人原发性肝癌组织中α-L-岩藻糖苷酶的纯化与鉴定
Purification and characterization of alpha-L-fucosidase from human primary hepatocarcinoma tissue.
作者信息
Li Chao, Qian Jie, Lin Ju-Sheng
机构信息
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.
出版信息
World J Gastroenterol. 2006 Jun 21;12(23):3770-5. doi: 10.3748/wjg.v12.i23.3770.
AIM
To purify and characterize alpha-L-fucosidase from human liver cancer tissue and to detect the localization of alpha-L-fucosidase in tumor tissue.
METHODS
Cation exchange chromatography on CM-52 and ultrafiltration were used to separate alpha-L-fucosidase (AFU) from crude extract of liver cancer tissue. 4-methylumbelliferyl-alpha-L-fucopyranoside was used as a fluorescent substrate to quantify the purified AFU activity in each step. A polyclonal antibody (pAb) against the purified AFU was obtained by anion exchange chromatography on DEAE-52 after ammonium sulfate fractionation and ultrafiltration. Immuohistochemical staining was used to observe the expression of AFU in malignant and adjacent liver tissues.
RESULTS
Human alpha-L-fucosidase was purified 74-fold to apparent homogeneity with 15% yield. SDS-PAGE indicated the presence of one subunit of molecular weight of 55 Ku. The specific activity of AFU in pooled fraction by chromatography was 10085 IU/mg. Western blot analysis indicated that the pAb could recognize one protein band of molecular weight of 55 Ku. The expression of AFU was observed in cytoplasm membrane of liver cancer tissue but not in that of adjacent tissue.
CONCLUSION
The purified alpha-L-fucosidase from primary hepatocarcinoma (PHC) is different in its properties from alpha-L-fucosidase in human other organs. The polyclonal antibody prepared in this experiment can be applied to the diagnosis of PHC.
目的
从人肝癌组织中纯化并鉴定α-L-岩藻糖苷酶，并检测α-L-岩藻糖苷酶在肿瘤组织中的定位。
方法
采用CM-52阳离子交换层析和超滤从肝癌组织粗提物中分离α-L-岩藻糖苷酶（AFU）。以4-甲基伞形酮-α-L-岩藻糖苷为荧光底物，定量各步骤纯化后的AFU活性。经硫酸铵分级分离和超滤后，通过DEAE-52阴离子交换层析获得针对纯化后AFU的多克隆抗体（pAb）。采用免疫组织化学染色观察AFU在恶性肝组织及癌旁肝组织中的表达情况。
结果
人α-L-岩藻糖苷酶纯化了74倍，达到表观均一性，产率为15%。SDS-PAGE显示存在一条分子量为55 Ku的亚基。层析合并组分中AFU的比活性为10085 IU/mg。Western blot分析表明，该pAb可识别一条分子量为55 Ku的蛋白条带。AFU在肝癌组织的细胞质膜中有表达，而在癌旁组织中未表达。
结论
从原发性肝癌（PHC）中纯化得到的α-L-岩藻糖苷酶在性质上与人其他器官中的α-L-岩藻糖苷酶不同。本实验制备的多克隆抗体可应用于PHC的诊断。
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