Characterization and partial purification of three glycosidases from castor bean endosperm

Abstract

α-Mannosidase and β- N-acetylhexosaminidase, which could function in the cleavage of glycosidic linkages in the native Ricinus communis lectins, and β-galactosidase were purified some 100-fold from the endosperm tissue of castor bean seedlings. The procedure used ammonium sulphate precipitation followed by chromatography on CM-cellulose, hydroxyapatite and Sephacryl S-300 to separate the three activities. All three glycosidases were present, with the lectins, in the protein bodies of dry seed and increased in activity during the time that lectins are broken down in the vacuoles. The enzymes show optimal activity in the range pH 3–5.5. The α-mannosidase had a K m of 0.77 mM for p- nitrophenyl-α- D-mannopyranoside. The β-galactosidase showed a K m of 1.39 mM for p-nitrophenyl-β- D-galactopyranoside. The β- N-acetylhexasominidase had a K m of 0.47 mM for p-nitrophenyl- N-acetyl-β- N-glucosamide and a K m of 0.33 mM for p-nitrophenyl- N-acetyl-β- D-galactosamide. Effects of competitive inhibitors and cations were described.