Generation and release of nitrotyrosine O-sulfate by HepG2 human hepatoma cells upon SIN-1 stimulation: identification of SULT1A3 as the enzyme responsible.
Generation and release of nitrotyrosine O-sulfate by HepG2 human hepatoma cells upon SIN-1 stimulation: identification of SULT1A3 as the enzyme responsible.
复制标题
DOI：
10.1042/bj20060536
复制
发表时间：
2007-01
期刊：
影响因子：
--
通讯作者：
S. Yasuda;S. Idell;Ming-Cheh Liu
中科院分区：
文献类型：
--
作者：
S. Yasuda;S. Idell;Ming-Cheh Liu
除了作为氧化/硝化应激的生物标志物外，硝基酪氨酸水平升高还显示会导致DNA损伤或触发细胞凋亡。人体是否具有保护机制，以防止潜在的有害硝基酪氨酸仍然是未知的。本研究旨在研究硫酸化作为硝基酪氨酸代谢/调节途径的可能性。使用代谢标记，发现硝基酪氨酸O-[35 S]硫酸盐在硝基酪氨酸存在下产生并释放到用[35 S]硫酸盐标记的HepG 2人肝癌细胞的培养基中。为了鉴定负责硝基酪氨酸硫酸化的酶，对所有11种已知的人细胞溶质SULT（磺基转移酶）进行了系统研究。在测试的11种酶中，仅SULT 1A 3显示出对硝基酪氨酸的硫酸化活性。测定了SULT 1A 3与硝基酪氨酸或多巴胺作为底物的pH依赖性和动力学常数。为了检查硝基酪氨酸的硫酸化是否在细胞生理学背景下发生，用[35 S]硫酸盐标记的HepG 2细胞用SIN-1（吗啉苷酮亚胺）（一种过亚硝酸盐产生剂）处理。在用较高浓度的SIN-1处理的HepG 2细胞的培养基中检测到硝基酪氨酸O-[35 S]硫酸盐的增加。为了深入了解硝基酪氨酸硫酸化的生理相关性，使用SIN-1处理的[3 H]酪氨酸标记的HepG 2细胞进行了时间过程研究。这些发现证实了细胞内游离[3 H]硝基酪氨酸的大部分以非缀合形式存在。随着时间的推移，培养基中硫酸化[3 H]硝基酪氨酸的比例急剧增加，这意味着硫酸化可能在游离硝基酪氨酸的代谢中发挥重要作用。
In addition to serving as a biomarker of oxidative/nitrative stress, elevated levels of nitrotyrosine have been shown to cause DNA damage or trigger apoptosis. Whether the body is equipped with mechanisms for protecting against the potentially harmful nitrotyrosine remains unknown. The present study was designed to investigate the possibility that sulfation serves as a pathway for the metabolism/regulation of nitrotyrosine. Using metabolic labelling, nitrotyrosine O-[35S]sulfate was found to be produced and released into the medium of HepG2 human hepatoma cells labelled with [35S]sulfate in the presence of nitrotyrosine. To identify the enzyme(s) responsible for nitrotyrosine sulfation, a systematic study of all eleven known human cytosolic SULTs (sulfotransferases) was performed. Of the 11 enzymes tested, only SULT1A3 displayed sulfating activity toward nitrotyrosine. The pH-dependence and kinetic constants of SULT1A3 with nitrotyrosine or dopamine as substrate were determined. To examine whether the sulfation of nitrotyrosine occurs in the context of cellular physiology, HepG2 cells labelled with [35S]sulfate were treated with SIN-1 (morpholinosydnonimine), a peroxynitrite generator. Increments of nitrotyrosine O-[35S]sulfate were detected in the medium of HepG2 cells treated with higher concentrations of SIN-1. To gain insight into the physiological relevance of nitrotyrosine sulfation, a time-course study was performed using [3H]tyrosine-labelled HepG2 cells treated with SIN-1. The findings confirm that the bulk of free [3H]nitrotyrosine inside the cells was present in the unconjugated form. The proportion of sulfated [3H]nitrotyrosine increased dramatically in the medium over time, implying that sulfation may play a significant role in the metabolism of free nitrotyrosine.