Isolation of Opiate Binding Components by Affinity Chromatography and Reconstitution of Binding Activities (Opiate Binding Activity/Opiate Receptor/Acidic Lipids) T

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. Each acidic lipid spot was collected gent, which may interfere with binding, was effectively re- and its amount was determined. The proportion of total acidic moved from the binding material by the column. lipids was CerSO4/P3-Ins/PtdIns/PtdSer = 2.0:1.5: 1.0: 1.0. Isolation of the binding material was then attempted using The mixture of acidic lipids by itself did not show significant excess affinity resin. Since such a column separates detergent opiate binding activity. The reason for this could be contami- from solubilized binding material, we assumed that the resin nation by morphine in the organic solvent eluate. Therefore, would also bind- the latter. The supernatant of detergent-sol- the interfering morphine was removed by chromatography on ubilized binding material from eight rats was applied to an af- Sephadex LH-20 as previously used to remove free and bound finity column (2.5 x 20 cm) that had been equilibrated with 50 levorphanol (4). However, the acidic lipid mixture still showed mM Tris HCI (pH 7.4) and then washed with 1 liter of the same little opiate binding activity (Fig. 2A). buffer. Neither the void volume nor the Tris buffer eluate ex- Reconstitution of Opiate Binding Material. While none of hibited significant opiate binding activity. Morphine or nalox- the fractions eluted from the affinity column showed any sig- one (0.1 ,uM, 10 ,AM, and 1 mM) was then added to the column nificant binding activity, the possibility remained that the col- in an attempt to elute the binding material; each eluate was umn separated the binding material into different components, concentrated by using the Amicon filter method and dialyzed each of which was unable to bind opiate in the absence of the other. Consequently, the opiate binding activity to mixtures of the individual peak proteins with the acidic lipid fraction was determined. Fig. 2A shows [3H]diprenorphine binding to the

0 15 r B E

E0 ._ 2

0 z

10 20 30 Fraction number 50 100 50 100 150 200 250 Acidic lipids, pg Protein, ,g FIG. 1. Chromatogram of proteins eluted from a 6-succinylmor- phine affinity column by NaCl. Brain membranes were solubilized and FIG. 2. Specific [3Hldiprenorphine binding to the specific isolated applied to a 2.5 x 20 cm column. Material was eluted with a linear gra- peak A protein/lipid mixture. Data are means of duplicate experi- dient of 250 ml of 50 mM Tris-HCl (pH 7.4) and 250 ml of 50 mM Trs5- ments carried out at three separate times. (A) The peak A protein (40 HCl, pH 7.4/1 M NaCl. Eight-milliliter fractions were collected. Pro- Mg) was mixed with various amounts of an acidic lipid fraction (CerSO4/ tein was measured according to the method of Lowry et al. (20). P3-Ins/PtdIlns/PtdSer, 2:1.5:1:1). (B) The acidic lipid fraction (27.5 Mg) Protein; ---, NaCl. was mixed with various amounts of the peak A protein. Downloaded by guest on September 25, 2021 5178 Biochemistq: Cho et aL Proc. Natl. Acad. Sci. USA 80 (1983)

100 protein/lipid mixture extensively, we substituted for this na- I.R_ (+)-Morphine tive lipid fraction a mixture of acidic lipids having a similar _ composition-i.e., CerSO4/P3-Ins/Ptdlns/PtdSer = 2:1.5: 1: 1. . 80 D [3H]Opiate binding to this lipid mixture with peak A protein Ga _ U 0 60 A

I00

i 40

- 0 80k . 20-_ c

0 -60 0 10-6 Drug, M 40 FIG. 3. Stereoselectivity of opiate binding to.the isolated protein/ lipid mixture. Binding experiments were carried out using [3H]di- prenorphine (final concentration, 2 nM), 27.5 pug of acidic lipid (CerSO4/ 20 P3-Ins/PtdIns/PtdSer, 2:1.5:1: 1), and 40 Ag ofthe isolatedpeak A pro- tein and various concentrations of (+)- or (-)-morphine. Specific bind- ing of controls, defined as that displaced by 1 11M unlabeled dipren- 10- l0-' 10-6 10-5 10-4 10-3 10-2 + are orphine, was 5,260 150 cpm. Nonspecific binding and blank 300 Drug M cpm ±+50 cpm and 250 cpm ± 40 cpm, respectively. Data are means of duplicate experiments carried out at three separate times. B 100 acidic lipids in the presence and absence of a constant amount oftthe isolated protein in peak A. Binding of the tritiated ligand increased in a lipid concentration-dependent manner and was at all lipid concentrations much higher in the presence of the _c protein than in its absence. Ligand binding to a constant amount .c -of lipid was also increased with increasing amounts of the peak c A protein (Fig. 2B). Thepeak B protein did not exhibit [3H]- -.a diprenorphine binding to the acidic lipid mixture (data not shown). c0 Since the amount of acidic lipid extracted from the column E was-insufficient to-study the binding properties of the peak A

0.25-

10-7 10-6 A B lo-,D o-5 10-4 10-3 Drug M

0.20_ C

0r

\ 10-8M KD 25X KD 34X 108M 1. Cyclorphan 2. GPA-2163 3. Levorphanol 0.15- 80k 4. GPA-2443 5. n-allylmetozocine

0) 6. GPA- 1657 60h 7. Cyclazocine 8. Pentazocine C0.10

40

0.05- 20k

10 10 10-6 10-5 10-4 10-3 5 10 5 10 Drug M Bound,nM Bound, nM FIG. 5. Displacement of [3H]diprenorphine binding to the peak A FIG. 4. Scatchard plots of opiate binding to the protein/lipid mix- protein/lipid mixture. Binding experiments were carried out using 2 ture. Forty micrograms of the peak A protein and 27.5 ;kg of the acidic nM (final concentration) [3H]diprenorphine, 40 ,g of the peak A pro- lipid mixture were used. Data are means of duplicate experiments car- tein, and 27.5 ,g ofthe acidic lipid mixture. Control binding was 5,260 ried out at three separate times. (A) [3H]Diprenorphine. (B) [3H]Etor- 150 cpm. Data are means of triplicate experiments carried out at a phine. single time. DADL, [D-Ala2-D-Leu5]enkephalin. Downloaded by guest on September 25, 2021 Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5179 Table 1. IC,50 values of opiate and opioid peptides for the P2 was found to be similar to that of the extracted lipids. Further fraction and the isolated lipid/protein mixture studies revealed the binding to be stereospecific, with the af- IC50, riM finity for (-)-morphine being two orders of magnitude greater than that for (+)-morphine (Fig. 3). The Kd for diprenorphine Lipid/protein was 25 x 10-9 (Fig. 4A) and that for etorphine was 30 x 10-9 Drug P2 fraction* mixturet (Fig. 4B). Etorphine 1.1 85 The peak A protein at lipid saturation bound about 60% more Diprenorphine 0.6 40 diprenorphine per mg of protein than the membrane-bound Buprenorphine 0.5 50 receptor. To compare further the opiate binding properties of Cyprenorphine 1.5 75 these lipid/protein-mixtures with those of the membrane-bound Oxymorphone 52 850 opiate receptor, the displacement of bind- Naloxone 23 1,200 [3H]diprenorphine Naltrexone 7 250. ing by a variety of opiates (Fig. 5) was studied. The concen- Nalmexone 100 1,500 trations (IC values) of the unlabeled drugs required to inhibit Nalbuphine 35 800 50% of the labeled drug binding are summarized in Table 1. Morphine 80 560 Linear regression analysis of the data showed that there was Dihydromorphone 42 1,300 excellent correlation (r = 0.99) between these IC50 values and Nalorphine 30 400 those for binding to intact membrane receptors, (Fig. 6), even Dihydrodihydroxycodeinone 1,700 35,000 though the affinities for the isolated material were one to two Codeine 40,000 380,000 orders of magnitude lower (Table 1). Interestingly, the 8 ag- Ketobemidone 270 7,000 onist ([D-Ala-D-Leu5]enkephalin), K agonists (ethylketocycla- N-Amylnorketobemidone 150 4,000 zocine and ketocyclazocine), and ou agonist (SKF-10047) were Anileridine 1,000 11,000 relatively weak in displacing [3H]diprenorphine binding to the Profadol 1,000 16,000 isolated material (Table 1 and Fig. 6). It should be pointed out a-Prodine 2,000 21,000 that, in this system, cyclorphan, cyclazocine, and GPA-2443 Levorphanol 11 800 show multiphasic concentration-inhibition curves (Fig. 6C), Levallorphan 5 600 suggesting the possibility that more than one class or site is in- Cyclorphan 2.3 300 volved in the binding of [3H]diprenorphine. SKF 10,047 25 5,000 Opiate binding to the lipid/protein mixture was highly sen- Pentazocine 200 25,000 sitive to trypsin and N-ethylmaleimide. Thus, incubation with GPA-2163 56 2,000 trypsin (1.0 ,g/ml) for 30 min in 50 mM Tris HCI (pH 7.4) de- GPA-2443 300 6,000 stroyed 90% of the binding and incubation with N-ethylmale- Bremazocine 230 3,000 imide ,B3Endorphin 200 2,000 diminished 80% of the binding activity (unpublished data). [D-Ala2-D-Leu5]Enkephalin 200 70,000 Competition of the Lipid-Protein Complex with Mem- Ethylketocyclazocine 35 10,000 brane-Bound Opiate Receptors. A particularly powerful test.of Ketocyclazocine 70 the authenticity of isolated receptors is their ability to compete 10,000 for ligand with membrane-bound opiate receptors (21). As shown * Unpublished data. in Fig. 7, [3H]diprenorphine binding to the membrane-bound t Taken from Fig. 5. opiate receptor (P2 fraction) was inhibited by the lipid-protein complex in a concentration-dependent manner. Using 80 ,g of lipid and 200 ug of peak A protein, which produces 50% in-

* Codeine

.O..._ 1Q-4 DADL 0 c Codeine Pentozocine 0 0 a-prodine 2 D Profodo Ketocyclozocine. 0 * Anileridine. _ Kelobemidone 0._CL SKF 10,074-o0 G6PA-2443 ~0 I 0- N-mnvlnorketobemidone Ethoketocychzoci o n - - e mazocine GPA- 2163 0 *- ,-endorphin Nalmexone Nolosuone *______* Dihydromorphone S 10-6 Levorphonol * * '-O Ox morphone 0 Levollorphan -M Morphine- Nalbuphe *- Nolorphine Cyclorphon-e c Naltrexone

Etorphne-0 Cyprenorphine 0 Buprenorpthine Diprenorphine

10-8 III 100 10-9 10C8 10-7 co-6 10-5 10-4 IC50 for P. fraction FIG. 6. Correlation between IC50 values of opiate binding to P2 fraction(s) and to the peak A protein/lipid mixture. Data are taken from Table 1 and were analyzed by linear regression. Correlation coefficient = 0.99; n = 31. Downloaded by guest on September 25, 2021 5180 Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 80 (1983) 12 ture can compete with membrane receptors for opiates (Fig. 7), the affinities of 8, K, and oa ligands for the reconstituted ma- A F B / I0 terials are much weaker than those of 1L ligands (Table 1 and '0 £~~~~~~~~~~~~l Fig. 6). This is consistent with the idea that these ligands bind to distinct receptors that can be separated independently. I? 6 However, our data do not rule out the possibility of a close as- sociation of the binding sites in a single complex, as recently A suggested (23). 80060 3 ~~~~2O~ ~ ~ ~ ~ The selectivity of our purification procedure is indicated by C~~~~ the fact that only 1-2% of the total protein in the solubilized material was eluted in peak A. Furthermore, based on the data in Fig. 4, which indicate about 100 pmol of opiate bound per mg of protein, we have achieved about 420-fold purification. Nevertheless, NaDodSO4 gel electrophoresis of this material Protein, jug P2 fraction, mg of wet brain revealed a very broad profile containing most of the peptide FIG. 7. Inhibition of [3H]diprenorphine binding to the P2 fractions bands present in the original P2 membranes. This is not sur- by the isolated peak A protein/lipid mixture. Binding experiments were prising in light of the nonspecific manner in which this fraction carriedoutby the filtrationmethiod as described in Fig. 5. Data are means was eluted. At least some specificity is suggested, however, by ofduplicate experiments carried out at two separate times. (A) Various the fact that protein material isolated from opiate-insensitive amounts ofthe peak protein and 80 /lgoflipidwere used. (B)A constant tissue-cerebellum and liver-has distinctly different prop- amount of the protein (200 yg)/lipid (80 ,ug) mixture was used. erties. In the presence of acidic lipid, it does bind opiate, but with an affinity that is about 1% of that of the whole brain peak hibition, we observed a shift in the binding curve to. the right A/lipid mixture. and by about 3-fold (Fig. 7B); this indicates that the isolated 1. Simon, E. J., Hiller, J. M. & Edelman, I. (1973) Proc. Nad Acad. reconstituted lipid-protein complex competes directly with the Sci. USA 70, 1947-1949. membrane-bound receptor for this ligand. The-binding of the 2. Pasternak, G. W. & Snyder, S. (1975) MoL PharmacoL 10, 183- 8 ligand [3H][D-Ala2-D-Leu5]enkephalinl to brain membrane, in 193. contrast, was not significantly inhibited (data not shown). Fi- 3. Law, P. Y., Fisher, G., Loh, H. H. & Herz, A. (1979) Biochem. nally, neither lipid nor protein alone was able to inhibit di- PharmacoL 28, 2257-2262. prenorphine binding to brain membrane (data not shown). 4. Loh, H. H., Cho, T. M., Wu, Y. C. & Way, E. L. (1974) Life Sci. 14, 2231-2245. 5. Cho, T. M., Cho, J. S. & Loh, H. H. (1979) MoL PharmacoL 16, DISCUSSION 393-405. 6. Abood, L. G. & Hoss, W. (1975) Eur. J. PharmacoL 32, 66-75. These findings represent the successfull isolation- and reconsti- 7. Hasegawa, J., Nagaoka, S., Niwa, M., Nozaki. M. & Fujimura, tution- of opiate binding material that can be related to phar- H. (1981) Advances in Endogenous and Exogenous Opioids, Pro- macologic activity and, in particu-lar, to the ,u recepftor. Our ability ceedings of the International Narc. Res. Conference, Kyoto, Ja- pan (Kodansha, Tokyo), pp. 83-85. to achieve this task was dependent on several factors. First, al- 8. Wu, Y. C., Cho, T. M., Loh, H. R. & Way, E. L. (1976) Biochem. though the detergent-solubilized material could not be eluted Pharmacol. 25, 1551-1553. from the resin with excess free ligand, as is customary, it was 9. Bidlack, J. M. & Abood, L. G. (1981) Life Sci. 27, 331-340. possible to use NaCl at high concentration to elute the protein. 10. Simonds, W. F., Koski, G., Streaty, R. A., Hjelmeland, L. M. & Although the procedure is nonspecific, 98% of the original pro- Klee, W. A. (1980) Proc. Natl Acad. Sci. USA 77, 4623-4627. tein had been eluted from the column prior to this step so the 11. Howells, R. D., Gioannini, T. L., Hiller, J. M. & Simon, E. J. (1982) J. Pharmacol Exp. Ther. 222, 629-634. amount of impurity possibly removed with receptor was con- 12. Bidlack, J. M., Abood, L. G., Osel-Gylmah, P. & Archer, S. (1981) siderably reduced. The second key step was the addition of lipid Proc. Natl. Acad. Sci. USA 78, 636-639. to the peak A mixture, resulting in binding properties similar 13. Simon, E. J., Dole, W. P. & Hiller, J. M. (1972) Proc. Natt Acad. to those of solubilized opiate receptors reported by others (10, Sci. USA 69, 1835-1837. 21). It may be relevant that the nicotine receptor exhibits a sim- 14. Cho, T. M., Yamato, C., Cho, J. S. & Loh, H. H. (1981) Life Sci. ilar loss in activation after purification (22). 28, 2651-2657. 15. Kean, E. L. (1968) J. Lipid Res. 9, 319-327. These results suggest that the removal during solubilization 16. Bartlett, G. R. (1959) J. BioL Chem. 234, 466-468. of some of the receptor or its needed membrane enevironment 17. Tovey, K. C., Oldham, K. G. & Whelan, J. A. (1974) Clin. Chim. may lead to a decrease of affinity. In this respect, the isolated Acta 56, 221-234. materials may be only a part of the receptor. However, other 18. Cuatrecasas, P. (1972) Proc. Natl Acad. Sci. USA 69, 318-322. possibilities, at the present time, cannot be ruled~out. Further 19. Hummel, J. P. & Dreyer, W. J. (1962) Biochim. Biophys. Acta 63, purification of the protein in peak A and of the acidic lipids and 530-532. 20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. eventual coupling of this isolated material in a subsequent bio- (1951)J. Biol Chem. 193, 265-271. chemical step with, for example, the adenylate cyclase system 21. Cuatrecasas, P. (1973) Biochemistry 12, 3547-3557. may be necessary. 22. Chang, H. W. & Bock, E. (1979) Biochemistry 18, 172-179. Interestingly, although the reconstituted lipid/protein mix- 23. Lee, N. M. & Smith, A. P. (1980) Life Sci. 26, 1459-1464. Downloaded by guest on September 25, 2021