Hellberg C B, Boggs S E, Lapetina E G
Molecular Cardiovascular Research Center, Case Western Reserve, University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio, 44106-4958, USA.
Biochem Biophys Res Commun. 1998 Nov 18;252(2):313-7. doi: 10.1006/bbrc.1998.9581.
A major mechanism of injury associated with the production of nitric oxide (NO*) in vivo is due to its diffusion-limited reaction with superoxide to form peroxynitrite, which in turn may cause nitration of protein tyrosine residues. To assess the physiological role of tyrosine nitration, it is crucial to identify the proteins that become nitrated. Therefore, we treated lysates from RAW 264.7 cells with 1 mM peroxynitrite and immunoprecipitated tyrosine nitrated proteins. This treatment resulted in the nitration of several proteins, with molecular weights ranging from 60-250 kD. One of these proteins was immunologically identified as the p85 regulatory subunit of the phosphatidylinositol 3-kinase, a key enzyme involved in the signal transduction cascade initiated by many agonists including growth factors. Treatment of RAW 264.7 macrophages with the NO* donor spermine NONOate also induced a nitration of the p85 subunit, demonstrating that this covalent modification also occurs in intact cells. Immunoprecipitation of the p110 catalytic subunit of the phosphatidylinositol 3-kinase co-immunoprecipitated p85 in control lysates. However, p85 could not be detected in the same immunoprecipitates when the lysates had been preincubated with 1 mM peroxynitrite, indicating that the nitration of the p85 subunit may abrogate its interaction with the p110 subunit.
体内一氧化氮（NO*）产生相关的主要损伤机制是由于其与超氧化物发生扩散受限反应形成过氧亚硝酸盐，而过氧亚硝酸盐进而可能导致蛋白质酪氨酸残基的硝化。为了评估酪氨酸硝化的生理作用，识别被硝化的蛋白质至关重要。因此，我们用1 mM过氧亚硝酸盐处理RAW 264.7细胞的裂解物，并免疫沉淀酪氨酸硝化的蛋白质。这种处理导致几种蛋白质发生硝化，分子量范围为60 - 250 kD。其中一种蛋白质经免疫鉴定为磷脂酰肌醇3激酶的p85调节亚基，磷脂酰肌醇3激酶是一种关键酶，参与由包括生长因子在内的许多激动剂引发的信号转导级联反应。用NO*供体精胺NONOate处理RAW 264.7巨噬细胞也诱导了p85亚基的硝化，表明这种共价修饰也发生在完整细胞中。在对照裂解物中，磷脂酰肌醇3激酶的p110催化亚基的免疫沉淀共沉淀了p85。然而，当裂解物与1 mM过氧亚硝酸盐预孵育时，在相同的免疫沉淀中检测不到p85，这表明p85亚基的硝化可能消除其与p110亚基的相互作用。
