| Summary: | 碩士 === 國立交通大學 === 生物科技研究所 === 87 === Purification and Characterization of phingomyelinase from Helicobacter pylori
student：Chih-Chieh Chang Advisor：C. Allen Chang
Err-Cheng Chan
Institute of Biological and Technology
ABSTRACT
Sphingomyelinase (SMase) catalyzes the hydrolysis of membrane sphingomyelin to generate lipid moiety ceramide and water solube phosphocholine. Sphingomyelin and ceramide are key component of the signaling pathway in cytokine-and stress-induced cellular responses. In this study, we report the purification and characterization of membrane bound and magnesium-dependent SMase(N-SMase) from Helicobacter pylori. SMase was purified 2083-fold to homogeneity from the microaerophilic human gastric bacterium, Helicobacter pylori.
The SMase isolation procedure included an acid-glycine extraction step、80% ammonium sulfate precipitation、CM Sepharose、Mono Q、Sephadex G-75 column chromatography and gel filtration high performance liquid chromatography(HPLC). The purified enzyme exhibited a Km of 3.97μM and a Vmax of 16.47μmol of SMase hydrolyzed/hr/mg of protein at 37 oC in 10 mM Tris-HCl contain 7.5 mM Mg2+. The isoelectric point was 7.1±0.05. Molecular was estimated by SDS-PAGE and HPLC for the enzyme was 32,200 daltons. By western blot analysis, the membrane bound SMase of H. pylori and Bacillus cereus were shown to be antigenically related. Thus, the Smase of H. pylori shows similarities to SMase of B. cereus. We used the purified SMase as antigen for serodiagnosis of H. pylori infected persons to demonstrate infected persons, serum have SMase antibodies. Therefor, should SMase exist in H. pylori, it may play a potential role in pathogenesis of gastric ulcer diseases.
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