Electron-Sepectroscopic-Imaging Studies of the Damage to Membranes by Pore-Forming-Toxins

应用电子光谱成像技术（ESI）研究了细菌前体毒素链球菌溶血素O（SLO）和肉毒杆菌溶血素（BLY）对细胞膜的损伤作用。新发展的电喷雾离子化技术即使在支持膜上也能提供低信噪比的精细分子图像。成像板（IP）结合ESI将生物样品上的电子束暴露降低到百分之一，因为它们非常敏感。SLO形成的环是一个同轴的双环结构。两种类型的SLO分子构成一个环是相同的，不同的等电点（pI）。虽然红细胞膜上形成的BLY环被观察为SLO环的类似结构，但通过免疫电泳评估，它显然是由单一类型的BLY分子构成的。电喷雾电离技术的应用还提供了大量生物样品如人发、铁蛋白颗粒和羟基磷灰石晶体的成功数据。为了观察生物样品的碳图像，我们研制了一种由二氧化硅制成的非碳支撑膜。该胶片连续提供了大肠杆菌超低温切片的碳像。杆菌由于非碳膜的强度足以支撑一个完整的细菌E. coli，膜的表面粒度较细。通过冷冻断裂的膜痉挛的方法也是理想的，以评估胆固醇在膜损伤中与成孔毒素的作用。结合以上三种方法，阐明细胞毒素致孔机理的研究取得了进展。

The damage to cell membranes by bacterial fore-forming-toxins such as streptolysin O (SLO) and botulinolysin (BLY) was studied ultrastructurally by electron spectroscopic imaging (ESI). The newly developed technique of ESI provides the fine molecular images with a low noise-to-signal ratio even if it is on a supporting film. An imaging plate (IP) combined with ESI decreases the electron beam exposure on the biological specimens to one hundredth since they are very susceptible. The ring formed by SLO is a double and coaxial ring-shaped structure. The SLO molecules of two types which construct one ring are identical by the different iso-electric point (pI). While the BLY ring formed on erythrocyte membranes is observed as a similar structure of SLO ring, it is obviously constructed by BLY molecules of a single type evaluated by immuno-electrophoresis. The application of ESI offers additionally the successful data from the extensive biological specimens such as human hair, ferritin particles and hydroxyapatite crystals. To observe a carbon image of biological specimens, we developed a non-carbon supporting film made by silicon dioxide. The film offers successively an carbon image of ultra-cryosection of E. coli. It is expected to use the non-carbon film to the study of the pore-forming toxins in the future since the film is strong enough to support a whole bacterium, E. coli, the granularity of surface of the film is fine. A method of membrane-cramp by freeze-fracture is also ideal to evaluate the role of cholesterol in membrane damage with the pore-forming toxins. As combining above three methods, the study to clarify the mechanism of pore-formation with cytolitic toxins has been progressing.