[en] Previous studies on protein turnover in ³H-labelled L-cell cultures have shown recovery of total ³H at the end of a three-day experiment to be always significantly in excess of the ³H recovered at the beginning of the experiment. A number of possible sources for this error in measuring radioactivity in cell proteins has been reviewed. ³H-labelled proteins, when dissolved in NaOH and counted for radioactivity in a liquid-scintillation spectrometer, showed losses of 30-40% of the radioactivity; neither external or internal standardization compensated for this loss. Hydrolysis of these proteins with either Pronase or concentrated HCl significantly increased the measured radioactivity. In addition, 5-10% of the cell protein is left on the plastic culture dish when cells are recovered in phosphate-buffered saline. Furthermore, this surface-adherent protein, after pulse labelling, contains proteins of high radioactivity that turn over rapidly and make a major contribution to the accumulating radioactivity in the medium. These combined errors can account for up to 60% of the total radioactivity in the cell culture. Similar analytical errors have been found in studies of other cell cultures. The effect of these analytical errors on estimates of protein turnover in cell cultures is discussed. (author)

[en] In previous studies on protein turnover in labeled L-cells the authors observed that the accumulation of the /sup 3/H-leucine in the medium during the chase period exceeded the loss of label from the cells by a factor of 2-3. This resulted in a progressive increase in the total radioactivity (RA) recovered over the course of a 3-day experiment. Of concern, the methods used to recover the cells from the dishes and to prepare the proteins for counting were commonly used techniques: scraping the cell monolayer carefully X3 into buffer, precipitating the protein with cold TCA, and solubilizing the protein in NaOH before counting. Estimates of protein turnover were markedly affected by the different results obtained from the medium RA and from the cell RA. Finally, this problem appeared to be particularly severe in certain types of cell cultures, e.g., some tumor cell lines and senescent cells, making it virtually impossible to measure accurately the effects of these pathologic states on protein turnover. In this study, the authors have critically reviewed the methodologies used in the recovery and preparation of cell proteins for measuring RA