CHARACTERIZATION OF MAMMALIAN ADP-RIBOSYLTRANSFERASES

单adp核糖基化是一种蛋白质的翻译后修饰，其中NAD的adp核糖部分被转移到蛋白质中，并负责某些细菌毒素的毒性（例如霍乱毒素，百日咳毒素）。与霍乱毒素类似，一些哺乳动物ADP-核糖基转移酶特异性地使用精氨酸的胍基作为ADP-核糖受体。从不同组织中克隆了5种哺乳动物NAD：精氨酸ADP-核糖基转移酶（ART）。ART1被证明是一种细胞表面蛋白，通过糖基磷脂酰肌醇（GPI）锚点连接。转移酶似乎在哺乳动物组织中选择性表达。ART-1存在于骨骼肌、心肌和淋巴样细胞中，ART-2存在于淋巴细胞中，ART-4存在于脾脏，art -5存在于睾丸中。ART-5主要是一种NAD糖水解酶，而NAD：精氨酸ADP-核糖基转移酶则明显较少。在标准实验条件下与NAD孵育后，在存在或不存在精氨酸类似物agmatine的情况下，NAD糖水解酶活性逐渐下降；相反，adp -核糖基转移酶的活性在短暂的延迟后，速度增加，然后，大约一小时后，似乎在几个小时内保持不变。NAD糖水解酶活性的丧失和adp -核糖基转移酶活性的增加与十二烷基硫酸钠-聚丙烯酰胺凝胶上出现自adp -核糖基化形式的ART-5有关。一个adp -核糖基化似乎足以引起NAD糖水解酶和自adp -核糖基转移酶活性的剧烈变化。然而，孵育超过一小时，会导致adp核糖片段的进一步增加。为了验证自动adp核糖化的ART-5在酶活性上是否存在差异，将转移酶与NAD孵育1或8小时，然后通过两根PD-10凝胶渗透柱进行顺序层析纯化。与对照组相比，自ADP-核糖基化酶表现出ADP-核糖基转移酶活性增加和NAD糖水解酶活性降低。质谱法证实了与NAD孵育的ART-5被adp -核糖修饰的事实，其质量单位增加了541个。这些数据支持了自ADP-核糖基化可以发挥调节作用并决定NAD糖水解酶和ADP-核糖基转移酶活性的相对水平的假设。-单ADP-核糖基化，ADP-核糖基转移酶，细菌毒素

Mono-ADP-ribosylation is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to proteins and is responsible for the toxicity of some bacterial toxins (e.g., cholera toxin, pertussis toxin). Similar to cholera toxin, some mammalian ADP- ribosyltransferases specifically use the guanidino group of arginine as an ADP-ribose acceptor. Five mammalian NAD: arginine ADP- ribosyltransferases (ART) were cloned from various tissues. ART1 was shown to be a cell surface protein, linked through a glycosylphosphatidylinositol (GPI) anchor. The transferases appear to be selectively expressed in mammalian tissues. ART-1 is found in skeletal and cardiac muscle and lymphoid cells, ART-2 in lymphocytes, ART-4 in spleen andART-5 in testis. ART-5 is primarily an NAD glycohydrolase, with significantly less NAD: arginine ADP- ribosyltransferase. Upon incubation with NAD under standard assay conditions, in the presence or absence of agmatine, an arginine analog, the NAD glycohydrolase activity gradually declines; in contrast, the ADP-ribosyltransferase, activity after a brief delay, increases in velocity, and then, after about one hour, appears to be constant for several hours. The loss of NAD glycohydrolase activity and the increase in ADP-ribosyltransferase activity is associated with the appearance on sodium dodecyl sulfate-polyacrylamide gels of an auto-ADP-ribosylated form of ART-5. One ADP-ribosylation appears to be sufficient for the dramatic changes in NAD glycohydrolase and auto-ADP-ribosyltransferase activities. Incubation for greater than one hour, however, results in the further addition of ADP-ribose moieties. To verify that the auto- ADP-ribosylated ART-5 exhibited differences in enzymatic activities, the transferase was incubated with NAD for one or eight hours, then purified by sequential chromatography over two PD-10 gel permeation columns. The auto-ADP-ribosylated enzyme exhibited an increase in ADP- ribosyltransferase activity and decrease in NAD glycohydrolase when compared to control. The fact that ART-5 incubated with NAD was modified with ADP-ribose was verified by mass spectrometry, which demonstrated an increase of 541 mass units. These data support the hypothesis that the auto-ADP-ribosylation can serve a regulatory role and determine the relative levels of NAD glycohydrolase and ADP- ribosyltransferase activities. - Mono-ADP-ribosylation, ADP- ribosyltransferases, bacterial toxins