Measurement of the rate of protein synthesis and compartmentation of heart phenylalanine

Abstract

Calculation of rates of protein synthesis, based upon incorporation of [14C]phenylalanine into protein, depended upon use of the specific activity of phenylalanyl-tRNA. At a perfusate phenylalanine concentration of 0.01 mM, the specific activity of phenylalanyl-tRNA was 65 and 155% of extracellular and intracellular specific activities, respectively. At this concentration, the rate of protein synthesis was overestimated if calculated using the intracellular specific activity, but underestimated if the extracellular specific activity was employed. Thus, neither the extracellular nor total intracellular pool of phenylalanine served as the sole precursor for protein synthesis. When the concentration of perfusate phenylalanine was increased from 0.01 to 3.6 mM, the concentration of intracellular phenylalanine increased linearly. At a perfusate phenylalanine concentration of 0.4 mM, specific activities of extracellular, intracellular, and tRNA-bound phenylalanine were the same, and the calculated rate of protein synthesis was 106 nmol of phenylalanine incorporated/g of heart/h. The same rate was obtained using the specific activity of V2lphenylalanyLtRNA, when perfusate phenylalanine was 0.01 mM. Thus, protein synthesis did not depend on extracellular phenylalanine concentration over this range. Similarly, raising perfusate phenylalanine from 0.01 to 3.6 mM had no effect on the incorporation of [14Clhistidine. Addition of insulin to the perfusate did not modify the relationship between the specific activities of extracellular, intracellular, and tRNA-bound phenylalanine at perfusate phenylalanine levels of either 0.01 or 0.4 mM, but increased the rate of protein synthesis approximately 100%. Specific activities of heart phenylalanine and phenylalanyl-tRNA equilibrated with perfusate phenylalanine (0.01 mM) within 3 to 5 min. Addition of insulin resulted in even faster equilibration. Two models of amino acid compartmentation involving (a) aminoacylation of tRNA from both the extracellular and intracellular compartments or (b) aminoacylation of tRNA from a compartmented intracellular pool were consistent with the steady state and transient data. Inclusion of an intracellular pool of nonradioactive phenylalanine was not required to fit the data. Preferential aminoacyla-