A spectrophotometric assay for alpha-mannosidase activity.
A simple and versatile spectrophotometric assay for alpha-mannosidase activity, which can be used with unlabelled natural substrate or synthetic substrates, was developed. The reducing mannose released from the substrate by the enzyme is quantitated using glucose oxidase, peroxidase and o-dianisidine. Using recombinant alpha 1,2-mannosidase obtained from Saccharomyces cerevisiae and Man9, GlcNAc, the spectrophotometric assay yielded values of 0.3 mM for Km and 15 mU/microgram for V(max), which are comparable to those obtained using the traditional radiochemical assay. The assay was also used to evaluate some alternative oligosaccharides as substrates for the enzyme. Man5-O(CH2)8-COOCH3 shows potential as an alternative synthetic substrate as the enzyme retained its specificity for a single alpha 1,2-mannose residue. Kinetic results suggest that the lower 1,3 linked arm of Man9GlcNAc is more critically involved in substrate recognition than the upper 1,6 linked arm.
Scaman
C H
CH
Department of Chemistry, University of Alberta, Edmonton, Canada.
Lipari
F
F
Herscovics
A
A
D002240:Carbohydrate Sequence; D005069:Evaluation Studies as Topic; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008351:Mannans / Q000737:chemistry; D008361:Mannosidases / Q000032:analysis* / Q000378:metabolism; D008362:Mannosides / Q000737:chemistry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011854:Radiochemistry; D011994:Recombinant Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013053:Spectrophotometry / Q000379:methods*; D013379:Substrate Specificity
D008351:Mannans; D008362:Mannosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; C057328:mannosyl(9)-N-acetylglucosamine; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase