title: Partial purification and characterization of a novel endo-beta-mannosidase acting on N-linked sugar chains from Lilium longflorum thumb.
abstract: An enzyme catalyzing the hydrolysis of the Manbeta1-4GlcNAc linkage of N-linked sugar chains was partially purified and characterized. Endo-beta-mannosidase activity was detected using pyridylaminated (PA-) Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc as the substrate in a homogenate of lily flowers (Lilium longflorum Thumb). The enzyme was partially purified by ammonium sulfate precipitation, and Q-Sepharose, Superdex 200, hydroxyapatite, Poros PE/M, Mono Q, and Superdex 200 column chromatographies. The optimum pH was 5.0 and the estimated molecular weight of the enzyme was 78,000, as determined by gel filtration. The Km value found for Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA was 1.4 mM. The enzymatic activity was not influenced by the addition of 10 mM EDTA or 2 mM Ca2+. Experiments on the hydrolysis of several PA-N-linked sugar chains revealed that the enzyme hydrolyzed MannManalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) into a mixture of MannManalpha1-6Man and GlcNAcbeta1-4GlcNAc-PA, indicating that it is an endoglycosidase in nature. However, the enzyme did not hydrolyze beta1-4mannohexaose or p-nitrophenyl beta-mannopyranoside.
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Sasaki
authors.forename: M
authors.initials: M
authors.lastname: Yamagishi
authors.forename: T
authors.initials: T
authors.lastname: Mega
authors.forename: S
authors.initials: S
authors.lastname: Norioka
authors.forename: S
authors.initials: S
authors.lastname: Natsuka
authors.forename: S
authors.initials: S
authors.lastname: Hase
mesh_terms: D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000302:isolation & purification; D019668:Liliaceae / Q000201:enzymology*; D008969:Molecular Sequence Data; D013379:Substrate Specificity