Cytoplasmic extracts of green monkey kidney-Vero M 3 cells that have been grown to high cell density and have entered the stationary phase of growth lose the capacity to synthesize proteins after they have been frozen and thawed. The same loss of protein synthetic capacity is not observed when cytoplasmic extracts of low cell density Vero M3 or HeLa S-3 cells are frozen and thawed before assay, nor when high cell density Vero M3 cell extract is assayed without having been frozen. The loss of the protein synthesis capacity of the frozen and thawed extracts of stationary phase Vero M 3 cells is accounted for by the appearance of an inhibitor. This inhibitor is precipitated in the 30 to 70% ammonium sulfate fraction of the 100,000 g supernatant. It is inactivated by heat and is non-dialyzable. It blocks the transfer of amino acids from tRNA to growing peptide chains.
The amount of this inhibitor in the extract (measured by relative inhibition of in vitro protein synthesis) increases as a function of the density of the cells from which the extract was made, and the increase can be correlated with the progressive turnoff of protein synthesis in whole cells.