Identification of prosomatostatin in pancreatic islets.

Abstract

A 12.5-kilodalton protein not related to insulin or glucagon was detected in pulse-chase-labeled rat islets of Langerhans. Although this protein reacted poorly with various somatostatin antisera, analysis of two-dimensional peptide maps showed that it contains all of the tryptic fragments of somatostatin, which is located at its COOH terminus. Proteolytic conversion of the putative prosomatostatin, which took place parallel to the processing of proinsulin and proglucagon in pulse-chase experiments, coincided with the appearance of newly synthesized somatostatin and proceeded without the apparent involvement of major intermediate forms.The tetradecapeptide somatostatin was isolated from hypothalamic extracts in 1973 (1) and has since been found to be present in other parts of the central nervous system, as well as in the gastrointestinal tract and in the D-cells of pancreatic islets (2-6). Because the formation of such a small peptide hormone is likely to proceed via the biosynthesis of a prohormone, many attempts have been made to identify larger forms of immunoreactive somatostatin in tissue extracts and several "large" somatostatins have been reported (7)(8)(9)(10)(11)(12)(13)(14).However, most of these studies have not provided clear evidence for a somatostatin precursor that is processed on a time scale similar to that for the conversion of proinsulin (15), proglucagon (16), or other hormone precursors (17, 18). Only studies on isolated fish islets have demonstrated the probable formation of somatostatin via the processing of cysteine-and tryptophan-labeled precursor material slightly larger in size than proinsulin (13).The data reported here demonstrate that a distinct rapidly synthesized protein of 12.5 kilodaltons (kDal) in rat pancreatic islets contains the tryptic peptides of somatostatin and that its cellular conversion is accompanied by the formation of tetradecapeptide somatostatin. MATERIALS AND METHODSLabeling of Islet Proteins. Isolation of pancreatic islets from Sprague-Dawley rats has been described (19,20). Samples of 100 isolated islets were incubated at 370C in 100,ul of Hank's buffer supplemented with 2.5 mM glucose and 50 ,Ci (1 Ci = 3.7 X 1010 becquerels) of the indicated radioactively labeled amino acid (New England Nuclear, Boston, MA, or the Radiochemical Centre, Amersham, England). For subsequent chase incubations, 5 ml of Hanks' buffer containing 1 mM unlabeled amino acid was added. Washing and lysis of islets and electrophoresis of lysates on NaDodSO4/polyacrylamide gels followed by fluorography were performed as described (21).Elution of Proteins from Electrophoresis Gels. Protein bands were excised from dried and fluorographed electrophoresis gels of lysates of 1000-1200 islets that had been incubated for 2 hr with the indicated radioactive amino acid as described above. Proteins were eluted by shaking the chopped gel bits twice for 48 hr at 370C either in 2 ml of 0.1 M NH4HCO3 (proteins < 6 kDal) or in 2 ml of 25 mM Tris/200 mM glycine/0.1% NaDodSO4 (proteins > 6 kDal). Bo...

A high molecular weight form of somatostatin-28 (1-12)-like immunoreactive substance without somatostatin-14 immunoreactivity in the rat pancreas. Evidence that somatostatin-14 synthesis can occur independently of somatostatin-28.

“…With respect to higher molecular weight S-14 LI, two and possibly three forms were identified corresponding to sizes of 18,000, 11,000, and 9,000 daltons, respectively, but each was present in very small concentrations. The 9,000-11,000 mol wt material is of similar size to the 12,500 mol wt precursor identified by pulse labeling of rat islets (5) and also corresponds in size to the recently sequenced rat prosomatostatin molecule (10,11 The most significant finding of our study is the identification of high concentrations of a high molecular weight form of S-28(1-12) LI of M, = 9,000-11,000. This material comprised 35-47% of total S-28(1-12) LI.…”

“…The relative abundance of the 9,000-11,000 mol wt S-28(112) LI material was unchanged following removal of S-14 LI from pancreatic extracts by affinity chromatography before gel filtration. Serial dilutions of fractions containing 9-11,000 and 1,200 mol wt materials exhibited parallelism with synthetic S-28 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The total pancreatic concentration of S-28(1 12) LI was 1.56 pmol/mg protein, of which S-28 (1 12) accounted for 0.83 pmol/mg protein and 9-11,000 S-28(1 12) LI comprised 0.55 pmol/mg protein.…”

“…INTRODUCTION Tetradecapeptide somatostatin (somatostatin-14, S-14)' was the first of the somatostatin-related peptides to be isolated and characterized (1). Subsequent studies from a number of laboratories demonstrated the existence of at least two additional higher molecular weight forms of S-14-like immunoreactive (S-14 LI) peptides of M, 3,000 (3,000 mol wt S-14 LI), and Mr 12,000-15,000 (12-15,000 mol wt S-14 LI) in mammalian tissues (2)(3)(4)(5)(6). A molecule corresponding to 3,000 mol wt S-14 LI has been isolated from the mammalian hypothalamus and intestine as somatostatin-28 (S-28), a 14-amino acid N-terminally extended form of S-14 connected to S-14 by dibasic (Arg-Lys) residues (7-9).…”

“…1). 12-15,000 mol wt S-14 LI, or prosomatostatin, appears to be a biosynthetic precursor for S-14 and S-28 (5,13,14). Using antisera directed against the NH2-terminal half of the S-28 molecule, the S-28(1 12) fragment has recently been isolated from the rat pancreas and hypothalamus and shown to be a major molecular species in these and other somatostatin-containing tissues (15,16).…”

The major proglucagon fragment: An abundant islet protein and secretory product

“…Again this labeled protein is almost absent in 'duodenal' islets as well as the other easily discernible components of glucagon biosynthesis which are the two bands of proglucagon and a likely intermediate of prohormonal processing of Mr 13 OOO- 14 000. Extended documentation on the identification of these and other precursors of islet hormones has been presented in [ 1,3,9]. The identity of the stained and the radioactively labeled 10 000 MI protein is substantiated by twodimensional electrophoretic analysis ( fig.2).…”