Salman-Tabcheh S, Guérin M C, Torreilles J
INSERM U 58, Montpellier, France.
Free Radic Biol Med. 1995 Nov;19(5):695-8. doi: 10.1016/0891-5849(95)00075-9.
We measured the amounts of tyrosine and 3-nitrotyrosine (NO2-tyrosine) in proteins of plasma and polymorphonuclear leukocytes (PMN) from human whole blood before and after activation with phorbol ester (PMA) or calcium ionophore (A 23187). In unstimulated blood, no significant nitration of tyrosine was detected into PMN proteins, but a NO2-tyrosine/tyrosine ratio of 0.7% was detected in plasma proteins. When blood was activated with PMA, the NO2-tyrosine/tyrosine ratio stayed at 0.7% in plasma proteins, but it increased to 1.4% in PMN proteins, indicating a peroxynitrite production within the cells. In blood activated with calcium ionophore, the NO2-tyrosine/tyrosine ratio was 1.2% in plasma proteins and 2.1% in PMN proteins. Incubation of blood with a NO-synthase inhibitor before stimulation inhibited such a protein tyrosine nitration. To ensure that NO2-tyrosine detected in intracellular proteins did not result from the enzymatic posttranslational tyrosylation of PMN proteins, the incorporation of 14C labeled tyrosine into PMN proteins after activation with PMA or A23187 was studied. The addition of a 10 fold excess of NO2-tyrosine did not modify the course of protein tyrosylation. Because tyrosine nitration is an irreversible reaction, NO2-tyrosine could be accumulated into proteins and could act as a cumulative index of peroxynitrite production.
我们测量了人全血中血浆和多形核白细胞（PMN）蛋白质中酪氨酸和3-硝基酪氨酸（NO2-酪氨酸）的含量，这些全血在用佛波酯（PMA）或钙离子载体（A 23187）激活之前和之后进行检测。在未受刺激的血液中，未检测到PMN蛋白质中酪氨酸有明显的硝化作用，但在血浆蛋白质中检测到NO2-酪氨酸/酪氨酸比值为0.7%。当血液用PMA激活时，血浆蛋白质中的NO2-酪氨酸/酪氨酸比值保持在0.7%，但在PMN蛋白质中增加到1.4%，表明细胞内产生了过氧亚硝酸盐。在用钙离子载体激活的血液中，血浆蛋白质中的NO2-酪氨酸/酪氨酸比值为1.2%，PMN蛋白质中为2.1%。在刺激前用一氧化氮合酶抑制剂孵育血液可抑制这种蛋白质酪氨酸硝化作用。为确保在细胞内蛋白质中检测到的NO2-酪氨酸不是由PMN蛋白质的酶促翻译后酪氨酸化产生的，研究了在用PMA或A23187激活后14C标记的酪氨酸掺入PMN蛋白质的情况。加入10倍过量的NO2-酪氨酸并没有改变蛋白质酪氨酸化的过程。由于酪氨酸硝化是一种不可逆反应，NO2-酪氨酸可以积累到蛋白质中，并可作为过氧亚硝酸盐产生的累积指标。
