Purification of the Opiate Receptor from Rat Brain
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Proc. Nati Acad. Sci. USA Vol. 78, No. 1, pp. 636-639, January 1981 Neurobiology Purification ofthe opiate receptor from rat brain (affinity chromatography/solubilized receptor/opiate binding) JEAN M. BIDLACK*, LEO G. ABOOD*, PETER OSEI-GYIMAHt, AND SYDNEY ARCHERt *Center for Brain Research, University ofRochester Medical Center, Rochester, New York 14642; and tDepartment ofChemistry, Rensselaer Polytechnic Institute, Troy, New York 12181 Communicated by Marshall D. Gates, September 11, 1980 ABSTRACT The opiate receptor was urified from a Triton- without the cerebellum was homogenized in 20 ml of 50 mM solubilized preparation ofrat neural membranes by the use ofaf- Tris-HCI (pH 7.5). The homogenate was centrifuged at 100,000 finity chromatography. The affinity gel was prepared by coupling 14-3bromoacetamidomorphine, anewly synthesized l d,toa- x g for 30 min, yielding a neural membrane preparation that aminohexyl-Sepharose. After elution of the nonspecific proteins was suspended at a concentration of 10 mg/ml in 50 mM Tris with 50mM Tris (pH 7.5), the receptor proteins were eluted with (pH 7.5). Triton X-100 was added to the suspension to yield a 1 ImM levorphanol or etorphine. NaDodSO4/polyacrylamide gel concentration of 1.0%. After incubation on ice for 15 min, the electrophoresis revealed three major proteins associated with the suspension was centrifuged at 100,000 x g for 30 min. The re- opiate receptor, having molecular weifhts of43,000, 35,000, and sulting supernatant was added to Biobeads SM-2 (Bio-Rad), that 23,000. Thepurifiedreceptorbinds 10- molofdihydromorphine/ had been prewashed with 50 mM Tris (pH 7.5), at a concentra- per mg ofprotein, with a Kd of 3.8 x 10-9 M. Other opiates, na- loxone, and methionine-enkephalin, inhibit [3H]dihydromorphine tion of0.4 g ofBiobeads per ml ofsupernatant. After stirring for binding in a manner similar to that observed with intact and sol- 2 hr at 4TC, the supernatant was separated from the Biobeads ubilized neural membranes. and concentrated on an Amicon PM-10 membrane to a concen- tration of30-50 mg ofprotein per ml. Protein concentration was Purification ofthe opiate receptor is a key step in determining determined by the method ofLowry et al. (6). its chemical identity and in characterizing the molecular nature Preparation of 14-fi-Bromacetamidomorphine Hydrochlo- of the reactive sites. A major difficulty has been the preparation ride (1). The synthesis of 14-/3-bromoacetamidomorphine hy- of a stable, active, soluble product from brain tissue. Initially, drochloride (1) started with the reduction of 14-f3nitrocodein- Simon et al. (1) reported the solubilization ofan etorphine-opiate one (2) (7) with sodium borohydride to give 14 /3-nitrocodeine receptor complex. By molecular sieve chromatography, this (3, mp 156-1580C). Acetylation of3 afforded 14-.3-nitrocodeine complex had a molecular weight of370,000. Zukin and Kream 6-acetate (4, mp 229-231'C). O-Demethylation of4 with boron (2) covalently bound the [3H]enkephalin-receptor complex by bromide followed by acetylation gave 14-,-nitromorphine 3,6- crosslinking the solubilized noncovalent complex, which, by diacetate (5, mp 149-150'C). Reduction of the nitro group with molecular sieve chromatography, had a molecular weight of zinc dust followed by acylation with bromoacetyl bromide in the 370,000. NaDodSO4 gel electrophoresis, however, revealed presence oftriethylamine gave 14-13-bromoacetamidomorphine the major radioactive peak to have a molecular weight of 3,6-diacetate (6, mp 2350C dec.). Compound 6 was hydrolyzed 35,000. Neither of these complexes could bind opiates after in 1 M HC1 to yield the desired compound (1, mp >270'C). The solubilization. free base of 1 gave a positive reaction for active halogens when Recently we succeeded in solubilizing an active opiate recep- treated with 4-(p-nitrobenzyl)pyridine according to the method tor from rat neural membranes with the use ofTriton X-100 (3). ofBaker et al. (8). The solubilized material, which was composed of protein and lipid, exhibited properties similar to those ofthe membrane re- NCH3 NCH3 ceptor. Ruegg et al. (4) recently reported on the solubilization ofan active opiate receptor from toad neural membranes by us- V NHCOCH2Bi ing the detergent digitonin. Simonds et al. (5) subsequently sol- ubilized the opiate receptor from rat and beef neural mem- branes and membranes from a neuroblastoma-glioma hybrid HO R'O 0 R (NG108-15 cells) by using a zwitterionic derivative of cholic acid. 1 This communication describes the purification of the opiate 2 R, O; R', CH3 receptor by use of an affinity column prepared by conjugating H 14-,B-bromoacetamidomorphine to Sepharose beads. The puri- fied material, which consists mainly of three protein bands, 3 R,& ; R', CH3 binds opiates stereospecifically with high affinity and exhibits OH all the characteristics ofthe membrane-bound receptor. H MATERIALS AND METHODS 4 R, ; R', CH3 Solubilization. Neural membranes were prepared from rat OCOCH3 brains without the cerebellum and were solubilized with Triton H X-100 as described by Bidlack and Abood (3). Briefly, a rat brain 5 R,< , R', COCH3 The publication costs ofthis article were defrayed in part by page charge OCOCH3 payment. This article must therefore be hereby marked "advertise- nent" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: IC50, concentration for 50% inhibition. 636 Downloaded by guest on October 2, 2021 Neurobiology: Bidlack et aL Proc. Nati Acad. Sci. USA 78 (1981) 637 i A.M .,, 0.2e Mis Preparation of Affinity Column. After 3 g of w-aminohexyl- 1.0 levorphanol~~~~~~1/kmI NaC w Sepharose 4B (Pharmacia) was swollen in 40 ml of 0.1 M NaHCO3, it was suspended in 500 ml of0.5 M NaCl and filtered 0.8- through a glass fiber filter. After the gel was suspended in 25 ml 0.6- of cold 0.1 M NaHCO3, 40 mg of 14-(3-bromoacetamidomor- phine hydrochloride (1) was added, and the suspension was 0.4- shaken gently at 40C for 16 hr. After the gel had settled and ex- 0.2- cess buffer had been decanted, 25 ml of 0.1 M NaHCO3 was added. To block the free NH2 groups still associated with the 0 - gel, 100 mg of 1-ethyl-3-(3-dimetliylaminopropyl)carbodiimide 0 5 10 15 20 25 30 35 40 45 (Pierce) and 80 1.L ofglacial acetic acid were added to the gel sus- Fraction pension. The pH remained between 5 and 6. After shaking at FiG. 1. Profile ofelution from affilnity column. Protein was applied 250C for 18 hr, the gel suspension was poured into a 0.7 x 15cm to the column at the start offraction 1. The column was eluted with 50 column to a total volume of 5.7 ml. The gel was then washed mM Tris (pH 7.5). Buffer was switched where indicated. Each fraction with 300 ml of50 mM Tris (pH 7.5) and was ready for use. was 5 ml. Purification. Prior to application to the column, 30-50mg of the solubilized preparation in 0.5-1.0 ml of50mM Tris (pH 7.5) presence of 1 AM levorphanol. In experiments to determine was incubated at 370C for 30 min. The sample was then applied drug sensitivity, varying concentrations of drugs were used to to the column and eluted with 50 mM Tris (pH 7.5). The eluant compete with 4 nM [3H]dihydromorphine. The concentrations was collected in 5.0-ml fractions and absorbance at 225 nm was for 50% inhibition (IC,% values) were determined from monitored. The elution with 50mM Tris (pH 7.5) continued un- probit-logarithm plots ofthe data. til the absorbance at 225 nm had been near zero for at least five tubes; generally, this required 125 ml. The opiate receptor was RESULTS eluted from the column with either 1 puM levorphanol or 1 After solubilization of an active opiate receptor, purification of ,uMetorphine in 50 mM Tris (pH 7.5). The absorbance at 225 the receptor was accomplished in one step by affinity chroma- nm was monitored and a small peak was seen about four tubes tography using a bromoacetamide derivative of morphine. This after the eluant was changed. About 0.5 mg ofprotein bound to ligand has the same opioid potency as dihydromorphine as de- the column nonspecifically and was eluted with 0.2 M NaCl. termined by its ability to compete with [3H]dihydromorphine The various fractions of interest were combined and concen- for binding to rat neural membranes. A protein elution pattern trated on an Amicon PM-10 membrane. from the affinity column is shown in Fig. 1. Most ofthe protein NaDodSO4 Slab Gel Electrophoresis. To determine the pro- applied to the column eluted in the first three fractions. Elution tein composition of each fraction, NaDodSO4polyacrylamide with 50 mM Tris (pH 7.5) was continued until the absorbance at slab gel electrophoresis was carried out according to the method 225 nm remained stable and close to zero for at least five frac- ofLaemmli and Favre (9>. Slab gels (1.5 x 120 mm) consisted of tions, a procedure which required washing the column with at a 12% acrylamide running gel with a6% acrylamide stacking gel. least 20 column volumes of buffer. The opiate receptor was The samples were prepared for electrophoresis by dialysis eluted from the column with either 1 uM levorphanol or 1 pLM against water followed by lyophilization and then solubilization etorphine in 50 mM Tris (pH 7.5). The elution of the receptor in 2% NaDodSOJ62.5 mM Tris-HCl, pH 6.8/1 mM EDTA/3 could be monitored by noting a slight increase in the absorbance mM LiCV5% (vol/vol) mercaptoethanol/10% (vol/vol) glycerol/ at 225 nm.