SOURCE OF AMINO-ACIDS FOR TRANSFER-RNA ACYLATION - IMPLICATIONS FOR MEASUREMENT OF PROTEIN-SYNTHESIS
SOURCE OF AMINO-ACIDS FOR TRANSFER-RNA ACYLATION - IMPLICATIONS FOR MEASUREMENT OF PROTEIN-SYNTHESIS
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DOI：
10.1042/bj2830583
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发表时间：
1992-04-15
影响因子：
4.1
通讯作者：
KLASING, KC
中科院分区：
文献类型：
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作者：
BARNES, DM;CALVERT, CC;KLASING, KC
使用放射性同位素估计蛋白质合成速率需要准确测量随着时间的推移标记在蛋白质和前体池中的特定放射性。虽然细胞外和细胞内的氨基酸池最容易采样，但tRNA池是直接的前体，是合适的采样池。为了测试细胞内或细胞外池是否反映tRNA特异性放射性，鸡巨噬细胞系在含有0.23mM-亮氨酸和14.5mU-Ci的[H-3]亮氨酸(示踪剂剂量)或2.3mU-M-亮氨酸+145.0 mU-Ci的[H-3]亮氨酸(洪泛剂量)的培养液中孵育。在两种亮氨酸水平下，tRNA比放射性迅速达到平台期，但在30min内与胞外和胞内的比放射性均未达到平衡，并保持在接近蛋白质的水平。在第二个实验中，用[H-3]亮氨酸标记鸡巨噬细胞中的蛋白质，为期2天。去掉标记介质，洗去细胞中残留的[H-3]亮氨酸，并与含有0.23 mM或2.3 mM亮氨酸的培养液(未标记)孵育。亮氨酰-tRNA亮氨酸的比放射性在2分钟内达到平台期，并在至少60分钟内与蛋白质中的比放射性比胞内池和胞外池中的比放射性更接近。这些结果表明，蛋白质降解产生的氨基酸是tRNA充电的主要来源。当通过标记掺入来估计蛋白质合成速率时。使用细胞外或细胞内的特定放射性活度值会导致明显低估。
Estimates of protein-synthesis rates using radioisotopes require accurate measurement of the specific radioactivity of the label in protein and in the precursor pool over time. Although the extracellular and intracellular pools of amino acids are easiest to sample, the tRNA pool is the direct precursor and is the appropriate pool for sampling. To test if the intracellular or extracellular pools reflect the tRNA specific radioactivity, a chicken macrophage cell line was incubated in medium containing either 0.23 mm-leucine and 14.5-mu-Ci of [H-3]leucine (tracer dose) or 2.3-mu-M-leucine plus 145.0-mu-Ci of [H-3]leucine (flooding dose). At both leucine levels, the tRNA specific radioactivity reached a plateau quickly, but did not equilibrate with either the extracellular or intracellular specific radioactivity within 30 min, and remained closer to that of protein. In a second experiment, proteins in chicken macrophages were labelled with [H-3]leucine for 2 days. Labelling medium was removed, and the cells were washed free of residual free [H-3]leucine and incubated with medium containing either 0.23 mM- or 2.3 mm-leucine (unlabelled). The specific radioactivity of leucyl-tRNA leuCine reached a plateau within 2 min and remained considerably closer to that in the protein than that in intracellular or extracellular pools for at least 60 min. These results suggest that amino acids from protein degradation are a primary source for charging tRNA. When protein-synthesis rates are estimated by label incorporation. use of extracellular or intracellular specific-radioactivity values result in a marked underestimation.