Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase.
Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase.
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DOI：
10.1021/bi9909445
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发表时间：
1999-08
期刊：
影响因子：
2.9
通讯作者：
R. Viner;T. Williams;C. Schöneich
中科院分区：
文献类型：
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作者：
R. Viner;T. Williams;C. Schöneich
骨骼肌的收缩和舒张是通过体内活性氧-氮与肌浆网蛋白硫醇的反应来有效调节的。然而，功能上重要的修饰的确切位置目前尚不清楚。在这里，我们通过HPLC-MS确定，通过过氧亚硝酸盐修饰肌浆网（SR）Ca-ATP酶亚型SERCA 1的一个（24个中的）Cys残基，Cys（349）足以调节酶活性。尽管SR Ca-ATP酶（一种110 kDa膜蛋白）的大小和性质不同，但通过用4-（二甲氨基）苯基偶氮苯基-4 '-马来酰亚胺（DABMI）和/或N-（2-碘乙基）三氟乙酰胺（IE-TFA）标记，然后进行彻底的胰蛋白酶消化和在线HPLC-UV-电喷雾MS分析，实现了Cys修饰的鉴定和定量。与IE-TFA的反应产生氨乙基半胱氨酸，一种新的胰蛋白酶切割位点，其允许产生特异性肽片段，所述肽片段对于IE-TFA标记是诊断性的，通过质谱法方便地鉴定。SR Ca-ATPase暴露于低浓度1 mM）的过氧亚硝酸盐导致Cys在位置344、349、471、498、525和614处的完全可逆的化学修饰（观察到Cys（344）和Cys（349）的亚硝基化），而较高浓度的过氧亚硝酸盐（0.45 mM）另外影响位置636、670和674处的Cys残基。当SR Ca-ATP酶暴露于0.45 mM过氧亚硝酸盐在5.0 mM谷胱甘肽（GSH）的存在下，巯基修饰变得部分可逆和S-谷胱甘肽被检测到Cys残基的位置344，349，364，498，525和614。酶失活的程度（先前确定）与（i）单个Cys残基和（ii）含有Cys（344）和Cys（349）的胰蛋白酶片段的标记效率损失定量相关。早期的结果已经表明，Cys（344）的独立选择性修饰在功能上是不重要的[Kawakita，M.，Yamashita，T.（东京）102，103-109]。因此，我们的结论是，只有半胱氨酸（349）的修饰是负责调制SR Ca-ATP酶活性的过氧亚硝酸盐。
Skeletal muscle contraction and relaxation is efficiently modulated through the reaction of reactive oxygen-nitrogen species with sarcoplasmic reticulum protein thiols in vivo. However, the exact locations of functionally important modifications are at present unknown. Here, we determine by HPLC-MS that the modification of one (out of 24) Cys residue of the sarcoplasmic reticulum (SR) Ca-ATPase isoform SERCA1, Cys(349), by peroxynitrite is sufficient for the modulation of enzyme activity. Despite the size and nature of the SR Ca-ATPase, a 110 kDa membrane protein, identification and quantitation of Cys modification was achieved through labeling with 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABMI) and/or N-(2-iodoethyl)trifluoroacetamide (IE-TFA) followed by an exhaustive tryptic digestion and on-line HPLC-UV-electrospray MS analysis. The reaction with IE-TFA generates aminoethylcysteine, a new trypsin cleavage site, which allows the production of specific peptide fragments that are diagnostic for IE-TFA labeling, conveniently identified by mass spectrometry. Exposure of the SR Ca-ATPase to low concentrations (0.1 mM) of peroxynitrite resulted in the fully reversible chemical modification of Cys at positions 344, 349, 471, 498, 525, and 614 (nitrosylation of Cys(344) and Cys(349) was seen), whereas higher concentrations of peroxynitrite (0.45 mM) additionally affected Cys residues at positions 636, 670, and 674. When the SR Ca-ATPase was exposed to 0.45 mM peroxynitrite in the presence of 5.0 mM glutathione (GSH), thiol modification became partially reversible and S-glutathiolation was detected for Cys residues at positions 344, 349, 364, 498, 525, and 614. The extent of enzyme inactivation (determined previously) quantitatively correlated with the loss of labeling efficiency (i) of a single Cys residue and (ii) of the tryptic fragment containing both Cys(344) and Cys(349). Earlier results had shown that the independent selective modification of Cys(344) is functionally insignificant [Kawakita, M., and Yamashita, T. (1987) J. Biochem. (Tokyo) 102, 103-109]. Thus, we conclude that modification of only Cys(349) is responsible for the modulation of the SR Ca-ATPase activity by peroxynitrite.