Isolation and characterization of beta-endorphin-like peptides from bovine brains.

Abstract

ABSTRACT, Four fl-endorphin-like peptides from bovine brain extracts have been identified hy their behavior in CMcellulose chromatography, gel filtration, high-performance liquid chromatography, and radioimmunoassay. Two of them have been isolated in sufficient quantity for amino acid analysis, radioimmunoassay, and radioreceptor assay. One peptide has an amino acid com osition nearly identical to that of ,-endorphin with 56% of the radioimmunoreactivity and i.3%of the potency in the radioreceptor assay of P-endorphin. The amino acid content of the other P-endorphin-like peptide is very different from that of bovine P-endorphin but it has 47% of the radioimmunoreactivity and 1% of the potency in the radioreceptor assay of P-endorphin.The early isolation, sequence determination, and characterization of 3-endorphin (f3-EP) were performed on material from pituitary glands of camel (1), pig (2, 3), sheep (4), cow (5), rat (6), and human (4, 7). Among opioid peptides, f3-EP is the most potent in every biological system including analgesic and behavioral activity. Recent studies by radioimmunoassay and immunocytochemical techniques have shown the presence of immunoreactive f3-EP in the brain (8-11). We report here the isolation and characterization of two j3-EP like peptides, one of which has an amino acid composition nearly identical to that of f3-EP. In addition, two additional peptides with molecular size and immunoreactivity similar to those of f3-EP are described. MATERIALS AND METHODSBovine brains were collected from a local slaughterhouse. The cerebral hemispheres were dissected, rinsed to remove any blood, frozen on dry ice, and stored at -20'C before use.Acid acetone powder (AAP) was prepared as described (12). From 1 kg of bovine brains, approximately 12 g of AAP was obtained. Six grams of AAP was dissolved in 100 ml of 0.01 M NH4OAc (pH 4.6) at 4VC, and the insoluble material was removed by centrifugation. The supernatant was applied to a CM-cellulose column (3.06 X 56 cm) and fractionated in a stepwise manner by using a sequence of three eluents. Elution was with 0.01 M NH4OAc (pH 4.6) for the first 50 fractions (8 ml), with 0.2 M NH4OAc for the next 50 fractions and with 0.5 M NH4OAc for the last 100 fractions. Gel filtrations on a Sephadex G-100 column (2.7 X 88 cm) add a G-50 column (2.14 X 56 cm) were carried out in 0.1 M NH4OAc (pH 5.0) and 0.01 M NH4OAc (pH 4.6), respectively. A second CM-cellulose chromatographic step used a small coldiihn (1.5 X 28 cm) with gradient elutions as described for the isolation of camel melanotropins (13) and 3-EP (1).The final step in the isolation procedure consisted of highperformance liquid chromatography (HPLC) on a reversephase column (Altex; 10 X 500 mm) packed with Lichrosorb C8 (10 L). The proteins were dissolved in 0.5 ml of 1.0 M pyridine/0.5 M HOAc, pH 5.5, containing 18% n-proponal and injected via a 1.0-ml loading loop. Chromatography was performed by isocratic elution using the same buffer with a flow rate of 1.0 ml/min. The column effluent was collected i...

Stress-Induced Changes in Plasma, Pituitary and Hypothalamic Immunoreactive β-Endorphin: Effects of Diurnal Variation, Adrenalectomy, Corticosteroids, and Opiate Agonists and Antagonists

“…; recent evidence suggests that POMC is simi larly produced by hypothalamic cells [29,45]. As the end products of POMC vary significantly between these tissues [9,31,43,50], different fragments with P-endorphin-like immunoreactivity may serve different physiological roles in different tissues of origin.…”