中文摘要：

N-乙酰葡萄糖胺（O-linkedN-acetylglucosamine,O-GlcNAc）修饰是一种重要的蛋白翻译后修饰,参与生物体中的多种细胞活动.O-GlcNAcase（OGA）是生物体内唯一水解蛋白O-GlcNAc修饰的糖苷酶,但该酶的糖苷酶活性中心一直存在争论.实验利用PCR的方法,获得3种不同缺失的人源OGA片段（OGA1：aa1～916;OGA2：aa1～677;OGA3：aa1～350）,并将3种片段构建到质粒pET28a中;重组质粒转化大肠杆菌BL21后,进行诱导表达、纯化;以4-甲基伞形酮2-乙酰氨基-2-脱氧葡萄糖为底物测定不同OGA片段的糖苷酶酶学特征常数.结果发现OGA3仍保持糖苷酶的活性,因此OGA的糖苷酶活性中心位于N端1-350aa.

英文摘要：

O-linked N-acetylglucosamine （O-GlcNAc） is an important protein post-transla-tional modification, which is involved in many cellular processes. O-GlcNAcase （OGA） is the only enzyme responsible for removal of O-GlcNAc from protein. However, to date no experimental data clearly indicates the glycosidase catalytic site of O-GlcNAcase. Here, three coding sequences of human OGA fragments （OGA1 ： aal-916： OGA2 ： aal-677 ; OGA3. aal-350） were amplified by PCR and cloned into plasmid pET28a. After transformed three recombinant plas- mids into Escherichia coli BL21, His-tagged recombinant protein were expressed and purified. Using 4-MU-GlcNAc （ 4-methylumbelliferyl 2-acetamido-2-deoxy-fl-D-glucopyrano-side ） as substrate, the enzymatic characteristics of three OGA fragments were determined. This results indicated that OGA3 （1-350 aa） retained glycosidase catalytic activity and N-terminal aal-350 is the glycosidase domain.