MECHANISM OF ACTION OF C PERFRINGENS ENTEROTOXIN

产气荚膜梭菌肠毒素（CPE）是一种单链多肽 35，000人的CPE先生已被证明会产生与以下疾病相关的所有症状： C.产气荚膜杆菌食物中毒和最近的研究强烈表明， CPE也可能涉及其他形式的严重胃肠道疾病。 疾病CPE具有独特的作用，与所有其他 肠毒素CPE产生膜渗透性的变化，导致 二次效应，如抑制大分子合成。 拟议研究的主要目标是扩大对 CPE在生物化学/分子水平上的作用。为了实现这一 目的：提出以下研究：1）识别和 通过使用受体纯化纯化CPE受体， 抗体反应性研究和CPE：CPE受体交联。（二） 将CPE插入肠刷状缘膜（PFM）中， 进一步研究使用光反应膜探针和蛋白酶- 释放研究。在CPE行动中插入的重要性将是 测定3)将研究CPE的作用，以确定CPE是否 除了插入之外还有其他作用这将由低- 温度研究、蛋白脂质体实验和 结肠细胞（这些细胞结合但不响应CPE）。4)完整 将克隆CPE基因并进行DNA测序。克隆的基因和基因 片段将用作Southern或北方印迹中的探针 实验研究CPE的合成和调控。5)定义的突变 将被工程化到CPE基因或基因片段中， 表达，这些突变对CPE活性的影响， 特别是在绑定，将被确定，以帮助解剖步骤在CPE 行动上 这些研究应该大大增加我们对CPE如何 作用于分子水平。增加对CPE行动的了解应 允许CPE作用与其他途径使用的有价值的比较 引起肠道疾病的病原体。由于CPE的膜作用， 澄清，CPE也应该成为一个有用的探针检查 肠BBM的结构/功能关系这些发现 对许多寻求 了解正常和疾病条件下的肠道生理学。 此外，这些研究可能有助于改进诊断分析 并可能导致CPE疫苗的开发。

Clostridium perfringens enterotoxin (CPE) is a single polypeptide chain of 35,000 Mr. CPE has been shown to produce all symptoms associated with C. perfringens food poisoning and recent studies strongly suggest that CPE may also be involved in other forms of serious gastrointestinal disease. CPE has a unique action distinguishable from all other enterotoxins. CPE produces changes in membrane permeability which lead to secondary effects, such as inhibition of macromolecular synthesis. The principal goal of the proposed research is to extend understanding of the action of CPE to the biochemical/molecular level. To accomplish this goal the following research is proposed; 1) Identification and purification of the CPE receptor by use of receptor purification, antibody reactivity studies and CPE: CPE receptor crosslinking. 2) Insertion of CPE into intestinal brush border membranes (PFMs) will be further investigated using photoreactive membrane probes and protease- release studies. The importance of insertion in CPE action will be determined. 3) The action of CPE will be investigated to determine if CPE has any action beyond insertion. This will be determined by low- temperature studies, proteoliposome experiments and experiments with colonic cells (these cells bind but do not respond to CPE). 4) The intact CPE gene will be cloned and DNA sequenced. The cloned gene and gene fragments will be used as a probe in Southern or Northern blotting experiments to study CPE synthesis and regulation. 5) Defined mutations will be engineered into the CPE gene or gene fragments and, after expression, the effects of these mutations on CPE activities, particularly on binding, will be determined to help dissect steps in CPE action. These studies should significantly increase our understanding of how CPE acts at the molecular level. Increased knowledge of CPE action should allow valuable comparisons of CPE action with pathways used by other agents producing intestinal disease. As the membrane action of CPE is clarified, CPE should also become a useful probe to examine structure/function relationships in intestinal BBMs. These findings would have long-term significance to numerous medical disciplines seeking to understand intestinal physiology under normal and disease conditions. Additionally, these studies may contribute to improved diagnostic assays for CPE and possibly lead to development of a CPE vaccine.