Proc. Nati. Acad. Sci. USA
Vol. 74, No. 7, pp. 3052-3055, July 1977
Medical Sciences
Isolation and characterization of the opioid peptides from rat pituitary: fl-Lipotropin (fluorescence analysis/high performance liquid chromatography)
M. RUBINSTEIN, S. STEIN, L. D. GERBER, AND S. UDENFRIEND Roche Institute of Molecular Biology, Nutley, New Jersey 07110
Contributed by Sidney Udenfriend, May 10, 1977
ABSTRACr Rat ,-lipotropin was isolated from 40 anterior pituitaries using high efficiency chromatography columns and sensitive fluorometric methods. The P-lipotropin was characterized as to molecular weight, amino acid composition, and generation of opioid activity by trypsinization. Other opioid peptides, as well as a precursor to opioid peptides with a molecular weight larger than that of P-lipotropin, were observed in this tissue. The series of peptides a-, fi-, and y- endorphin and methionine-enkephalin, presumably fragments of fl-lipotropin, have been demonstrated to possess opioid activity in vitro (1-5). Furthermore, fl-endorphin has been reported to have potent analgesic activity in (6, 7). All the above were isolated from tissues of large animals after slaughter or from human cadavers. However, the physiology and pharmacology of the opioid peptides will most likely be elucidated in small laboratory animals, preferably the rat, in which most analgesic and behavioral studies are performed. Since human and bovine fl-lipotropins and fl-endorphins have already been shown to differ in structure (8), it appeared likely that the opioid peptides in rats would also have their own unique structures and would have to be characterized. Utilizing sensitive fluorometric procedures for protein, peptide, and amino acid assay developed in this laboratory (9-11), as well as a receptor binding assay for opioid peptides, we have been able to isolate and characterize opioid peptides in rat pituitaries. In this paper we report the purification to homogeneity of rat ,f-lipotropin from 40 rat pituitaries and its characterization as to molecular weight, amino acid analysis, and biological activation by trypsin.
servative (Pierce Chemical Co., Rockford, IL) to prevent growth of microorganisms. All peptide solutions and column effluents were stored in polypropylene tubes or in Siliclad (Clay Adams, Parsippany, NJ)-treated glassware. An automatic fluorescence detection system was used for column monitoring of peptides (9). In this system a discontinuous sampling valve allowed typically 2-10% of the column effluent to react with fluorescamine, while the remainder was directed to a fraction collector. An LKB (Hicksville, NY) gradient mixer, a Milton Roy (Riviera Beach, FL) minipump, and an Altex Scientific (Berkeley, CA) 7000 lbs./inch2 sample injection valve were the major components of the chromatography system. Aliquots of the collected fractions were hydrolyzed in constant boiling hydrochloric acid that had been redistilled over sodium dichromate (12). Thioglycolic acid (0.1% vol/vol) was added to prevent oxidation of cysteine (13). Amino acid analyses were performed on an instrument that used fluorescamine for detection at the picomole level as previously reported (10). Trypsin (twice crystallized) was obtained from Sigma (St. Louis, MO). Bioassay of opioid activity was carried out with neuroblastoma X glioma hybrid cells (NG 108CC15) (14), which were generously provided by M. Nirenberg, National Institutes of Health, Bethesda, MD. Opioid activity was measured by competitive binding with [3H]Leu-enkephalin (Amersham/Searle) to these cells, and activity is reported in terms of Leu-enkephalin equivalents. Details of the binding assay will be published elsewhere. Other experimental details are given in the figure legends and in Results.
MATERIALS AND METHODS Male Wistar rats (250 g) were obtained from Marland Farms (Hewitt, NJ). The anterior lobes of the pituitaries were removed and frozen within 30 sec of decapitation. Human fl-endorphin, camel #-endorphin, and sheep fl-lipotropin were the generous contributions of C. H. Li from the Hormone Research Laboratory, University of California, San Francisco. Other peptides were obtained from Peninsula Laboratories, Inc. (San Carlos, CA), and, when needed, were purified by high performance liquid chromatography. Fluorescamine (Fluramw, Hoffmann-La Roche, Inc., Nutley, NJ) was dissolved in distilled acetone (Burdick and Jackson Laboratories, Inc., Muskegon, MI). Water was purified by a system obtained from Hydro Services and Supplies (Durham, NC). Pyridine and acetic acid were distilled twice over ninhydrin. Sephadex G-75 columns (Pharmacia Fine Chemicals, Inc., Piscataway, NJ) were used for initial fractionations. Partisil SCX (Whatman, Clifton, NJ) and Lichrosorb RP-18 (Altex Scientific, Berkeley, CA) columns were used for resolving the peptides. Bio-Gel P-30 (Bio-Rad Laboratories, Richmond, CA) columns were used for determination of molecular weights. All column buffers contained thiodiglycol (0.01%) as an antioxidant and pHix Buffer Pre-
RESULTS Anterior pituitaries from 40 rats were extracted with acid/ acetone according to the procedure of Li et al. (15), with some modifications. The tissue was homogenized in 2.5 ml of cold 75% acetone/25% 0.2 M hydrochloric acid containing 0.01% thiodiglycol and 0.001% phenylmethylsulfonyl fluoride (a protease inhibitor). The following steps were performed at room temperature. Acetone was removed under a stream of nitrogen and the volume was brought to 2 ml by the addition of water. Lipids were extracted with three 1-ml portions of ethyl acetate/ether (3:1 vol/vol). The aqueous phase was fractionated on a Sephadex G-75 column and the effluent was monitored for fl-lipotropin by the following procedure. Aliquots (25 Ml) from each collected fraction were dried and then incubated with 10 Mg of trypsin in 50 ,l of 0.05 M Tris buffer (pH 8.5) at 370 for 18 hr. Opioid activity was then determined by the competitive binding assay. A minor peak of activity was found in the 30,000- to 60,000-dalton range (fractions 20-25) while the major activity was found in fractions 34-37, which corresponded to the sheep 03-lipotropin standard (Fig. 1). Fractions 34-37 were combined, lyophilized, and resolved on Partisil SCX (a strong cation exchange column), which was also monitored
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Vol. 74, No. 7, pp. 3052-3055, July 1977
Medical Sciences
Isolation and characterization of the opioid peptides from rat pituitary: fl-Lipotropin (fluorescence analysis/high performance liquid chromatography)
M. RUBINSTEIN, S. STEIN, L. D. GERBER, AND S. UDENFRIEND Roche Institute of Molecular Biology, Nutley, New Jersey 07110
Contributed by Sidney Udenfriend, May 10, 1977
ABSTRACr Rat ,-lipotropin was isolated from 40 anterior pituitaries using high efficiency chromatography columns and sensitive fluorometric methods. The P-lipotropin was characterized as to molecular weight, amino acid composition, and generation of opioid activity by trypsinization. Other opioid peptides, as well as a precursor to opioid peptides with a molecular weight larger than that of P-lipotropin, were observed in this tissue. The series of peptides a-, fi-, and y- endorphin and methionine-enkephalin, presumably fragments of fl-lipotropin, have been demonstrated to possess opioid activity in vitro (1-5). Furthermore, fl-endorphin has been reported to have potent analgesic activity in (6, 7). All the above were isolated from tissues of large animals after slaughter or from human cadavers. However, the physiology and pharmacology of the opioid peptides will most likely be elucidated in small laboratory animals, preferably the rat, in which most analgesic and behavioral studies are performed. Since human and bovine fl-lipotropins and fl-endorphins have already been shown to differ in structure (8), it appeared likely that the opioid peptides in rats would also have their own unique structures and would have to be characterized. Utilizing sensitive fluorometric procedures for protein, peptide, and amino acid assay developed in this laboratory (9-11), as well as a receptor binding assay for opioid peptides, we have been able to isolate and characterize opioid peptides in rat pituitaries. In this paper we report the purification to homogeneity of rat ,f-lipotropin from 40 rat pituitaries and its characterization as to molecular weight, amino acid analysis, and biological activation by trypsin.
servative (Pierce Chemical Co., Rockford, IL) to prevent growth of microorganisms. All peptide solutions and column effluents were stored in polypropylene tubes or in Siliclad (Clay Adams, Parsippany, NJ)-treated glassware. An automatic fluorescence detection system was used for column monitoring of peptides (9). In this system a discontinuous sampling valve allowed typically 2-10% of the column effluent to react with fluorescamine, while the remainder was directed to a fraction collector. An LKB (Hicksville, NY) gradient mixer, a Milton Roy (Riviera Beach, FL) minipump, and an Altex Scientific (Berkeley, CA) 7000 lbs./inch2 sample injection valve were the major components of the chromatography system. Aliquots of the collected fractions were hydrolyzed in constant boiling hydrochloric acid that had been redistilled over sodium dichromate (12). Thioglycolic acid (0.1% vol/vol) was added to prevent oxidation of cysteine (13). Amino acid analyses were performed on an instrument that used fluorescamine for detection at the picomole level as previously reported (10). Trypsin (twice crystallized) was obtained from Sigma (St. Louis, MO). Bioassay of opioid activity was carried out with neuroblastoma X glioma hybrid cells (NG 108CC15) (14), which were generously provided by M. Nirenberg, National Institutes of Health, Bethesda, MD. Opioid activity was measured by competitive binding with [3H]Leu-enkephalin (Amersham/Searle) to these cells, and activity is reported in terms of Leu-enkephalin equivalents. Details of the binding assay will be published elsewhere. Other experimental details are given in the figure legends and in Results.
MATERIALS AND METHODS Male Wistar rats (250 g) were obtained from Marland Farms (Hewitt, NJ). The anterior lobes of the pituitaries were removed and frozen within 30 sec of decapitation. Human fl-endorphin, camel #-endorphin, and sheep fl-lipotropin were the generous contributions of C. H. Li from the Hormone Research Laboratory, University of California, San Francisco. Other peptides were obtained from Peninsula Laboratories, Inc. (San Carlos, CA), and, when needed, were purified by high performance liquid chromatography. Fluorescamine (Fluramw, Hoffmann-La Roche, Inc., Nutley, NJ) was dissolved in distilled acetone (Burdick and Jackson Laboratories, Inc., Muskegon, MI). Water was purified by a system obtained from Hydro Services and Supplies (Durham, NC). Pyridine and acetic acid were distilled twice over ninhydrin. Sephadex G-75 columns (Pharmacia Fine Chemicals, Inc., Piscataway, NJ) were used for initial fractionations. Partisil SCX (Whatman, Clifton, NJ) and Lichrosorb RP-18 (Altex Scientific, Berkeley, CA) columns were used for resolving the peptides. Bio-Gel P-30 (Bio-Rad Laboratories, Richmond, CA) columns were used for determination of molecular weights. All column buffers contained thiodiglycol (0.01%) as an antioxidant and pHix Buffer Pre-
RESULTS Anterior pituitaries from 40 rats were extracted with acid/ acetone according to the procedure of Li et al. (15), with some modifications. The tissue was homogenized in 2.5 ml of cold 75% acetone/25% 0.2 M hydrochloric acid containing 0.01% thiodiglycol and 0.001% phenylmethylsulfonyl fluoride (a protease inhibitor). The following steps were performed at room temperature. Acetone was removed under a stream of nitrogen and the volume was brought to 2 ml by the addition of water. Lipids were extracted with three 1-ml portions of ethyl acetate/ether (3:1 vol/vol). The aqueous phase was fractionated on a Sephadex G-75 column and the effluent was monitored for fl-lipotropin by the following procedure. Aliquots (25 Ml) from each collected fraction were dried and then incubated with 10 Mg of trypsin in 50 ,l of 0.05 M Tris buffer (pH 8.5) at 370 for 18 hr. Opioid activity was then determined by the competitive binding assay. A minor peak of activity was found in the 30,000- to 60,000-dalton range (fractions 20-25) while the major activity was found in fractions 34-37, which corresponded to the sheep 03-lipotropin standard (Fig. 1). Fractions 34-37 were combined, lyophilized, and resolved on Partisil SCX (a strong cation exchange column), which was also monitored
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