Prasad S C, Soldatenkov V, Notario V, Smulson M, Dritschilo A
Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007, USA.
Electrophoresis. 1999 Mar;20(3):618-25. doi: 10.1002/(SICI)1522-2683(19990301)20:3<618::AID-ELPS618>3.0.CO;2-B.
Caspace-mediated proteolysis of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) (EC 2.4, 2.30) is a biochemical marker of cell death in response to various apoptotic stimuli. Anti-PARP antibodies identifying the 89 kDa polypeptide from the C-terminus as well as the 113 kDa native enzyme are often used to demonstrate evidence of apoptosis-associated, interleukin converting enzyme (ICE)-mediated limited cleavage. Recent evidence points to redundancy of caspases, heterogeneity of their cleavage sites, and a possibility of generating distinct context-specific, and cell-specific PARP fragments. In the present study, we employed antibodies directed to multiple sites in PARP and probed two-dimensionally resolved proteins of the estrogen receptor negative MDA-MB-468 breast tumor cells, induced to undergo apoptosis by ionizing radiation (IR). Our results revealed that the 24 kDa apoptotic fragment of PARP, from the N-terminus, consists of at least three isoforms, located at a p/more basic than the full length enzyme. We also report a hitherto unrecognized feature of an anti-PARP antiserum, VIC-5, detecting both the 89 kDa and the 24 kDa caspase-generated fragments of PARP. Thus, application of two-dimensional electrophoresis combined with antisera directed to multiple sites would be valuable in distinguishing PARP cleavage site- and inhibitor specificities of proteases during apoptosis.
胱天蛋白酶介导的核酶聚（ADP - 核糖）聚合酶（PARP）（EC 2.4, 2.30）的蛋白水解是细胞对各种凋亡刺激作出反应时细胞死亡的生化标志物。识别来自C端的89 kDa多肽以及113 kDa天然酶的抗PARP抗体常被用于证明凋亡相关的白细胞介素转化酶（ICE）介导的有限切割的证据。最近的证据表明胱天蛋白酶存在冗余，其切割位点具有异质性，并且有可能产生不同的上下文特异性和细胞特异性PARP片段。在本研究中，我们使用针对PARP多个位点的抗体，对雌激素受体阴性的MDA - MB - 468乳腺肿瘤细胞经电离辐射（IR）诱导发生凋亡后的二维分辨蛋白进行检测。我们的结果显示，来自N端的PARP的24 kDa凋亡片段至少由三种异构体组成，其位置比全长酶更偏碱性。我们还报告了一种抗PARP抗血清VIC - 5迄今未被认识的特性，它能检测到PARP的89 kDa和24 kDa胱天蛋白酶生成的片段。因此，二维电泳结合针对多个位点的抗血清的应用，对于区分凋亡过程中蛋白酶的PARP切割位点和抑制剂特异性将是有价值的。
