Quantification of protein turnover in primary cultures of rat hepatocytes.
1. A radioactive tracer method, based on [3H]valine, for determination in parallel experiments of degradation of cellular proteins and synthesis of both cellular and secreted proteins in cultured rat hepatocytes was developed with special emphasis on methods of calculation, the number of protein pools and maintenance of nitrogen balance. 2. An extracellular concentration of 2 mM valine ensured a specific activity of the precursor pool for protein synthesis, which was constant during the experimental period and practically identical to that of extracellular valine. 3. Amino acid concentrations in the culture medium used were not rate-limiting for the synthesis of proteins. 4. The rate of labelling of the cellular protein pool during 8 days was in accordance with first-order saturation kinetics, which together with a constant ratio between labelling of soluble and membrane bound proteins, is compatible with a single pool of cellular proteins. 5. Protein degradation can be accurately measured by the release from prelabelled proteins of [3H]valine in the presence of 2 mM extracellular valine, if a 1 h chasing period is included in the experimental design. 6. The constancy of the degradation constant (kd) during protein labelling for up to 8 days, is in accordance with the existence of only one pool of cellular protein. 7. The cultured hepatocytes were in nitrogen balance during the experimental period of 8 days as reflected in a constant protein content per cell. The absolute rates of degradation and synthesis of cellular protein were identical, which confirm the validity of the method described.
Elsner
P
P
Department of Medical Biochemistry and Genetics, University of Copenhagen, Denmark.
Dich
J
J
Grunnet
N
N
D000818:Animals; D002478:Cells, Cultured; D005260:Female; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011849:Radioactive Tracers; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D014633:Valine / Q000378:metabolism
D008565:Membrane Proteins; D011506:Proteins; D011849:Radioactive Tracers; D014633:Valine; D009584:Nitrogen