IDENTIFICATION OF DEOXYRIBONUCLEASE-II AS AN ENDONUCLEASE INVOLVED IN APOPTOSIS
IDENTIFICATION OF DEOXYRIBONUCLEASE-II AS AN ENDONUCLEASE INVOLVED IN APOPTOSIS
复制标题
DOI：
10.1006/abbi.1993.1060
复制
发表时间：
1993-01-01
影响因子：
3.9
通讯作者：
EASTMAN, A
中科院分区：
文献类型：
--
作者：
BARRY, MA;EASTMAN, A
细胞死亡发生在细胞程序性缺失和暴露于细胞毒性物质后的凋亡过程中。细胞凋亡的核心机制是核小体间DNA被内源性核酸内切酶消化，这被认为介导细胞死亡。凋亡的一个公理是，所涉及的内切酶是Ca2+/Mg2+依赖的内切酶。在纯化中国仓鼠卵巢细胞内切酶的过程中，我们发现Ca2+/Mg2+依赖性内切酶活性很小，但在pH 7以下有大量内切酶活性。这种酸性内切酶在完整细胞中通过质子离子载体将细胞内pH值降低到7以下而被激活。这种活性产生了核小体间DNA的消化，这是细胞凋亡的特征。核提取物含有一种不依赖阳离子的内切酶，具有相同的ph依赖性活性。我们将酸性内切酶与牛脱氧核糖核酸酶II （DNase II）进行了比较，发现它们在所有测试中几乎相同，包括对各种抑制剂的敏感性，通过相同的色谱步骤纯化，以及对牛酶的抗体识别。在分离的细胞核中加入酸性核酸内切酶或牛DNA酶II，可诱导核小体间DNA酶切直至pH 6.5。这些数据表明，DNase II可以介导核小体间DNA消化，这是细胞内酸化后细胞凋亡的特征。此外，这些数据质疑Ca2+/Mg2+依赖性内切酶是唯一参与细胞凋亡的内切酶的前提。
Cell death occurs by apoptosis during programmed deletion of cells and following exposure to cytotoxic agents. Central to the mechanism of apoptosis is internucleosomal DNA digestion by an endogenous endonuclease which is thought to mediate cell death. An axiom of apoptosis is that the endonuclease involved is a Ca2+/Mg2+-dependent endonuclease. During purification of endonucleases from Chinese hamster ovary cells, we found little Ca2+/Mg2+-dependent endonuclease activity, but large amounts of an endonuclease active below pH 7. This acidic endonuclease was activated in intact cells by reducing intracellular pH values below 7 with a proton ionophore. This activity generated internucleosomal digestion of DNA characteristic of apoptosis. Nuclear extracts contained a cation-independent endonuclease with identical pH-dependent activity. We have compared the acidic endonuclease to bovine deoxyribonuclease II (DNase II) and have found them nearly identical by all tests, including sensitivity to various inhibitors, purification by the same chromatographic steps, and recognition by antibody raised against the bovine enzyme. Addition of either the acidic endonuclease or bovine DNase II to isolated nuclei induced internucleosomal DNA digestion up through pH 6.5. These data demonstrate that DNase II can mediate internucleosomal DNA digestion characteristic of apoptosis following intracellular acidification. Furthermore, these data question the premise that the Ca2+/Mg2+-dependent endonuclease is the only endonuclease involved in apoptosis.