ANALYSIS OF ADP-RIBOSYLATION ON MOUSE LYMPHOCYTES USING BIOTINYL NAD

以5-(N-琥珀酰亚胺氧甲酰基)戊基D-生物胺和N6[[(6-氨基己基)氨基甲酰基]甲基]NAD为原料合成了生物素化NAD，经高效液相色谱分离纯化，证实为鸡和小鼠ADP-核糖基转移酶催化ADP-核糖化的底物。当以生物素NAD为底物检测鸡血浆中Tuftsin的AD-核糖化时，生物素-亲和素-过氧化酶反应不能检测到该多肽的修饰。小鼠淋巴细胞与生物素NAD孵育后，用FITC-亲和素染色并用细胞分选仪进行分析。在标记反应中加入ADP-核糖、NAD或精氨酸，或在标记后加入ADP-核糖精氨酸水解酶，细胞的荧光标记强度不会降低。没有ADP-核糖基转移酶的细胞也有类似程度的停滞。因此，在我们用于细胞染色和洗涤的条件下，标记并不能准确地反映淋巴细胞上发生的ADP再糖基化，这可能是因为生物素NAD的非特异性和非共价结合。要确定具有其目标蛋白的ADP核糖基转移酶的淋巴细胞亚群，可能需要更严格的条件来标记和洗涤。

Biotinyl NAD was prepared from 5- (N-succinimidyloxycarbonyl) pentyl D-biotinamide and N6 [[(6-aminohexyl) carbamoyl] methyl] NAD,purified by HPLC and confirmed to be a substrate for ADP-ribosylation catalyzed by chicken and mouse ADP-ribosyltransferases. When biotinyl NAD was used as a substrate for detection of specific AD-ribosylation of tuftsin in chicken plasma, the modification of the peptide was not detected by biotin-avidin-peroxydase reaction. After incubated with biotinyl NAD,mouse lymphocytes were stained with FITC-avidin and analyzed by cell sorter. The intensity of the fluorescent labeling of the cells did not decrease by addition of ADP-ribose, NAD or arginine in the labeling reaction or ADP-ribosylarginine hydrolase treatment after the labeling. The cells which do not have the ADP-ribosyltransferase were also stianed to a similar extent. Thus, the labeling does not exactly reflect the ADP-reibosylation occurring on the lymphocytes under the conditions we used for staining and washing of cells, probably because of non-specific and non-covalent binding of biotinyl NAD.To determine the subset of lymphocytes which have ADP-ribosyltransferase of its target protein, more stringent conditions may bed needed for labeling and washing.