Purification and Characterization of Porcine Liver Lysosomal Membrane Sialidase

• Main Authors: Cheng-Yen Hung, 洪承延
• Other Authors: Chin-Hsiang Chien
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/50787697478939002358

碩士 === 國立陽明大學 === 生物化學研究所 === 88 === Sialidase (nanH) is a group of glycohydrolytic enzyme, widely distributed in nature, that cleaves sialic acid residues from the oligosaccharide components of glycoconjugates. In this study, the porcine liver sialidase was purified by Fractogel TSK CM650 gel, DEAE Sephacel, p-aminophenyloxamic acid agarose, and heparin agarose chromatographies. We obtained two sialidase preparations, SIAⅠand SIAⅡ, which showed the molecular weight of 55 kDa and 49 kDa, respectively, on the SDS-polyacrylamide gel electrophoresis. By Western blotting, using antibody against the SIA Ⅰ, we found that 49 kDa is a protein truncated from 55 kDa in the purification processes. The two enzyme preparations showed an acidic optimum pH, and hydrolyzed gangliosides not only GM3 and GD1a but also GM1 and GM2, which resemble the characteristics of lysosomal membrane sialidase. By immunohistological staining of porcine liver section observed with transmission electron microscope, we confirmed that the sialidase is localized on the lysosomal membrane. The sialidase of Micromonospora viridifaciens has three domains: active site, galactose-binding domain, and immunoglobulin module. In our purification process, we also found that porcine liver sialidase was bound by protein G column, which suggests that the porcine liver sialdase has an immunoglobulin module similar to that of the bacterial sialidase. We have cloned the full-length cDNA of human liver lysosomal matrix sialidase by RT-PCR for the purpose of expressing the human liver lysosomal matrix sialidase in E. coli . The purified sialidase will be used for studying the 3D structure by X-ray crystallography in the future. 英文摘要 --------------------------- 3 緒論 --------------------------- 4 材料與方法 --------------------------- 11 結果 --------------------------- 22 討論 --------------------------- 27 參考文獻 --------------------------- 32 圖表 --------------------------- 38 附錄 --------------------------- 57