REGULATION OF CYCLIC NUCLEOTIDE METABOLISM

毒素似乎在一定程度上导致了临床表现 有些疾病是由于细菌感染引起的。百日咳毒素，一种分泌物 百日咳杆菌产物与百日咳发病机制的关系 咳嗽，和霍乱毒素(CT)，霍乱毒素的病原体，施加 它们通过鸟嘌呤核苷酸的ADP-核糖化作用对细胞产生影响。 结合(G)蛋白是细胞表面信号传递的关键 细胞内靶标的受体(如腺苷环化酶)。 霍乱毒素由一个A亚基组成，它是一种ADP- 核糖基转移酶和五个将毒素与其细胞结合的B亚基 表面受体，神经节苷脂GM1。A亚基的底物 疾病似乎是刺激性G蛋白Gs的阿尔法亚单位 腺酰环化酶系统和一个可能调节离子的G蛋白 流量。霍乱毒素还催化简单的ADP-核糖化 胍基化合物(如胍丁胺、精氨酸)，与 Gsalpha，可能是由于存在一种可接近的精氨酸 在自动-ADP-核糖化反应中，残基和它自己的A亚基。全 ARF增强了毒素催化的反应，这与其 建议充当CTA1的变构激活剂。目前的研究是 采用体外诱变技术检测氨基酸残留量 这可能参与了霍乱毒素对G蛋白的识别。 假设对Gsalpha的承认应该需要 比ADP更多的蛋白质结构的参与- 简单的胍基化合物的核糖化，如胍丁胺。基于 这个模型假设CTA1可能存在突变， 会优先影响Gs的ADP-核糖化，而不是ADP- 胍丁胺的核糖化。基于霍乱之间的结构类比 毒素和其他细菌毒素，如百日咳毒素，一些 在大肠杆菌中合成了CTA1突变。检查了突变情况 它们对ADP-核糖基化苷类、Gsalpha和转导蛋白的能力(或 Gtalpha)以及各种形式的自动ADP-核糖化 磷脂/洗涤剂混合物。几个突变(例如，在位置 7、44和112)导致酶活性的总体丧失。 然而，天冬酰胺替代组氨酸-70降低了ADP-70的活性。 核糖基-Gsalpha的合成多于ADP-核糖苷丁胺的合成。 这些和其他数据表明，组氨酸-70可能参与了 霍乱毒素对Gsalpha的特异性识别。

Toxins appear to be responsible in part for the clinical presentation of some diseases due to bacterial infection. Pertussis toxin, a secretory product of Bordetella pertussis involved in the pathogenesis of whooping cough, and cholera toxin (CT), an etiologic agent in cholera toxin, exert their 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). Cholera toxin consists of one A subunit, which is an ADP- ribosyltransferase, and five B subunits which bind the toxin to its cell surface receptor, ganglioside GM1. A substrate for the A subunit in disease appears to be the alpha subunit of Gs, the stimulatory G protein of the adenylyl cyclase system and a G protein that may regulate ion flux. Cholera toxin also catalyzes the ADP-ribosylation of simple guanidino compounds (e.g., agmatine, arginine), proteins unrelated to Gsalpha, presumably due to the presence of an accessible arginine residue, and its own A subunit in an auto-ADP-ribosylation reaction. All toxin-catalyzed reactions are enhanced by ARF, consistent with its proposed role as an allosteric activator of CTA1. The present study was undertaken to determine, using in vitro mutagenesis, amino acid residues that may participate in recognition of the G protein by cholera toxin. The assumption was made that recognition of Gsalpha should require the participation of more of the protein structure than would ADP- ribosylation of a simple guanidino compound such as agmatine. Based on this model it was hypothesized that mutations of CTA1 might exist which would preferentially affect ADP-ribosylation of Gs rather than ADP- ribosylation of agmatine. Based on structural analogies between cholera toxin and other bacterial toxins such as pertussis toxin, a number of CTA1 mutations were synthesized in E. coli. Mutations were examined for their ability to ADP-ribosylate agmatine, Gsalpha, and transducin (or Gtalpha) as well as to auto-ADP-ribosylate in various phospholipid/detergent mixtures. Several mutations (e.g., at positions 7, 44, and 112) resulted in overall loss of enzymatic activity. Substitution of asparagine for histidine-70, however, diminished ADP- ribosyl-Gsalpha formation more than it did ADP-ribosylagmatine synthesis. These and other data suggest that histidine-70 may be involved in specific recognition of Gsalpha by cholera toxin.