title: Purification and characterization of an alpha-1,2-mannosidase involved in processing asparagine-linked oligosaccharides.
abstract: A calcium-dependent alpha-1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides was purified to homogeneity from rabbit liver microsomes. N-terminal amino acid analysis was consistent with the presence of a homogeneous protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and nonreducing conditions, revealed a single protein band with an apparent molecular weight of 52,000. Gel filtration and sedimentation analysis under nondenaturing conditions suggested that the purified enzyme is a monomeric protein. The mannosidase is a glycoprotein based on the presence of protein-linked sugar and specific binding of the enzyme to concanavalin A-Sepharose. Purified mannosidase was optimally active between pH 5.0 and 6.0. The enzyme was inactive with p-nitrophenyl-alpha-D-mannopyranoside and was inhibited by deoxymannojirimycin but not by swainsonine. The enzyme was specifically activated by Ca2+, with half-maximal activation occurring at concentrations of 10 microM or less and was inhibited by Mn2+, Co2+, Ba2+, and Zn2+. Calcium ions protected the enzyme against inactivation by p-chloromercuribenzoate. Rabbit liver mannosidase hydrolyzed alpha-1,2-mannosyl-mannose linkages in a variety of substrates including methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (Schutzbach, J. S. (1987) Anal. Biochem. 167, 279-283), ovalbumin glycopeptide IV, and the high mannose chains of thyroglobulin and phytohemagglutinin-P. Approximately 70% of the alpha-1,2-linked mannosyl units in the oligosaccharides of thyroglobulin were accessible to rabbit liver alpha-mannosidase, whereas most of the alpha-1,2-mannosyl units in phytohemagglutinin were resistant to digestion prior to heat denaturation of the plant lectin.
authors.affiliation: Department of Microbiology, University of Alabama, Birmingham 35294.
authors.forename: W T
authors.initials: WT
authors.lastname: Forsee
authors.forename: C F
authors.initials: CF
authors.lastname: Palmer
authors.forename: J S
authors.initials: JS
authors.lastname: Schutzbach
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001216:Asparagine; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002845:Chromatography; D006023:Glycoproteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D010835:Phytohemagglutinins / Q000378:metabolism; D011817:Rabbits; D013954:Thyroglobulin / Q000378:metabolism; D043323:alpha-Mannosidase; D020245:p-Chloromercuribenzoic Acid
chemical_list: D000596:Amino Acids; D002729:Chloromercuribenzoates; D006023:Glycoproteins; D010835:Phytohemagglutinins; D020245:p-Chloromercuribenzoic Acid; D001216:Asparagine; D013954:Thyroglobulin; D008361:Mannosidases; D043323:alpha-Mannosidase