Isolation, Structure and Synthesis of a Heptapeptide with In Vitro ACTH-Releasing Activity from Porcine Hypothalamus

Chang, Robert C.; Huang, Wei; Arimura, Akira; Redding, Tommie W.; Coy, D. H.; Saffran, Murray; Kong, Ann; Hamilton, Joshua W.; Cohn, David V.; Schally, A. V.

doi:10.1055/s-2007-1019228

Cited by 14 publications

(8 citation statements)

References 9 publications

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“…The molecular weight of small CRF has been reported to be 1,000-1,500 [2,6,21]. We also reported that the MEE showed two CRF activities, big CRF and small CRF, on Sephadex gel filtration [10].…”

Section: Discussionmentioning

confidence: 52%

“…It remains to be clarified whether these small immu noreactive CRF peaks are the fragments of rat CRF and re sponsible for a part of small CRF bioactivity. Schally et al [20] reported that tetradecapeptides or heptapeptide [2] ob tained from porcine hypothalamus showed CRF activity, but they concluded that these small peptides were not ge nuine CRF.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Rat Hypothalamic Corticotropin-Releasing Factor by Reversed-Phase, High-Perf ormance Liquid Chromatography with Synthetic Ovine Corticotropin-Releasing Factor as a Marker

Hashimoto¹,

Murakami²,

Ohno³

et al. 1983

Neuroendocrinology

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Reversed-phase, high-performance liquid chromatography (HPLC) was carried out to characterize rat hypothalamic corticotropin-releasing factor (CRF) using synthetic ovine CRF as a marker. Samples were injected onto a stainless steel column (4 × 250 mm) packed with Hitachi gel 3053. The column was eluted using a gradient elution of increasing acetonitrile concentration, in a mixture of NaCl-HCl at a flow rate of 1.0 ml/min, monitoring the column effluent at 220 nm with an UV detector. Fractions eluted every 1–2 min were collected and lyophilized for subsequent CRF bioassay and radioimmunoassay. When various neuropeptide mixtures including synthetic ovine CRF were injected onto the column, synthetic ovine CRF was well separated from the other neuropeptides with a gradient of 0–60% acetonitrile in 0.1 M NaCl-0.01 N HCl or 0–80% acetonitrile in 0.05 M NaCl-0.01 N HCl. The median eminence extracts showed two main peaks of CRF bioactivity on HPLC. One (small CRF) coeluted with arginine vasopressin and oxytocin markers, and the other (big CRF) appeared near the position of synthetic CRF and was divided into two peaks. One coeluted with synthetic ovine CRF and the second eluted after synthetic CRF, showing high CRF activity. Three or four peaks of CRF immunoreactivity appeared on HPLC and the main peak appeared after synthetic ovine CRF marker. Our results suggest that rat CRF is different from ovine CRF, and the total lipophilicity of amino acid residues of rat CRF may be higher than that of ovine CRF.

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Section: Discussionmentioning

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Characterization of Rat Hypothalamic Corticotropin-Releasing Factor by Reversed-Phase, High-Perf ormance Liquid Chromatography with Synthetic Ovine Corticotropin-Releasing Factor as a Marker

Hashimoto¹,

Murakami²,

Ohno³

et al. 1983

Neuroendocrinology

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“…However, the purification of these materials led only to a heptapeptide with weak CRF activity (10). In the course of our work to isolate the hypothalamic growth hormone-releasing factor, we again detected major CRF activity in fractions eluted near the void volume and behaving like 5000-dalton molecules on gel-filtration.…”

mentioning

confidence: 71%

“…The materials eluted near the void volume were repurified on Sephadex G-50. All the purification procedures have been described in detail (10)(11)(12)(13).…”

Section: Methodsmentioning

confidence: 99%

High molecular weight peptide with corticotropin-releasing factor activity from porcine hypothalami.

Schally¹,

Chang²,

Arimura³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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The presence of a corticotropin-releasing factor (CRF) behaving as a peptide with a molecular weightofabout 5000 was established after purification ofporcine hypothalamic extracts by gel filtration on Sephadex G-25 Although the presence, in hypothalamic extracts, of a physiological stimulator of corticotropin (ACTH) release, called the "corticotropin-releasing factor (CRF)" was demonstrated more than 25 years ago (1, 2), up to now the structure ofthis substance has not been identified. The search for CRF has been severely hampered by various problems such as the presence in hypothalamic extracts of vasopressin, which stimulates ACTH release in several systems (3), or of ACTH and ACTH-like molecules (4-6), which obscure CRF activity, and the loss of CRF activity during the purification. These-problems have been reviewed (7-9). In spite ofintensive effort by several laboratories, these difficulties so far have prevented the elucidation of the structure of CRF. The hypothesis that CRF is a small peptide influenced many attempts at its isolation.The presence of CRF activity in retarded fractions (RF = 0.82-0.7) from Sephadex G-25 has been demonstrated (8, 9, *). However, the purification of these materials led only to a heptapeptide with weak CRF activity (10). In the course of our work to isolate the hypothalamic growth hormone-releasing factor, we again detected major CRF activity in fractions eluted near the void volume and behaving like 5000-dalton molecules on gel-filtration. Renewed efforts were made to reexamine and characterize these 'high molecular weight CRF fractions. This paper reports our findings. MATERIALS AND METHODSPurification. A total of 470,000 pig hypothalami were dissected, defatted, and extracted as reported (11,12). The extract was the same as that used for the isolation of somatostatin (13). The extracts were purified by preparative gel filtration on Sephadex G-25 (11-13). The materials eluted near the void volume were repurified on Sephadex G-50. All the purification procedures have been described in detail (10-13).Inactivation Experiments. In all experiments, 500-to 800-Ag aliquots ofCRF purified on Sephadex G-50 were digested with 30 jig of the enzyme in a volume of 1 ml at 370C for 19 hr. The digestions with trypsin (Worthington, lot TRL 9FR) and chymotrypsin (Armour, lot X IN) were carried out in 0.1 M ammonium acetate buffer (pH 8.1). The incubation with pepsin (Worthington, PM SEB) was performed in 0.01 M hydrochloric acid. Control samples of CRF were incubated under the same conditions except that the enzymes were omitted. Samples of enzymes without CRF were also incubated and used as additional controls in the CRF assays. Before the assay, aliquots of tryptic and chymotryptic digests were acidified with acetic acid, boiled for 10 min to inactivate the enzymes, and then lyophilized twice. The peptic digest was neutralized to pH 7 and subjected to the same procedure.CRF Assays. The stimulation ofthe release ofACTH in vitro was measured in three different systems: isolated ra...

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“…Schallyet al [19] have reported the partial pu rification of six fractions with CRF activity, one of which, a heptapeptide. has been sequenced and synthesized [6]. Large-scale extraction and partial purification of CRF from Received: April 28, 1982 Accepted after revision: August 31, 1982 porcine hypothalami [2] indicated that proteolytic degrada tion may account for the heterogeneity of CRFs in previous reports.…”

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confidence: 99%

Differences in the Elution Profiles of Corticotropin-Releasing Activity in Rat Plasma and Hypothalamic Extracts Following High Performance Liquid Chromatography

et al. 1983

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Extracts of rat plasma and hypothalami were fractionated by reversed-phase high performance liquid chromatography (HPLC) and the fractions were assayed in an in vitro bioassay for corticotropin-releasing activity. Corticotropin releasing factor (CRF) bioactivity was found in many fractions with retention times of 20-50 min. In contrast, material derived from the plasma of rats stressed by ether anaesthesia yielded only one peak of CRF bioactivity, with a retention time of 47-48 min. These results suggest that ACTH can be released from isolated pituitary cells by a number of components derived from HPLC of extracts of rat hypothalami. It is possible, however, that some of these are artefacts of the extraction process from post-mortem material. Because the CRF activity found in plasma was homogeneous by HPLC, plasma can be used to obtain physiologically active CRF

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Isolation, Structure and Synthesis of a Heptapeptide with In Vitro ACTH-Releasing Activity from Porcine Hypothalamus

Cited by 14 publications

References 9 publications

Characterization of Rat Hypothalamic Corticotropin-Releasing Factor by Reversed-Phase, High-Perf ormance Liquid Chromatography with Synthetic Ovine Corticotropin-Releasing Factor as a Marker

Characterization of Rat Hypothalamic Corticotropin-Releasing Factor by Reversed-Phase, High-Perf ormance Liquid Chromatography with Synthetic Ovine Corticotropin-Releasing Factor as a Marker

High molecular weight peptide with corticotropin-releasing factor activity from porcine hypothalami.

Differences in the Elution Profiles of Corticotropin-Releasing Activity in Rat Plasma and Hypothalamic Extracts Following High Performance Liquid Chromatography

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