In vitro Inhibition of Peptide Synthesis and GTP Hydrolysis by Cycloheximide and Reversal of Inhibition by Glutathione

Abstract

THE inhibition of protein synthesis by cycloheximide seems to occur at some stage later than the formation of aminoacyl-tRNA and its subsequent transfer to the ribosome¹. Studies of the action of cycloheximide on intact cells^2,3 have led to the proposal that the inhibitor has two sites of action at the ribosome level, one involving chain initiation and the other chain elongation. Using a cell-free protein-synthesizing system, we⁴ have observed that cycloheximide can inhibit polysome aggregation in much smaller doses than are needed to prevent chain elongation. With this system, we also observed that the action of the inhibitor on chain elongation could be partially reversed by adding high concentrations of glutathione to the incubation medium. This communication presents evidence that the site of this action of cycloheximide and its reversal by glutathione is the sulphydryl-dependent enzyme transferase II. Skogerson and Moldave⁵ have suggested that transferase II is identical with translocase, an enzyme responsible for transferring the peptidyl-tRNA from the acceptor to the peptidyl site on the ribosome, with concomitant hydrolysis of GTP. We have now demonstrated that this specific hydrolysis of GTP is inhibited by cycloheximide, and that this effect can also be partially reversed by raising the concentration of glutathione in the medium.