Zn2+와 Ni2+에 의한 톨라신 용혈활성 저해효과의 pH 의존성

Pseudomonas tolaasii에 의해 분비되는 톨라신은 펩티드 독소로서, 버섯 자실체 구조와 세포를 파괴하여 갈반병을 일으킨다. 톨라신의 독성은 용혈활성을 측정함으로서 평가하며, 이는 톨라신 분자가 적혈구 막에 pore를 형성하여 세포 구조를 파괴하기때문이다. 이전 연구에서, Zn2+ 뿐만 아니라 Ni2+이 톨라신의 세포독성에 억제효과를 가짐을 확인하였다. Ni2+은 농도가 증가함에 따라 톨라신에 의한 용혈작용을 저해하였으며, 이의 Ki 값은 1.8 mM이었다. 용혈활성은 10 mM 이상의 농도에서 완전히저해되었다. Ni2+의 효과는 pH에 따라 크게 변하지 않았으나, Zn2+의 톨라신 세포독성 억제 효과는 염기성 pH에서 크게 증가하였다. 완충액의 pH를 7에서 9로 증가시키면, 50% 용혈작용이 일어나는 시간인 T50은 1 mM Ni2+에 의해 조금 증가하였으나 100 μM Zn2+에서는 크게 증가하였다. Zn2+와 Ni2+을 반응용액에 동시에 처리하였을 때, 두 양이온의 상승효과는 모든 pH 에서 나타났다. 서로 다른 pH 의존성을 보이는 두 금속이온의분자적 설명은 톨라신의 pore 형성과 세포 독성에 관한 기작의이해에 기여할 것이다. 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 Zn2+ and Ni2+. Ni2+ inhibited the tolaasin-induced hemolysis in a dose-dependent manner and its Ki value was 1.8 mM. The hemolytic activity was completely inhibited at the concentration higher than 10 mM. The inhibitory effect of Zn2+ on tolaasininduced hemolysis was increased in alkaline pH, while that of Ni2+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 (T50) was increased greatly by 100 μM Zn2+; however, it was slightly increased by 1 mM Ni2+ at all pH values. When the synergistic effect of Zn2+ and Ni2+ 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. KCI Citation Count: 0