Regulation of protein accumulation in cultured cells

Abstract

1. A technique is described whereby protein synthesis, protein breakdown and net protein accumulation are measured separately in monolayer cultures of mammalian cells. All rates are expressed as microgram of protein per 18 h incubation. 2. Under most incubation conditions with either L6 rat myoblasts or T47D human breast carcinoma cells the rates of protein accumulation, determined directly, agreed with the rates obtained by subtracting protein breakdown from protein synthesis. 3. Foetal calf serum, human and bovine colostrum, human milk and insulin increased protein accumulation in both cell lines, mainly as a consequence of effects on protein synthesis. 4. NH4Cl, in addition to inhibiting protein breakdown in both cell lines in the presence and in the absence of serum, stimulated protein synthesis in L6 myoblasts. 5. Leupeptin slightly inhibited protein breakdown without affecting protein-synthesis rates. 6. Cycloheximide almost completely inhibited protein synthesis, but restricted the net loss of cell proteins under most conditions because protein-breakdown rates were also decreased. 7. The assumptions, limitations and potential application of this technique for evaluating changes in protein turnover are described.