Ferrington D A, Krainev A G, Bigelow D J
Department of Biochemistry, Haworth Hall, University of Kansas, Lawrence, Kansas 66045, USA.
J Biol Chem. 1998 Mar 6;273(10):5885-91. doi: 10.1074/jbc.273.10.5885.
We have measured the in vivo protein turnover for the major calcium regulatory proteins of the sarcoplasmic reticulum from the skeletal muscle of young adult (7 months) and aged (28 months) Fischer 344 rats. From the time course of the incorporation and decay of protein-associated radioactivity after a pulse injection of [14C]leucine and correcting for leucine reutilization, in young rats, the apparent half-lives for calsequestrin, the 53-kDa glycoprotein, and ryanodine receptor are 5.4 +/- 0.4, 6.3 +/- 1.3, and 8.3 +/- 1.3 days, respectively. A half-life of 14.5 +/- 2.5 days was estimated for the Ca-ATPase isolated from young muscle. Differences in protein turnover associated with aging were determined using sequential injection of two different isotopic labels ([14C]leucine and [3H]leucine) to provide an estimate of protein synthesis and degradation within the same animal. The Ca-ATPase and ryanodine receptor isolated from aged muscle exhibits 27 +/- 5% and 25 +/- 3% slower protein turnover, respectively, relative to that from young muscle. In contrast, the 53-kDa glycoprotein exhibits a 25 +/- 5% more rapid turnover in aged SR, while calsequestrin exhibits no age-dependent alteration in turnover. Statistical analysis comparing the sensitivity of various methods for discriminating different rates of protein turnover validates the approach used in this study and demonstrates that the use of two isotopic labels provides at least a 6-fold more sensitive means to detect age-related differences in protein turnover relative to other methods.
我们测量了年轻成年（7个月）和老年（28个月）Fischer 344大鼠骨骼肌肌浆网主要钙调节蛋白的体内蛋白质周转率。在脉冲注射[14C]亮氨酸后，根据蛋白质相关放射性的掺入和衰减时间进程，并校正亮氨酸再利用情况，在年轻大鼠中，钙结合蛋白、53 kDa糖蛋白和兰尼碱受体的表观半衰期分别为5.4±0.4天、6.3±1.3天和8.3±1.3天。从年轻肌肉中分离出的钙ATP酶的半衰期估计为14.5±2.5天。通过顺序注射两种不同的同位素标记物（[14C]亮氨酸和[3H]亮氨酸）来确定与衰老相关的蛋白质周转率差异，以估计同一动物体内的蛋白质合成和降解情况。从老年肌肉中分离出的钙ATP酶和兰尼碱受体的蛋白质周转率分别比年轻肌肉中的慢27±5%和25±3%。相比之下，53 kDa糖蛋白在老年肌浆网中的周转率快25±5%，而钙结合蛋白的周转率没有年龄依赖性变化。比较各种区分不同蛋白质周转率方法敏感性的统计分析验证了本研究中使用的方法，并表明使用两种同位素标记物相对于其他方法提供了至少高6倍的检测蛋白质周转率年龄相关差异的灵敏手段。
