Ultrastructural Analysis of Mechanism of Protein-Domain-Assembly on Cell Membrane of Specific Lipid-Binding Toxin.

链球菌溶血素O（Streptolysin O，SLO）是由A组链球菌产生的一种膜损伤毒性蛋白。使用SLO的突变形式[SLO（C/A）-SS和天然SLO]对SLO的孔形成和溶血机制进行超微结构分析。负染色制备物的电子显微照片显示，SLO（C/A）-SS不能穿透红细胞膜，这是由于结构域之间的二硫键导致的固定化的结果。结合到膜上的SLO（C/A）-SS分子形成许多单层环状结构，这不会导致膜上的孔。这些结构与天然SLO在0℃下形成的结构相似。在用二硫苏糖醇（DTT）处理后，与膜结合的SLO（C/A）-SS在膜上形成具有孔的双层环，如天然SLO在室温下所做的那样。双层环状结构的数量与形成的孔隙和DTT治疗的持续时间之间存在相关性。从这些结果中，得出以下两点：（1）以前提出的SLO（J.Bacteriol. 1993）由在环中缔合的单个SLO分子组成。(2)通过SLO毒素的溶血涉及两个确定的步骤;温度非依赖性步骤和温度依赖性步骤。在结构域3插入宿主细胞膜后，温度的升高对于构象变化的发生和双层环的形成是必要的。

Streptolysin O (SLO) is a membrane-damaging toxic protein produced by group A streptococci. An ultrastructural analysis of pore formation and the mechanism of hemolysis by SLO, using a mutant form of SLO [SLO(C/A)-SS and native SLO. Electron micrographs of negatively stained preparations revealed that SLO(C/A)-SS was unable to penetrate the erythrocyte membrane as a consequence of immobilization that was due to a disulfide bond between domains. The SLO(C/A)-SS molecules that bound to membranes formed numerous single-layered ring-shaped structures that did not result in pores on the membranes. These structures were similar to the structures formed by native SLO at 0℃. After treatment with dithiothreitol (DTT), SLO(C/A)-SS that had bound to membranes formed double-layered rings with pores on the membranes, as does native SLO at room temperature. There was a correlation between the number of double-layered ring structure with pores that formed and the duration of treatment with DTT. From these results, the following two points were concluded ; (1) Each inner and outer layer of the previously proposed ring model of SLO (J.Bacteriol. 1993) was composed of single SLO molecules associated in a circle. (2) Hemolysis by SLO toxin involves two defined steps; a temperature-independent step and a temperature-dependent step. An increase in temperature is necessary for the occurrence of conformational changes and the formation of the double-layered ring after the insertion of domain 3 into the host cell membrane.