Purification and characterization of neutral sphingomyelinase from Helicobacter pylori.
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.
Chan
E C
EC
School of Medical Technology, Chang Gung University, Taoyuan, Taiwan. chanec@mail.cgu.edu.tw
Chang
C C
CC
Li
Y S
YS
Chang
C A
CA
Chiou
C C
CC
Wu
T Z
TZ
D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D002413:Cations, Divalent / Q000494:pharmacology; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000276:immunology; D016481:Helicobacter Infections / Q000175:diagnosis / Q000276:immunology / Q000382:microbiology; D016480:Helicobacter pylori / Q000201:enzymology* / Q000276:immunology / Q000472:pathogenicity; D006461:Hemolysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010714:Phosphatidylethanolamines / Q000378:metabolism; D012680:Sensitivity and Specificity; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism; D013276:Stomach Ulcer / Q000175:diagnosis / Q000276:immunology / Q000382:microbiology; D013379:Substrate Specificity; D013816:Thermodynamics; D014774:Virulence
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D002413:Cations, Divalent; D010714:Phosphatidylethanolamines; D013109:Sphingomyelins; D013108:Sphingomyelin Phosphodiesterase