Radioactive puromycin formation as an assay for the proportion of active ribosomes in mammalian cells: correlation with polysome profiles under different physiological conditions.

We have established optimal conditions for the in vitro formation of peptidyl-[3H] puromycin by mammalian ribosomes. The growth conditions of cultured Ehrlich ascites tumor cells were manipulated to produce changes in the polysome profiles. The correlation between polysome content and peptidyl-[3H] puromycin formation was linear and excellent when different cell densities were compared. The percentage of ribosomes actively engaged in protein synthesis, calculated from the number of 3H-peptide bonds formed, was similar in rapidly growing Ehrlich cells (47%) and in young rat gastrocnemius muscle (44%). Starvation resulted in a 50% reduction in the number of puromycin-reactive ribosomes in rat gastrocnemius.