Carter W J, Faas F H, Wynn J
J Biol Chem. 1975 May 25;250(10):3588-94.
The observation that thyroxine stimulated in vitro protein synthesis in the absence of mitochondria (Carter, W.J., Faas, F.H., and Wynn, J (1971) J. Biol. Chem. 246, 4973-4977) has been disputed on the basis that radioactivity incorporated into protein did not represent peptide synthesis but incorporation of labeled contaminants present in the L-(U-14C) valine precursor (Sokoloff, L., and Roberts, P.A. (1972 Fed. Proc. 31, 1525). The question of mitochondrial requirement is important in determining whether thyroxine has a direct action on the polysome or causes the release of stimulatory factors from mitochondria. In this paper, thyroxine stimulation of peptide synthesis in mitochondria-free systems has been confirmed. Peptide synthesis is required for the thyroxine effect since it is dependent on the presence of polysomes and an energy source in the reaction mixture and is abolished by puromycin. The thyroxine effect is not due to incorporation of labeled contaminants since hydrolysis of labeled protein recovered from control and thyroxine-treated reaction mixtures yields the labeled amino acid precursor as the only radioactive product. Thyroxine stimulates polyuridylic acid-directed polyphenylalanine synthesis, providing further evidence that thyroxine is stimulating peptide synthesis rather than incorporation of radioactive contaminants by mechanisms other than peptide synthesis. Although thyroxine stimulates polyphenylalanine synthesis, it does not influence polyuridylic acid hydrolysis measured in the same reaction. Therefore, thyroxine stimulation of peptide synthesis is not due to prevention of hydrolysis of nucleic acid components of the reaction mixture. Thyroxine does not influence the size or specific activity of the free valine pool in the reaction mixture, indicating that observed increases in valine incorporation reflect increased peptide synthesis rather than increased specific activity of the valine precursor. The fact that thyroxine stimulates peptide synthesis using (14C)aminoacyl-tRNA precursors strengthens this conclusion. Therefore, thyroxine stimulation of protein labeling is dependent on the presence of peptide synthesis and cannot be explained by incorporation of labeled contaminants, prevention of RNA hydrolysis, or change in the specific activity of the amino acid precursor. Thyroxine causes a genuine increase in peptide synthesis by a direct action at the polysomal level.
在缺乏线粒体的情况下，甲状腺素能刺激体外蛋白质合成（卡特，W.J.，法斯，F.H.，和韦恩，J（1971年）《生物化学杂志》246卷，4973 - 4977页），但这一观察结果受到了质疑，理由是掺入蛋白质中的放射性并非代表肽合成，而是L -（U - 14C）缬氨酸前体中存在的标记污染物的掺入（索科洛夫，L.，和罗伯茨，P.A.（1972年）《联邦会议记录》31卷，1525页）。线粒体需求问题对于确定甲状腺素是直接作用于多核糖体还是导致线粒体释放刺激因子至关重要。在本文中，甲状腺素对无线粒体系统中肽合成的刺激作用得到了证实。肽合成是甲状腺素发挥作用所必需的，因为它依赖于反应混合物中多核糖体和能量源的存在，并且会被嘌呤霉素所抑制。甲状腺素的作用并非由于标记污染物的掺入，因为从对照和甲状腺素处理的反应混合物中回收的标记蛋白质水解后，产生的标记氨基酸前体是唯一的放射性产物。甲状腺素刺激多聚尿苷酸指导的多聚苯丙氨酸合成，进一步证明甲状腺素是在刺激肽合成，而不是通过肽合成以外的机制掺入放射性污染物。尽管甲状腺素刺激多聚苯丙氨酸合成，但它并不影响在同一反应中测得的多聚尿苷酸水解。因此，甲状腺素对肽合成的刺激并非由于防止反应混合物中核酸成分的水解。甲状腺素不影响反应混合物中游离缬氨酸池的大小或比活性，这表明观察到的缬氨酸掺入增加反映的是肽合成增加，而不是缬氨酸前体比活性的增加。甲状腺素使用（14C）氨酰 - tRNA前体刺激肽合成这一事实强化了这一结论。因此，甲状腺素对蛋白质标记的刺激依赖于肽合成的存在，不能用标记污染物的掺入、RNA水解的防止或氨基酸前体比活性的变化来解释。甲状腺素通过在多核糖体水平的直接作用导致肽合成真正增加。
