Purification of the Opiate Receptor from Rat Brain

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, naloxone, 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 concentration 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 solubilized the opiate receptor from rat and beef neural membranes 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 purified 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-bromoacetamidomorphine 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. Approximately 0.5% ofthe protein applied to the col- 0.001% bromophenol blue. After the samples had incubated at umn remained nonspecifically bound to the column and was 37°C for 1 hr, they were electrophoresed for 2 hr at constant eluted with 0.2 M NaCl. voltage (150 V). Gels were stained in 0.1% Coomassie blue/ 10% (vol/vol) acetic acid/50% (vol/vol) methanol overnight and diffusion destained in 10% acetic acid. Apparent molecular weights were calculated from a graph ofrelative mobilities ver- sus logarithm of molecular weight. Molecular weight standards used were phosphorylase a (94,600), bovine serum albumin (68,000), catalase (57,500), lactate dehydrogenase (35,000), car- bonic anhydrase (30,000), trypsin inhibitor (21,500), lysozyme (14,400), and cytochrome c (12,600). 43 Opioid Binding. The fractions eluted with either levorphanol 35 or etorphine were combined and concentrated to 1-2 ml. The i~40 samples were dialyzed against 4 liters of 50 mM Tris (pH 7.5) for 24 hr at 25°C with two changes ofbuffer, which removed all of 23 the opioid ligand present as determined by the addition of radioactive etorphine. Opioid binding to the purified receptor and other fractions was measured by equilibrium dialysis as described by Bidlack and Abood (3). A solution containing 2-10 ug of the receptor in 0.2 ml of 50 mM Tris (pH 7.5) was dialyzed A B C D E F against 50 mM Tris (pH 7.5) containing [3H]dihydromorphine (Amersham; 76 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) for 5 hr FIG. 2. NaDodSO4/polyacrylamide gels of the column fractions at 37°. Competing drugs were added to the outside solution. from Fig. 1. Lanes: A, solubilized supernatant that was applied to the in 5 ml of column; B, fractions 1-3, the protein that passes rapidly through the Aliquots ofinside and outside solutions were assayed column; C, fractions 20-24; D, fractions eluted with 1 aM levorphanol, Aquasol-2 (New England Nuclear). All counts were converted 25-34; E, fractions 35-40; F, another preparation of the opiate recep- to dpm. Stereospecific binding was defined as the number of tor, eluted from the affinity column with 1 /iM levorphanol. Molecular dpm in the presence of 1 pM dextrorphan minus that in the weights are shown x 10-3. Downloaded by guest on October 2, 2021 638 Neurobiology: Bidlack et aL Proc. Natl. Acad. Sci. USA 78 (1981)

Table 1. Stereospecific binding to affinity column fractions Table 2. Relative potencies ofdrugs in decreasing [3Hjdifhydromorphine Stereospecific binding of binding to the purified receptor Fraction [3H]dihydromorphine, Drug ICro, M x 109 no. mol x 10n4/mg Etorphine 0.5 Supernatant 8 Naloxone 2 1-2 2 [Met]Enkephalin 3 14-24 0 Levorphanol 30' 25-34 2100 Dextrorphan 10,000 35-40 2 The concentration of[3Hldihydromorphine was 4 nM. The concentration of[3H]dihydromorphine was 4 nM. The concentration ofdextrorphan or levorphanol was 1 ,M. The fraction numbers re- which the Kd for stereospecific binding ofdihydromorphine was fer to the fractions in Fig. 1. 3.5 nM (3). At saturating levels of dihydromorphine, the purified receptor bound 4 x 10-11 mol ofopiate per mg ofprotein, NaDodSOpolyacrylamide gels of the various column frac- which represents a 2000-fold purification from the starting ma- tions are shown in Fig. 2. The pattern in lane A is of the total terial. Triton-solubilized proteins applied to the column and is identi- The relative potencies of naloxone and various opiate ago- cal to that ofthe neural membrane preparation. The gel pattern nists, including [Met]enkephalin, to inhibit dihydromorphine ofthe proteins that pass quickly through the column in the first binding to the purified receptor are shown in Table 2. As with three fractions is shown in lane B. As seen from lane C, few pro- neural membranes and the solubilizied receptor, etorphine was teins were eluted in fractions 20-24. The gel pattern of the ma- the most potent opiate in inhibiting dihydromorphine binding terial eluted with 1 AM levorphanol (fractions 25-34) shows to the purified receptor having an IC50 value of 5 x 10-1o M. three major bands, with molecular weights of 43,000, 35,000, Naloxone and [Met]enkephalin were quite potent, having IC50 and 23,000. The minor protein contaminants represent nonspe- values in the low nanomolar range. Levorphanol had only 10% cifically bound proteins that eluted gradually from the column of the potency of these latter two compounds, having an IC,% and were completely removed after prolonged elution. Lane E value of3 x 10-8 M. shows the protein pattern of the numerous nonspecifically bound proteins eluted with 0.2 M NaCl. Lane F shows a similar DISCUSSION gel pattern ofanother preparation ofthe opiate receptor, eluted The use ofa 14-,B3bromoacetamidomorphine coupled to Sepha- from the column with 1 AM levorphanol; the three major pro- rose 4B beads has permitted an apparent purification ofthe op- teins in lane D are present in this gel to at least 95% purity. Ifit iate receptor in a one-step process. Three majorproteins appear is assumed that the three major bands constitute the receptor, to be associated with the eluted receptor, but it remains to be it can be inferred that the receptor comprises much less than 1% determined whether opiate binding involves one or a complex ofthe membrane protein. Approximately 100 Ag ofpure recep- of the proteins. Martin et al. (10) proposed that there. are sub- tor can be obtained when 40 mg of the Triton-solubilized solu- classes ofopiate receptors, designated IL, K, and a, as suggested tion is applied to the affinity column. by the prototype agonists that act at each site-namely, mor- Both levorphanol and NaCl were removed from the fractions phine, ketocyclazocine, and SKF 10,047 (N-allylnormetazo- by dialysis against 50 mM Tris (pH 7.5) for 1 day. The stereospe- cine), respectively. Several laboratories have observed that cific binding of [3H]dihydromorphine to each fraction is shown higher concentrations of some opiate alkaloids are required to in Table 1. The affinity column retained about 75% ofthe opiate compete for opiate receptors when radiolabeled opioid peptides receptor applied, as evidenced by the fact that the initial column are used as ligands, a finding that points to the existence ofsep- peak bound 2 x 10-'4 mol/mg ofprotein. In the fractions from arate "peptide" and "alkaloid" opiate receptors (11, 12). Re- the latter part ofthe column wash, there was essentially no op- cently, Pert et al. (13) proposed type 1 and type 2 opiate recep- iate binding. The purified opiate receptor bound 2 X 10-" mol tors, based on their sensitivity and insensitivity to GTP. ofopiates per mg ofprotein. The binding to this fraction was [Leu]Enkephalin is the ligand with the most marked preference 100% stereospecific: with 1 AM levorphanol, [3H]dihydro- for the type 2 receptors, the GTP-insensitive receptors; nalox- morphine binding was completely inhibited. one prefers the GTP-sensitive receptors. To demonstrate that The stereospecific binding of [3H]dihydromorphine to the the three protein bands are separate receptors would require purified receptor was saturable at 9 nM, with half-maximal the use ofspecific covalent ligands capable ofdifferentiating the binding occurring at 3.8 nM (Fig. 3). These data are in agree- receptors. On the basis of size, our molecular weight, 35,000 ment with those obtained with the solubilized supernatant, in protein corresponds to the protein that was labeled by Zukin and Kream (2) when they covalently bound enkephalin to the solubilized receptor complex. Insofar as the solubilized receptor is eluted in the void volume ofa Sephadex G-200 column (data °3- not shown), itwould appear, as suggested by others (1, 2, 5), that the opiate receptor exists as a higher molecular weight complex; E 2- however, the evidence is inadequate for such a conclusion. The purified receptor binds 4 x 10-11 mol of dihydromor- x° - phine per mg of protein at saturating concentrations ofligand. a'6 If it is assumed that the molecular weight of the receptor complex is between 100,000 and 500,000 and that the receptor binds Q O(. ,.. 1 mol ofopiate per mol ofreceptor, one would expect binding in the range of10-9 mol ofopiate per mg ofprotein. Ifthe receptor DHM, nM has a lower molecular weight (30,000-40,000), then the density FIG. 3. Saturation curve for the binding of [3H]dihydromorphine ofbinding sites would be somewhat greater. There are a number (DHM) to the purified receptor. ofpossible explanations for the failure to obtain this high level of Downloaded by guest on October 2, 2021 Neurobiology: Bidlack et al. Proc. Natl. Acad. Sci. USA 78 (1981) 639

binding. It is conceivable that the receptor may not bind 1 mol 2. Zukin, R. S. & Kream, R. M. (1979) Proc. NatL Acad. Sci. USA 76, ofopiate per mol of receptor protein or that the receptor com- 1593-1597. plex was partially inactivated during the purification procedure. 3. Bidlack, J. M. & Abood, L. G. (1980) Life Sci. 27, 331-340. 4. Ruegg, U. T., Hiller, J. M. & Simon, E. J. (1980) Eur.J. Pharm. 64, A cofactor required for optimal binding may be missing from the 367-368. purified complex. 5. Simonds, W. F., Koski, G., Streaty, R. A., Hjelmeland, L. M. & The high affinity and drug sensitivity ofthe purified receptor Klee, W. A. (1980) Proc. NatL Acad. Sci. USA 77, 4623-4627. are similar to those observed for intact and solubilized neural 6. Lowry, D. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. membranes (3). All preparations had a Kd for [3H]dihy- (1951)J. BioL Chem. 193, 265-275. dromorphine of4 X 10' M. Naloxone exhibited a higher affin- 7. Archer, S. & Osei-Gyimah, P. (1979)J. HeterocycL Chem. 16, 389. 8. Baker, B. R., Santi, D. V., Coward, J. K., Shapiro, M. S. & Jor- ity than morphine or levorphanol in the solubilized and purified daan, J. H. (1966)J. HeterocycL Chem. 3, 425-434. preparations and a lesser affinity than either agonist in intact 9. Laemmli, U. K. & Favre, M. (1973)J. MoL BioL 80, 575-599' membranes. [Met]Enkephalin had a higher affinity than mor- 10. Martin, W. R., Eacles, C. G., Thompson, J. A. Huppler, R. E. & phine in all three preparations. Gilbert, P. E. (1976)J. PharmnacoL Exp. Ther. 197 517-532. 11. Snyder, S. H., Childers, S. R. & Creese, I. (1979) in Advances in Biochemistry and Psychopharmacology (Raven, New York), Vol. This research was supported by U.S. Public Health Service Grants 20, pp. 543-552. DA 00464, DA-01674, and NS 15345. 12. Lord, J. A. H., Waterfield, A. A., Hughes, J. & Kosterlitz, H. W. (1977) Nature (London) 267, 495-499. 13. Pert, C. B., Taylor, D. P., Pert, A., Herkenham, M. A. & Kent, J. 1. Simon, E. J., Hiller, J. M. & Edelman, I. (1975) Science 190, L. (1980) in Advances in Biochemistry and Psychopharmacology 389-390. (Raven, New York), Vol. 22, pp. 581-589. Downloaded by guest on October 2, 2021