Glutathione. VIII. The effects of glutathione disulfide on initiation of protein synthesis

1. 1. Glutathione disulfide (GSSG) additions (1 · 10^-4-2· 10^-4 M or less) to rabbit reticulocyte lysates containing glutathione (GSH) in normal amounts (0.7 · 10^-3-3 · 10^-3 M) have no immediate effect on the rate of protein synthesis at 33°C but cause a rapid decrease in synthesis rate after 6-9 min (the lag period), followed by complete cessation of protein-synthesizing activity. 2. 2. The lag period varies with temperature; the length of the lag period and the time dependence of the decrease in synthesis provide important information for the interpretation of the GSSG effect on protein synthesis. 3. 3. Reduction of GSSG to GSH before or during the lag period prevents inhibition of protein synthesis. Such reduction has no effect on the behavior of the system if carried out after the lag period. 4. 4. Cessation of protein synthesis is accompanied by a conversion of polysomes to monosomes, with little dissociation into subunits. 5. 5. A ribosome-free supernatant is effective in reversing the inhibition of protein synthesis caused by GSSG. Fractionation of the supernatant leads to a protein (precipitated by 40-70 % (NH₄)₂SO₄) which reverses the GSSG inhibition. Further fractionation yields a high molecular weight fraction with inhibition-reversing activity. 6. 6. Factor Q is the name given to the material with inhibition-reversing activity, the inhibition-reversal factor. A scheme for the behavior of Factor Q in protein synthesis is proposed. 7. 7. The effect of the antibiotic, cycloheximide, on initiation of protein synthesis is interpreted in light of the GSSG effect. 8. 8. A small rise in the usual GSSG concentration of a cell is recognized as a potentially important event, one requiring careful scrutiny.