Purification of the opiate receptor from rat brain.

Proc. Nati Acad. Sci. USA Vol. 78, No. 1, pp. 636-639, January 1981 Neurobiology

Purification of the 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 of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12181

Communicated by Marshall D. Gates, September 11, 1980

ABSTRACT The opiate receptor was urified from a Tritonsolubilized preparation of rat neural membranes by the use of affinity chromatography. The affinity gel was prepared by coupling 14-3bromoacetamidomorphine, a newly synthesized l d,to aaminohexyl-Sepharose. After elution of the nonspecific proteins with 50mM Tris (pH 7.5), the receptor proteins were eluted with 1 ImM levorphanol or etorphine. NaDodSO4/polyacrylamide gel electrophoresis revealed three major proteins associated with the opiate receptor, having molecular weifhts of 43,000, 35,000, and 23,000. Thepurifiedreceptorbinds 10- molofdihydromorphine/ per mg of protein, with a Kd of 3.8 x 10-9 M. Other opiates, naloxone, and methionine-enkephalin, inhibit [3H]dihydromorphine binding in a manner similar to that observed with intact and solubilized neural membranes.

without the cerebellum was homogenized in 20 ml of 50 mM Tris-HCI (pH 7.5). The homogenate was centrifuged at 100,000 x g for 30 min, yielding a neural membrane preparation that was suspended at a concentration of 10 mg/ml in 50 mM Tris (pH 7.5). Triton X-100 was added to the suspension to yield a concentration of 1.0%. After incubation on ice for 15 min, the suspension was centrifuged at 100,000 x g for 30 min. The resulting supernatant was added to Biobeads SM-2 (Bio-Rad), that had been prewashed with 50 mM Tris (pH 7.5), at a concentration of 0.4 g of Biobeads per ml of supernatant. After stirring for 2 hr at 4TC, the supernatant was separated from the Biobeads and concentrated on an Amicon PM-10 membrane to a concentration of30-50 mg of protein per ml. Protein concentration was determined by the method of Lowry et al. (6). Preparation of 14-fi-Bromacetamidomorphine Hydrochloride (1). The synthesis of 14-/3-bromoacetamidomorphine hydrochloride (1) started with the reduction of 14-f3nitrocodeinone (2) (7) with sodium borohydride to give 14 /3-nitrocodeine (3, mp 156-1580C). Acetylation of 3 afforded 14-.3-nitrocodeine 6-acetate (4, mp 229-231'C). O-Demethylation of 4 with boron bromide followed by acetylation gave 14-,-nitromorphine 3,6diacetate (5, mp 149-150'C). Reduction of the nitro group with zinc dust followed by acylation with bromoacetyl bromide in the presence of triethylamine gave 14-13-bromoacetamidomorphine 3,6-diacetate (6, mp 2350C dec.). Compound 6 was hydrolyzed in 1 M HC1 to yield the desired compound (1, mp >270'C). The free base of 1 gave a positive reaction for active halogens when treated with 4-(p-nitrobenzyl)pyridine according to the method of Baker et al. (8).

Purification of the opiate receptor is a key step in determining its chemical identity and in characterizing the molecular nature ofthe reactive sites. A major difficulty has been the preparation of a stable, active, soluble product from brain tissue. Initially, Simon et al. (1) reported the solubilization of an etorphine-opiate receptor complex. By molecular sieve chromatography, this complex had a molecular weight of 370,000. Zukin and Kream (2) covalently bound the [3H]enkephalin-receptor complex by crosslinking the solubilized noncovalent complex, which, by molecular sieve chromatography, had a molecular weight of 370,000. NaDodSO4 gel electrophoresis, however, revealed the major radioactive peak to have a molecular weight of 35,000. Neither of these complexes could bind opiates after

solubilization. Recently we succeeded in solubilizing an active opiate receptor from rat neural membranes with the use of Triton X-100 (3). The solubilized material, which was composed of protein and lipid, exhibited properties similar to those ofthe membrane receptor. Ruegg et al. (4) recently reported on the solubilization of an active opiate receptor from toad neural membranes by using 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 (NG108-15 cells) by using a zwitterionic derivative of cholic acid. This communication describes the purification of the opiate receptor by use of an affinity column prepared by conjugating 14-,B-bromoacetamidomorphine to Sepharose beads. The purified material, which consists mainly of three protein bands, binds opiates stereospecifically with high affinity and exhibits all the characteristics of the membrane-bound receptor.

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MATERIALS AND METHODS Solubilization. Neural membranes were prepared from rat brains without the cerebellum and were solubilized with Triton X-100 as described by Bidlack and Abood (3). Briefly, a rat brain

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