Selective control of the degradation of normal and aberrant proteins in Reuber H35 hepatoma cells.
1. Rates of degradation of normal and abnormal protein were measured in hepatoma cells after labelling first for 16h with [14C]leucine plus L-arginine and then for 3h with [3H]-leucine plus the arginine analogue, L-canavanine. 2. Over the first 2h of the degradation period, canavanine-containing proteins were degraded at approximately 5 times the average degradation rate of normal proteins. 3. Degradation of normal proteins was inhibited by about 30% by insulin, cycloheximide, puromycin, leupeptin, antipain and foetal calf serum, whereas these agents had a negligible effect on the breakdown of canavanine-containing proteins. 4. Other compounds inhibited degradation of both classes of protein to equal extents. 5. Combination experiments showed no additional inhibitory effects on the degradation of normal proteins over degradation measured in the presence of a single selective inhibitor. 6. In contrast with the results with a 16 h labelling period, the degradation of normal proteins labelled for only 3 h was not inhibited by insulin. 7. These results are explained by a model with two distinct pathways of protein turnover. The first of these pathways involves the formation of autophagic vacuoles and would be completely inhibited by each of the selective inhibitors. Normal and canavanine-containing proteins would be catabolized by this pathway at equal rates. We propose that degradation by a second pathway is not regulated by the agents tested, but by the inherent stability of each protein.
D000596:Amino Acids / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism; D002172:Canavanine / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D005312:Fetal Blood; D007328:Insulin / Q000494:pharmacology; D007930:Leucine / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008113:Liver Neoplasms / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D009374:Neoplasms, Experimental / Q000378:metabolism; D010436:Pepstatins / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology
D000596:Amino Acids; D007328:Insulin; D007976:Leupeptins; D009363:Neoplasm Proteins; D010436:Pepstatins; D002172:Canavanine; D011691:Puromycin; D010664:Phenylmethylsulfonyl Fluoride; D001120:Arginine; D003513:Cycloheximide; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D007930:Leucine