Shiokawa D, Ohyama H, Yamada T, Tanuma S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Tokyo 162, Japan.
Biochem J. 1997 Sep 15;326 ( Pt 3)(Pt 3):675-81. doi: 10.1042/bj3260675.
We previously identified three distinct DNA endonucleases, DNases alpha, beta and gamma, present in rat thymocyte nuclei. On the basis of their enzymic and biochemical properties, gamma-type DNase was regarded as a candidate for the apoptotic endonuclease. Here we purified DNase gamma to apparent homogeneity from apoptotic rat thymocyte nuclei induced by X-irradiation and characterized its properties in detail. The purified DNase gamma exhibited one predominant protein band on SDS/PAGE and an endonuclease activity in a zymography with an estimated molecular mass of 33 kDa. The molecular mass of the native form determined by G2000SW gel-filtration HPLC was 30 kDa. Amino acid analysis showed that the amino acid composition of DNase gamma was similar to that of rat DNase I (molecular mass 32 kDa) but different with regard to alanine and lysine residues. The N-terminal amino acid sequence of DNase gamma was revealed to be not identical with that of rat DNase I. In accordance with previous studies, homogeneously purified DNase gamma requires both Ca2+ and Mg2+ for activity. This requirement could be partially supplied by Mn2+. Of the bivalent metal ions tested, Co2+, Ni2+, Cu2+ and Zn2+ inhibited DNase gamma activity. These bivalent cations also suppressed apoptotic DNA fragmentation in rat thymocytes irradiated by X-rays. The same order of inhibitory ability was observed for these bivalent metal ions in vivo (in intact cells) and in vitro, suggesting that the suppression of apoptotic DNA fragmentation at the cellular level is due to the inhibition of DNase gamma. DNase gamma activity was found to exist at high levels in spleen, lymph node, thymus, liver and kidney, but little was present in brain, heart or pancreas. On the basis of these findings, together with previous data, we conclude that DNase gamma is a novel DNase I-like endonuclease responsible for internucleosomal cleavage of chromatin during thymic apoptosis.
我们之前在大鼠胸腺细胞核中鉴定出三种不同的DNA内切酶，即α型、β型和γ型脱氧核糖核酸酶（DNase）。基于它们的酶学和生化特性，γ型DNase被认为是凋亡内切酶的候选者。在此，我们从经X射线照射诱导凋亡的大鼠胸腺细胞核中纯化出了近乎纯一的γ型DNase，并详细表征了其特性。纯化后的γ型DNase在SDS/PAGE上呈现出一条主要蛋白带，在酶谱分析中具有内切酶活性，估计分子量为33 kDa。通过G2000SW凝胶过滤HPLC测定的天然形式的分子量为30 kDa。氨基酸分析表明，γ型DNase的氨基酸组成与大鼠DNase I（分子量32 kDa）相似，但在丙氨酸和赖氨酸残基方面有所不同。γ型DNase的N端氨基酸序列与大鼠DNase I不同。与先前的研究一致，高度纯化的γ型DNase的活性需要Ca²⁺和Mg²⁺。这种需求可部分由Mn²⁺提供。在所测试的二价金属离子中，Co²⁺、Ni²⁺、Cu²⁺和Zn²⁺抑制γ型DNase的活性。这些二价阳离子也抑制经X射线照射的大鼠胸腺细胞中的凋亡DNA片段化。在体内（完整细胞中）和体外观察到这些二价金属离子具有相同的抑制能力顺序，这表明细胞水平上凋亡DNA片段化的抑制是由于γ型DNase的抑制。发现γ型DNase活性在脾脏、淋巴结、胸腺、肝脏和肾脏中含量较高，但在脑、心脏或胰腺中含量很少。基于这些发现以及先前的数据，我们得出结论，γ型DNase是一种新型的类似DNase I的内切酶，在胸腺细胞凋亡过程中负责染色质的核小体间切割。
