REGULATION OF CYCLIC NUCLEOTIDE METABOLISM

细菌毒素，霍乱毒素和百日咳毒素， 分别与霍乱和百日咳的发病机制有关。 的 毒素通过鸟嘌呤的ADP-核糖基化作用对细胞发挥作用 核苷酸结合（G）蛋白，其对于从 其细胞内靶的受体（例如，腺苷酸环化酶）。 与细菌毒素类似的ADP-核糖基转移酶存在于 动物细胞 还存在ADP-核糖基精氨酸水解酶、酶 切断ADP-核糖-蛋白质的连接 因此，看来， 在动物细胞中，存在ADP-核糖基化循环，其催化 蛋白质的可逆修饰。 水解酶活性观察'， 在多种动物物种中，大鼠和 小鼠比豚鼠和小牛。 在大鼠组织中， 脑、脾和睾丸中的含量高于肺、心、肝和骨骼 肌肉. 大鼠脑水解酶纯化约20，000倍， 在十二烷基硫酸钠-聚丙烯酰胺凝胶上显示一条主带 与大约39 kDa的蛋白质一致。 兔抗大鼠 水解酶多克隆抗体识别约30 kDa的蛋白质 在来自大鼠、小鼠和小牛的部分纯化的水解酶制剂中 大脑和火鸡红细胞。 基于从以下获得的氨基酸序列： 纯化蛋白、寡核苷酸和聚合酶链反应 合成PCR产生的cDNA探针，并用于筛选大鼠脑 Lambda ZAP库。 从两个独立的克隆中分离得到一个 复合开放阅读框为1086 bp。 表达了该水解酶cDNA 在大肠大肠杆菌作为融合蛋白，表现出Mg 2 +-和二硫苏糖醇- 依赖活性类似于大鼠脑酶。 一 PCR产生的编码区cDNA与北方上的1.7 kb mRNA杂交 分析大鼠和小鼠的poly（A）+ RNA，但不分析人、牛或 兔组织和细胞。 数据与部分兼容 在动物物种中保持水解酶结构。

The bacterial toxins, cholera toxin and pertussis toxin, are responsible in part for the pathogenesis of cholera and pertussis, respectively. The toxins exert their effects on cells through the ADP-ribosylation of guanine nucleotide-binding (G) proteins that are critical for signaling from receptors to their intracellular targets (e.g., adenylyl cyclase). ADP-ribosyltransferases similar to the bacterial toxins are present in animal cells. Also present are ADP-ribosylarginine hydrolases, enzymes that cleave the ADP-ribose-protein linkage. It would thus appear that, in animal cells, an ADP-ribosylation cycle exists which catalyzes the reversible modification of proteins. Hydrolase activity was observed 'in a variety of animal species, with significantly higher levels in rat and mouse than in guinea pig and calf. In rat tissues, specific activity was higher in brain, spleen and testis than in lung, heart, liver and skeletal muscle. The rat brain hydrolase was purified approximately 20,000-fold and exhibited one major band on sodium dodecyl sulfate-polyacrylamide gels consistent with a protein of approximately 39 kDa. Rabbit anti-rat hydrolase polyclonal antibodies recognized proteins of approximately 30 kDa in partially purified hydrolase preparations from rat, mouse, and calf brains and turkey erythrocytes. Based on amino acid sequence obtained from the purified protein, oligonucleotide and polymerase chain reaction (PCR)-generated cDNA probes were synthesized and used to screen a rat brain Lambda ZAP library. Inserts from two independent clones yielded a composite open reading frame of 1086 bp. The hydrolase cDNA was expressed in E. coli as a fusion protein that exhibited a Mg2+- and dithiothreitol- dependent activity similar to that of the rat brain enzyme. A PCR-generated coding region cDNA hybridized to a 1.7 kb mRNA on Northern analysis of poly(A)+ RNA from rat and mouse but not human, bovine, or rabbit tissues and cells. The data are compatible with partial conservation of hydrolase structure across animal species.