| Description |
Methods have been developed for the study of amino acid incorporation into a specific protein under established conditions of net synthesis. The results of these studies have been reported and discussed. Synthesis and storage of the secretory proteins of mouse pancreas have been shown to occur between fifteen to twenty-one hours after pilocarpine injections. The concentration of amylase, ribonuclease, and total pancreatic proteins has been shown to increase rapidly during that period. Deoxyribonucleic acid as well as ribonucleic acid content of the pancreatic tissue does not increase during the period of protein synthesis. Injection of the labeled amino acid, valine, into pilocarpine treated mice eighteen hours after stimulation, followed by chromatographic resolution of the components of an extract of pancreas, has allowed a demonstration of radioactive labeling of the enzyme ribonuclease under conditions of net synthesis. The ratio of radioactivity to enzymatic activity has been used to define a specific activity for that enzyme. Analyses of the total radioactivity present in the pancreatic tissue as well as the total radioactivity of each of four cell fractions have been carried out. It was found that ten minutes after intraperitoneal injection of isotope approximately ten per cent of the radioactivity is present in the pancreas. Radioactivity appears most rapidly in the microsomal fraction of the cell during the first ten minutes but at later periods the soluble portion of the cell becomes more intensely labeled. The distribution of labeled ribonuclease in the various parts of the pancreatic cells has been studied as a function of time. The data indicate that the microsomal portion of the acinar cells is the site of formation of the secretory enzyme, ribonuclease, in the living mouse. The distribution of labeled ribonuclease in the other cell fractions suggests that the newly formed enzyme is transferred to the zymogen granules by way of the soluble portion of the cell. From a consideration of the rates of isotope uptake into the total homogenate and the microsomal fraction, as well as the rate of appearance of labeled ribonuclease in the microsomal fraction, it has been estimated that the labeled amino acid is present in the microsomes for about three minutes before it appears in the pancreatic enzyme. It has been proposed that this time lag is the period required for the elaboration of the finished, enzymatically active protein, ribonuclease, in the microsomal portion of the cells. |
