Regulation of amino acid accumulation in isolated rat diagphragm: effect of puromycin and insulin.

Abstract

Abstract 1. 1. The effect of puromycin and insulin on the accumulation of amino acids by isolated rat diaphragm was studied. 2. 2. Puromycin added in vitro inhibited the incorporation of radioactivity from [14C]leucine into protein of muscle; significant inhibition occurred with as little as 5 μg/ml of the antibiotic and appeared maximal with 100 μg/ml or more. The inhibition was apparent as early as 5 min after the start of incubation. 3. 3. When the concentration of puromycin was 50 μg/ml or less, insulin stilll stimulated amino acid incorporation into protein; when the concentration was greater, no effect of the hormone was observed. 4. 4. The fraction of the total radioactivity from [14C]phenylalanine added to the medium that was incorporated into protein was found to be 12.1% and was increased to 18.9% by insulin. Of the actual amount of 14C that appeared in the cell water of diaphragm muscle, 12 to 76.9%, depending on the particular amino acid, was incorporated into protein. It was concluded that a significant portion of the [14C]amino acid added to medium was utilized for protein synthesis. 5. 5. In the presence of puromycin insulin increased the accumulation in isolated diaphragm of radioactivity from 14C-labeled isoleucine and arginine; without puromycin the accumulation of the two amino acids was not increased by the hormone. 6. 6. When diaphragm was incubated in medium containing a single radioactive amino acid, the addition of insulin increased the disappearance of radioactivity from the medium; however, less radioactivity was present in the intracellular water of diaphragm and for that reason the concentration ratio (radioactivity in the intracellular water to that in the extracellular water) was unchanged. 7. 7. It was concluded: (1) that insulin probably increases the entry into muscle of most, if not all, amino acids; (2) that the insulin stimulation of amino acid transport, and of protein synthesis, are independent of each other.