Cell-free synthesis of rat preproinsulins: characterization and partial amino acid sequence determination.

Abstract

Whole nucleic acid fractions of isolated rat islets of Langerhans greatly stimulate incorporation of radioactive amino acids into protein in a wheat germ ribosomal system. Approximately 30% of the synthetic product is precipitated with antisera to insulin or proinsulin. Characterization of this material by gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a molecular mass of 11,500 daltons. Trypsin digestion releases intact A chain as well as tryptic fragments of the C-peptides and B chains of the two rat proinsulins. Automated sequence determination of labeled cell-free product purified by immunoprecipitation discloses the presence of 23 additional amino acids NH-terminal to the B chain sequence of proinsulin. The partial amino acid sequence of this extension is as follows: NH2-XLeu (Lys) Met-X-Phe-Leu-Phe-Leu-Leu (Lysy Leu-Leu-X-Leu-X-X-X-X-X-X-X-X-proinsulin. On the basis of the above evidence we have designated this peptide preproinsulin. Studies on the biosynthesis of insulin have shown that this hormone is synthesized initially as the larger, single-chain polypeptide precursor, proinsulin (1). During labeling of beta cells in vitro, only proinsulin is initially detected (2). Thus, if larger precursors than proinsulin exist, they must be rapidly cleaved, perhaps before the polypeptide chain is completed (1).Recent improvements in methods for large scale isolation of pancreatic islets now permit the isolation of sufficient tissue for the preparation of small amounts of messenger-RNA-containing fractions. These fractions can prime the cell-free synthesis of protein in a ribosomal system derived from wheat germ, yielding sufficient quantities of proinsulin-like peptides for radiochemical characterization and amino acid sequence determination. This system thus provides the means for a direct experimental examination of the nature of the polypeptide encoded within the structural gene for insulin. The results reported here show that the initial translation product of the messenger RNA for proinsulin is a larger polypeptide than proinsulin with a molecular weight of approximately 11,500. The additional mass can be accounted for by an amino-terminal extension of 23 residues. The structure and properties of this preproinsulin are similar to those of several other presecretory proteins that have been described. Chemically synthesized rat C-peptides I and II and guinea pig antisera to the synthetic rat C-peptide I were generous gifts of Dr. Noburo Yanaihara, Shizuoka, Japan.Preparation of Islet Nucleic Acid. Islets were isolated from Sprague-Dawley rats by collagenase digestion as described elsewhere (A. Lernmark, A. Nathans, and D. F. Steiner, manuscript in preparation). Batches of 2-4 thousand islets were rapidly lysed in 1.5 ml of hot (800) buffer containing 0.1 M Tris-HCl (pH 9), 0.01 M EDTA, 0.1 M dithiothreitol, and 2% NaDodSO4 (wt/vol); diluted at room temperature with an equal volume of phenol-chloroform-isoamyl alcohol (50:50:2) previously equilibrated with 0.1...

Establishment of rat pancreatic endocrine cell lines by infection with simian virus 40

“…The results obtained with the B7C-induced tumours (Fig. 2) pointed to the possibility that SV40-transformed pancreatic cells might produce a protein antigenically related to insulin, but larger than preproinsulin (11500 daltons) (Chan et al, 1976). Such a protein might be insoluble in acid/ethanol and thus escape detection when acid/ethanol is used to extract insulin, proinsulin and preproinsulin (Chan et al, 1976).…”

“…2) pointed to the possibility that SV40-transformed pancreatic cells might produce a protein antigenically related to insulin, but larger than preproinsulin (11500 daltons) (Chan et al, 1976). Such a protein might be insoluble in acid/ethanol and thus escape detection when acid/ethanol is used to extract insulin, proinsulin and preproinsulin (Chan et al, 1976). SV40-transformed cultures of rat pancreatic cells, both cloned and uncloned, were therefore sonicated in glycine buffer and the 30000g supernatants were assayed for immunoreactive insulin.…”

A large product of cell-free translation of messenger RNA coding for corticotropin.

“…These results indicate that corticotropin messenger RNA directs the cell-free synthesis of a product that contains the amino acid sequence of corticotropin but is much larger than this hormone. Recent studies on a variety of endocrine peptides have indicated that the primary gene products differ significantly from the known peptide hormones (1)(2)(3)(4)(5)(6)(7). These peptides appear to be initially synthesized from larger precursors, which are then converted to their respective peptides by post-translational cleavage.…”

“…Abbreviations: ACTH, corticotropin (adrenocorticotropic hormone); ACTH, synthetic peptide with the 1-24 amino acid sequence of ACTH; (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)ACTH, synthetic peptide with the 2-18 amino acid sequence of ACTH and glycine at the amino terminus; IgG, immunoglobulin G; NaDodSO4, sodium dodecyl sulfate.…”

Adventures with Insulin in the Islets of Langerhans

“…Space does not permit me to cover this aspect of precursor processing in any detail except to say that, when such an extension was first found in a nascent immunoglobulin light chain and then in the in vitro translation product of parathyroid hormone mRNA in the early 1970s, we were all very excited and quickly looked into this possibility. My colleague Shu Jin Chan, then a graduate student in my laboratory, became one of the first to translate islet mRNA in cell-free systems and succeeded in identifying and characterizing rat preproinsulin (42). Shortly afterward, Dennis Shields, working with Blobel, identified several preproinsulins in teleost fishes (43).…”