Biosynthesis of Insulin in Bovine Fetal Pancreatic Slices: The Incorporation of Tritiated Leucine Into a Single-Chain Proinsulin, a Double-Chain Intermediate, and Insulin in Subcellular Fractions

Abstract

Abstract.-This communication reports the biosynthesis of insulin in the bovine fetal pancreatic slices in vitro. Double-chain proinsulin and insulin were found as major components in the mitochondrial-granule fraction of bovine fetal pancreas. Tritiated leucine was incorporated into a single-chain proinsulin, a double-chain proinsulin, and insulin. Subcellular fractionation of the slices incubated with tritiated leucine showed that radioactive single-chain proinsulin was present in the deoxycholate-soluble microsomal fraction, deoxycholateinsoluble microsomal fraction, and mitochondrial-granule fraction. Labeled double-chain proinsulin and insulin were present in the deoxycholate-soluble microsomal and mitochondrial-granule fractions. These results are consistent with the hypothesis that insulin is synthesized as a single-chain polypeptide on the ribosomes, and that intracellular proteolysis in the subcellular membranous organelles and beta-granules converts the single-chain proinsulin to insulin via a double-chain intermediate.Introduction.-A precursor protein in the biosynthesis of insulin has been demonstrated in human islet adenoma tissue, in isolated rat islets of Langerhans,1-3 and in slices of bovine fetal pancreas.4 This precursor protein is a single-chain polypeptide larger than insulin, is immunologically reactive with anti-insulin antibody, and is convertible to desalanyl insulin by limited tryptic hydrolysis. Bovine proinsulin has been obtained from crystalline insulin preparations in several laboratories,5-8 and the amino acid sequence of porcine proinsulin is now known.9 A double-chain proinsulin has also been obtained from crystalline bovine insulin.5' I It is possible that both proteins are involved in the biosynthesis of insulin, the single-chain being the precursor and the doublechain an intermediate. Experiments reported here were undertaken to study (1) the incorporation of radioactive leucine by slices of bovine fetal pancreas into these two proteins and into insulin and (2) the distribution of these proteins

Biosynthesis of Avian Glucagon: Evidence for a Possible High Molecular Weight Biosynthetic Intermediate

“…Our findings suggested that glucagon, like insulin (Steiner, Cunningham, Spiegelman and Aten 1967, Steiner and Oyer 1967, Tung and Yip 1968, Tung and Yip 1969, Grant and Reid 1968, and parathyroid hormone (fIabener, Kemper, Potts and Rich 1972) may be synthesized via a high molecular weight intermediate. Dur data from Sephadex G-IOO, gel-filtration and SDS polyacrylantide gel electrophoreses suggested a molecular size of approximate- and tryptic peptides from porcine glucagon, shown in shaded zones.…”

Synthesis and release of proinsulin and insulin by isolated rat islets of Langerhans

“…The preferential release of insulin over proinsulin may represent the preferential release of granule contents over nongranular hormone. Evidence reported by Tung and Yip (22) and Howell, Kostianovsky, and Lacy (23) suggests that only insulin and double-chain proinsulin intermediate forms are present in granules.…”

Fine Structure of the Pancreatic Islets in Domestic Fowl with Special Reference to the Cell Type and Secretion

“…The study of insulin biosynthesis in the bovine fetal pancreatic slices (TUNG and YIP, 1969) indicated the hypothesis that insulin was synthetized as a single-chain polypeptide on the ribosomes, and that intracellular proteolysis in the subcellular membranous organelles and B granules converted the single-chain proinsulin to insulin by means of a double-chain intermediate.…”