Proc. Nati. Acad. Sci. USA Vol. 80, pp. 5176-5180, September 1983 Biochemistry Isolation of opiate binding components by affinity chromatography and reconstitution of binding activities (opiate binding activity/opiate receptor/acidic lipids) T. M. CHO, B. L. GE, C. YAMATO, A. P. SMITH, AND H. H. LOH Departments of Pharmacology and Psychiatry, University of California, San Francisco, California 94143 Communicated by Vincent P. Dole, April 15, 1983 ABSTRACT Rat brain membranes exhibiting stereospecific MATERIALS AND METHODS opiate binding activity were solubilized by sonication and deter- gent treatment. The active material could be bound to an affinity Chemicals. [3H]Etorphine (35 Ci/mmol; 1 Ci = 37 GBq) column containing 6-succinylmorphine but could not be eluted with and [3H]diprenorphine (38 Ci/mmol) were purchased from free agonist. Although two protein peaks could be eluted with NaCl, Amersham. Other opioids were donated by the following com- neither possessed binding activity; however, one of the peaks (A), panies: naloxone, naltrexone, nalbuphine, and oxymorphine by in combination with an acidic lipid fraction, eluted subsequently DuPont; diprenorphine, etorphine, and buprenorphine by from the column, showed stereospecific binding. Opiate ligands Reckett-Coleman; nalorphine by Merck Sharp & Dohme; mor- of the 1L type bound to this protein/lipid mixture with an order phine was obtained from Mallinckrodt. CerSO4 was isolated from of affinities closely correlating with those of the original mem- bovine brain by using a Sephadex LH-20 column (4). Thin-layer brane but one to two orders of magnitude lower; binding of 8, K, chromatography showed it to be more than 90% pure. Other and .or prototype opioids was considerably less. The protein/lipid lipids were purchased: PtdIns from Analabs (North Haven, CT); mixture also competed with the membranes for ,u ligands. These P3-Ins from Sigma; and cholesterol, phosphatidylcholine, phos- results suggest that the isolated protein-lipid complex may be a phatidylethanolamine, ganglioside, PtdSer, and cerebroside from component of the opiate receptor and, specifically, the ,j receptor Supelco (Bellefonte, PA). Liquiscint was purchased from Na- or binding site. However, because of the lower affinities of ,u opi- tional Diagnostics (Somerville, NJ). ates for this complex, it is conceivable that some essential mem- Coupling of 6-Succinylmorphine to Affi-Gel 102. 6-Succi- brane component is still missing. Preliminary analysis of peak A nylmorphine was prepared by the method of Simon et al. (13). indicates that it contains a broad spectrum of protein bands, but A solution of it (0.85 g/100 ml of 50% aqueous ethylene glycol) it remains to be seen which of these are essential for activity. was added to 100 ml of settled washed Affi-Gel 102 beads and then 2.0 g of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide was Despite extensive research, the chemical nature of the opiate added slowly over a 5-min period while maintaining the pH at receptor remains unresolved. A large amount of data is con- 4.7-5.0 with 1.0 M HCl. The mixture was shaken overnight sistent with it containing both protein and certain lipid com- and then washed thoroughly first with 50% ethylene glycol and ponents. Thus, opiate binding to brain membranes in vitro is then with 50 mM Tris HCI (pH 7.4). inhibited by both protein-sensitive reagents such as trypsin and Affinity Chromatography. P2 fractions from eight rat brains N-ethylmaleimide (1) and lipolytic enzymes such as phospho- were solubilized by sonication as described (14) and treatment lipase A2 (2) and arylsulfatase (3). In addition, opiates can bind with 0.5% Triton X-100 (final concentration) in an ice-bath for stereospecifically to certain acidic lipids such as cerebroside 30 min (9). The undissolved material was sedimented at 100,000 sulfate (CerSO4) (4, 5), phosphatidylserine (PtdSer) (6), phos- x g for 1 hr and the supernatant, containing the solubilized re- phatidylinositol (PtdIns) (7), and triphosphoinositide (P3-Ins) (8). was loaded onto an column 20 To delineate the chemical properties of an opiate receptor, its ceptors, affinity (2.5 x cm). The isolation is essential. However, this requires solubilization and column was washed with 1 liter of 50 mM Tris HCl (pH 7.4) purification of an active form and progress with such an ap- and elution was then carried out with a linear gradient of 250 proach has been slow. Workers in two laboratories have re- ml of 50 mM Tris-HCl (pH 7.4) and 250 ml of 50 mM Tris-HCl, ported solubilization of active opiate receptors in low yields (9, pH 7.4/1 M NaCl. Fractions of 8 ml were collected. 10). A third group has reported solubilization in high yield of Isolation of Lipid Fraction from the Affinity Resin. After ,u type opiate receptors but the purification was not reported the removal of virtually all protein from the affinity column (by (11). Another group, working with a different preparation, has using NaCl as described above), the resin was washed with 500 reported purification (12), but this study has not been con- ml of distilled water, with 300 ml of methanol, and finally with firmed yet in other laboratories. 100 ml of chloroform/methanol (1:2). The lipid fraction was In light of these problems, it seemed to us that most solu- eluted with 500 ml of the same solvent containing 0.4 M po- bilization procedures might be removing from the receptor some tassium acetate. The lipid eluate was concentrated by evapo- factor crucial for binding in its native form. In support of this ration at reduced pressure and subjected to thin-layer chro- hypothesis, we now report solubilization and partial purifica- matography using silica gel H and chloroform/methanol/4 M tion of opiate binding material in high yield by reconstitution NH40H (9:7:2). Each lipid was visualized by treatment with of a protein fraction and a lipid fraction, neither of which alone iodine. CerSO4 and phospholipids were determined by the azure binds significantly. The protein-lipid combination represents method (15) and by phosphate measurement (16), respectively. a 420-fold purification of the opiate receptor. Binding Assay. Binding experiments were carried out ac- cording to the method of Tovey et aL (17) with slight modifi- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: P3-Ins, triphosphoinositide; PtdIns, phosphatidylinosi- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tol; PtdSer, phosphatidylserine; CerSO4, cerebroside sulfate. 5176 Downloaded by guest on September 25, 2021 Biochemistry: Cho et aL Proc. Nati. Acad. Sci. USA 80 (1983) 5177 cation. Briefly, 0.5-ml samples were allowed to stand at 250C against 50 mM Tris HCI to remove the free opiate. However, for 30 min in 50 mM Tris HCl, pH 7.4/2 nM [3H]diprenor- no binding activity was observed in any of the eluates. phine in the presence or absence of unlabeled drug (or re- As a further means of removing the material on the column, agent). Then, the mixture was cooled in an ice bath for 20 min, NaCi, which inhibits agonist receptor binding (1), was used at and 0.2 ml of the samne buffer containing 2.6% charcoal and 2% increasing concentration for elution. This salt would also be ex- albumin was added to remove unbound ligand. This mixture pected to elute much protein material in a nonspecific fashion. was shaken for 10 sec and centrifuged for 3.5 min in an Ep- Fig. 1 shows a chromatogram of material eluted by a linear pendorf 5413 centrifuge (Brinkmann). Finally, 0.5 ml of the NaCl concentration gradient procedure. Two protein peaks were supernatant was transferred to a vial containing 9 ml of Li- observed. Peak A eluted at approximately 0.35 M NaCl (frac- quiscint and the radioactivity was measured by liquid scintil- tions 10-20) and peak B, at 0.60 M NaCl (fractions 22-34). The lation spectrophotometry. Unfortunately, this assay cannot be fractions in each peak were pooled, concentrated by Amicon used in the presence of detergent. A Sephadex assay, on the ultrafiltration, and dialyzed overnight against 50 mM Tris-HCl other hand, is feasible but imprecise. Thus we were unable to (pH 7.4). Peaks A and B contained 1.8% and 0.7% of the total determine with any confidence the amount of binding to the protein loaded, respectively. The rest of the proteins were found solubilized receptor. in the void volume and in the Tris buffer eluate. Neither pro- tein peak A nor B, however, exhibited any opiate binding ac- tivity. RESULTS Afterthe elution of proteins with NaCl, the affinity resin still Isolation of Opiate Binding Components. Opiate binding to retained a yellowish color that was not detected in the original solubilized material from rat brain was assayed by several pro- unloaded resin. This material was extracted with organic sol- cedures, including gel filtration (14), charcoal precipitation (17), vent and, after separation by thin-layer chromatography [silica polyethylene glycol treatment (18), and the Hummel-Dreyer gel H, CHC13/CH30H/4 M NH40H (9:7:2)], the extracted method (19). Only the last procedure resulted in observable material showed spots that corresponded to morphine and to stereospecific binding, which may have resulted from two fea- the acidic lipids P3-Ins, PtdIns, PtdSer, and CerSO4; neutral tures of this method: (i) assay of rapidly dissociating ligand was lipids such as phosphatidylcholine, phosphatidylethanolamine, made possible by carrying out the binding experiments in the cerebroside, and cholesterol, which are abundant in the P2 frac- continuous presence of an equilibrating ligand and (ii) deter- tions, were not observed.