Purification and Characterization of Hen Oviduct l,2-Mannosidase

Abstract

An a-mannosidase capable of hydrolyzing three Man αl,2-residues from pyridylamine-(PA-) labeled Man⁹GlcNAc² was purified from hen oviduct. The purity of the preparation was analyzed by PAGE; its molecular weight was 42,000 by SDS-PAGE or 50,000 by gel filtration. The pH optimum was 6.5. The enzyme was inactivated with EDTA; enzyme activity was restored by the addition of Ca²⁺. The enzyme acitivity was inhibited by 1-deoxymannojirimycin, but not by swainsonine. The substrate specificity of the purified enzyme was analyzed using PA-oligomannose-type sugar chains. When Man⁹GlcNAc²-PA was digested, Manαl–6(Manαl–2Manαl–3)Manαl–6(Manαl–3)Manβl–4GlcNAc;β1–4Glc-NAc-PA was obtained as an end product, and the enzyme was incapable of hydrolyzing p-nitrophenyl α-D-mannoside and Man αl,3- or Man α,6-residues. Judging from these characteristics, the enzyme was classified as a Man²-mannosidase or Golgi mannosidase I and speculated to participate in the processing or catabolism of glycoproteins.