Tarentino A L, Gómez C M, Plummer T H
Biochemistry. 1985 Aug 13;24(17):4665-71. doi: 10.1021/bi00338a028.
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 but that prior denaturation in sodium dodecyl sulfate greatly decreased the amount of enzyme required for complete carbohydrate removal.
β-N-乙酰氨基葡萄糖苷酶F（内切糖苷酶F）和肽：N-糖苷酶F（PNGase F）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得到。该系统分离了这两种酶，并提供了高纯度的PNGase F，但分离的基础似乎是疏水相互作用而非分子大小。使用纯化的内切糖苷酶F和PNGase F与特定糖肽进行的研究表明，内切糖苷酶F在某种程度上与内切糖苷酶H相似，因为它能水解许多但并非所有的高甘露糖型和杂合型寡糖，以及复杂的双天线寡糖。相比之下，PNGase F能水解所检测的所有类型的天冬酰胺连接聚糖，前提是天冬酰胺残基的α-氨基和羧基都处于肽键连接状态。用PNGase F进行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，但在十二烷基硫酸钠中预先变性会大大减少完全去除碳水化合物所需的酶量。
