides [M. R. Hardy and R. R. Townsend, Proc.

Natl. Acad. Sci. U.S.A., 85 (1988) 3289-3293].

This report describes separations involving a total of nineteen different high-mannose, hybrid and complex-type oligosaccharides isolated after peptide N-glycosidase F (PNGase F) or endo-beta-N-acetylglucosaminidase H digestion of glycoproteins. Separations were carried out at a constant base concentration M NaOH) using linear gradients from to M sodium acetate. The applicability of this chromatography for profiling the N-linked oligosaccharides of glycoproteins was demonstrated by generating oligosaccharide maps of PNGase F-liberated oligosaccharides from recombinant human tissue plasminogen activator, ribonuclease b, human transferrin, and bovine fetuin. Methods for recovering salt-free oligosaccharides after this chromatography were also investigated. On-line ion suppression with an anionic micromembrane suppressor cartridge was found to be capable of effective desalting up to a total sodium ion concentration of5 M at a flow-rate of 1 mlmin. After high-pH anion-exchange chromatography with ion suppression, collected oligosaccharides were analyzed by fast-atom bombardment mass spectrometry after conversion to permethyl derivatives or after reductive amination with rho-aminobenzoic acid ethyl ester.Endoglycosidase and glycoamidase release of N-linked oligosaccharides.

Carbohydrate chain modifications are often used to monitor glycoprotein movement through the secretory pathway. This is because stepwise sugar-chain processing is unidirectional and generally corresponds to the forward or anterograde movement of proteins. Seebio lacto-n-neotetraose offers a group of techniques that will help analyze the general structure of carbohydrate chains on a protein and, therefore, oligosaccharide processing mileposts. The sugar chains themselves are not analyzed, but their presence and structure are inferred from gel mobility differences after one or more enzymatic digestions. This approach is most often used in combination with [35S]Met pulse-chase metabolic labeling protocols, but they can be applied to any suitably labeled protein (e.g., biotinylated or Sugar residues content and distribution in atrophic and hyperplastic postmenopausal human endometrium lectin histochemistry.

A lectin histochemical study was performed to investigate the glycoconjugate saccharidic moieties of the human postmenopausal endometrium (14 atrophic and 15 hyperplastic). For this purpose a battery of seven horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, WGA, ConA, LTA and UEA I) was used. No differences in lectin binding between atrophic and hyperplastic endometria were observed.
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