A peptide has been isolated from ovine hypothalamus which, at 1 x 10(-9)M, inhibits secretion in vitro of immunoreactive rat or human growth hormones and is similarly active in vivo in rats. Its structure is H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH The synthetic replicate is biologically active.

The primary structure of ovine hypothalamic hypophysiotropic luteinizing hormone-releasing factor, LRF, has been established as pGlu-His-Trp-SerTyr-Gly-Leu-Arg-Pro-Gly-NH2 by hydrolysis of the peptide with chymotrypsin or pyrrolidone-earboxylylpeptidase and by analysis of the products by an Edman-dansylation sequencing technique, as well as by mass spectrometry of the derived phenylthiohydantoins. A decapeptide with the proposed primary structure, prepared by total synthesis, gave the same result on sequencing. The synthetic decapeptide possesses the same biological activities as the native ovine LRF. The amino-acid sequence of ovine LRF is identical to that already published for porcine LRF.Various areas of the central nervous system participate in the fine regulation of the secretion of all adenohypophysial hormones. The ultimate integrator of information originating in the central nervous system is the hypothalamus. The final information from the hypothalamus to the adenohypophysis is not transmitted in the form of nerve impulses, but is carried in the form of specific hypothalamic hypophysiotropic substances, the hypothalamic releasing factors, that are carried through the hypothalamo-hypophysial portal system of capillaries from the median eminence region of the ventral hypothalamus to the cells of the adenohypophysis. There is good physiological evidence that such a hypothalamic control is involved in the secretion of the gonadotropin, luteinizing hormone. In the early 1960s, several investigators reported experimental results that were best explained by proposing the existence of substances that specifically stimulated the secretion of luteinizing hormone, and that were probably polypeptides, in crude aqueous extracts of hypothalamic tissues of various mammalian species (1-3). Preparations of LRF, active at 1 /Ag per dose in animal bioassays, were obtained by gel filtration and ion-exchange chromatography on carboxymethylcellulose (4), an observation that was confirmed by similar methods by several investigators (5, 6). In spite of the vagaries of the various bioassay methods available, several laboratories reported preparations of LRF of increased potency (5, 6). Several of these early publications led to contradictory statements regarding purification and separation of LH-releasing factor (LRF), from a follicle-stimulating hormone releasing factor (5, 7). Two laboratories independently reported the isolation of porcine LRF (8) and ovine LRF (9), both groups concluding that LRF from either species was a nonapeptide containing, on the basis of acid hydrolysis, 1 His, 1 Arg, 1 Ser, 1 Glu, 1 Pro, 2 Gly, 1 Leu, 1 Tyr. Earlier results with the pyrrolidone-carboxylylpeptidase prepared by Fellows and Mudge (10) had led us to conclude (11) that the Nterminal residue of LRF was Glu in its cyclized pyroglutamic (pGlu) form, as in the case of hypothalamic TRF, (pGluHis-Pro-NH2).While our own studies on the amino-acid sequence of ovine LRF were in progress, Matsuo et al. (12) reported that porcine LRF contai...

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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

The isolation and primary structure of two peptides with morphinomimetic activity, obtained from an extract of porcine hypothalamus-neurohypophysis, are described. The amino acid sequence of the two peptides, named a-endorphin and 'y-endorphin, was determined by mass spectrometry and dansyl-Edman methods to be H-Tyr-GlylyPhe-Met-ThrSerGlu-Lysl-erln-Thr-Pro-Leu-Val-Thr OH and H-Tyr Gly-Gly-Phe-Met-ThrSer-lu-Lys-Ser-Gln-Thr-Pro-LeuVal-Thr-Leu-OH, respectively. These correspond to the amino acid sequences present between residues 61 and 76 and residues 61 and 77 of the various P-lipotropins. A third peptide also obtained in pure form in these studies was found to be an unstable salt of a-endorphin.In a preliminary note (1) we have reported isolating from a crude extract of (porcine) hypothalamus-neurohypophysis three peptides named endorphins with morphine-like activity in a bioassay and in a synaptosomal opiate-receptor binding assay. In that same note we presented the primary structure of one of these peptides, a-endorphin, to be that of the hexadecapeptide H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-SerGln-Thr-Pro-Leu-Val-Thr-OH. The present communication will describe in detail: (i) the starting material utilized, (ii) the method of isolation of these peptides, (iii) the methods used to establish the amino acid sequence of both a-endorphin and y-endorphin, and (iv) evidence that the third isolated peptide is, in fact, an unstable salt of a-endorphin. MATERIALS AND METHODSAssay of Biological Activity. It was decided that morphinomimetic substances would be defined as those substances which would decrease linearly the amplitude of the muscle contractions electrically induced in vitro in the myenteric plexus-longitudinal muscle of the guinea pig ileum (2) only when this biological activity would be reversed or prevented by naloxone, a morphine-analogue antagonist.Starting Material. We first confirmed reports by others (3-5) that aqueous extracts of whole brain or of several specific anatomical brain structures (caudate nucleus, hypothalamus) or of the pituitary gland (6) did contain naloxone-reversible morphinomimetic activity. Calculations based on simple assumptions relating expected specific activity of the substances to be characterized to their apparent concentration in these extracts indicated that several hundreds of kg of fresh tissues would have to be procured and handled to provide a reasonable chance of characterizing the postulated substances again within a reasonable time schedule. Searching through materials available to us in large quantities from our earlier isolation program of the hypothalamic-hypophysiotropic factors, we found a partially purified extract of (porcine) neurohypophysis-hypothalamus to be already considerably enriched in morphinomimetic activity (half maximal activity in the bioassay at about 10 ,ug of the dry powder per ml of incubation fluid). This material was used for the isolation of the endorphins; it is an acetic acid-acetone extract corresponding to fraction G of the Kam...

Two analogs of the hypothalamic luteinizing hormone releasing factor modified at the histidine-2 position were tested for biological activity (secretion of luteinizing hormone) in cultures of dispersed rat anterior pituitary cells. The analog in which glycine was substituted for histidine at position 2, [Gly(2)]LRF, behaves as a partial agonist releasing less than 50 percent of the luteinizing hormone secreted at maximum concentrations of the releasing factor, while the analog in which histidine at position 2 is deleted has no significant agonist activity at any of the doses tested. When added to the cultured cells at molar ratios 10(3) to 10(4) times that of the luteinizing hormone releasing factor, both analogs decrease the amount of luteinizing hormone secreted in response to the releasing factor.