Soluble forms of α-D-mannosidases from rat liver.

We have previously reported the substrate specificity of the rat liver cytosolic alpha-D-mannosidase [Haeuw, J. F., Strecker, G., Wieruszeski, J. M., Montreuil, J. & 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) (NH[sub4])[sub2]SO[sub4] 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 Co[sup2+], was strongly inhibited by swainsonine (K[subi] = 3.7 μM) but not by deoxymannojirimycin and was active with p-nitrophenyl alpha-D-mannoside (K[subm] = 0.072 mM). Man[sub9]GlcNAc was hydrolyzed by the purified enzyme down to a Man[sub5]GlcNAc structure, i.e. Man(α 1-2)Man(α 1-2)Man(α 1-3)[Man(alpha 1-6)]Man(β 1-4) GlcNA c, which represents the Man[sub5] oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. [ABSTRACT FROM AUTHOR]