REGULATION OF CYCLIC NUCLEOTIDE METABOLISM

细菌毒素，霍乱毒素和百日咳毒素，是 部分原因分别是霍乱和百日咳的发病机制。这个 毒素通过鸟嘌呤的ADP-核糖化作用对细胞产生影响 核苷酸结合(G)蛋白是信号转导的关键 细胞内靶标的受体(如腺苷环化酶)。 与细菌毒素类似的ADP-核糖基转移酶存在于 动物细胞。也存在ADP-核糖精氨酸水解酶，酶 这会切断ADP-核糖-蛋白质的连接。这样看来，在 动物细胞中，存在一个ADP-核糖化循环，它催化 蛋白质的可逆修饰。观察到水解酶活性 多种动物物种，其中大鼠和 小白鼠高于豚鼠和小牛。在大鼠组织中，比活性是 脑、脾和睾丸高于肺、心、肝和骨骼 肌肉。大鼠脑水解酶纯化约20,000倍， 十二烷基硫酸钠-聚丙烯酰胺凝胶显示一条主带 与大约39 kDa的蛋白质一致。兔抗鼠抗体 水解酶多克隆抗体识别约30 kDa的蛋白质 在部分纯化的大鼠、小鼠和小牛的水解酶制剂中 大脑和火鸡红细胞。根据从以下来源获得的氨基酸序列 纯化蛋白、寡核苷酸和聚合酶链式反应 合成了(聚合酶链式反应)产生的cDNA探针，并用于筛选大鼠脑 Lambda ZAP库。来自两个独立克隆的插入产生了一个 1086个碱基的复合开放阅读框。表达了水解酶基因 在大肠杆菌中作为融合蛋白，显示出镁离子和二硫苏糖醇- 依赖活性类似于大鼠脑酶的活性。一个 聚合酶链式反应产生的编码区基因在Northern上与1.7kb的mRNA杂交 分析大鼠和小鼠的Poly(A)+RNA，但不包括人、牛或 兔组织和细胞。数据与部分数据兼容 动物物种间水解酶结构的保守性。

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.