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ANALYSIS OF ADP-RIBOSYLATION ON MOUSE LYMPHOCYTES USING BIOTINYL NAD

使用生物素 NAD 分析小鼠淋巴细胞上的 ADP-核糖基化

基本信息

批准号：
08680687
负责人：
TSUCHIYA Mikako
金额：
$ 1.54万
依托单位：
SHIMANE MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680687/
关键词：
ADP-ribosyltransferase ADP-ribosylation biotinyl NAD lymphocytes

项目摘要

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.

生物素NAD由5-(N-琥珀酰亚胺氧基羰基)戊基D-生物素酰胺和N6[[(6-氨基己基)氨基甲酰基]甲基]NAD制备，通过HPLC纯化，并被证实是鸡和小鼠ADP-核糖基转移酶催化的ADP-核糖基化的底物。当使用生物素NAD作为底物检测鸡血浆中tuftsin的特异性AD-核糖基化时，生物素-亲和素-过氧化物酶反应未检测到肽的修饰。与生物素NAD孵育后，用FITC-亲和素对小鼠淋巴细胞进行染色并通过细胞分选仪进行分析。通过在标记反应中添加ADP-核糖、NAD或精氨酸或在标记后进行ADP-核糖精氨酸水解酶处理，细胞的荧光标记强度不会降低。不具有ADP-核糖基转移酶的细胞也被染色至类似程度。因此，标记并不能准确反映在我们用于细胞染色和洗涤的条件下淋巴细胞上发生的 ADP-重糖基化，可能是因为生物素 NAD 的非特异性和非共价结合。为了确定其靶蛋白具有 ADP-核糖基转移酶的淋巴细胞子集，可能需要更严格的条件进行标记和洗涤。