Isolation and characterization of somatostatin from pigeon pancreas.

Abstract

Most of the somatostatin-like activity from pigeon pancreas was found to correspond to a small species with an apparent molecular weight of 1500-2500. This species was isolated under conditions minimizing intermolecular interactions and protease activities. The isolated product was characterized by two somatostatin radioimmunoassays, a bioassay, endgroup determination, and amino acid analysis. The structure of the isolated compound was determined to be H-Ala-Glycyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Additionally, small amounts of des-Ala'-somatostatin, a possible degradation product of pancreatic somatostatin, and a large somatostatin-like species with an apparent molecular weight of 11,000-12,500 were detected. It is concluded that the main somatostatin-like polypeptide isolated from pigeon pancreas is identical to the mammalian hypothalamic tetradecapeptide somatostatin. In 1973, the amino acid sequence of somatostatin (SS) from ovine hypothalamus was reported (1, 2). The sequence for porcine hypothalamic SS was shown to be identical (3). Biologic and immunologic SS-like activities (SSLA) have also been detected in extrahypothalamic parts of the brain (4, 5), pancreas (6, 7), and gastrointestinal tract (7). Although these activities had been observed by bioassay and by immunologic techniques with several antisera directed against different portions of the SS molecule (8), the chemical identities of extrahypothalamic SS-like substances had not been established. In two earlier reports (9, 10) we described the initial purification of SS-like substances from pigeon pancreas, which was chosen as a source of extrahypothalamic SS-like species because of its high content of SSLA (8). We report here details of the isolation and characterization of SS from pigeon pancreas.METHODS AND MATERIALS Purification. Male pigeons were obtained from Carpenter Squab Ranch (Ventura, CA). For one experiment, 5-20 pigeons were decapitated. The pancreata were removed and submitted to either one of the following procedures:(A) The pancreata were frozen in liquid nitrogen within 1 min after removal from the animal. The frozen material was then pulverized in liquid nitrogen. The powder was almost totally dissolved in 8 M guanidine hydrochloride/2 M ammonium acetate, pH 2.5, at room temperature and centrifuged for 20 min at 48,000 X g. The supernatant was defatted with an equal volume of diethyl ether/hexane (2:1, vol/vol) and submitted to column chromatography (Bio-Gel P-100, Bio-Rad), which was performed in the presence of 6 M guanidine hydrochloride/i M ammonium acetate, pH 2.5. The chromatographic elution pattern was characterized by the following molecular weight markers: bovine serum gamma globulin, bovine ribonuclease A, cytochrome c (horse heart), lima bean trypsin inhibitor, bovine pancreatic trypsin inhibitor, porcine insulin, porcine '25I-labeled glucagon (New England Nuclear), [125I-Tyrl'SS, bacitracin, and tyrosine. All elution volumes were calculated as partition coefficients KD, referring to the ...

Isolation and Characterization of Immunoreactive Somatostatin from Fish Pancreatic Islets

“…5 mg of pancreatic somatostatin was isolated from islets obtained from 400 lb of fish (-100-150 fish). On the basis of previously published estimates (11)(12)(13) fish somatostatins are bigger. Furthermore, the sequence of fish insulins differs from mammalian insulin in up to 50% of the amino acids, yet they maintain considerable biological activity in mammalian systems (28 A small amount of somatostatin comigrated with the tetradecapeptide.…”

“…It was noted that both big and small somatostatin from crude pancreas and stomach extracts displaced tracer in a manner parallel to that of synthetic somatostatin, indicating that the gastric and pancreatic somatostatin-like substances were immunologically indistinguishable from the somatostatin tetradecapeptide. Spiess et al (13) found most of the somatostatin-like activity of pigeon pancreas was 1,500-2,500 mol wt, but a small amount of material 11,000-12,500 mol wt was also found. Noe et al (29) incubated slices of anglerfish islets for several hours, then chromatographed acid extracts on Bio-Gel P-10.…”

“…The majority of that extracted from rat pancreas migrated in the void volume of a Sephadex G-25 column (11). Recently, a pancreatic somatostatin was isolated from pigeon pancreas that was identical to tetradecapeptide somatostatin from ovine hypothalamus (13).…”

“…Rat (11), chicken (12), and pigeon (13) pancreas extracts gave an inhibition curve in a radioimmunoassay which paralleled that of SRIF. The majority of somatostatin-like immunoreactivity from human tumor (14), chicken (12), and pigeon pancreas extracts (13) chromatographed with SRIF by gel filtration, but small amounts of larger forms were also reported. The majority of that extracted from rat pancreas migrated in the void volume of a Sephadex G-25 column (11).…”

Sequence analysis of a cDNA coding for a pancreatic precursor to somatostatin.

“…Pradayrol et aL (8) determined the amino acid sequence of a 28-residue somatostatin precursor from porcine intestine, and Schally et aL (9) and Esch et aL (10) have determined the sequence of a 28-residue peptide from bovine and ovine hypothalami, respectively. These precursors to somatostatin have amino-terminal extensions and share the common 14-amino acid sequence found in ovine (1), porcine (11), pigeon (12), and angler fish (13) somatostatin. Recently the amino acid sequence of a somatostatin isolated from channel catfish was reported (14).…”