REGULATION OF CYCLIC NUCLEOTIDE METABOLISM

霍乱毒素(CT)是霍乱的一种病原体，对 通过鸟嘌呤核苷酸结合的ADP核糖化作用的细胞(G) 细胞表面受体信号转导的关键蛋白质 它们的细胞内靶标(如腺苷环化酶)。这种毒素由 一个A亚基是ADP-核糖基转移酶，五个B亚基 亚基，将毒素与细胞表面神经节苷脂GM1结合。这个 A亚基的酶活性是潜伏性的，活性需要 蛋白质的降解和单一二硫键的还原导致 形成具有催化活性的A1蛋白和小得多的A2 从A亚基的羧基末端衍生的蛋白质。活动 由20 kDa ADP-核糖化因子或 ARF，以依赖GTP的方式。(1)确定潜伏期是否 该毒素表达ARF激活位点，这是一种不活跃的突变形式 大肠杆菌不耐热肠毒素(LT)，含有一种关键的谷氨酸 A亚基第112位的赖氨酸取代被用作 含有限制性ARF的霍乱毒素检测中的竞争对手。这个 全菌毒素不能显著抑制ARF刺激的血管紧张素转换酶 CT A亚基ADP-核糖基转移酶活性。胰酶消化和 LT的降低导致了一种有效的抑制作用；单独降低LT并不能。 胰酶消化的LT不能抑制ARF对纯化的、烷化的 CTA1蛋白，它的活性不依赖于还原。这些 研究结果与CT上ARF部位为 在潜伏的毒素中是不容易接近的。释放CT A1蛋白，这是 是表达ADP-核糖基转移酶活性所必需的，也是 出现ARF结合位点所必需的。(2)霍乱毒素 A1蛋白ADP-核糖化G蛋白中的精氨酸残基。哺乳动物 细胞含有被称为ADP核糖精氨酸水解酶的酶， 切断ADP-核糖-精氨酸键，释放游离精氨酸，并可能 在霍乱的康复过程中发挥作用。检查结构的步骤 水解酶的特性和保守性，它们是从大鼠中克隆的， 老鼠和人类的来源。这些物种的水解酶表现出 相当大的氨基酸同一性。

Cholera toxin (CT), an etiologic agent in cholera, exerts its effect on cells through the ADP-ribosylation of guanine nucleotide-binding (G) proteins that are critical for signaling from cell surface receptors to their intracellular targets (e.g., adenylyl cyclase). The toxin consists of one A subunit, which is an ADP-ribosyltransferase and five B subunits, which bind the toxin to cell surface ganglioside GM1. The enzymatic activity of the A subunit is latent, and activity requires proteolysis and reduction of a single disulfide resulting in the formation of a catalytically active A1 protein and a much smaller A2 protein derived from the carboxy terminus of the A subunit. The activity of the A1 protein is enhanced by 20 kDa ADP-ribosylation factors or ARFs, in a GTP-dependent manner. (1) To determine if the latent form of the toxin expresses an ARF activation site, an inactive mutant form of E. coli heat-labile enterotoxin (LT), with a critical glutamate to lysine substitution at position 112 of the A subunit, was utilized as a competitor in a cholera toxin assay containing limiting ARF. The holotoxin did not significantly inhibit the ARF-stimulated ADP-ribosyltransferase activity of CT A subunit. Trypsinization and reduction of LT resulted in a potent inhibitor; reduction alone did not. Trypsinized LT did not inhibit ARF stimulation of a purified, alkylated CTA1 protein, which is not dependent on reduction for activity. These studies are consistent with the hypothesis that the ARF site on CT is not accessible in the latent toxin. Release of the CT A1 protein, which is necessary for expression of ADP-ribosyltransferase activity, is also required for appearance of the ARF binding site. (2) The cholera toxin A1 protein ADP-ribosylates arginine residues in G proteins. Mammalian cells contain enzymes, termed ADP-ribosylarginine hydrolases, that cleave the ADP-ribosyl-arginine bond, releasing free arginine and could play a role in recovery from cholera. To examine the structural features and conservation of hydrolases, they were cloned from rat, mouse and human sources. The hydrolases from these species exhibited considerable amino acid identity.