Primary structure of ovine hypothalamic somatostatin-28 and somatostatin-25.

Abstract

The primary structure of the NH2terminally extended somatostatins isolated from ovine hypothalamic extracts, one containing 28 residues and the other 25, has been determined. The structure of somatostatin-28 is Ser-AlaAsn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-GlyCys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cts-OH; the shorter one, somatostatin-25, has the same sequence as somatostatin-28 except that the first three NH2-terminal residues are deleted. The two peptides as isolated were found to be oxidized at the methionine residue to the methionine sulfoxide. Their structures were established by subjecting the native peptides to direct sequence analysis in a Beckman 890C sequencer and identifying the released phenylthiohydantoin derivatives by high-performance liquid chromatography. Their structures were confirmed by trypsin digestion and isolation of all the tryptic peptides, followed by amino acid analysis of the tryptic fragments. Moreover, some of the tryptic peptides were matched with their respective synthetic replicates on high-performance liquid chromatography.The tetradecapeptide somatostatin was first isolated and characterized from ovine hypothalamus (1) on the basis of its ability to inhibit the secretion of growth hormone from primary cultures of dispersed rat anterior pituitary cells. The identical tetradecapeptide was subsequently isolated and characterized from porcine hypothalamus (2) as well as pigeon (3) and anglerfish (4) pancreases. In addition, several laboratories (2, 3, 5-8) have observed, in the hypothalamic and pancreatic extracts, bioactive and immunoreactive somatostatin-like substances having molecular weights larger than the tetradecapeptide. Pradayrol et al. (9) reported the isolation (from porcine intestinal extracts) and primary structure of an NH2-terminally extended somatostatin containing 28 amino acids which was designated SS-28. We have also isolated two bioactive somatostatins containing 28 and 25 residues from ovine hypothalamic extracts. The primary structure of the larger ovine hypothalamic somatostatin is Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-AlaPro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe -Phe -TrpLys-Thr-Phe-Thr-Ser-Cys-OH, which is identical to the structure of the intestinal SS-28 isolated by Pradayrol et al. (9). The shorter ovine hypothalamic peptide, SS-25, has the same structure as SS-28 except that the first three NH2-terminal residues are deleted.We report here in detail the determination of the primary structure of the two larger hypothalamic somatostatins.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6827MATERIALS AND METHODS The starting material for the isolation work was the combined Sephadex G-25 excluded fractions from extracts of 350,000 ovine hypothalami used in our purification of the thyrotropin-releasing factor (10). Details of the isolation procedure will be published elsewher...

MOLECULAR EVOLUTION OF GPCRS: Somatostatin/urotensin II receptors

“…SS1 has subsequently been demonstrated to exist in two forms, SS-14, composed of 14 amino acids (Brazeau et al 1973), and SS-28, its N-terminally extended form, composed of 28 residues, first characterized in porcine gut (Esch et al 1980, Pradayrol et al 1980. Both peptides are generated from the same precursor molecule (called prepro-SS1 or PSS1) through post-translational processing at two cleavage sites, a dibasic Arg-Lys site and a monobasic Arg site respectively (Patel & Galanopoulou 1995).…”

Biosynthesis of immunoreactive somatostatin by hypothalamic neurons in culture.

“…We have previously characterized SLI in rat hypothalamus and identified three forms of immunoreactivity: one with a molecular weight of 15,000, comprising -5% of total SLI (15,000-mol-wt SLI); a second form of Mr = 3,000 (3,000-mol-wt SLI) accounting for -25% of total SLI; and a third, dominant form of Mr = 1,600 (-70% of total SLI), which corresponds to SRIF (6). Recent amino acid analyses have established the identity of 1,600-mol-wt SLI with SRIF (16) and of 3,000-mol-wt SLI with that of somatostatin-28 (S-28) (17), a biologically active 14 amino acid N-terminally extended form of SRIF isolated from mammalian hypothalamus and intestine (18)(19)(20). Our earlier investigations on the processing of 15,000-mol-wt SLI and S-28 by hypothalamic enzymes support a possible role for both peptides as precursors for S-14 (21,22).…”

“…In particular, the experiments described here were designed both to demonstrate biosynthesis in nerve cells and to provide insight into the biosynthetic relationship between the three molecular forms of SLI. [17][18][19][20][21]. The cells were suspended in 20 ml of Dulbecco's modified Eagle's medium (DME) and plated in 35-mm-Diam plastic Petri dishes (Falcon Labware, Div.…”

Evidence for the presence of somatostatin 28 in plasma

“…A 28-amino-acid form of somatostatin S-28 has recently been isolated from porcine intestine 1 and ovine 2 and porcine hypothalamus. 3 It is also released by isolated pancreatic islets in perifusion experiments.…”