Soluble forms of alpha-D-mannosidases from rat liver. Separation and characterization of two enzymic forms with different substrate specificities.
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) (NH4)2SO4 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 (Ki = 3.7 microM) but not by deoxymannojirimycin and was active with p-nitrophenyl alpha-D-mannoside (Km = 0.072 mM). Man9GlcNAc was hydrolysed by the purified enzyme down to a Man5GlcNAc 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 Man5 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 (Ki = 28.4 microM) but not by deoxymannojirimycin and was active with p-nitrophenyl alpha-D-mannoside (Km = 0.633 mM). Man9GlcNAc was broken by this enzymic activity down to Man8GlcNAc and Man7GlcNAc structures. Similitaries with endoplasmic reticulum alpha-D-mannosidase exist and this enzyme could be the cytosolic form of the endoplasmic reticulum alpha-D-mannosidase.
Laboratoire de Chimie Biologique, (Unité mixte de Recherche du Centre National de la Recherche Scientifique no. 111), Université des Sciences et Technologies de Lille, France.
T
T
Grard
A
A
Saint-Pol
J F
JF
Haeuw
C
C
Alonso
J M
JM
Wieruszeski
G
G
Strecker
J C
JC
Michalski
D000818:Animals; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D012995:Solubility; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D008361:Mannosidases; D043323:alpha-Mannosidase