Purification and Characterization of Neutral Sphingomyelinase from Helicobacter pylori

详细信息    查看全文

• 作者：Err-Cheng Chan ; Chih-Chieh Chang ; Yi-Shuane Li ; C. Allen Chang ; Chiuan-Chian Chiou ; and Tzong-Zeng Wu
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：April 25, 2000
• 年：2000
• 卷：39
• 期：16
• 页码：4838 - 4845
• 全文大小：138K
• 年卷期：v.39,no.16(April 25, 2000)
• ISSN：1520-4995

文摘

Phospholipase activities of human gastric bacterium, Helicobacter pylori, are regarded as thepathogenic factors owing to their actions on epithelial cell membranes. In this study, we purified andcharacterized neutral sphingomyelinase (N-SMase) from the superficial components of H. pylori strainsfor the first time. N-SMase was purified 2083-fold with an overall recovery of 37%. The purificationsteps included acid glycine extraction, ammonium sulfate precipitation, CM-Sepharose, Mono-Q, andSephadex G-75 column chromatography. Approximate molecular mass for the native N-SMase was around32 kDa. When N--trinitrophenylaminolauryl sphingomyelin (TNPAL-SM) was used as a substrate, thepurified enzyme exhibited a K_m of 6.7 M and a V_max of 15.6 nmol of TNPAL-sphingosine/h/mg ofprotein at 37 C in 50 mM phosphate-buffered saline, pH 7.4. N-SMase reaches optimal activity at pH7.4 and has a pI of 7.15. The enzyme activity is magnesium dependent and specifically hydrolyzedsphingomyelin and phosphatidylethanolamine. The enzyme also exhibits hemolytic activity on humanerythrocytes. According to Western blot analysis, a rabbit antiserum against purified N-SMase from H.pylori cross-reacted with SMase from Bacillus cereus. Sera from individuals with H. pylori infection butnot uninfected ones recognizing the purified N-SMase indicated that it was produced in vivo. In enzyme-linked immunosorbent assays, the purified N-SMase used as an antigen was as effective as crude proteinantigens in detecting human antibodies to H. pylori.