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

采用电子能谱成像（ESI）技术对细菌形成前毒素链溶素O （SLO）和肉毒杆菌素（BLY）对细胞膜的损伤进行了超微结构研究。新开发的ESI技术即使在支撑膜上也能提供低信噪比的精细分子图像。成像板（IP）与ESI相结合，使生物标本的电子束暴露减少到百分之一，因为它们非常容易受到影响。SLO形成的环为双同轴环形结构。构成一个环的两类SLO分子因等电点（pI）不同而相同。虽然在红细胞膜上形成的BLY环与SLO环结构相似，但经免疫电泳鉴定，它显然是由单一类型的BLY分子构建的。ESI的应用还为人类头发、铁蛋白颗粒和羟基磷灰石晶体等广泛的生物标本提供了成功的数据。为了观察生物标本的碳像，我们研制了一种由二氧化硅制成的无碳支撑膜。该胶片连续提供了大肠杆菌超低温切片的碳图像。由于膜的强度足以支撑整个细菌大肠杆菌，膜表面粒度细，因此有望在未来将非碳膜用于成孔毒素的研究。冻裂致膜痉挛的方法也是评价胆固醇在成孔毒素致膜损伤中的作用的理想方法。随着以上三种方法的结合，阐明胞质毒素的成孔机制的研究不断取得进展。

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.