PARTIAL-PURIFICATION AND CHARACTERIZATION OF AN ENDO-ALPHA- N-ACETYLGALACTOSAMINIDASE FROM THE CULTURE-MEDIUM OF STREPTOMYCES SP OH-11242
PARTIAL-PURIFICATION AND CHARACTERIZATION OF AN ENDO-ALPHA- N-ACETYLGALACTOSAMINIDASE FROM THE CULTURE-MEDIUM OF STREPTOMYCES SP OH-11242
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DOI：
10.1042/bj2880475
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发表时间：
1992-12-01
影响因子：
4.1
通讯作者：
OMURA, S
中科院分区：
文献类型：
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作者：
ISHIIKARAKASA, I;IWASE, H;OMURA, S
用于从链霉菌属培养基中纯化新型内切-α-N-乙酰半乳糖胺酶。 OH-11242（内切-GalNAc-ase-S）[Iwase、Ishii、Ishihara、Tanaka、Omura 和 Hotta (1988) Biochem。生物物理学。资源。交流。 151, 422-428]，建立了测定酶活性的方法。以纯化的猪胃粘液糖蛋白（PGM）为底物，对PGM中游离的寡糖进行吡啶胺化，并采用高压液相色谱法对大于Galbeta1-3GalNAc的寡糖的还原末端糖进行分析。将内切-GalNAc-ase-S 的粗酶从浓缩培养基中制备为 80% (w/v) 硫酸铵沉淀物。该酶通过凝胶色谱聚焦和随后的 DEAE-Toyopearl 色谱进行部分纯化。内酶活性在凝胶色谱聚焦柱上约 pl 4.8 洗脱，并在 DEAE-Toyopearl 柱上用 0.19-0.25 m-NaCl 洗脱。在获得的酶级分中，没有检测到外切糖苷酶或蛋白酶。通过凝胶过滤测得该酶的分子量为105 kDa，最适pH为5.5。 Endo-GalNAc-ase-S 水解 H-3 标记和未标记的脱唾液酸胎球蛋白中 GalNAc 和 Ser (Thr) 之间的 O-糖苷键，释放二糖 (Galbeta1-3GalNAc) 和四糖 [Galbeta1-3(Galbeta1-4GlcNAcbeta1-6)GalNAc]。当来自产碱菌属的内切-α-N-乙酰半乳糖胺酶时。 (endo-GalNac-ase-A) 与 H-3 标记和未标记的脱唾液酸胎球蛋白一起孵育，仅释放二糖 (Galbeta1-3GalNAc)。
For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Galbeta1-3GalNAc were analysed by h.p.l.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80 % (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pl 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 m-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in H-3-labelled and unlabelled asialofetuin, liberating both the disaccharide (Galbeta1-3GalNAc) and the tetrasaccharide [Galbeta1-3(Galbeta1-4GlcNAcbeta1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with H-3-labelled and unlabelled asialofetuin, only the disaccharide (Galbeta1-3GalNAc) was liberated.