Purification and Characterization of -N-Acetylhexosaminidase from the Ascidian, Halocynthia roretzi

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Abstract
β-N-Acetylhexosaminidase [EC 3.2.1.30] was purified 820-fold from the viscera of Halocynthia roretzi by Sephadex G-200 gel filtration and chromatography on columns of DEAE-Sephadex and CM-Sephadex. The final preparation was sufficiently free from α-N-acetylglucosaminidase, α-N-acetylgalactosaminidase, α- and β-gluco-sidases, α- and β-galactosidases, α- and β-mannosidases, and α-L-fucosidase, and gave one protein band on disc gel electrophoresis. Two different molecular weight forms which depended upon the pH were observed on Sephadex gel filtration. At pH 7.0, a species with a molecular weight of 170,000 was observed, whereas at pH 4.5, an enzyme of 330,000 daltons was seen. The enzyme was active at pH 4.5 but inactive at pH7.0. The optimum pH and the Km were pH4.2 and 1.9mM for p-nitrophenyl β-N-acetylglucosaminide and pH 4.0 and 0.9 m for p-nitrophenyl -N-acetylgalactosaminide. The terminal β-N-acetylhexosamine of glycolipids such as globoside I, GM2 and asialo GM2 was cleaved by the ascidian β-N-acetylhex osaminidase though GM2 was less susceptible to the enzyme.