Subcellular Distribution of Proteolytically Generated Valine in Isolated Rat Hepatocytes

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Abstract
1 A small fraction of the intracellular acid-soluble radioactivity in [14C]valine-labelled hepatocytes remained non-extracted even after repeated incubations in isotope-free medium at 37°C. This non-extracted radioactivity was only present when the cells had been labelled under conditions allowing protein synthesis. 2 Approximately two-thirds of the non-extracted radioactivity was recovered in material with a molecular weight larger than free valine, i.e. presumably acid-soluble peptides. The intracellular contents of these peptides were unaffected by inhibitors of hepatocytic protein degradation, and they would therefore seem to represent primary synthesis products rather than products of proteolysis. 3 The remaining one-third of the radioactivity was free [14C]valine, derived from intracellular protein degradation as indicated by the reduced levels seen in the presence of inhibitors of lysosomal (ammonia, methylamine, leupeptin, chymostatin) and non-lysosomal (chymostatin) proteolysis. The intracellular levels of twelve other amino acids were similarly reduced upon inhibition of protein degradation. 4 The contents of free [14C]valine in subcellular fractions were compatible with a uniform distribution of the amino acid throughout the cell. There was no evidence for any enrichment of [14C]valine in a purified lysosomal fraction, separated from mitochondria by means of isotonic metrizamide gradients. 5 It can be concluded that the non-extracted valine in hepatocytes may represent a steady-state level, maintained by intracellular protein degradation, of amino acid in transit through the cell. The non-extractability, i.e. the maintenance of a concentration gradient towards the extracellular medium, can be calculated to be compatible with a limitation of valine efflux by the concentration-dependent valine transport system. Since no specific subcellular compartmentation is indicated, intracellular valine can provisionally be regarded as a single, uniform pool.