Viner R I, Ferrington D A, Williams T D, Bigelow D J, Schöneich C
Department of Pharmaceutical Chemistry, University of Kansas, Simons Building, 2095 Constant Avenue, Lawrence, KS 66047, USA.
Biochem J. 1999 Jun 15;340 ( Pt 3)(Pt 3):657-69.
The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (approximately 3.5+/-0. 7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation.
共价修饰蛋白的积累是生物衰老的一个重要标志，但相对较少的研究涉及导致特定蛋白质靶点修饰的详细分子化学变化和过程。最近，纳拉亚南等人[纳拉亚南、琼斯、徐和于（1996年）《美国生理学杂志》271卷，C1032 - C1040页]报道，衰老对骨骼肌功能的影响具有肌肉特异性，慢肌中由ATP支持的Ca2 +摄取活性有显著的年龄依赖性变化，而快肌则没有。在此，我们详细表征了大鼠骨骼肌肌浆网（SR）Ca2 + - ATP酶同工型SERCA1和SERCA2a分别来自快肌和慢肌的年龄依赖性功能和化学修饰。我们发现，Ca2 + - ATP酶活性（相对于Ca2 + - ATP酶含量降低26%）和Ca2 +摄取速率存在显著的年龄依赖性下降，特别是在主要从慢肌而非快肌分离的SR中。蛋白质免疫印迹和氨基酸分析表明，选择性地，SERCA2a同工型随着年龄增长逐渐积累大量硝基酪氨酸（约3.5±0.7摩尔/摩尔SR Ca2 + - ATP酶）。两种Ca2 + - ATP酶同工型都出现了年龄依赖性的半胱氨酸还原态损失，然而，这在功能上并不显著。在体外，用过氧亚硝酸根（ONOO - ）（而非NO/O2）孵育快肌和慢肌SR，结果仅导致SERCA2a的选择性硝化，这表明ONOO - 可能是体内硝化剂的来源。体外和体内SR Ca2 + - ATP酶活性与共价蛋白修饰的相关性表明，酪氨酸硝化可能影响Ca2 + - ATP酶活性。通过用胰蛋白酶或金黄色葡萄球菌V8蛋白酶对纯化的SERCA2a进行部分和完全蛋白水解消化，随后进行蛋白质印迹、氨基酸和高效液相色谱 - 电喷雾 - 质谱（ESI - MS）分析，我们将大部分年龄依赖性酪氨酸硝化定位到SERCA2a的M4 - M8跨膜结构域中的Tyr294 - Tyr295序列，靠近Ca2 +转运的关键位点。
