pH-dependence in the inhibitory effects of Zn²⁺and Ni²⁺ on tolaasin-induced hemolytic activity

Abstract

Tolaasin secreted by Pseudomonas tolaasii is a peptide toxin and causes brown blotch disease on the cultivated mushrooms by collapsing cellular and fruiting body structure. Toxicity of tolaasin was evaluated by measuring hemolytic activity because tolaasin molecules form membrane pores on the red blood cells and destroy cell membrane structure. In the previous studies, we found that tolaasin cytotoxicity was suppressed by Zn 2+ and Ni 2+. Ni 2+ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and its K i value was 1.8 mM. The hemolytic activity was completely inhibited at the concentration higher than 10 mM. The inhibitory effect of Zn 2+ on tolaasininduced hemolysis was increased in alkaline pH, while that of Ni 2+ was not much dependent on pH. When the pH of buffer solution was increased from pH 7 to pH 9, the time for 50% hemolysis (T 50) was increased greatly by 100 µM Zn 2+ ; however, it was slightly increased by 1 mM Ni 2+ at all pH values. When the synergistic effect of Zn 2+ and Ni 2+ on tolaasin-induced hemolysis was measured, it was not dependent on the pH of buffer solution. Molecular elucidation of the difference in pH-dependence of these two metal ions may contribute to understand the mechanism of tolaasin pore formation and cytotoxicity.