IDENTIFICATION AND CHARACTERIZATION OF HELICOBACTER-PYLORI PHOSPHOLIPASE-C ACTIVITY

We analyzed 11 H. pylori isolates from humans using the artificial chr omogenic substrate paranitrophenylphosphorylcholine to detect phosphol ipase C (PLC) activity. The range of PLC in sonicates was 8.8-92.3 (Me an 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 s onicate and purified PLC from C. perfringens (PLC1) but not purified P LC from B. cereus (PLC3). H. pylori sonicates had little acid phosphat ase and no detectable alkaline phosphatase activity, and H. pylori PLC showed markedly different biochemical characteristics from either pho sphatase. In total, these studies indicate that activity measured in H . pylori sonicate by PLC assay is due to PLC and not phosphatase activ ity. The temperature optimum for PLC activity of H. pylori sonicate wa s 56-degrees-C and for PLC1 was 65-degrees-C. For H. pylori PLC and PL C1, optimal activity occurred at pH 8. Despite multiple similarities b etween H. pylori PLC and PLC1, known PLC inhibitors show different int eractions with each enzyme. Although PLC activity is present in many s ubcellular constituents of H. pylori, including culture supernatants a nd water extracts, highest specific activity is associated with a memb rane-enriched fraction.