PURIFICATION AND CHARACTERIZATION OF HEN OVIDUCT ALPHA-1,2-MANNOSIDASE

An alpha-mannosidase capable of hydrolyzing three Man alpha 1,2-residu es from pyridylamine(PA-) labeled Man(9)GlcNAc(2) was purified from he n oviduct. The purity of the preparation was analyzed by PAGE; its mol ecular 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 activ ity was restored by the addition of Ca2+, The enzyme acitivity was inh ibited by 1-deoxymannojirimycin, but not by swainsonine. The substrate specificity of the purified enzyme was analyzed using PA-oligomannose -type sugar chains, When Man(9)GlcNAc(2)-PA was digested, Man alpha 1- 6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4G lcNAc beta 1-4GlcNAc-PA was obtained as an end product, and the enzyme was incapable of hydrolyzing p-nitrophenyl alpha-D-mannoside and Man alpha 1,3- or Man alpha 1,6-residues. Judging from these characteristi cs, the enzyme was classified as a Man(9)-mannosidase or Golgi mannosi dase I and speculated to participate in the processing or catabolism o f glycoproteins.