Characterization of a novel alpha-D-mannosidase from rat brain microsomes.
A new alpha-D-mannosidase has been identified in rat brain microsomes. The enzyme was purified 70-100-fold over the microsomal fraction by solubilization with Triton X-100, followed by ion exchange, concanavalin A-Sepharose, and hydroxylapatite chromatography. The purified enzyme is very active towards mannose-containing oligosaccharides and has a pH optimum of 6.0. Unlike rat liver endoplasmic reticulum alpha-D-mannosidase and both Golgi mannosidases IA and IB, which have substantial activity only towards alpha 1,2-linked mannosyl residues, the brain enzyme readily cleaves alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannosyl residues present in high mannose oligosaccharides. The brain enzyme is also different from liver Golgi mannosidase II in that it hydrolyzes (Man)5GlcNAc and (Man)4GlcNAc without their prior N-acetylglucosaminylation. Moreover, the facts that the ability of the enzyme to cleave GlcNAc(Man)5GlcNAc, the biological substrate for Golgi mannosidase II, is not inhibited by swainsonine, and that p-nitrophenyl alpha-D-mannoside is a poor substrate provide further evidence for major differences between the brain enzyme and mannosidase II. Inactivation studies and the co-purification of activities towards various substrates suggest that a single enzyme is responsible for all the activities found. In view of these results, it seems possible that, in rat brain, a single mannosidase cleaves asparagine-linked high mannose oligosaccharide to form the core Man3GlcNAc2 moiety, which would then be modified by various glycosyl transferases to form complex type glycoproteins.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002845:Chromatography; D004355:Drug Stability; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007700:Kinetics; D008297:Male; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D009844:Oligosaccharides; D011134:Polysaccharides; D008361:Mannosidases; D043323:alpha-Mannosidase; D008358:Mannose