Tanigawa Y, Shimoyama M
J Biol Chem. 1983 Aug 10;258(15):9184-91.
A novel endonuclease from adult hen liver nuclei has been purified to a homogeneous state through salt extraction, ammonium sulfate fractionation, gel filtration, acetone fractionation, and successive chromatography of 1) hydroxyapatite and DNA Sepharose and 2) hydroxyapatite and isoelectric focusing. The endonuclease has a pH optimum at 9.0 and requires Mg2+ for activity. The enzyme hydrolyzes more rapidly in the order of polynucleotide: denatured DNA = rRNA greater than poly(dA) = poly(dT) greater than poly(dC) = poly(dG) greater than native DNA. This endonuclease degrades denatured DNA about 20 times more rapidly than does the native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini and all four deoxynucleotides are present while dGMP is predominant. The enzyme cleaves the circular duplex PM2 DNA, endonucleotically, via single strand scission. The isoelectric point is 10.2 +/- 0.2 and the molecular weight is 43,000 +/- 2,000, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. Pyridoxal 5'-phosphate and 2,3-butanedione inhibit the catalytic activity, respectively. The inhibition of DNA binding activity was also seen with former, but not with the latter. Purified Mg2+-dependent alkaline endonuclease was used to investigate the nature of poly(ADP-ribose) inhibition of the enzyme. In contrast to the Ca2+/Mg2+-dependent endonuclease (Yoshihara, K., Tanigawa, Y., Burzio, L., and Koide, S. S. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 289-293), ADP-ribosylation of the endonuclease protein was not observed. When 100 ng of the poly(ADP-ribose) having four to five ADP-ribose units per molecule were added to the nuclease assay system (total volume of 0.2 ml) 14% inhibition was observed, and increase in the chain length increased the inhibition. When 100 ng of poly(ADP-ribose) consisting of 20 or more units of the ADP-ribose per mol were added, the inhibition was over 95%. The possible role of the poly(ADP-ribose)-sensitive endonuclease is discussed.
一种来自成年母鸡肝细胞核的新型核酸内切酶，通过盐提取、硫酸铵分级分离、凝胶过滤、丙酮分级分离以及以下连续层析步骤被纯化至均一状态：1）羟基磷灰石和DNA琼脂糖凝胶层析，以及2）羟基磷灰石和等电聚焦。该核酸内切酶的最适pH为9.0，活性需要Mg2+。该酶对多核苷酸的水解速度顺序为：变性DNA = rRNA大于聚（dA） = 聚（dT）大于聚（dC） = 聚（dG）大于天然DNA。这种核酸内切酶降解变性DNA的速度比天然DNA快约20倍。产物含有5'-磷酸和3'-羟基末端，并且所有四种脱氧核苷酸都存在，其中dGMP占主导。该酶通过单链断裂在内切核酸酶作用下切割环状双链PM2 DNA。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和凝胶过滤测定，其等电点为10.2±0.2，分子量为43000±2000。磷酸吡哆醛和2,3-丁二酮分别抑制催化活性。前者也能抑制DNA结合活性，而后者则不能。使用纯化的依赖Mg2+的碱性核酸内切酶来研究聚（ADP-核糖）对该酶的抑制性质。与依赖Ca2+/Mg2+的核酸内切酶（吉原，K.，谷川，Y.，布尔齐奥，L.，和小出，S.S.（1975年）《美国国家科学院院刊》72，289 - 293）不同，未观察到该核酸内切酶蛋白的ADP-核糖基化。当向核酸酶测定系统（总体积0.2 ml）中加入每分子含有4至5个ADP-核糖单元的100 ng聚（ADP-核糖）时，观察到14%的抑制率，并且链长增加会增强抑制作用。当加入每摩尔由20个或更多ADP-核糖单元组成的100 ng聚（ADP-核糖）时，抑制率超过95%。文中讨论了聚（ADP-核糖）敏感核酸内切酶的可能作用。
