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.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000503:physiopathology*; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D007700:Kinetics; D051379:Mice; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D011691:Puromycin / Q000031:analogs & derivatives* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D014316:Tritium
C031736:peptidyl-puromycin; D014316:Tritium; D011691:Puromycin