Assessment of protein turnover in perfused rat liver. Evidence for amino acid compartmentation from differential labeling of free and tRNA-gound valine.
Assessment of protein turnover in perfused rat liver. Evidence for amino acid compartmentation from differential labeling of free and tRNA-gound valine.
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评估灌注大鼠肝脏中的蛋白质周转率。
DOI：
10.1016/s0021-9258(17)33750-x
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发表时间：
1976
期刊：
影响因子：
--
通讯作者：
G. E. Mortimore
中科院分区：
文献类型：
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作者：
E. Khairallah;G. E. Mortimore
通过测量基于连接到tRNA的缬氨酸的比活性的缬氨酸掺入速率来测定喂食大鼠的灌注肝脏中的总蛋白质合成。当灌流液缬氨酸从0.40增加到5 mM时，速率没有显著改变，并且与早先从浓度为15 mM的细胞外缬氨酸的比活性计算的值相似。这与在加入放线菌酮后5 - 15分钟之间发生的游离缬氨酸的增加密切对应。在后面的实验中，利用了这样的事实，即先前建立的放线菌酮对蛋白水解的抑制作用最初并不开始于合成的抑制，而是在15分钟后开始。因此，假定5至15分钟的缬氨酸释放代表在加入放线菌酮之前的蛋白质降解速率。因此，在这些蛋白质代谢的独立评估中发现的密切一致性似乎消除了肝脏蛋白质周转定量中的大部分先前不确定性。在这些研究的过程中，我们注意到缬氨酰-tRNA的比活性达到稳态值，其介于细胞外和细胞内池的比活性之间，但似乎比细胞内缬氨酸更快达到稳态。为了更精确地评估这些早期事件，在一系列单程灌注实验中测量tRNA和细胞内池中缬氨酸的比活性，其中细胞外缬氨酸浓度和比活性保持恒定。细胞内的缬氨酸比活性上升的半衰期为1.2分钟。相比之下，在比活性的缬氨酰-tRNA的上升是双相的：缬氨酰-tRNA曲线的初始阶段是快速的，而第二阶段的半衰期等于细胞内的缬氨酸。这些数据表明，在生理浓度的缬氨酸，缬氨酰-tRNA从细胞外和细胞质池中获得其氨基酸，并且至少一些tRNA在与细胞内氨基酸池混合之前被细胞外氨基酸充电，可能来自细胞膜处或附近的前体池。
Total protein synthesis in perfused livers of fed rats was determined by measuring the rate of valine incorporation based on the specific activity of valine attached to tRNA. Rates were not significantly altered when perfusate valine was increased from 0.40 to 5 mM and were similar to values calculated earlier from the specific activity of extracellular valine at a concentration of 15 mM. Overall protein degradation, computed from the sum of the rates of synthesis and the total increase of free intra- and extracellular valine, corresponded closely to the increase of free valine that occurred between 5 and 15 min after the addition of cycloheximide. In the latter experiments advantage was taken of the fact that the previously established suppressive effect of cycloheximide on proteolysis does not begin initially with the inhibition of synthesis, but 15 min later. Thus, the release of valine from 5 to 15 min was assumed to represent rates of protein degradation in effect prior to the addition of cycloheximide. The close agreement found among these independent assessments of protein metabolism thus appears to eliminate much of the previous uncertainty in the quantitation of hepatic protein turnover. In the course of these studies we noted that the specific activity of valyl-tRNA attained steady state values that were intermediate between specific activities of the extracellular and intracellular pools, but appeared to reach a steady state sooner than that of intracellular valine. To evaluate these early events more precisely, the specific activity of valine in tRNA and the intracellular pool was measured in a series of single-pass perfusion experiments where extracellular valine concentration and specific activity were held constant. The intracellular valine specific activity rose with a half-life of 1.2 min. By contrast, the rise in the specific activity of valyl-tRNA was biphasic: the initial phase of the valyl-tRNA curve was rapid, while the second phase had a half-life equal to that of intracellular valine. These data show that at physiological concentrations of valine, valyl-tRNA derives its amino acids from both the extracellular and cytoplasmic pools, and that at least some tRNA is charged by extracellular amino acids before they mix with intracellular amino acid pools, possibly from a precursor pool at or near the cell membrane.