Lysosomal alpha-D-mannosidase of rat liver. Purification and comparison with the golgi and cytosolic alpha-D-mannosidases.
Rat liver contains alpha-D-mannosidases in lysosomes, Golgi membranes, and cytosol. The lysosomal enzyme has now been purified approximately 30,000-fold over the crude extract and is free of at least 13 other lysosomal hydrolases. The enzyme has an apparent molecular weight of 335,000 by molecular sieve chromatography and 200,000 by sucrose density centrifugation. It is a glycoprotein, as evidenced by its binding to a concanavalin A affinity column and by a positive periodic acid-Schiff stain. The enzyme has a pH optimum near 4.6. Although it is generally insensitive to a large variety of inorganic salts, chelating agents, and sulfhydryl reagents, prolonged exposure to ethylenediaminetetraacetic acid caused loss of activity, which could be restored by the addition of ZnSO4. Substrate specificity studies were performed on the purified lysosomal alpha-D-mannosidase, as well as on the purified Golgi and cytosolic alpha-D-mannosidases. The three enzymes exhibited only very limited activity on native glycoproteins, but were found to be active on glycopeptides and oligosaccharides, hydrolyzing 1 yields 2 and 1 yields 3 linkages, except that the Golgi enzyme had negligible activity towards the latter linkage. Immunological comparisons by antibody precipitation tests and double-diffusion plates indicated that the three enzymes are not immunologically related. The alpha-D-mannosidase isolated from rat epididymis was found to be immunologically very similar, if not identical, to the lysosomal enzyme isolated from rat liver.
D000818:Animals; D000937:Antigen-Antibody Reactions; D002413:Cations, Divalent; D003600:Cytosol / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D002413:Cations, Divalent; D008361:Mannosidases