Study on recognition mechanism of heat-stable enterotoxin by its receptorprotein

耐热肠毒素(STS)是由产肠毒素大肠杆菌产生的由18或19个氨基酸残基组成的多肽，已知可引起人和动物的急性腹泻。ST的生物学作用是通过与肠上皮细胞膜上的ST受体蛋白结合而启动的。ST受体复合体的形成导致鸟苷环化酶的刺激，随之而来的是细胞内GMP浓度的增加和细胞内液体的分泌。本研究拟鉴定ST与ST结合的受体蛋白结合部分，提纯并结晶ST与其受体蛋白的复合物j，用于其X射线结晶学分析，以阐明ST与其受体蛋白的相互识别机制，这是ST通过细胞膜上的受体蛋白进行信号转导的第一步。为了达到这一目的，我合成了各种ST类似物，并用荧光团标记了这些ST类似物，并用层析的方法检测了它们的结合能力。其中有两个ST类似物与受体蛋白的结合能力与天然ST相当，其中一个类似物与大鼠肠道膜上的受体蛋白形成了络合物。在考察了络合物的增溶实验条件后，将几种高效液相色谱联用，得到了部分纯化的荧光络合物，了解了受体蛋白与ST的结合部分，用荧光团标记的ST交联的蛋白质的纯化正在进行中。

Heat-stable enterotoxins (STs) are peptides of 18 or 19 amino acid residues produced by enterotoxigenic Escherichia coli and are known to cause acute diarrhea in men and animals. The biological action of ST is initiated by the binding to its receptor protein on the membrane of intestinal epithelial cell. The formation of a ST-receptor complex leads to stimulation of guanylate cyclase and is followed by increase in GMP concentration in the cells and fluid secretion from the cells. This study is planned to identify the binding part of the receptor protein in its binding to ST, purify and crystallize the complex j of ST with its receptor protein for its X-ray crystallographic analysis in order to elucidate the co-recogniton mechanism between ST and its receptor protein which is the first step of signal transduction by ST via the receptor protein on the membrane of cells. To accomplish this purpose, I synthesized a variety of ST analogues were labeled with the fluorophore and cific binding abilties of these ST analogues were examined by means of chromatographic method. Among these, there were two ST analogues with the specific binding ability to the receptor protein corresponding to that of the native ST.One of these analogues was used to form the complex with the receptor protein on the membrane prepared from rat intestine. After solubilization experimental conditions to purify the complex were examined and the conbination of several kinds of HPLC lead to obtain a partially purified complex with fluorescence to get the knowledge about the binding part of receptor protein to ST, the purification of the protein crosslinked with ST labeled with the fluorophore is in progress.