In vivo aging of rat skeletal muscle sarcoplasmic reticulum Ca-ATPase. Chemical analysis and quantitative simulation by exposure to low levels of peroxyl radicals.
In vivo aging of rat skeletal muscle sarcoplasmic reticulum Ca-ATPase. Chemical analysis and quantitative simulation by exposure to low levels of peroxyl radicals.
复制标题
大鼠骨骼肌肌浆网Ca-ATP酶的体内衰老。
DOI：
10.1016/s0005-2736(97)00125-9
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Schöneich,C
中科院分区：
文献类型：
--
作者：
Viner,RI;Ferrington,DA;Aced,GI;Miller-Schlyer,M;Bigelow,DJ;Schöneich,C
分析了年轻成年（5个月）和老年（28个月）Fischer 344雄性大鼠骨骼肌的肌浆网（SR） ca - atp酶的翻译后修饰，这是由于生物老化及其潜在的功能后果。氨基酸组成的显著差异是，与年轻大鼠相比，老年大鼠ca - atp酶的巯基含量低6.8%，Arg残基含量低约4%。基于总共24个Cys残基，蛋白质硫醇的差异对应于体内衰老导致的1.5 mol Cys/mol ca - atp酶的损失。尽管老化的ca - atp酶对热失活、聚集和胰蛋白酶消化更敏感，但胱氨酸残基的损失并不伴随着酶活性的丧失。对所有SR蛋白的总巯基含量进行比较，发现从老年大鼠中分离的SR小泡的含量降低了13%。与Cys和Arg的变化相比，随着年龄的增长，蛋白质羰基的增加幅度很小，可能在生理上不显著，即每mol Ca-ATPase从0.32 mol羰基增加到0.46 mol羰基。当幼龄大鼠SR囊泡暴露于aaph衍生的过氧基自由基中时，每4 mg SR蛋白/ml损失约1.38×10−4M个SR巯基（相当于约25%），每1.6×10−5M个起始过氧基损失9.6×10−5M个ca - atp酶巯基（相当于约31%），表明每起始aaph衍生的过氧基氧化的化学统计量为≥6个氧化巯基。除了Cys外，暴露于aaph衍生的自由基还会导致ca - atp酶Arg、Met和Ser残基的轻微损失。最重要的是，暴露在这种低浓度过氧自由基下的SR ca - atp酶显示出与老年大鼠分离的SR ca - atp酶相当的物理和功能特性，即没有立即丧失活性，增加了对热失活、聚集和胰蛋白酶消化的敏感性。此外，通过hplc -电喷雾质谱和n端测序对早期色氨酸片段的动力学比较发现，“衰老”和aaph氧化的ca - atp酶产生了类似的肽片段，而不是“年轻”ca - atp酶产生的，这表明ca - atp酶由于衰老和aaph暴露而发生了一些构象变化。除了一个外，所有这些肽都起源于远离核苷酸结合位点和钙结合位点的位置。后一个结果表明，衰老和aaph暴露可能针对类似的Cys残基，主要是在远离核苷酸结合位点和钙结合位点的位置，这证明了Cys氧化不会立即导致ca - atp酶失活的事实。我们的研究结果提供了活性氧的净浓度的定量估计，这里是过氧自由基，它诱导SR ca - atp酶的物理和化学变化，定量上与体内衰老诱导的变化相当。
Sarcoplasmic reticulum (SR) Ca-ATPase of young adult (5 months) and aged (28 months) Fischer 344 male rat skeletal muscle was analyzed for posttranslational modifications as a result of biological aging and their potential functional consequences. The significant differences in the amino acid composition were a 6.8% lower content of sulfhydryl groups and a ca. 4% lower content of Arg residues of the Ca-ATPase from old as compared to young rats. Based on a total of 24 Cys residues the difference in protein thiols corresponds to a loss of 1.5 mol Cys/mol Ca-ATPase as a result of in vivo aging. The loss of Cys residues was not accompanied by a loss of enzyme activity though the `aged' Ca-ATPase was more sensitive to heat inactivation, aggregation, and tryptic digestion. A comparison of the total sulfhydryl content of all SR proteins present revealed a 13% lower amount for SR vesicles isolated from aged rats. Compared to the alterations of Cys and Arg, there was only a slight and probably physiologically insignificant increase of protein carbonyls with aging, i.e. from 0.32 to 0.46 mol carbonyl groups per mol of Ca-ATPase. When SR vesicles from young rats were exposed to AAPH-derived peroxyl radicals, there was a loss of ca. 1.38×10−4M total SR sulfhydryl groups per 4 mg SR protein/ml (corresponding to ca. 25%) and a loss of 9.6×10−5M Ca-ATPase sulfhydryl groups (corresponding to ca. 31%) per 1.6×10−5M initiating peroxyl radicals, indicating that the stoichiometry of sulfhydryl oxidation was ≥6 oxidized thiols per initiating AAPH-derived peroxyl radical. Besides Cys, the exposure to AAPH-derived radicals caused a slight loss of Ca-ATPase Arg, Met, and Ser residues. Most importantly, the SR Ca-ATPase exposed to this low concentration of peroxyl radicals displayed physical and functional properties quantitatively comparable to those of SR Ca-ATPase isolated from aged rats, i.e. no immediate loss of activity, increased susceptibility to heat inactivation, aggregation, and tryptic digestion. Moreover, a comparison of kinetically early tryptic fragments by HPLC-electrospray MS and N-terminal sequencing revealed that similar peptide fragments were produced from `aged' and AAPH-oxidized Ca-ATPase which were not (or kinetically significantly later) generated from the `young' Ca-ATPase, suggesting some conformational changes of the Ca-ATPase as a result of aging and AAPH-exposure. All except one of these peptides originated from locations remote from the nucleotide-binding and calcium-binding sites. The latter results suggest that aging and AAPH-exposure may target similar Cys residues, mainly at locations remote from the nucleotide-binding and calcium-binding sites, rationalizing the fact that Cys oxidation did not immediately cause inactivation of the Ca-ATPase. Our results provide a quantitative estimate of a net concentration of reactive oxygen species, here peroxyl radicals, which induces physical and chemical alterations of the SR Ca-ATPase quantitatively comparable to those induced by in vivo aging.