Detection of sequence-specific tyrosine nitration of manganese SOD and SERCA in cardiovascular disease and aging.
Detection of sequence-specific tyrosine nitration of manganese SOD and SERCA in cardiovascular disease and aging.
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检测心血管疾病和衰老中锰 SOD 和 SERCA 的序列特异性酪氨酸硝化。
DOI：
10.1152/ajpheart.01293.2005
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Cohen,RichardA
中科院分区：
文献类型：
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作者：
Xu,Shanqin;Ying,Jia;Jiang,Bingbing;Guo,Wei;Adachi,Takeshi;Sharov,Viktor;Lazar,Harold;Menzoian,James;Knyushko,TatyanaV;Bigelow,Diana;Schöneich,Christian;Cohen,RichardA
蛋白质酪氨酸残基（nY）的硝化是氧化应激的标志，并且可以改变修饰的蛋白质的生物活性。本研究的目的是开发针对位点特异性的nY修饰蛋白的抗体，并使用组织化学和免疫印迹来证明组织中的蛋白质硝化。开发了针对MnSOD nY-34中具有已知nY位点的肽和肌质内质网钙ATP酶（SERCA 2 di-nY-294，295）中具有两个相邻nY位点的肽的亲和纯化的多克隆抗体。用已知MnSOD nY的ANG II输注大鼠的肾脏和动脉粥样硬化兔的主动脉以及已知SERCA di-nY的老化大鼠骨骼和心脏肌浆网用作阳性对照。MnSOD nY-34染色在远端肾小管和集合管中最强烈。在动脉粥样硬化斑块中，动脉粥样硬化主动脉的SERCA 2 di-nY染色最强烈。老年大鼠骨骼肌和糖尿病患者动脉粥样硬化的主动脉和心房也呈阳性染色。染色减少连二亚硫酸钠，其化学还原硝基酪氨酸氨基酪氨酸，和抗原性nY-肽阻断染色的每个相应的nY网站，但不为其他。如前所述，免疫印迹法未能检测到这些修饰的蛋白质在整个组织裂解物，但当蛋白质被浓缩。特异性nY修饰的酪氨酸残基的免疫组织化学染色提供了评估与病理条件相关的氧化应激对特定组织部位功能可能受影响的单个蛋白质的影响的能力。
Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.