Ca2+/Mg(2+)-dependent endonuclease from human spleen: purification, properties, and role in apoptosis.
Ca2+/Mg(2+)-dependent endonuclease from human spleen: purification, properties, and role in apoptosis.
复制标题
来自人脾脏的 Ca2 /Mg(2 ) 依赖性核酸内切酶：纯化、特性和在细胞凋亡中的作用。
DOI：
10.1021/bi00086a018
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Carson,DA
中科院分区：
文献类型：
--
作者：
Ribeiro,JM;Carson,DA
19939年6月15日收到的修订稿件摘要：细胞凋亡中的一个重要事件是染色质被消化成寡核体片段。然而，负责DNA降解的酶还没有得到很好的表征。在这里，我们报道了一种从人脾细胞核中纯化的核酸内切酶，该酶可能在细胞凋亡中负责DNA的消化。用灵敏的荧光法测定酶的活性，用来评估质粒DNA从超螺旋形式到开放形式的转化。用100~350 mM的氯化钠从分离的细胞核中提取核酸内切酶，用磷酸纤维素柱层析、Superdex 75柱层析、螯合琼脂糖柱层析(锌形式)进一步纯化。经凝胶过滤，表观分子质量为22-26 kDa；经SDS-聚丙烯酰胺凝胶电泳法测定，纯化后的酶呈现单一的27 kDa条带。该酶的活性同时需要镁离子(最适浓度为5 mM)和钙离子(最适浓度为2 mM)。这种作用可被锌离子(50 PM时100%抑制)和高浓度(10 MM)的钙离子所抑制。金杂羧酸、精胺、对羟基汞苯甲酸酯和TV-乙基马来酰亚胺也是核酸内切酶抑制剂。碘乙酰胺、G-肌动蛋白或核苷S对此无抑制作用。S的-双磷酸盐。最适pH为8.0。当加入不含核酸内切酶的人CCRF-CEM淋巴母细胞核时，纯化的脾酶将染色质消化成单核和寡核体片段，这是细胞凋亡的特征。根据这一结果，以及观察到纯化的内切酶的激活物和抑制物与影响细胞凋亡的那些密切相关，似乎该酶参与了DNA人类淋巴细胞的凋亡降解。细胞凋亡(Kerr等人，1972)是一个生理过程，通过这个过程，细胞在正常的胚胎发生、分化和变态期间从活组织中删除(Wyllie等人，1980；Gerschenson&Rotello，1992)。细胞凋亡在淋巴细胞中的作用是特别重要的，并且已经被非常详细地研究[在Cohen等人(1992)中回顾]。它被认为参与了发育过程中未成熟的自身反应性淋巴细胞的缺失(Smith等人，1989；Murphy等人，1990)，HIV感染引起的淋巴细胞枯竭(Terai等人，1991；Groux等人，1992)，以及细胞毒性T淋巴细胞(CTL)(Duke等人，1983；Ucker，1987)。细胞凋亡素也可以由病理性刺激诱导，例如电离辐射(Berger，1985；Carson等人，1986b)、氧化剂
Revised Manuscript Received June 15, 19939 abstract: A major event in apoptosis is the digestion of chromatin into oligonucleosomal fragments. However, the enzymes responsible for the DNA degradation have not been well characterized. Here we report the purification of an endonuclease from human spleen cell nuclei that is likely to be responsible for DNA digestion in apoptosis. Enzyme activity was measured by a sensitive fluorometric assay, which assesses the conversion of plasmid DNA from a supercoiled to an open form. Theendonuclease was extracted from isolated nuclei with NaCl between 100 and 350 mM and was further purified by chromatography on columns of phosphocellulose, Superdex 75, and chelating Sepharose (Zn2+ form). By gel filtration, the apparent molecular mass was 22-26 kDa; on SDS-polyacrylamide gel electrophoresis, the purified enzyme showed a single 27-kDa band. The enzyme required both Mg2+(optimum, 5 mM) and Ca2+(optimum, 2 mM) for activity. It was inhibited by Zn2+(100% inhibition at 50 pM) and by high (> 10 mM) concentrations of Ca2+. Aurintricarboxylic acid, spermine, p-(hydroxymercuri) benzoate, and TV-ethylmaleimide were also endonuclease inhibitors. No inhibition was observed with iodoacetamide, G-actin, or nucleoside S'. S'-bisphosphates. An optimum pH of 8.0 was found. When added to human CCRF-CEM lymphoblast nuclei, that do not contain the endonuclease, the purified splenic enzyme digested the chromatininto the mono-and oligonucleosomal fragments that are characteristic of apoptosis. On the basis of this result, and the observation that the activators and inhibitors of the purified endonuclease closely parallel those that affect apoptosis, it seems likely that this enzyme is involved in the apoptotic degradation of DNA inhuman lymphocytes.Apoptosis (Kerr et al., 1972) is a physiological process by which cells are deleted from living tissues during normal embryogenesis, differentiation, and metamorphosis (Wyllie et al., 1980; Gerschenson & Rotello, 1992). The role of apoptosis in lymphocytes is especially important andhas been studied in great detail [reviewed in Cohen et al.(1992)]. It is considered toparticipate in the deletion of immature autoreactive lymphocytes during development (Smith et al., 1989; Murphy et al., 1990), in lymphocyte depletion induced by HIV infection (Terai et al., 1991; Groux et al., 1992), and in cell death effected by cytotoxic T-lymphocytes (CTL)(Duke et al., 1983; Ucker, 1987). Apoptosisin lymphocytes may also be induced by pathological stimuli, such as ionizing radiation (Berger, 1985; Carson et al., 1986b), oxidants