Purification and Characterization of Endo- -N-Acetylglucosaminidase from Hen Oviduct

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Abstract
Endo-β-N-acetylglucosaminidase from hen oviduct (Endo-HO) was purified to homogeneity by ammonium sulfate fractionation and then by column chromatographies on DEAE-Sephacel, hydroxyapatite, Octyl-Sepharose CL-4B, Co2+-chelating Sepharose FF, and YMC-Pack Diol-200G. Partial purification of the enzyme was reported previously [Tar-entino, A.L. and Maley, F. (1976) J. BioL Chem. 251,6537–6543]. The molecular weight was 54,000 by gel filtration and 52,000 by SDS-PAGE in the presence of 2-mercaptoethanol, indicating that Endo-HO is composed of a single polypeptide chain. The optimum pH was 6.5, and the Km value was 25 μM when pyridylaminated Man8GlcNAc2 was used as a substrate. EDTA and metal cations tested, except Hg2+, had no effects on Endo-HO activity. Substrate specificity results using pyridylaminated N-linked sugar chains revealed that Endo-HO hydrolyzed oligomannose-type sugar chains faster than complex- and hybrid-type chains, and that sugar chains containing the Manα1-2Manα1-3Manα1-4GlcNAcα1-GlcNAc structure were good substrates for the enzyme. These findings suggest that in cytosol the enzyme contributes to the production of a free oligosaccharide with one reducing end N-acetylglucosamine residue in cooperation with neutral a-mannosidase, an enzyme that specifically hydrolyzes oligosaccharides to Manα1-2Manα1-2Manα1-3(Manα1-6)Man-α1-4GlcNAc.

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