体外和体内合成的聚（ADP - 核糖）的免疫学测定及大小表征

Immunological determination and size characterization of poly(ADP-ribose) synthesized in vitro and in vivo.

作者信息

Affar E B, Duriez P J, Shah R G, Winstall E, Germain M, Boucher C, Bourassa S, Kirkland J B, Poirier G G

机构信息

Poly(ADP-ribose) Metabolism Group, Health and Environment Unit, Laval University Medical Research Center, CHUQ, 2705, Boul. Laurier, Ste-Foy, Quebec G1V 4G2, Canada.

出版信息

Biochim Biophys Acta. 1999 Aug 5;1428(2-3):137-46. doi: 10.1016/s0304-4165(99)00054-9.

DOI:10.1016/s0304-4165(99)00054-9

PMID:10434031

Abstract

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells.

摘要

聚（ADP - 核糖）聚合酶是一种DNA断裂检测酶，在基因组完整性监测中发挥作用。聚（ADP - 核糖）是在DNA损伤剂作用下由β - NAD迅速且短暂合成的。为了研究聚（ADP - 核糖）代谢的生理意义，我们开发了免疫方法，这些方法能使我们在不干扰细胞代谢和完整性的情况下研究内源性聚（ADP - 核糖）。为此，我们制备了一种高度特异性的抗聚（ADP - 核糖）多克隆抗体，它能与聚合物和寡聚物发生免疫反应。除了我们最近描述的免疫斑点印迹法（Affar等人，《分析生物化学》259（1998）280 - 283），还在细胞中研究了其他应用：（i）通过酶联免疫吸附测定法检测聚（ADP - 核糖）；（ii）使用聚合物的高分辨率凝胶电泳对聚（ADP - 核糖）大小进行表征，随后将其转移到带正电荷的膜上并用抗聚（ADP - 核糖）抗体进行检测；（iii）免疫细胞化学和流式细胞术分析，可在单个细胞水平上研究聚（ADP - 核糖）。