Biosynthesis of glucagon in isolated pigeon islets

Tung, A. K.; Zerega, F.

doi:10.1016/0006-291x(71)90831-x

Cited by 30 publications

(20 citation statements)

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“…In addition, four immunoreactive fractions have been observed in the plasma of normal or de-pancreatized animals [22], suggesting that they are derived from pancreatic or extrapancreatic sources. In addition, incorporation of 3H-L-tryptophan into peptides larger than glucagon has been detected in pancreatic islets from several animal species [14,[23][24][25]. Our results also indicate that the incorporation of radioactive tryptophan into polypeptides of smaller molecular weight increases with the period of incubation, more than 2 h of incubation being necessary to obtain a significant amount of radioactive immunoreactive glucagon.…”

Section: Discussionsupporting

confidence: 65%

“…Appearance of the label in glucagon takes more than 4 h in the perfused rat pancreas [27] and more than 6 days in cultured guinea pig islets [28]. Although the nature of the very large immunoreactive glucagon and its possible rrle in glucagon production is not well understood, Tung and Zerega [23] have described the incorporation of 3H-L-tryptophan into a 69 K polypeptide by pigeon islets, which retains its size after treatment with dissociative agents and contains peptides of molecular size and chromatographic properties identical to porcine glucagon. We have found that isolated cells of the rat submaxillary glands incorporate 3H-L-tryptophan into large polypeptides, which are related to glucagon because of their specific immunoprecipitation with a C-terminal glucagon antiserum.…”

Section: Discussionmentioning

confidence: 99%

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Evidence of glucagon biosynthesis involving protein intermediates in rat salivary glands

Pérez-Castillo

Blázquez

1984

Diabetologia

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Summary.In an attempt to determine the ability of rat submaxillary glands to synthesise glucagon via protein intermediates, isolated cells from these glands were incubated in vitro with 3H-L-tryptophan and the acid-ethanol extracts of the cells were purified on Bio-Gel P-30 columns. Aliquots of the eluates were incubated with a C-terminal glucagon antiserum (30K) and the radioactivity bound to the glucagon antibody appeared to be distributed among proteins of Molecular weight > 40.14 and 3.5 Kdaltons. A similar elution pattern was obtained in the presence of urea (7 mol/1) and guanidine hydrochloride (6 tool/l). To determine the molecular weight of the immunoreactive material eluting before the 3.5 Kdalton polypeptide, aliquots of the cell extracts were immunoprecipitated and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polypeptides of 125.8, 63.1, 42.6 and 14.4 Kdaltons were obtained. These polypeptides incorporate more radioactive tryptophan with increase in the time of incubation. Pulse-chase experiments with unlabelled tryptophan, cycloheximide-treatment of isolated cells and limited tryptic digestion of the larger glucagon immunoreactive component, transform it into a 3.5 Kdalton polypeptide with immunological characteristics indistinguishable from pancreatic glucagon. These results suggest that the larger molecule contains glucagon and thus may serve as a precursor or an intermediate of extrapancreatic glucagon biosynthesis.Key words: Glucagon biosynthesis, protein intermediates, rat submaxillary glands.Our knowledge concerning the location of cells that produce and secrete polypeptide hormones has changed rapidly during the last few years. Gastrointestinal hormones have been detected in unforeseen locations [1] and peptides of neural origin have been found in pancreatic and gastrointestinal endocrine cells [2]. Glucagon, a hormone previously considered to be exclusively a product of pancreatic islets, has recently been detected in several extrapancreatic locations [3][4][5][6], where it could represent synthesised hormone, trapped or stored as an aggregate. However, cells similar to pancreatic A cells have been identified in the stomach [7,8], where large amounts of immunoreactive glucagon is released in response to specific stimuli [9][10][11]. Demonstration of active hormonal biosynthesis could offer conclusive evidence for the existence of extrapancreatic glucagon. In previous reports we have described that isolated cells of human [12] and rat submaxillary glands [5] incorporate 3H-L-tryptophan into a 3,500dalton polypeptide with specific immune reaction with a C-terminal glucagon antiserum, suggesting that salivary glands actively synthesise glucagon rather than selectively trap or store this hormone. Therefore, the present study was designed in order to determine the ability of isolated rat submaxillary gland cells to synthesise glucagon, with special regard to defining the different forms of immunoreactive glucagon and the probable precursor-product relationship. Material ...

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Section: Discussionsupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Evidence of glucagon biosynthesis involving protein intermediates in rat salivary glands

Pérez-Castillo

Blázquez

1984

Diabetologia

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“…Furthermore, biosynthetic studies using [3H]tryptophan have suggested that a precursor to glucagon is synthesized in islets of anglerfish (8), pigeons (9), and guinea pigs (10). The biosynthesis of these largely uncharacterized proteins appears to be under a control similar to that serving for glucagon secretion since high glucose concentrations inhibit both processes (8)(9)(10). Based on reports in the literature, the precursor appears to have a molecular weight of at least 9000.…”

Section: Discussionmentioning

confidence: 99%

“…Accumulated evidence has suggested the existence of higher molecular weight precursor forms for several polypeptide hormones including insulin (1,2), parathyroid hormone (3,4), gastrin (5,6), and glucagon (7)(8)(9)(10). In 1970, Rigopoulou et al reported that extracts of canine pancreas contain two components with glucagon-like immunoreactivity, the larger having a molecular weight of about 9000 (7).…”

mentioning

confidence: 99%

“…Studies of the biosynthesis of glucagon in islets of the anglerfish by Noe and Bauer showed that a 11,400-dalton component possessed several of the characteristics required for a biosynthetic precursor to glucagon (8,11). Other workers also have suggested the existence of biosynthetically labeled glucagon precursors in islets of pigeon (9) and guinea pig (10). None of these high molecular weight, glucagon-like substances, however, has been isolated or characterized chemically.…”

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confidence: 99%

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Isolation of a Glucagon-containing Peptide: Primary Structure of a Possible Fragment of Proglucagon

Tager

Steiner

1973

Proc. Natl. Acad. Sci. U.S.A.

151

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The heterogeneity of crystalline bovine (ox)/porcine glucagon has been examined by gel filtration and ion-exchange chromatography. A strongly basic peptide that reacted well with antibodies to bovine/porcine glucagon was isolated and its primary structure was determined. The amino-acid sequence of the NH2-terminal 29 residues of the 4500-dalton peptide is identical with that of intact bovine or porcine glucagon. The remaining eight residues at its COOH-terminus are Lys-Arg-Asn-AsnLys-Asti-Ile-Ala. Small amounts of other glucagon-immunoreactive peptides having molecular weights ranging from 3700 to 9000 were also detected in crystals of bovine/ porcine glucagon. We propose that the 37-residue peptide is a fragment of bovine or porcine proglucagon.Accumulated evidence has suggested the existence of higher molecular weight precursor forms for several polypeptide hormones including insulin (1, 2), parathyroid hormone (3, 4), gastrin (5, 6), and glucagon (7-10). In 1970, Rigopoulou et al. reported that extracts of canine pancreas contain two components with glucagon-like immunoreactivity, the larger having a molecular weight of about 9000 (7). Studies of the biosynthesis of glucagon in islets of the anglerfish by Noe and Bauer showed that a 11,400-dalton component possessed several of the characteristics required for a biosynthetic precursor to glucagon (8,11). Other workers also have suggested the existence of biosynthetically labeled glucagon precursors in islets of pigeon (9) and guinea pig (10). None of these high molecular weight, glucagon-like substances, however, has been isolated or characterized chemically.Since proinsulin and several of its partially converted intermediate forms cocrystallize with insulin during the commercial preparation of that hormone (12, 13), we considered the possibility that small amounts of a glucagon precursor might be present in crystalline preparations of glucagon. Bromer et al. recently examined the heterogeneity of crystalline glucagon by ion-exchange chromatography (14), but limited their attention to the isolation and characterization of desamidoglucagon. Our studies proceeded from gel-filtration of crystalline bovine/porcine glucagon so that we might examine only those components larger than glucagon itself. MATERIALS AND METHODSMaterials. The glucagon used during these studies was a mixture of the bovine and porcine hormones. The crystalline preparation was purchased from ELANCO Products Co., Indianapolis, Ind. (lot no. 6PK72B) and is of pharmaceutical quality.Isolation of Peptides. 1 g of crystalline glucagon was filtered through Bio-Gel P-30 with 3 M propionic acid as the eluting solvent. Two early-eluting peaks of UV absorbance were separately pooled, and the fractions were concentrated by rotary evaporation. Each of the pools was then chromatographed on a column of SP-Sephadex C-25 using 5 mM sodium acetate buffer (pH 4.0), 8 M in freshly deionized urea, with a series of salt gradients eventually reaching 1 M in NaCl.Appropriate peaks of UV absorbance were poole...

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