SOLUBLE FORMS OF ALPHA-D-MANNOSIDASES FROM RAT-LIVER - SEPARATION AND CHARACTERIZATION OF 2 ENZYMATIC FORMS WITH DIFFERENT SUBSTRATE SPECIFICITIES

We have previously reported the substrate specificity of the rat liver cytosolic alpha-D-mannosidase [Haeuw, J. E, Strecker, G., Wieruszeski, J. M., Montreuil, J. and Michalski, J.-C. (1991) Eur. J. Biochem. 202, 1257-1268]. Here, we report the characterization and the purification of this alpha-D-mannosidase and the presence of two soluble forms of alpha-D-mannosidases from rat liver. The cytosolic alpha-D-mannosidase was purified nearly 660-fold with 2.66% recovery to a state approaching homogeneity using: (a) (NH4)(2)SO4 precipitation; (b) concanavalin-A-Sepharose chromatography; (c) affinity chromatography on a cobalt-chelating Sepharose column; (d) ion-exchange (DEAE-trisacryl M) column chromatography; (e) molecular-size chromatography (Sephacryl S 200). The enzyme was eluted from the final column at an apparent molecular mass of 113 kDa. SDS/PAGE analysis yielded a major protein band at 108 kDa. Moreover, the purification allowed to distinguish two mannosidase activities with different kinetic properties. The first cytosolic activity retained on the cobalt-chelating column was optimally active at neutral pH, was activated by Co2+, was strongly inhibited by swainsonine (K-i = 3.7 mu M) but not by deoxymannojirimycin and was active with p-nitrophenyl alpha-D-mannoside (K-m = 0.072 mM). Man(9)GlcNAc was hydrolysed by the purified enzyme down to a Man(5)GlcNAc structure, i.e. Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNA c, which represents the Man, oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The second activity not retained on the cobalt-chelating column was optimally active at neutral pH, was inhibited by swainsonine (K-i = 28.4 mu M) but not by deoxymannojirimycin and was active with p-nitrophenyl alpha-D-mannoside (K-m = 0.633 mM). Man(9)GlcNAc was broken by this enzymic activity down to Man(8)GlcNAc and Man(7)GlcNAc structures. Similitaries with endoplasmic reticulum alpha-D-mannosidase exist and this enzyme could be the cytosolic form of the endoplasmic reticulum alpha-D-mannosidase.