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

Abstract

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...

Isolation and amino acid sequence of corticotropin-releasing factor from pig hypothalami.

“…After gel filtration, CRF activity was eluted in several fractions. CRF activity in retarded fractions has been described (20). This paper reports the characterization of high molecular weight fractions with CRF activity, which emerged near the void volume.…”

“…This work was followed by the isolation and identification of rat CRF (15,16) and deduction of the structure of human CRF from the cDNA sequence (17). Schally and coworkers have described the presence of CRF in porcine hypothalami and its partial purification (18,19) and in 1981, reported (20) that porcine CRF is a polypeptide of Mr 5000. This paper gives an account of the final stages of the structural characterization of porcine CRF.…”

“…A total of 470,000 pig hypothalami were dissected, defatted, and extracted with 2 M acetic acid as reported (4,7). The extracts (lyophilized weight, 3.6 kg) were purified by preparative gel filtration on 20). After gel filtration, CRF activity was eluted in several fractions.…”

Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami.

“…A total of 470,000 porcine hypothalami were dissected, defatted, and extracted with 2 M acetic acid as reported (10, 11). The extracts were purified by preparative gel filtration on 12). Ten grams of Sephadex fractions 557-656 with Rf = 0.82-0.7 (see figure 1 of ref.…”

“…Ten grams of Sephadex fractions 557-656 with Rf = 0.82-0.7 (see figure 1 of ref. 12), from 16,600 hypothalami, was dissolved in 1 liter of buffer A [0.1% trifluoroacetic acid (F3CCOOH) in water] containing 0.2% 2-mercaptoethanol and centrifuged at 17,500 x g for 20 min. The supernatant was pumped onto a 2.5 x 50 cm glass column, packed with SynChroprep RP-P packing material (SynChrom, Linden, IN), and eluted as described (9).…”