Ida Chieri, Yamashita Sachiko, Tsukada Masaki, Sato Teruaki, Eguchi Takayuki, Tanaka Masakazu, Ogata Shin, Fujii Takahiro, Nishi Yoshisuke, Ikegami Susumu, Moss Joel, Miwa Masanao
Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan; Department of Applied Life Studies, College of Nagoya Women's University, Nagoya-shi, Aichi 467-8610, Japan.
Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
Anal Biochem. 2016 Feb 1;494:76-81. doi: 10.1016/j.ab.2015.10.014. Epub 2015 Nov 6.
PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.
多聚ADP核糖基化由多聚（ADP-核糖）（PAR）聚合酶（PARP）介导，可能参与多种细胞活动，包括染色体稳定性、DNA修复、转录、细胞死亡和分化。由于PARP能快速合成PAR，且PAR会被包括多聚（ADP-核糖）糖苷水解酶（PARG）和ADP-核糖水解酶3在内的PAR降解酶分解，因此完整细胞中PAR的生理水平难以确定。在培养细胞裂解过程中，可能会出现PAR的人为合成和/或降解。我们开发了一种灵敏的酶联免疫吸附测定（ELISA）方法来测量培养细胞中PAR的生理水平。我们立即使催化PAR合成和降解的酶失活。我们验证了三氯乙酸适用于在细胞裂解过程中使培养细胞中参与PAR代谢的PARP、PARG和其他酶失活。与用三氯乙酸裂解相比，用标准放射免疫沉淀测定缓冲液收获的细胞中的PAR水平增加了450倍，这可能是由于细胞裂解过程中发生的DNA损伤激活了PARP。这种ELISA可用于分析培养细胞在各种生理条件下多聚ADP核糖基化的生物学功能。
