Purification and characterization of the alpha-D-mannosidase of rat liver cytosol.
Three forms of alpha-D-mannosidase have previously been identified in rat liver, and each is localized in a different subcellular fraction: lysosomes, Golgi membranes, and cytosol. This communication reports the purification and characterization the cytosolic form. The enzyme was purified 12,000-fold in good yield to approximately 90% purity with the aid of the competitive inhibitor mannosylamine and dithioerythritol as stabilizers. The molecular weight of the enzyme is in the range of 372,000 to 490,000 depending on the method used. Since the subunit molecular weight is 110,000 by sodium dodecyl sulfate polyacrylamide electrophoresis, the enzyme is probably a tetramer. The pH optimum was shown to be between 5.5 and 5.9 (in the presence of 1 mM CoCl2) with the substrate p-nitrophenyl-alpha-D-mannoside. Normal Michaelis-Menten kinetics were observed with a Km of 0.14 mM. Mannosylamine was a competitive inhibitor with a Ki of 0.007 mM. The purified enzyme, stabilized by Co2+, Mn2+, and Fe2+ under some conditions, was unstable at low protein concentrations. Since an electrophoresed sample showed a positive periodic acid-Schiff stain, the enzyme may contain carbohydrate. The availability of purified cytosolic alpha-D-mannosidase should now make it possible to carry out substrate specificity, immunological, and structural studies which may shed light on the biological role of this enzyme.
D000818:Animals; D002413:Cations, Divalent; D003600:Cytosol / Q000201:enzymology; D004186:Disaccharidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008361:Mannosidases / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats
D002413:Cations, Divalent; D046911:Macromolecular Substances; D004186:Disaccharidases; D008361:Mannosidases