Knyushko Tatyana V, Sharov Victor S, Williams Todd D, Schöneich Christian, Bigelow Diana J
Cell Biology and Biochemistry Group, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Biochemistry. 2005 Oct 4;44(39):13071-81. doi: 10.1021/bi051226n.
In the aging heart, decreased rates of calcium transport mediated by the SERCA2a isoform of the sarcoplasmic reticulum (SR) Ca-ATPase are responsible for the slower sequestration of cytosolic calcium and consequent prolonged muscle relaxation times. We report a 60% decrease in Ca-ATPase activity in the senescent Fischer 344 rat heart relative to that of young adult hearts; this functional decrease can be attributed, in part, to the 18% lower abundance of SERCA2a protein. Here, we show that the additional loss of activity is a result of increased 3-nitrotyrosine modification of the Ca-ATPase. Age-dependent increases in nitration of cardiac SERCA2a are identified using multiple analytical methods. In the young (adult) heart 1 molar equivalent of nitrotyrosine is distributed over at least five tyrosines within the Ca-ATPase, identified as Tyr(122), Tyr(130), Tyr(497), Tyr(586), and Tyr(990). In the senescent heart, the stoichiometry of nitration increases by more than two nitrotyrosines per Ca-ATPase, coinciding with the appearance of nitrated Tyr(294), Tyr(295), and Tyr(753). The abundant recovery of native analogues for each of the nitrated peptides indicates partial modification of multiple tyrosines within cardiac SERCA2a. In contrast, within skeletal muscle SERCA2a, a homogeneous pattern of nitration appears, with full site (1 mol/mol) nitration of Tyr(753), in young, with additional nitration of Tyr(294) and Tyr(295), in senescent muscle. The nitration of these latter vicinal sites correlates with diminished transport function in both striated muscle types, suggesting that these sites provide a mechanism for downregulation of ATP utilization by the Ca-ATPase under conditions of nitrative stress.
在衰老心脏中，肌浆网（SR）Ca - ATP酶的SERCA2a亚型介导的钙转运速率降低，导致胞质钙隔离减慢，进而使肌肉舒张时间延长。我们报告，衰老的Fischer 344大鼠心脏中Ca - ATP酶活性相对于年轻成年心脏降低了60%；这种功能下降部分可归因于SERCA2a蛋白丰度降低了18%。在此，我们表明活性的额外丧失是Ca - ATP酶3 - 硝基酪氨酸修饰增加的结果。使用多种分析方法确定了心脏SERCA2a硝化作用的年龄依赖性增加。在年轻（成年）心脏中，1摩尔当量的硝基酪氨酸分布在Ca - ATP酶内至少五个酪氨酸上，分别为Tyr(122)、Tyr(130)、Tyr(497)、Tyr(586)和Tyr(990)。在衰老心脏中，每个Ca - ATP酶的硝化化学计量增加超过两个硝基酪氨酸，同时出现了硝化的Tyr(294)、Tyr(295)和Tyr(753)。每种硝化肽的天然类似物大量回收表明心脏SERCA2a内多个酪氨酸发生了部分修饰。相比之下，在骨骼肌SERCA2a中，硝化模式较为均匀，在年轻骨骼肌中Tyr(753)完全位点（1摩尔/摩尔）硝化，在衰老骨骼肌中Tyr(294)和Tyr(295)额外硝化。后两个相邻位点的硝化与两种横纹肌类型的转运功能降低相关，表明这些位点提供了一种机制，在硝化应激条件下通过Ca - ATP酶下调ATP利用。
