Primary Structure of Somatostatin, A Hypothalamic Peptide That Inhibits the Secretion of Pituitary Growth Hormone

Abstract

Somatostatin, a peptide isolated from ovine hypothalamic tissue that inhibits the release of radioimmunoassayable growth hormone in vitro from rat or human pituitary cells or in vivo in rats, has the primary structure H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-LysThr-Phe-Thr-Ser-Cys-OH. The structure was established by submitting the carboxymethylated peptide, the carboxymethylated tryptic digest, and the chymotryptic digest of the peptide to Edman degradation. Degradation products were analyzed by amino-acid analysis, as well as in some cases by determination of N-termini by dansylation or by determination of phenylthiohydantoins by mass spectrometry.We have recently reported the isolation from (sheep) hypothalamic extracts of a peptide that specifically inhibits at ) 1 nM the in vitro secretion of immunoreactive rat or human growth hormones, and that is similarly active in vivo in rats (1). This peptide will be referred to as somatostatin (see ref.1). We report here details of the determination of the primary sequence of this peptide. MATERIALS AND METHODSThe starting material was a side fraction obtained from the purification of extracts of sheep hypothalamic fragments for the characterization of the releasing factors of the gonadotropins, in a program similar to that previously described for luteinizing hormone-releasing factor (LRF) (2). Details of the isolation procedure will be published elsewhere (Brazeau et al., in preparation). In summary, an ethanol-chloroformacetic acid-water 810:100:5:90 extract of about 500,000 sheep hypothalamic fragments (2 kg) was partitioned in the system 0.1% acetic acid-n-butyl alcohol-pyridine 11:5:3, and the material from the organic phase was then partitioned in n-butyl alcohol-acetic acid-water 4: 1: 5. The aqueous phase from the second distribution was subjected to ion-exchange chromatography on carboxymethyl-cellulose, whereupon a basic fraction with somatostatin activity was well separated from the luteinizing hormone-releasing factor zone. The biologically active fraction was further purified by gel filtration on "Sephadex G-25" in 0.5 M acetic acid (RF of somatostatin = 0.5), and finally by liquid partition chromatography (3) on a "Sephadex used with a 56 X 0.9-cm column of Beckman AA-15 resin at 55°. Buffers were prepared as described in Beckman/Spinco bulletin A-TB-020G with two modifications: the first buffer of the three-buffer system was adjusted to pH 3.26 rather than 3.49 and 5% (v/v) n-propyl alcohol was added to the third buffer (pH 6.41) to obtain more rapid elution of Trp. At a flow rate of 70 ml/hr, buffer changes were set at 110 min for the switch from pH 3.26 (0.2 M Na ion) to 4.14 (0.2 M Na ion) buffer and at 172 min for the switch from pH 4.14 to 6.41 (1.6 M Na ion) buffer.Determination of Free Sulfhydryl Groups. Quantitative analysis of free SH groups with 5,5'-dithiobis-(2-nitrobenzoic acid), Ellman's reagent, (8), was as described by Habeeb (9). 684

Isolation, primary structure, and synthesis of α-endorphin and γ-endorphin, two peptides of hypothalamic-hypophysial origin with morphinomimetic activity

“…Sequential degradation of peptides was performed using the Edman method followed by dansylation with [14C]dansyl chloride and identification of Pthderivatives by mass spectrometry as previously described (8,10).…”

“…Material of pool 2-(i) was dissolved in 100 ml of 0.001 M NH4OAc, pH 4, and then applied to a SP-Sephadex C-25 column (5 X 40 cm, Vbed = 800 ml) equilibrated in 0.001 M NH4OAc, pH 4 Amino Acid Analysis. Amino acid composition of peptides was determined as previously described (8) after hydrolysis in 6 M HCI-0.5% thioglycollic acid or enzymatic digestion with papain and leucineaminopeptidase (9).…”

Function and expression of somatostatin receptors of the endocrine pancreas

“…There are two major circulating SST-isoforms, which consist of 14 and 28 amino acids, respectively, which are processed from a common precursor protein in a cell-and tissue-specific pattern (Brazeau et al, 1973;Burgus et al, 1973;Pradayrol et al, 1980). In the adult, the primary secretory product of pancreatic D-cells is SST-14 (Noe 1981;Patel et al, 1981), which contributes to less than 5% of circulating SST (Taborsky, Jr. and Ensinck 1984).…”

Structure of octreotide, a somatostatin analogue

“…1), a cyclic tetradecapeptide, has been isolated from ovine hypothalamus (Brazeau et al, 1973;Burgus, Ling, Butcher & Guillemin, 1973), but is also present in many other organs. It inhibits the release of many peptide hormones including glucagon, insulin, secretin and growth hormone (Gerick & Lorenzi, 1978).…”