Open AccessPurification and characterization of a neutral processing mannosidase from calf liver acting on (Man) 9 (GlcNAc) 2 oligosaccharides
Author(s) -
SCHWEDEN Jürgen,
LEGLER Günter,
BAUSE Ernst
Publication year - 1986
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1986.tb09703.x
Subject(s) - mannosidase , characterization (materials science) , chemistry , biochemistry , glycoprotein , nanotechnology , materials science
A processing mannosidase acting on (Man) 9 (GlcNAc) 2 oligosaccharides, Man 9 mannosidase, has been purified 2190‐fold from calf liver crude microsomes by a four‐step procedure involving (a) differential salt/detergent extraction, (b) affinity chromatography on AH‐Sepharose 4B with N ‐5‐carboxypentyl‐1‐deoxy‐mannojirimycin as ligand, (c) ConA‐Sepharose and (d) DEAE‐Sephacel chromatography. (Man) 9 mannosidase has a subunit molecular mass of 56 kDa and does not bind to ConA‐Sepharose, indicating the absence of high‐mannose oligosaccharides. The enzyme has a pH optimum close to pH 6.0 and requires divalent cations for activity, Ca 2+ being most effective. It is inhibited by 1‐deoxymannojirimycin (dMM), N ‐methyl‐dMM and N ‐5‐carboxypentyl‐dMM with K i = 7 μM, 75 μM, and 140 μM, respectively. Man 9 mannosidase cleaves three of the four α1,2‐linked mannose residues from the (Man) 9 (GlcNAc) 2 oligosaccharide, does not hydrolyse the remaining (Man) 6 (GlcNAc) 2 structure and is not active against aryl α‐mannosides. This pronounced substrate specificity points to the participation of Man 9 mannosidase in the N‐linked processing pathway and, in addition, clearly distinguishes this enzyme from the mannosidases reported previously. As Man 9 mannosidase appears to act in the processing sequence immediately after the three glucose residues have been removed from the (Glc) 3 (Man) 9 (GlcNAc) 2 intermediate, we assume that the enzyme is located in the endoplasmic reticulum.