Compartmentation of intracellular amino acids in rat liver. Evidence for an intralysosomal pool derived from protein degradation.

The subcellular distribution of free amino acids was examined in 0.25 M sucrose homogenates of rat livers cycbtally perfused for 60 min with an unsupplemented (control) medium. An unexpectedly high (30%) proportion of total intracellular valine was found in the mitochondrial-lysosoma1 fraction; 57% was in the 100,000 x .o supernatant; and 9% and 4% were in the nuclear and microsomal fractions, respectively. Only 10% of the intracellular label was recovered in the mitochondrial-lysosmal fraction when livers were perfused for 15 min in the single-pass mode with I “Clvaline and unlabeled valine at concentrations of 0.37 and 3 mu, a distribution that could be accounted for by the water space of the pellet. The nonequilibrating mitochondrial-lysosomal pool, which comprised 20% of total intracellular valine, was fully labeled in livers from rats previously labeled with [‘4C]valine in I&O, thus indicating that the valine was derived from protein degradation. Similar subcellular distributions were found for 11 other amino acids, and the amounts recovered in the mitochondrial-lysosomal fraction (excluding the 10% pellet space) directly correlated (r = 0.92) with the molar content of the residues in acid hydrolysates of liver protein. Since total intraand extracellular valine relate linearly over a wide range of concentration, we extrapolated the slope to zero external valine to obtain from the intercept an approximation of the size of the nonequilibrating pool. This procedure eliminated the possibility of valine loss from particles during subcellular fractionation and provided a maximal estimate of the nonequilibrating pool. The calculated value was 574 f 144 nmol of valinelliver (100-g rat). A similar correlation between valine in the nuclear-mitochondrial-lysosomal pellet and external valine gave an intercept which was 40% of the maximal estimate. Additions of insulin and an incomplete mixture of amino acids during perfusion virtually abolished both intercepts (70 to 90% inhibition), an effect which directly substantiates our belief thatthe nonequilibrating pool is intralysosomal and is responsive to agents which regulate proteolysis.