Characterization of a specific alpha-mannosidase involved in oligosaccharide processing in Saccharomyces cerevisiae.
Fractionation of a crude extract from Saccharomyces cerevisiae X-2180 on Sepharose 6B in the presence of 0.5% Triton X-100 resolves two enzyme fractions containing alpha-mannosidase activity. Fraction I which is excluded from the gel contains alpha-mannosidase activity toward both p-nitrophenyl-alpha-D-mannopyranoside and Man9GlcNAc oligosaccharide as substrates, whereas Fraction II which is included in the gel contains only oligosaccharide alpha-mannosidase activity. The latter enzyme is very specific and removes a single mannose residue from Man9GlcNAc, whereas the alpha-mannosidase activity of Fraction I removes several mannose residues from Man9GlcNAc oligosaccharide. High resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the alpha-mannosidase of Fraction II showed only a single isomer with the following structure: (see formula; see text) This specific enzyme is most probably involved in processing of oligosaccharide during biosynthesis of mannoproteins. The mannose analog of 1-deoxynojirimycin (50-500 microM), dideoxy-1,5-imino-D-mannitol, inhibits the oligosaccharide alpha-mannosidase activities of Fractions I and II to about the same extent, but has no effect on the nonspecific alpha-mannosidase which acts on p-nitrophenyl-alpha-D-mannopyranoside.
D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008351:Mannans / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008362:Mannosides / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D043323:alpha-Mannosidase
D008351:Mannans; D008362:Mannosides; D009844:Oligosaccharides; C002731:4-nitrophenyl-alpha-D-mannopyranoside; D008361:Mannosidases; D043323:alpha-Mannosidase; D008358:Mannose