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REGULATION SYSTEM OF ADP-RIBOSYLTRANSFERASE ACTIVITY OF CHOLERA TOXING BY ARE AND ARI :

ARE和ARI对霍乱中毒的ADP-核糖基转移酶活性的调节系统：

基本信息

批准号：
03454180
负责人：
NODA Masatoshi
金额：
$ 4.03万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

Cholera toxin, a secretory product of Vibrio cholerae, causes the diarrheal syndrome characteristic of cholera by activating the adenylate cyclase of intestinal mucosal cells resulting in the elevatin of cell cAMP content that is responsible for effects on fluid and electroyte transport. Cholera toxin, by transferring ADP-ribose from NAD to a critical amino acid in the alpha subunit of Gs, enhances the ability of Gs to activate the cyclase catalytic unit. Toxin-catalyzed ADP-ribosylation of Gschi can be enhanced by several membrance or soluble factors. A 20 kDa protein termed ARF, ADP-ribosylation factor, that enhances the toxin-catalyzed modification in a reconstituted system containing purified Gsalpha was a guanine nucleotide-binding protein. It appears that ARF GTP, by interacting directly with A subunit of cholera toxin, alters the allosteric properties of the toxin resulting in increased catalytic activity with subsaturating substrates. A 20 kDa membrane protein termed ARI, ADP-ribosylation inhibitor, that inhibits cholera toxin-catalyzed modification was purified and the mechanism of inhibition of cholera toxin-catalyzed ADP-ribosylation by ARI was studied. To determine the mechanism of inhibitory effect of ARI on cholera toxin-catalyzed ADP-ribosylation, a Lineweaver-Burk analysis was performed specifically for observing NAD : agmatine ADP-ribosyltransferase activity of cholera toxin A subunit in the presence and absence of ARI.ARI had no effect on the Km for NAD, but significantly reduced the maximal velocity of the reaction. In addition to examining the effect of ARI on NAD, the kinetics of ADP-ribosylation using agmatine as a variable substrate was studied in the presence and absence of ARI.These was a significant increase in the Km for agmatine and a decrease in the maximal velocity of the reaction. These data show that ARI decreases reaction rate and substrate affinity of cholera toxin A subunit for agmatine and that ARI has no effect on the substrate

霍乱毒素是霍乱弧菌的一种分泌产物，它通过激活肠粘膜细胞的腺苷酸环化酶，导致细胞cAMP含量升高，从而影响液体和电解质的转运，从而引起霍乱特有的腹泻综合征。霍乱毒素通过将ADP-核糖从NAD转移到Gs α亚基中的关键氨基酸，增强Gs激活环化酶催化单位的能力。几种膜因子或可溶性因子可以增强毒素催化的Gschi ADP核糖基化。一个20 kDa的蛋白质称为ARF，ADP-核糖基化因子，增强了毒素催化的修饰，在含有纯化的Gsalpha的重建系统是一个鸟嘌呤核苷酸结合蛋白。看来，ARF GTP，通过直接与霍乱毒素的A亚基相互作用，改变了毒素的变构特性，导致与亚饱和底物的催化活性增加。纯化了一种能抑制霍乱毒素催化的ADP-核糖基化修饰的20 kDa膜蛋白ARI，并研究了ARI抑制霍乱毒素催化的ADP-核糖基化的机制。为探讨ARI抑制霍乱毒素催化ADP核糖基化反应的机理，采用Lineweaver-Burk法，观察了ARI对霍乱毒素A亚基NAD：胍丁胺ADP核糖基转移酶活性的影响，结果表明ARI对NAD的Km值无影响，但显著降低了反应的最大速率。除了研究ARI对NAD的影响外，还研究了ARI存在和不存在时胍丁胺作为可变底物的ADP-核糖基化动力学，胍丁胺的Km显著增加，反应的最大速度降低。这些数据表明ARI降低了霍乱毒素A亚单位对胍丁胺的反应速率和底物亲和力，而ARI对底物没有影响