Related Experiment Videos
Purification and characterization of neutral sphingomyelinase from Helicobacter pylori
1School of Medical Technology, Chang Gung University, Taoyuan, Taiwan. chanec@mail.cgu.edu.tw
Abstract:
Phospholipase activities of human gastric bacterium, Helicobacter pylori, are regarded as the pathogenic factors owing to their actions on epithelial cell 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-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM) was used as a substrate, the purified enzyme exhibited a K(m) of 6.7 microM 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 reaches optimal activity at pH 7.4 and has a pI of 7.15. 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.
Insights
We purified and characterized neutral sphingomyelinase (N-SMase) from Helicobacter pylori, identifying it as a potential diagnostic marker for H. pylori infection due to its in vivo production and antigenicity.
Area of Science:
- Microbiology
- Enzymology
- Pathogenesis
Background:
- Helicobacter pylori phospholipase activities are implicated in pathogenesis by damaging epithelial cell membranes.
- Neutral sphingomyelinase (N-SMase) is a key enzyme in sphingolipid metabolism.
Purpose of the Study:
- To purify and characterize neutral sphingomyelinase (N-SMase) from H. pylori for the first time.
- To investigate the enzyme's role in H. pylori infection and its potential as a diagnostic target.
Main Methods:
- Purification of N-SMase using acid glycine extraction, ammonium sulfate precipitation, and multiple chromatography steps (CM-Sepharose, Mono-Q, Sephadex G-75).
- Enzyme characterization including molecular mass determination, kinetic analysis (K(m), V(max)), optimal pH, pI, and substrate specificity.
- Western blot analysis and enzyme-linked immunosorbent assays (ELISA) to assess antigenicity and in vivo production.
Main Results:
- N-SMase was purified 2083-fold with 37% recovery; native enzyme molecular mass is approximately 32 kDa.
- The enzyme exhibits optimal activity at pH 7.4, is magnesium-dependent, and hydrolyzes sphingomyelin and phosphatidylethanolamine.
- N-SMase demonstrated hemolytic activity and cross-reacted with Bacillus cereus SMase. Sera from infected individuals recognized the purified N-SMase, indicating in vivo production.
- Purified N-SMase was effective as an antigen in ELISA for detecting H. pylori infection.
Conclusions:
- The study reports the first purification and characterization of H. pylori N-SMase.
- N-SMase is produced in vivo during H. pylori infection and shows potential as a reliable diagnostic antigen.