Purification and characterization of neutral sphingomyelinase from Helicobacter pylori

Abstract

Phospholipase activities of human gastric bacterium, Helicobacter pylori , are regarded as the pathogenic factors owing to their actions on epithelial eel membranes. In this study, we purified and characterized neutral sphingomyelinase (N-SMase) from the superficial components of H. pylori strains for the first time. N-SMase was purified 2083-fold with an overall recovery of 37%. The purification steps included acid glycine extraction, ammonium sulfate precipitation, CM-Sepharose, Mono-Q, and Sephadex G-75 column chromatography. Approximate molecular mass for the native N-SMase was around 32 kDa. When N-omega-trinitrophenylaminol auryl sphingomyelin (TNPAL-SM) was used as a substrate, the purified enzyme exhibited a K-m of 6.7 mu M and a V-max of 15.6 nmol of TNPAL-sphingosine/h/mg of protein at 37 degrees C in 50 mM phosphate-buffered saline, pH 7.4. N-SMase I-caches optimal activity at pH 7.4 and has a pi of 7.5. The enzyme activity is magnesium dependent and specifically hydrolyzed sphingomyelin and phosphatidylethanolamine. The enzyme also exhibits hemolytic activity on human erythrocytes. 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 but not 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 protein antigens in detecting human antibodies to H. pylori.