Mechanism of cycloheximide inhibition of protein synthesis in a cell-free system prepared from rat liver.

Sites of cycloheximide action on protein synthesis were examined using a cell-free system prepared from rat liver. I f all amino acids or aminoacyl transfer RNA were present at the start of incubation, the system appeared to incorporate W-leucine mainly by elongation of peptide chains. Under these conditions, high dose levels of cycloheximide were necessary in order to inhibit incorporation extensively. The inhibition could be prevented by raising the glutathione content of the reaction mixture, and particularly by preliminary incubation of a mixture of transferase I and II with high concentrations of glutathione before adding these enzymes to the system. Other sulfhydryl compounds were also effective in protecting against cycloheximide. It has been concluded that the inhibitory action of cycloheximide on peptide chain elongation involves inactivation of transferase II, an enzyme known to have a sulfhydryl requirement. High concentrations of glutathione were also found to prevent the inhibition of cell-free protein synthesis caused by streptovitacin A, a derivative of cycloheximide, but not inhibition caused by emetine or sparsomycin. If the protein-synthesizing system was first incubated without amino acids or aminoacyl-tRNA, polysomes present at the start of incubation underwent disaggregation. On addition of amino acids at this point, the polysomes became reaggregated and incorporation of 14C-leucine was stimulated, probably by a process involving chain initiation. The response of polysome aggregation and coincident 14C-leucine uptake could be inhibited by low doses of cycloheximide. Furthermore, this inhibitory action of cycloheximide could not be prevented by raising the glutathione content of the medium. This suggests that the action of cycloheximide on polysome aggregation differs from its effect on peptide chain elongation.