Biosynthesis of an insulin precursor by islet tissue of cod (Gadus callarias)

Abstract

1. At 15°, slices of cod islet tissue incorporated [U−¹⁴C]proline into proteins soluble in acid–ethanol at a linear rate for 6hr. 2. Initially, all the radioactivity was associated with a polypeptide that had a molecular weight of about 10000 and was appreciably more basic than cod insulin. After 1hr. there was also a significant and progressive increase in the radioactivity of insulin and of fractions intermediate in molecular size and basicity between the polypeptide and insulin. 3. O-Ethyl O-p-nitrophenyl phenylpropylphosphonate markedly decreased the radioactivity both of the intermediate fractions and of insulin, but had no significant effect on the biosynthesis of the polypeptide. In contrast, puromycin inhibited the incorporation of radioactivity into all the fractions. 4. The polypeptide had an activity of less than 0·2 international unit/mg. in the epididymal-fat-pad bioassay. Treatment with low concentrations of trypsin caused a progressive increase in the formation of an insulin-like material, judged by bioassay and ion-exchange chromatography of the digest. 5. Gel filtration of the polypeptide after oxidative sulphitolysis indicated that it was a single polypeptide chain. 6. The results suggest that the polypeptide is an insulin precursor whose formation is inhibited by puromycin and that the steps involved in the conversion of precursor into product are sensitive to O-ethyl O-p-nitrophenyl phenylpropylphosphonate.