Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H.
Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H.
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DOI：
10.1016/0003-2697(84)90080-0
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发表时间：
1984-09
影响因子：
2.9
通讯作者：
R. B. Trimble;Frank Maley
中科院分区：
文献类型：
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作者：
R. B. Trimble;Frank Maley
来自褶纹链霉菌的内切-β-乙酰氨基葡萄糖苷酶H（Endo H）从糖蛋白水解高甘露糖寡糖的能力受到许多因素的影响，包括底物糖蛋白的三级结构、所用Endo H的量、孵育时间以及是否存在影响蛋白质构型的试剂。无论孵育时间如何，低于10至20毫单位/ml的Endo H水平都可能不完全水解寡糖，因为即使酶保持活性，它也会被捕获或隔离并且不可用。在水解之前，首先在1.2倍重量过量的十二烷基硫酸钠中使底物糖蛋白变性，可以增强Endo H活性。然而，低水平的Endo H对十二烷基硫酸钠灭活敏感，当未结合的去污剂浓度超过蛋白质0.02%（0.2 mg/ml）时，4 h内会损失相当大的活性。其他变性剂，如Triton，两性离子，Brij系列，或辛基葡糖苷不增强或抑制低聚糖的Endo H去除，但离液盐硫氰酸钠在0.5 μ m增强Endo H对某些糖蛋白，特别是牛甲状腺球蛋白的作用。在变性条件下，蛋白水解污染物是一个潜在的问题。向Endo H孵育物中加入1 mM苯甲基磺酰氟完全抑制残留的Endo H相关蛋白酶。此外，Endo H不受可用于保护底物糖蛋白的多种蛋白水解抑制剂的影响。
The ability of endo-β-acetylglucosaminidase H (Endo H) from Streptomyces plicatus to hydrolyze high-mannose oligosaccharides from glycoproteins is influenced by numerous factors, including the tertiary structure of the substrate glycoproteins, the amount of Endo H used, the time of incubation, and the presence or absence of reagents that affect protein configuration. Endo H levels below 10 to 20 milliunits/ml may incompletely hydrolyze oligosaccharides, regardless of the incubation time, because even though the enzyme remains active, it becomes trapped or sequestered and is unavailable. Endo H activity can be potentiated by first denaturing substrate glycoproteins in a 1.2-fold weight excess of sodium dodecyl sulfate prior to hydrolysis. However, low levels of Endo H are sensitive to inactivation by sodium dodecyl sulfate, with considerable activity being lost over 4 h when the unbound detergent concentration exceeds protein by 0.02% (0.2 mg/ml). Other denaturants such as the Tritons, the zwittergents, the Brij series, or octylglucoside do not enhance or inhibit Endo H removal of oligosaccharides, but the chaotropic salt sodium thiocyanate at 0.5 m enhances Endo H action on some glycoproteins, particularly bovine thyroglobulin. Under denaturing conditions, proteolytic contaminants are a potential problem. Addition of 1 mm phenylmethylsulfonyl fluoride to Endo H incubations completely inhibits the residual Endo H-associated protease(s). Furthermore, Endo H is unaffected by a wide range of proteolytic inhibitors that may be used to protect substrate glycoproteins.