title: Purification and enzymatic properties of endo-alpha 1, 2-mannosidase from pig liver involved in oligosaccharide processing.
abstract: An endo-alpha 1,2-mannosidase, which is involved in N-linked oligosaccharide processing, has been purified to homogeneity from crude pig liver microsomes using conventional techniques. Two catalytically active polypeptides, of 48 kDa, have been isolated which degrade [14C]Glc3-1-Man9,-GlcNAc2 to [14C]Glc3-1-Man and a specific Man8-GlcNAc2 isomer. They are not, however, active on synthetic alpha-mannosides. [14C]Glc1-Man9-GlcNAc2 was found to be approximately sevenfold more rapidly hydrolyzed than the [14C]Glc2- and [14C]Glc3-homologues. The 48 kDa and 50 kDa proteins are not N-glycosylated and ran on Superdex 75 as monomers. Kinetic studies showed that these proteins had similar catalytic properties: (i) the pH optima were found to be close to 6.5; (ii) neither activity was metal ion dependent; (iii) hydrolysis of [14C]Glc3-Man9-GlcNAc2 was inhibited strongly by Glc-alpha 1,3-Man (app. Ki approximately 120 microM), but not by 1-deoxymannojirimycin or swainsonine. Other evidence, including immunological data, strongly suggests that the 48 kDa and 50 kDa polypeptides are proteolytic degradation products of a single endo-alpha 1,2-mannosidase, rather than distinct subunits of an oligomeric complex. Possible functions of the endo-alpha 1,2-mannosidase in N-linked oligosaccharide processing are discussed.
authors.affiliation: Institut für Physiologische Chemie, Rheinische Friedrich-Wilhelms -Universität, Bonn, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Bause
authors.forename: M
authors.initials: M
authors.lastname: Burbach
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism*; D013552:Swine
chemical_list: D009844:Oligosaccharides; D008361:Mannosidases; C051635:endo-alpha-D-mannosidase