Purification and characterization of endo-beta-N-acetylglucosaminidase from hen oviduct.
Endo-beta-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 [Tarentino, 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 microM when pyridylaminated Man6GlcNAc2 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 Manalpha1-2Manalpha1-3Manbeta1-4GlcNAcbeta1-GlcN Ac 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 alpha-mannosidase, an enzyme that specifically hydrolyzes oligosaccharides to Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)++ +Manbeta1-4GlcNAc.
Kato
T
T
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka.
Hatanaka
K
K
Mega
T
T
Hase
S
S
D000818:Animals; D002240:Carbohydrate Sequence; D002645:Chickens; D005260:Female; D006868:Hydrolysis; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010057:Oviducts / Q000201:enzymology*; D013379:Substrate Specificity
D007527:Isoenzymes; D009844:Oligosaccharides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase