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

热稳定肠毒素（ST）是由肠毒素性大肠杆菌产生的18或19个氨基酸残基的肽，并且已知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受体蛋白结合而启动的。ST-受体复合物的形成导致鸟苷酸环化酶的刺激，随后是细胞中GMP浓度的增加和细胞的液体分泌。本研究拟鉴定ST与受体蛋白结合的结合部位，纯化并结晶ST与受体蛋白的复合物j，进行X-射线晶体学分析，以阐明ST通过细胞膜上的受体蛋白进行信号转导的第一步--ST与受体蛋白的共结合机制。为了达到这个目的，我合成了一系列的ST类似物，用荧光团标记它们，并通过色谱法检测它们的特异性结合能力。其中，有两个ST类似物与受体蛋白的特异性结合能力对应的天然ST。这些类似物之一是用来形成复合物与受体蛋白的膜上制备的大鼠肠。通过增溶实验考察了复合物的纯化条件，并结合多种高效液相色谱技术，得到了部分纯化的具有荧光的复合物，以了解受体蛋白与ST的结合部位，荧光标记ST交联蛋白的纯化工作正在进行中。

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.