Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond.
Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond.
复制标题
来自杏仁的均质肽 N-糖苷酶的结构和化学表征。
DOI：
10.1021/bi00300a006
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
VanEtten,RL
中科院分区：
文献类型：
--
作者：
Taga,EM;Waheed,A;VanEtten,RL
尤拉齐奥湾Taga，* Abdul Waheed，and Robert L.货车Etten* 摘要：一种肽A-糖苷酶可催化糖肽和糖蛋白中N-连接低聚糖链的水解，已从杏仁乳蛋白和杏仁粉中纯化至同质。用离子交换层析、凝胶过滤层析和制备聚丙烯酰胺凝胶电泳从杏仁乳蛋白中纯化得到一种酶，纯化倍数超过700倍，产率为63%。更适合于有效的大规模纯化的替代方法使用离子交换、亲和和凝胶过滤色谱法。当纯化开始与杏仁emulsin，酶纯化1200倍，产率为37%，而当纯化开始与杏仁粉，酶纯化9000倍，产率为45%。该均相酶在10 mM醋酸钠（pH5.0）缓冲液中于4 ℃稳定数月.肽IV-糖苷酶本身被证明是一种糖蛋白，在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳上由分子量为66800的单一多肽链组成。天然分子的圆二色谱表明存在高（约80%）的α-螺旋含量。已经从细菌培养物中纯化了氨基酸和β-葡糖内切酶，并且已经发现它们在阐明糖蛋白的寡糖结构方面有很大帮助（Tarentino等人，1974; Tarentino & Maley，1974; Koide & Muramatsu，1974; Elder &亚历山大，1982）。在哺乳动物组织中也报道了相关的酶活性（Tachibana et al.，1982年）。内切糖苷酶如endo H、endo D和endo F切割高甘露糖，
Eulazio M. Taga,* Abdul Waheed, and Robert L. Van Etten* abstract: A peptide A-glycosidase that catalyzes the hy-drolysis of N-linked oligosaccharide chains from glycopeptides and glycoproteins has been purified to homogeneity from almond emulsin and from almond meal. Purification from almond emulsin using ion-exchange chromatography, gel filtration chromatography, and preparative polyacrylamide gel electrophoresis gave an enzyme which was purified more than 700-fold and with a yield of 63%. An alternative procedure, more suitable for efficient large scale purification, used ion-exchange, affinity, and gel filtration chromatography. When purification began with almond emulsin, the enzyme was purified 1200-fold with a 37% yield, while when purification began with almond powder, the enzyme was purified 9000-fold with a yield of 45%. The homogeneous enzyme is stable at 4 C for several monthsin 10 mM sodium acetate, pH 5.0, buffer. The peptide iV-glycosidase is itself shown to be a glycoprotein consisting of a single polypeptide chain with a molecular weight of 66800 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Circular dichroism spectra of the native molecule indicate the presence of a high (approximately 80%) a-helix content. The amino acid and^ Jumerous endoglycosidases have been purified from bac-terial cultures and have been found to be of great help in elucidating the structures of the oligosaccharides of glycoproteins (Tarentino et al., 1974; Tarentino & Maley, 1974; Koide & Muramatsu, 1974; Elder & Alexander, 1982). A related enzyme activity has also been reported in mammalian tissues (Tachibana et al., 1982). Endoglycosidasessuch as endo H, endo D, andendo F cleave high mannose, complex