Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from pre‐labelled protein. To reduce background radioactivity, the intracellular [14C]valine pool was depleted by serial extraction at 37 °C, effecting equilibration between the intracellular pool and the valine‐free extracellular medium. After extraction, a small, non‐equilibrating intracellular [14C]valine pool remained; this pool could only be labelled in the presence of ongoing protein synthesis, and might represent valine and valine‐containing oligopeptides derived from protein degradation. The [14C]valine released from degraded protein was not significantly re‐utilized for protein synthesis intracellularly (no effect of cycloheximide or high concentrations of unlabelled valine), reflecting the low rate of protein synthesis and the rapid transport of valine into the extracellular medium, both characteristic of isolated hepatocytes. From cells pre‐labelled for 24 h in vivo, [14C]valine was released linearly at a rate of 5%/h, probably representing the true over‐all protein degradation rate. The lysosomotropic inhibitor ammonia (10 mM NH4Cl) inhibited 70% of the degradation, presumably the contribution by the lysosomal pathway.From 1‐h pre‐labelled cells, [14C]valine was released at a declining rate, and ammonia inhibited degradation only by 45%, consistent with the view that the majority of short‐lived proteins are degraded by the non‐lysosomal pathway(s). Chloroquine and methylamine, which accumulate in lysosomes by virtue of their weak base properties, inhibited hepatocytic protein degradation to the same extent as ammonia, with no additivity. These compounds therefore seem to block the lysosomal pathway of protein degradation selectively and completely. Leupeptin, which binds to and inhibits the activity of certain lysosomal proteases, also inhibited protein degradation almost to the same extent as ammonia, but with a small part of the effect (< 20%) being additive to the NH3 effect and thus probably reflecting a slight inhibition of non‐lysosomal protein degradation as well. Of the four inhibitors tested, only the effect of ammonia was rapidly reversible within the experimental period (2 h). Leupeptin, on the other hand, was the only degradation inhibitor which did not also affect protein synthesis. Chloroquine caused significant cell death at concentrations above 0.2 mmol/l in this protein‐free medium, i.e. in the concentration range needed for maximal inhibition of protein degradation. Incubation of hepatocytes under anoxic conditions resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of ammonia, i.e. most of the degradation by the lysosomal pathway and more than one‐half of the degradation by the non‐lysosomal pathways appears to be energy‐dependent.

SEGLEN, P. O., GRINDE, B., & SOLHEIM, A. E. (1979). Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin. European Journal of Biochemistry, 95(2), 215–225. Portico.