Relationship of the Pool of Intracellular Valine to Protein Synthesis and Degradation in Cultured Cells

To explore the role of the pool of intracellular free valine in the processes of protein synthesis and protein degradation, cultured hepatoma (HTC) cells were incubated in media containing varying concentrations of L-valine, under conditions of constant rates of protein synthesis and protein breakdown, and at steady state levels of intracellular valine specific radioactivities. Two types of experiments were compared: in the first (designated "incorporation experiment"), unlabeled cells were exposed to [3H]valine for a short period of time. In the second (termed "reincorporation experiment"), cells were prelabled with [3H]valine and then incubated for a brief period with media containing different concentrations of unlabeled valine; reincorporation of [3H]valine was calculated by the difference between the release of [3H]valine from labeled cellular proteins at low valine concentrations, and the maximal rate of the release at high valine concentrations. In both types of experiments, the rates of [3H]valine incorporation or reincorporation were compared with the respective specific radioactivities of free intracellular valine. In the incorporation experiment, the rates of [3H]valine incorporation into protein calculated by the intracellular specific radioactivities were not constant, but showed an upward deviation at low valine concentrations. This is in agreement with the results of Mortimore, G.E., Woodside, K.H., and Henry, J.E. ((1972) J. Biol. Chem. 247, 2776-2784) in the perfused rat liver. By contrast, in the reincorporation experiment, the calculated rates of [3H]valine reincorporation based on intracellular specific radioactivities were constant throughout the range of valine concentrations. The constant value of calculated valine reincorporation was lower by 30 to 50% than the calculated rate of valine incorporation at high valine concentrations. The following model is proposed to explain these results. There is one common pool of free intracellular valine, but there are two sites where valyl-tRNA can be formed. The first is an internal site that utilizes valine from the intracellular pool, and the second is an external (possibly membranous) system that converts extracellular valine directly to valyl-tRNA. Valine originating from protein degradation flows into the intracellular pool, from which it can be reutilized by the internal system. According to these assumptions, in the incorporation experiment and at low valine concentrations, the specific activity of valyl-tRNA is higher than that of the intracellular pool of free valine, due to the contribution of the external system. On the other hand, in the reincorporation experiment the specific activity of extracellular valine is negligible in comparison with that of the intracellular pool. Therefore, in this case the specific activity of valyl-tRNA is proportional to that of the intracellular pool, with a constant dilution by unlabeled valine of extracellular origin...