来自克氏锥虫的聚（ADP-核糖）聚合酶的纯化及性质。DNA拓扑异构酶I的自身修饰和聚（ADP-核糖基）化作用。

Purification and properties of poly(ADP-ribose)polymerase from Crithidia fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I.

作者信息

Podestá Dolores, García-Herreros María I, Cannata Joaquín J B, Stoppani Andrés O M, Fernández Villamil Silvia H

机构信息

Bioenergetics Research Centre, CONICET, School of Medicine, University of Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina.

出版信息

Mol Biochem Parasitol. 2004 Jun;135(2):211-9. doi: 10.1016/j.molbiopara.2004.02.005.

DOI:10.1016/j.molbiopara.2004.02.005

PMID:15110462

Abstract

Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.

摘要

聚（ADP-核糖）聚合酶已从克氏锥虫中纯化了超过160000倍。这是首次从锥虫中分离出达到明显均一性的聚（ADP-核糖）聚合酶。纯化后的酶催化活性绝对依赖于DNA，当DNA与组蛋白的比例为1:1.3时，组蛋白可使其活性增强2.5倍。该酶不需要镁或任何其他金属离子作为辅因子。通过凝胶过滤测定，其表观分子量为111 kDa，这相当于两个相同的55 kDa亚基组成的二聚体。烟酰胺、3-氨基苯甲酰胺、茶碱、胸腺嘧啶、黄嘌呤和次黄嘌呤可抑制该酶的活性，但腺苷无此作用。该酶定位于细胞核。我们的研究结果表明，聚（ADP-核糖）聚合酶自身或DNA拓扑异构酶I的共价聚（ADP-核糖基）化导致其活性受到抑制，并首次对锥虫中的这种共价翻译后修饰进行了生化特性分析。