Purification and characterization of a novel broad‐specificity (α1 → 2, α1 → 3 and α1 → 6) mannosidase from rat liver

Abstract

We have identified a mannosidase in rat liver that releases α1 → 2, α1 → 3 and α1 → 6 linked manose residues from oligosaccharide substrates, MannGlcNAc where n= 4–9. The end product of the reaction is Manα1 → 3[Manα1 → 6]Manβ1 → 4GlcNAc. The mannosidase has been purified to homogeneity from a rat liver microsomal fraction, after solubilization into the aqueous phase of Triton X‐114, by anion‐exchange, hydrophobic and hydroxyapatite chromatography followed by chromatofocusing. The purified enzyme is a dimer of a 110‐kDa subunit, has a pH optimum between 6.1 and 6.5 and a Km of 65 μM and 110 μM for the Man5GlcNAc‐oligosaccharide or Man9GlcNAc‐oligosaccharide substrates, respectively. Enzyme activity is inhibited by EDTA, by Zn2+ and Cu2+, and to lesser extent by Fe2+ and is stabilized by Co2+. The pattern of release of mannose residues from a Man6GlcNAc substrate shows an ordered hydrolysis of the α1 → 2 linked residue followed by hydrolysis of α1 → 3 and α1 → 6 linked residues. The purified enzyme shows no activity against p‐nitrophenyl‐α‐mannoside nor the hybrid GlcNAc Man5GlcNAc oligosaccharide. The enzyme activity is inhibited by swainsonine and 1‐deoxymannojirimycin at concentrations 50–500‐fold higher than required for complete inhibition of Golgimannosidase II and mannosidase I, respectively. The data indicate strongly that the enzyme has novel activity and is distinct from previously described mannosidases. Copyright © 1991, Wiley Blackwell. All rights reserved