Demonstration of thyroxine-stimulated incorporation of amino acid into peptide linkage in mitochondria-free system.

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.