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

溶血素O （Streptolysin O， SLO）是由a群链球菌产生的一种破坏膜的毒性蛋白。利用SLO突变体[SLO(C/ a)-SS]和天然SLO，对SLO的孔隙形成和溶血机制进行超微结构分析。阴性染色的电子显微镜显示，SLO(C/A)-SS无法穿透红细胞膜，这是由于结构域之间的二硫键的固定所致。结合在膜上的SLO(C/A)-SS分子形成了许多单层环状结构，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，与天然SLO一样，与膜结合的SLO(C/A)-SS在室温下形成双层环，膜上有孔。形成带孔的双层环状结构的数量与DTT处理时间有关。从这些结果可以得出以下两点结论：(1)前人提出的SLO (J.Bacteriol.；（1993）是由单个SLO分子连接成一个圆圈。(2) SLO毒素溶血包括两个明确的步骤；一个与温度无关的步骤和一个与温度相关的步骤。结构域3插入宿主细胞膜后，构象变化的发生和双层环的形成需要温度的升高。

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.