Poly (ADP-Ribose) polymerase cleavage monitored in situ in apoptotic cells
Poly (ADP-Ribose) polymerase cleavage monitored in situ in apoptotic cells
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DOI：
10.2144/01304pf01
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发表时间：
2001-04-01
期刊：
影响因子：
2.7
通讯作者：
Riss, TL
中科院分区：
文献类型：
--
作者：
O'Brien, MA;Moravec, RA;Riss, TL
在细胞凋亡期间，半胱氨酸蛋白酶家族或半胱氨酸蛋白酶的激活导致多种底物的蛋白水解切割。对通过半胱天冬酶切割产生的蛋白片段上的新表位具有特异性的抗体探针提供了在单个细胞水平上监测半胱天冬酶活性的手段。聚（ADP-核糖）聚合酶（PARP），一种参与DNA修复的核酶，是细胞凋亡过程中caspase-3裂解的众所周知的底物。其裂解被认为是细胞凋亡的标志。在这里，我们证明，亲和纯化的多克隆抗体的PARP的p85片段是特定的凋亡细胞。Western印迹显示抗体识别PARP brit的85-kDa（p85）片段，而不是全长PARP。我们证明了一个时间过程中的PARP切割援助DNA片段原位使用PARP p85片段抗体和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）在Jurkat细胞处理抗Pas。此外，我们的研究结果表明，p85片段的PARP导致的半胱天冬酶裂解在凋亡过程中迅速定位外的浓缩染色质，但不是在细胞质中。
During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair; is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85)fragment of PARP brit not full-length PARP. We demonstrate a time course of PARP cleavage aid DNA fragmentation irt situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Pas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.