Isolation and characterization of #-endorphin-like peptides from bovine brains

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 METHODS Bovine 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 1.0M 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 in L.Q-ml fractions. An automatic sampling device diverted 20 ,l of effluent from the main stream once every minute, and its protein content was monitored by an automatic fluorescence-detection system (14). Radioimmunoassay was performed by using a specific rabbit antiserum to 13h-EP as described (15). The radioreceptor assay, using synaptic plasma membranes from rat brains and tritiated 3-EP as the primary ligand, was carried out as described (16). Amino acid analyses were performed according to the method of Spackman et al. (17) in a Beckman model 119C automatic amino acid analyzer. RESULTS Chromatography of AAP from bovine cerebral hemispheres on CM-cellulose column yielded three major protein peaks. The most retarded peak has been identified as being mostly myelin basic protein (12). Radioimmunoassay of the individual tubes revealed two peaks of immunoreactive f3-EP [fl-F.P(ir)] which coincided with each of the first two protein peaks and were designated CMC-A and CMC-B. From 100 g of.AAP, 5.2 g of CMC-A and 3.43 g of CMC-B were obtained. Each of these fractions was then subjected to chromatography on Sephadex G-100 (Fig. 1) and then on Sephadex G-50 (Fig. 2). In both instances, only a single peak of f3-EP(ir) was detected. Gel filtration on Sephadex G-100 yielded 820 and 520 mg of fractions G-100-A and G-100-B, respectively, from 100 g of AAP, whereas gel filtration on Sephadex G-50 yielded 160 mg of G-50-A and 410 mg of G-50-B. The Sephadex G-50 fractions were next subjected to a second CM-cellulose chromatographic step which again produced a single peak of 3-EP(ir), this running slightly more basic than tritiated 13h-EP standard (Fig. 3). The yields of CMC'-A and CMC'-B were 4.0 and 10.0 mg, respectively, from 100 g of AAP. These fractions were then subjected to HPLC and, in each instance, two peaks of f3-EP(ir) were found (Fig. 4). No fluorescent peaks were found associated with the 3-EP(ir) peaks (HPLC-C and HPLC-D) obtained from CMC'-B, but two quite distinct protein peaks (HPLC-A and HPLC-B) were found Abbreviations: AAP, acid acetone powder; HPLC, high-performance liquid chromatography; RIA, radioimmunoassay; f3-EP, fl-endorphin with subscripts h and b denoting as human and bovine, respectively; O-EP(ir), immunoreactive f3-EP. 230 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 77 (1980) 231