Identification and characterization of Helicobacter pylori phospholipase C activity.
We analyzed 11 H. pylori isolates from humans using the artificial chromogenic substrate paranitrophenylphosphorylcholine to detect phospholipase C (PLC) activity. The range of PLC in sonicates was 8.8-92.3 (Mean 56.9 +/- 6.5) nmol of substrate hydrolysed min-1 mg-1 protein; the amount of activity was not associated with urease or cytotoxin levels. Addition of sorbitol or glycerol enhanced PLC activity of H. pylori sonicate and purified PLC from C. perfringens (PLC1) but not purified PLC from B. cereus (PLC3). H. pylori sonicates had little acid phosphatase and no detectable alkaline phosphatase activity, and H. pylori PLC showed markedly different biochemical characteristics from either phosphatase. In total, these studies indicate that activity measured in H. pylori sonicate by PLC assay is due to PLC and not phosphatase activity. The temperature optimum for PLC activity of H. pylori sonicate was 56 degrees C and for PLC 1 was 65 degrees C. For H. pylori PLC and PLC1, optimal activity occurred at pH 8. Despite multiple similarities between H. pylori PLC and PLC1, known PLC inhibitors show different interactions with each enzyme. Although PLC activity is present in many subcellular constituents of H. pylori, including culture supernatants and water extracts, highest specific activity is associated with a membrane-enriched fraction.
Weitkamp
J H
JH
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2605.
Pérez-Pérez
G I
GI
Bode
G
G
Malfertheiner
P
P
Blaser
M J
MJ
D000438:Alcohols / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D003902:Detergents; D016481:Helicobacter Infections / Q000382:microbiology; D016480:Helicobacter pylori / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D010738:Type C Phospholipases / Q000187:drug effects / Q000378:metabolism*
D000438:Alcohols; D002413:Cations, Divalent; D003902:Detergents; D010744:Phosphoric Monoester Hydrolases; D010738:Type C Phospholipases