DEGLYCOSYLATION OF ASPARAGINE-LINKED GLYCANS BY PEPTIDE - N-GLYCOSIDASE-F
DEGLYCOSYLATION OF ASPARAGINE-LINKED GLYCANS BY PEPTIDE - N-GLYCOSIDASE-F
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DOI：
10.1021/bi00338a028
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
PLUMMER, TH
中科院分区：
文献类型：
--
作者：
TARENTINO, AL;GOMEZ, CM;PLUMMER, TH
通过硫酸铵沉淀，然后在 TSK HW-55(S) 上进行凝胶过滤，从脑膜败血黄杆菌培养物中纯化内切-β-N-乙酰氨基葡萄糖苷酶 F (Endo F) 和肽:N-糖苷酶 F (PNGase F)。该系统分离了两种酶并提供了高纯度的 PNGase F，但分辨率的基础似乎是疏水相互作用而不是分子大小。使用纯化的 Endo F 和 PNGase F 以及特定糖肽进行的研究表明，Endo F 与 Endo H 有点相似，因为它可以水解许多（但不是全部）高甘露糖和杂合寡糖，以及复杂的双触角寡糖。相反，只要天冬酰胺残基的α-氨基和羧基都处于肽键中，PNGase F 就会水解所检查的所有类别的天冬酰胺连接聚糖。 PNGase F 的去糖基化研究表明，许多天然构象的蛋白质对这种酶敏感，因为在十二烷基硫酸钠中预先变性大大减少了完全去除碳水化合物所需的酶量。
Endo-.beta.-N-acetylglucosaminidase F (Endo F) and peptide:N-glycosidase F (PNGase F) were purified from cultures of Flavobacterium meningosepticum by ammonium sulfate precipitation followed by gel filtration on TSK HW-55(S). This system separated the two enzymes and provided PNGase F in a high state of purity, but the basis for the resolution appeared to be hydrophobic interaction and not molecular size. Studies using purified Endo F and PNGase F with defined glycopeptides demonstrated that Endo F was somewhat similar to Endo H in that it hydrolyzed many, but not all, high-mannose and hybrid oligosaccharides, as well as complex biantennary oligosaccharides. PNGase F, in contrast, hydrolyzed all classes of asparagine-linked glycans examined, provided both the .alpha.-amino and carboxyl groups of the asparagine residue were in peptide linkage. Deglycosylation studies with PNGase F revealed that many proteins in their native conformation were susceptible to this enzyme by that prior denaturation in sodium dodecyl sulfate greatly decreased the amount of enzyme required for complete carbohydrate removal.