Biosynthesis of glycoproteins in Candida albicans: biochemical characterization of a soluble alpha-mannosidase.

Most alpha-mannosidase activity (80%) in C. albicans was found in a soluble form. Addition of protease inhibitors to explore proteolytic release from a particulate cell component during enzyme preparation did not change this distribution. Molecular mass, calculated from gel filtration chromatography, was 417 kDa. Optimum pH was 6.0 with 50 mM Mes-Tris when p-nitrophenyl-alpha-D-mannopyranoside was used as substrate. Optimum temperature was 42 degrees C with either 10 mM phosphate buffer (pH 6.8) or 50 mM Mes-Tris buffer (pH 6.0) and with 4-methylumbelliferyl-alpha-D-mannopyranoside as substrate. Apparent Km values for p-nitrophenyl-alpha-D-mannopyranoside and 4-methylumbelliferyl-alpha-D-mannopyranoside were 3.3 mM and 0.1 mM, respectively. 1 mM 1-deoxymannojirimycin and 0.3 mM swainsonine inhibited the hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside by 67% and 83%, respectively, whereas that of p-nitrophenyl-alpha-D-mannopyranoside was only slightly diminished (10-15%).