Reutilization of Amino Acids in Protein Synthesis in HeLa Cells

Abstract On the assumption that all amino acids entering protein must pass through the intracellular pool of free amino acids, reutilization of leucine, lysine, arginine, and alanine was determined by pulse labeling of HeLa cell protein, followed by a chase in unlabeled medium and measurement of the specific activities of the intracellular free pool and protein amino acids. The half-lives of the amino acids in protein were similar (approximately 72 hours), as were those of the intracellular free pool amino acids (approximately 25 hours). When specific activity values were compared between pool and protein at 10 hours, reutilization based on this assumption was found to be about 40% for lysine, leucine, and arginine and about 20% for alanine. Because the turnover rates of HeLa cell protein constituents differ widely, and because the truth of the stated assumption was doubtful, a more accurate measure of reutilization was sought. The intracellular pools of free lysine, leucine, and phenylalanine in unlabeled HeLa cells were equilibrated with the amino acids in labeled medium (40 min), and a protein (ferritin) was then induced over 3 hours, before the bulk of the protein had acquired significant labeling. Ferritin was then isolated by immunoprecipitation and its specific activity was measured and compared to that of the intracellular free pools and the medium. Reutilization of leucine was 81%, lysine, 86%, and phenylalanine, 84%. Moreover, the specific activity of leucine in newly induced ferritin was 30% lower than that of leucine in the free pool, and that of ferritin phenylalanine was 62% lower than pool phenylalanine, showing the separation into compartments of amino acids, part derived from protein breakdown and reutilized and part derived from the intracellular free pool of amino acids.