Isolation and structure of pro-somatostatin: a putative somatostatin precursor from pig hypothalamus.

Abstract

An octacosapeptide that we named pro-somatostatin has been isolated from acid extracts of porcine hypothalami and found to have the amino acid sequence 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-C's. This octacosapeptide possesses high somatotropin (growth hormone) and prolactin release-inhibiting activity in vitro. It also crossreacts strongly with antisera generated against the somatostatin tetradecapeptide. This octacosapeptide is most likely a precursor (pro-hormone) of somatostatin in the hypothalamus. The existence of still larger molecular size precursors of somatostatin was also observed.More than 5 years ago, our laboratory reported larger, more basic forms of somatostatin in fractions of pig hypothalamic extract (1, 2). We also recognized two types of immunoreactive somatostatin in extracts of rat pancreas, stomach, and duodenum (3). We suggested, on the basis of physicochemical, biological, and immunological data, that these substances represented precursors of somatostatin (1-3). Later, we postulated that the hypothalamic pro-somatostatin has an extension at the NH2 terminus containing one or more arginine residues (4-6). Manual degradation by the Edman-dansyl method showed the NH2 terminal sequence to be Ser-Ala-Asn-Ser. However, because of the small amounts available, we were unable to'complete the entire sequence. Recent developments in automated amino acid sequence analysis (7), which led to an increase in sensitivity of several thousandfold, permitted the successful elucidation of the whole sequence. In this paper we describe the isolation, biological and immunological characterization, and structural elucidation of putative pro-somatostatin from porcine hypothalami. The sequence of hypothalamic pro-somatostatin is identical with that of a putative precursor of somatostatin from pig intestine recently reported by Pradayrol et al. (8). We conclude that in the hypothalamus, as in the gut, somatostatin also exists in the form of the prohormone.MATERIALS AND METHODS Purification. A total of 630,000 pig hypothalami was dissected, defatted, and extracted as described (9, 10). The major part of this extract, corresponding to 470,000 hypothalami, was the same as that used for the isolation of porcine somatostatin (2) and pro-[Met]enkephalin (11). This extract was purified first by gel filtration on Sephadex G-25 (9, 10). Fractions containing immunoreactive somatostatin from the gel filtration were extracted with the upper phase of 0.1% acetic acid/1-butanol/ pyridine, 11:5:3 (vol/vol), (system I) and 1yophilized. This material was then further purified by preparative countercurrent distribution (CCD) in the same system by the C-2 model apparatus (H. 0. Post, New York), followed by chromatography on carboxymethyl (CM)-cellulose, another CCD in system I for 1800 transfers in the A-4 apparatus, chromatography on SESephadex, analytical gel filtration on Sephadex G-25, and rechromatography on SE-Sephadex. A smaller part of this extract, ...

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

“…3 It is also released by isolated pancreatic islets in perifusion experiments. 4 Its structure is that of the tetradecapeptide somatostatin (SRIF) with a 14-amino-acid amino terminal extension.…”

“…It has been suggested that S-28 is a precursor of SRIF. 3 However, since S-28 is more active than SRIF in some biologic systems, 5 it is also possible that S-28 may represent a naturally occurring form of somatostatin discrete from SRIF. S- 28 has not yet been identified in the circulation.…”

In vivo synthesis and processing of rat hypothalamic prosomatostatin

“…In the central nervous system, it is distributed throughout various brain areas [4] together with another form: somatostatin- [5]. Somatostatin-28, first discovered in both porcine intestine [6] and hypothalamus [7] and in ovine hypothalamus [8,9], corresponds to an NHz-terminally extended somatostatin-14 containing 28 amino acids.…”