Hildebran J N, Airhart J, Stirewalt W S, Low R B
Biochem J. 1981 Aug 15;198(2):249-58. doi: 10.1042/bj1980249.
Knowledge of the dynamics of collagen turnover requires information regarding rates of synthesis of this group of connective-tissue proteins. The relationship of various amino acid pools to the tRNA precursor pool used for protein synthesis is known to vary between different cell types and tissues, even for essential amino acids. We studied extracellular, intracellular and tRNA-proline pools in cultured human lung IMR-90 fibroblasts to determine the relationship between them as candidate proline precursor pools for total protein and collagen synthesis. Time-course experiments showed that the three proline pools attained distinctly different steady-state specific radioactivities (extracellular greater than intracellular greater than tRNA) at the extracellular proline concentration of 0.2 mM. The kinetics of radioisotope incorporation into cell protein and collagenase-digestible protein indicated that the intracellular free proline pool could not be used reliably as a precursor for calculating synthetic rates. However, tRNA-proline behaved isotopically as if it were the precursor and provided synthesis rates 2-3-fold higher than those calculated by using either free proline pool. The incorporation of labelled lysine and leucine was constant over a wide range of extracellular proline concentrations. Fractional rates of protein synthesis based on tRNA-amino acid were the same with [3H]phenylalanine as with [3H]proline. The specific radioactivity of cell-associated hydroxyproline reached a steady-state value 8-10h after radioisotope administration which matched the mean tRNA-proline specific radioactivity, suggesting that tRNA-proline is not isotopically compartmentalized. A model of cellular proline-pool relationship is presented and discussed.
了解胶原蛋白更新的动力学需要有关这类结缔组织蛋白合成速率的信息。已知不同细胞类型和组织中，各种氨基酸池与用于蛋白质合成的tRNA前体池之间的关系有所不同，即使是必需氨基酸也是如此。我们研究了培养的人肺IMR-90成纤维细胞中的细胞外、细胞内和tRNA-脯氨酸池，以确定它们之间的关系，将其作为总蛋白和胶原蛋白合成的候选脯氨酸前体池。时间进程实验表明，在细胞外脯氨酸浓度为0.2 mM时，三个脯氨酸池达到明显不同的稳态比放射性（细胞外大于细胞内大于tRNA）。放射性同位素掺入细胞蛋白和胶原酶可消化蛋白的动力学表明，细胞内游离脯氨酸池不能可靠地用作计算合成速率的前体。然而，tRNA-脯氨酸在同位素行为上就好像它是前体，并且提供的合成速率比使用任何一个游离脯氨酸池计算出的速率高2至3倍。在广泛的细胞外脯氨酸浓度范围内，标记赖氨酸和亮氨酸的掺入是恒定的。基于tRNA-氨基酸的蛋白质合成分数速率与[3H]苯丙氨酸和[3H]脯氨酸相同。给予放射性同位素后8-10小时，细胞相关羟脯氨酸的比放射性达到稳态值，与平均tRNA-脯氨酸比放射性相匹配，这表明tRNA-脯氨酸在同位素上没有被分隔。本文提出并讨论了细胞脯氨酸池关系的模型。
