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

链球菌溶血素O(SLO)是由A组链球菌产生的一种膜损伤性毒性蛋白。利用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了超微结构分析。负染样品的电子显微镜显示，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固定的结果。与膜结合的SLO(C/A)-SS分子形成许多单层环状结构，不会在膜上形成孔。这些结构与天然SLO在0℃时形成的结构相似。经二硫苏糖醇(DTT)处理后，结合在膜上的SLO(C/A)-SS形成双层环，膜上有孔，在室温下天然SLO也是如此。有孔形成的双层环状结构的数量与DTT治疗的持续时间相关。根据这些结果，得出以下两点：(1)以前提出的SLO(J.Bacteriol)环模型的每一内层和外层。1993)是由环状结合的单个SLO分子组成的。(2)SLO毒素的溶血包括两个明确的步骤：温度无关步骤和温度依赖步骤。在将结构域3插入宿主细胞膜后，温度的升高对于构象变化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.