Replacement perfusion of cultured eucaryotic cells: a method for the accurate measurement of the rates of growth, protein synthesis, and protein turnover.
The fractional rates of protein synthesis (ks) and degradation (kp) were studied in the myeloma cell line SP2/0-AG14 grown at different rates (kg). Cells in spinner flask suspension cultures were maintained at constant cellular density for prolonged periods by replacement perfusion of labeling medium at a rate equivalent to the rate of growth. Total protein synthesis was calculated from the specific radioactivity of labeled L-leucine in the precursor (medium) and cellular protein. Fractional synthesis rates determined by approach to equilibrium labeling were the same as those determined by equilibrium-pulse labeling kinetics and pulse-chase kinetics. The rate of protein degradation was determined from the established relationship Kg = ks - kp. Protein synthesis rates remained constant over a threefold range in the rate of cell growth. At relatively slow growth rates (kg = 0.017/hr) turnover represented a major fraction of total synthesis (kp = 0.032/hr = 0.65ks). At rapid growth rates (kg = 0.058/hr) the value of kp was less than 0.005/hr. No major difference was observed between the ks determined for individual cellular proteins (separated by SDS-polyacrylamide (7.5%) gel electrophoresis) from rapid- and slow-growing cultures. Thus, with an invariable ks, any change in growth rate is due to an inverse change in the rate of turnover. Since turnover is the balance between synthesis and degradation and since synthesis is unchanging, then changes in the growth rate of SP2/0-AG14 should be due to changes in the rate of protein degradation. Experiments were therefore performed to determine the origin of the degradative machinery, ie, cytosolic or lysosomal; autolysis of prelabeled cellular protein (in vitro) was observed only at acidic pH (4.2) and was totally inhibited by addition of leupeptin (10 microM) and pepstatin (2 microM), the specific inhibitors of lysosomal cathepsins B (&L) and D, respectively. Since growth rate appears to be regulated by the alterations in the rate of protein degradation and degradation (in vitro) in SP2/0-AG14 appears to be lysosomal, then one should be able to alter the rate of cellular growth by interfering with rate of lysosomal proteolysis. Indeed, when the lysosomotropic amine NH4Cl (10 mM) is added to cells growing with a kg of 0.018/hr +/- 0.001 (ks = 0.050/hr +/- 0.002) the growth rate increased to 0.051/hr +/- 0.002 without change in the rate of protein synthesis (ks = 0.049/hr +/- 0.003).(ABSTRACT TRUNCATED AT 400 WORDS)
D002477:Cells / Q000378:metabolism*; D002478:Cells, Cultured; D005057:Eukaryotic Cells / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010477:Perfusion; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*
D011506:Proteins; D010447:Peptide Hydrolases; D007930:Leucine