Hughes F M, Evans-Storms R B, Cidlowski J A
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Cell Death Differ. 1998 Dec;5(12):1017-27. doi: 10.1038/sj.cdd.4400418.
Chromatin degradation into oligonucleosomal and approximately 30-50 Kb fragments is a hallmark of apoptosis. Crude nuclear extract from apoptotic rat thymocytes is able to recapitulate both types of DNA fragmentation in an assay using HeLa cell nuclei as an exogenous substrate. Using size exclusion chromatography we have identified a novel activity (approximately 260 Kd) that produces only approximately 30-50 Kb DNA fragments, and a 25 Kd activity that generates both approximately 30-50 Kb and oligonucleosomal fragments. Both activities produced DNA fragments with 3'-OH termini, are dependent on Ca2+ and Mg2+ and are inhibited by N-ethyl-maleimide, sodium tetrathionate, aurintricarboxylic acid and sodium chloride, similar to other nucleases implicated in apoptosis. These activities were inhibited by the serine protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, but not by the serine protease inhibitor diisopropyl fluorophosphate, or by calpain inhibitors I or II, or the capsase inhibitors Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Tyr-Val-Ala-Asp-aldehyde, or Z-Val-Ala-Asp-fluoromethyl ketone. Both activities were insensitive to protease inhibitors when extracts were incubated with naked linear DNA, indicating the presence of both nuclease and protease activities in the preparation. Together, these observations suggest the involvement of non-caspase proteases in apoptosis which perhaps function by altering chromatin substructure and exposing it to nucleolytic attack.
染色质降解为寡核小体片段和约30 - 50 kb的片段是细胞凋亡的一个标志。凋亡大鼠胸腺细胞的粗核提取物能够在以HeLa细胞核作为外源底物的实验中重现这两种类型的DNA片段化。利用尺寸排阻色谱法，我们鉴定出一种仅产生约30 - 50 kb DNA片段的新活性（约260 Kd），以及一种产生约30 - 50 kb片段和寡核小体片段两者的25 Kd活性。这两种活性产生的DNA片段都具有3'-OH末端，依赖于Ca2+和Mg2+，并被N-乙基马来酰亚胺、连四硫酸钠、金精三羧酸和氯化钠抑制，这与其他参与细胞凋亡的核酸酶相似。这些活性被丝氨酸蛋白酶抑制剂N-对甲苯磺酰-L-苯丙氨酸氯甲基酮和Nα-对甲苯磺酰-L-赖氨酸氯甲基酮抑制，但不被丝氨酸蛋白酶抑制剂二异丙基氟磷酸、钙蛋白酶抑制剂I或II或半胱天冬酶抑制剂Ac-Asp-Glu-Val-Asp-醛、Ac-Tyr-Val-Ala-Asp-醛或Z-Val-Ala-Asp-氟甲基酮抑制。当提取物与裸露的线性DNA孵育时，两种活性对蛋白酶抑制剂均不敏感，这表明制剂中存在核酸酶和蛋白酶活性。总之，这些观察结果表明非半胱天冬酶蛋白酶参与了细胞凋亡，其可能通过改变染色质亚结构并使其暴露于核酸酶攻击而起作用。
