Inhibition of basal protein degradation in rat embryo fibroblasts by cycloheximide: Correlation with activities of lysosomal proteases

Rat embryo fibroblasts were grown in medium containing ¹⁴C-leucine and ³H-thymidine. After a 24-hour chase in nonlabeled medium, cultures were placed in either fresh growth medium or medium containing 10–20 μg/ml cycloheximide. Cell monolayers were processed at daily intervals for three days. Four hours prior to processing, cultures were placed in fresh medium and the accumulation rate of trichloracetic acid soluble ¹⁴C in the media assayed. Cycloheximide effects a progressive decrease in the fractional degradation rate of the labeled cell protein, primarily during the first 24 hours. The specific activities of cathepsin D, cathepsin B, and neutral protease correlate closely with the fractional degradation rate. Other lysosomal hydrolases show little change during this period. The activities of the lysosomal proteases approach a new steady state which is correlated with the new steady state leve of protein synthesis. A model is proposed which relates the rate of protein break-down in the cell to the level of protein synthesis. The data also suggests the possibility that subpopulations of high turnover and low turnover cells exist in these cultures.