PURIFICATION OF POLY(ADP-RIBOSE) GLYCOHYDROLASE AND DETECTION OF ITS ISOFORMS BY A ZYMOGRAM FOLLOWING ONE-DIMENSIONAL OR 2-DIMENSIONAL ELECTROPHORESIS
PURIFICATION OF POLY(ADP-RIBOSE) GLYCOHYDROLASE AND DETECTION OF ITS ISOFORMS BY A ZYMOGRAM FOLLOWING ONE-DIMENSIONAL OR 2-DIMENSIONAL ELECTROPHORESIS
复制标题
DOI：
10.1006/abio.1994.1177
复制
发表时间：
1994-05-01
影响因子：
2.9
通讯作者：
POIRIER, GG
中科院分区：
文献类型：
--
作者：
BROCHU, G;SHAH, GM;POIRIER, GG
聚腺苷二磷酸核糖代谢是一种翻译后修饰，涉及两种核酶。聚腺苷二磷酸核糖聚合酶（PARP）和聚腺苷二磷酸核糖糖水解酶（PARG）分别负责聚腺苷二磷酸核糖聚合物的固定和催化。尽管PARG的丰度低于PARP，但它是已知与DNA修复和其他细胞过程有关的聚合物代谢的关键决定因素。在这里，我们描述的修改，以提高纯化的PARG从小牛胸腺，在数量和质量方面，这将允许生化和免疫学研究。我们还开发了一个酶谱，以确定功能多肽表现出PARG活性。从小牛胸腺中提取纯化的和粗的酶制剂，在二维凝胶中进行凝胶电泳。样品在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上解析，所述凝胶电泳含有在非平衡pH梯度电泳后以自动修饰的PARP形式存在的聚合物底物。PARG在凝胶中复性后，在纯化的酶制剂中清楚地检测到四种异构体的活性。即使在组织的粗提物中，我们也可以观察到PARG的主要同种型。该技术将允许更好地理解聚（ADP-核糖）催化剂和更好地表征PARG亚型。(C)1994年出版社出版。
Poly(ADP-ribosyl)ation metabolism, a post-translational modification, involves two nuclear enzymes. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are responsible for the anabolism and catabolism of poly(ADP-ribose) polymer, respectively. PARG, despite being less abundant than PARP, is a crucial determinant of polymer metabolism which is known to be implicated in DNA repair and other cellular processes. Here, we describe modifications to improve the purification of PARG from calf thymus, in terms of both quantity and quality, which would allow biochemical and immunological studies. We also developed a zymogram to identify functional polypeptides exhibiting PARG activity. Purified and crude enzyme preparations from calf thymus were electrophoresed in two-dimensional gels. Samples were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing the polymer substrate in the form of automodified PARP after a nonequilibrium pH gradient electrophoresis. After renaturation of PARG in the gel, four isoforms of activity were clearly detected in the purified enzyme preparation. Even in the crude extract of the tissue, we could observe the major isoform of PARG. This technique will permit a better understanding of poly(ADP-ribose) catabolism and better characterization of PARG isoforms. (C) 1994 Academic Press, Inc.