Effect of Metal lons on Phospholipase C (Heat-Labile Hemolysin) from Pseudomonas aeruginosa

Abstract

Phospholipase C (PLC; heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ion exchange chromatography. Some chelating reagents and divalent metal ions were examined for their effects on the purified PLC activity. EDTA, ECTA, and o-phenanthroline did not inhibit the enzyme activity to p-nitrophenylphosphorylcholine (pNPPC) at concentrations below 10 mmol/L. Inclubation with Zn2+, Cu2+, Co2+ or Ni2+ caused a decrease in the enzyme activity. The activity of EDTA-treated PLC was most remarkably decreased in the presence of Zn2+, followed in descending order by Cu2+, Co2+ and Ni2+, while other metal ions (Mg2+, Ca2+, Ba2+, and Mn2+) did not significantly affect the enzyme activity using concentrations up to 10 mmol/L. Zn2+ competitively inhibited the enzyme activity against pNPPC, with a Ki value of 0.3 mmol/L. The hemolytic activity of PLC was also completely inhibited in the presence of 1 mmol/L Zn2+, and inactivation of the enzyme by Zn2+ was not reversed by concomtitant addition of other divalent metal ions. These results suggest that hemolytic PLC from P. aeruginosa has a unique property in that there is no metal ion requirement for its activity. Phospholipase C (PLC; heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ion exchange chromatography. Some chelating reagents and divalent metal ions were examined for their effects on the purified PLC activity. EDTA, ECTA, and o-phenanthroline did not inhibit the enzyme activity to p-nitrophenylphosphorylcholine (pNPPC) at concentrations below 10 mmol/L. Inclubation with Zn2+, Cu2+, Co2+ or Ni2+ caused a decrease in the enzyme activity. The activity of EDTA-treated PLC was most remarkably decreased in the presence of Zn2+, followed in descending order by Cu2+, Co2+ and Ni2+, while other metal ions (Mg2+, Ca2+, Ba2+, and Mn2+) did not significantly affect the enzyme activity using concentrations up to 10 mmol/L. Zn2+ competitively inhibited the enzyme activity against pNPPC, with a Ki value of 0.3 mmol/L. The hemolytic activity of PLC was also completely inhibited in the presence of 1 mmol/L Zn2+, and inactivation of the enzyme by Zn2+ was not reversed by concomtitant addition of other divalent metal ions. These results suggest that hemolytic PLC from P. aeruginosa has a unique property in that there is no metal ion requirement for its activity.