Chao Li1, Jie Qian, Ju-Sheng Lin. 1. Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.
Abstract
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.
AIM: To purify and characterize alpha-L-fucosidase from humanliver 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.
Authors: D Ayude; J Fernández-Rodríguez; F J Rodríguez-Berrocal; V S Martínez-Zorzano; A de Carlos; E Gil; M Páez de La Cadena Journal: Oncology Date: 2000-11 Impact factor: 2.935
Authors: Daniel Ayude; Maria Páez De La Cadena; Vicenta S Martínez-Zorzano; Almudena Fernández-Briera; Francisco J Rodríguez-Berrocal Journal: Oncology Date: 2003 Impact factor: 2.935