幽門螺旋桿菌鞘磷脂□的純化及其特性之研究

Abstract

幽門螺旋桿菌鞘磷脂□的純化及其特性之研究 學生：張志杰 指導教授：張 正 詹爾昌 國立交通大學生物科技研究所碩士班 中文摘要 鞘磷脂□(Sphingomyelinase，簡稱為SMase) ，為一種磷酸水解□，可以將細胞膜上的鞘磷脂(sphingomyelin)分解產生脂溶性的N-醯基鞘氨醇(ceramide)及水溶性的磷酸膽鹼(phosphocholine)兩種產物，而且在經由胞泌素(cytokine)和逆境(stress)所誘導引起的細胞訊息傳遞反應上，鞘磷脂和N-醯基鞘氨醇兩者皆扮演著重要的角色。本論文首先將純化幽門螺旋桿菌(Helicobacter pylori)細胞膜上的鞘磷脂□並且加以定量定性，幽門螺旋桿菌細胞膜上的鞘磷脂□在經由ㄧ系列的純化過程之後，最後可以得到專一活性為起始物的2083倍之鞘磷脂□。鞘磷脂□純化分離過程包括:利用酸性甘胺酸(acid-glycine pH2.2)緩衝溶液萃取幽門螺旋桿菌細胞膜上的鞘磷脂□、80%的硫酸銨(ammonium sulfate) 、CM Sepharose陽離子樹脂交換管柱層析、Mono Q陰離子樹脂交換管柱層析、Sephadex G-75 superfine膠體管柱層析和高效能液相管柱層析(gel filtration high performance liquid chromatography)。 經由純化過程所純化出的鞘磷脂□是屬於鎂離子依賴型(magnesium-dependent SMase)，其Km (Michaelis常數)值為 3.97μM、Vmax (酵素反應最大速率) 值為16.47μmolhr-1、等電點(isoelectric point)為7.1±0.05、再經由蛋白質電泳(SDS-PAGE)及高效能液相管柱層析得到其分子量為32,200道耳吞(daltons)。藉由西方墨點法分析(Western blot analysis)，得到幽門螺旋桿菌和蠟樣桿菌(Bacillus cereus)的鞘磷脂□具有相似性。 利用純化出的鞘磷脂□來當作抗原(antigen)，並且利用經由幽門螺旋桿菌感染之病人的血清作為抗體(antibody)，進行臨床血清免疫分析，實驗結果得知，經由幽門螺旋桿菌感染之病人的血液中含有可以和鞘磷脂□作用的抗體。因此，存在於幽門螺旋桿菌細胞膜上的鞘磷脂□對於造成腸胃方面的疾病(gastric ulcer diseases)可能扮演著極為重要的角色。

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.