Artifacts in studies of protein synthesis with radioactive amino acids. Invalidity of alleged stimulation of protein synthesis by thyroxine in vitro in absence of mitochondria.

Previous studies have demonstrated that thyroid hormones in vitro stimulate microsomal protein synthesis in cell-free systems, but the effect was found to be indirect and dependent on the presence of mitochondria in the incubation system. The mitochondrial requirement has recently been questioned by Carter ef al. ((1971) J. Biol. Chem. 246,4973), who claimed to observe direct stimulations by thyroxine in cell-free liver systems lacking mitochondria. The system used by them contained higher Mg2+ concentrations, lower microsomal concentrations, and uniformly labeled L-[“Clvaline of very high specific activity. We have examined this effect and confirmed the stimulation of 14C incorporation into protein but found that it bears no relationship to protein synthesis. The effect is not observed with tRNAJ%-amino acid as the source of 14C-amino acid. It is not inhibited by cycloheximide, puromycin, or ribonuclease, and is, in fact, enhanced in their presence. It exhibits no dependence on microsomes or ATP and is maximum in their absence. The effect is not diluted out by dilution of the L-[UJ4C]valine with unlabeled L-valine. Chromatographic analysis of hydrolysates of the labeled protein reveals that the thyroxine stimulation is on the incorporation not of the “C-amino acid but of a radioactive contaminant present in the L-[U-14C]valine. Purification of the %-amino acid by ion exchange chromatography before use reduces or eliminates both the base-line incorporation and the thyroxine effect. Similar spurious incorporation and thyroxine stimulations are observed with Land ~-[lJ~c]valine, but they are almost negligible compared to those observed with L-(UJ4C]valine. The thyroxine effect reported by Carter el al. is, therefore, not on protein synthesis but on the energy-independent incorporation of a radioactive contaminant of the L-UJ4C-amino acid into cell sap protein. It is, in fact, obscured by protein synthesis and was unmasked by them when they reduced protein synthesis by lowering the microsomal concentration and compensated for the reduced 14C incorporation by using L-U-14C-amino acid of high specific activity.