Isolation of Adenylate Cyclase-Free, F-Adrenergic Receptor from Turkey Erythrocyte Membranes by Affinity Chromatography

Proc. Nati. Acad. Sci. USA Vol. 74, No. 9, pp. 3710-3714, September 1977 Biochemistry

Isolation of adenylate cyclase-free, f-adrenergic receptor from turkey erythrocyte membranes by affinity chromatography (catecholamines/digitonin/alprenolol-agarose) GEORGES VAUQUELIN*, PHILIPPE GEYNETt, JACQUES HANOUNEt, AND A. DONNY STROSBERG* * Biochemical Pathology, Free University Brussels, V.U.B. Paardenstraat, 65, B-1640 St-Genesius-Rode, Belgium; and t Unite de Recherches INSERM U-99, Hbpital Henri Mondor, F-94010 Creteil, France Communicated by J. Brachet, June 13,1977

ABSTRACT The adenylate cyclase [ATP pyrophosphate- chloride (Burroughs Wellcome); and hydroxybenzyl pindolol lyase (cyclizing) EC 4.6.1.11 and ,-adrenergic receptor of plasma tartrate (Sandoz). The (-)-isomers of isoproterenol bitartrate, membranes of turkey erythrocytes were solubilized in an active epinephrine bitartrate, and norepinephrine bitartrate were form by treatment with either NaF or guanylylimidodiphosphate and digitonin. The solubilized enzyme was no longer purchased from Sigma. Sepharose 4B and Sephadex G-50 were stimulated by catecholamines, NaF, or guanine nucleotides. The from Pharmacia. Digitonin and polyethylene glycol-6000 were digitonin extract was chromatographed on an alprenolol-agarose obtained from Merck. Bovine gamma globulin was from Miles derivative. While the bulk of protein and all the adenylate cy- Laboratory, Inc. (Great Britain). 5'-Guanylylimidodiphosphate clase activity passed unretarded through the column, the re- [Gpp(NH)p] and creatine phosphokinase were purchased from ceptor was retained. It eluted free of enzyme activity with an Boehringer. alprenolol solution containing 1 M NaCl; the yield was 25-30%. Cyclic AMP and phosphocreatine were from The protein content of the alprenolol eluates was too low to be Calbiochem. [a-32PJATP (6-29 Ci/mmol) and (R)-(-)-[3H]- estimated by the Lowry technique and was assessed by a more dihydroalprenolol hydrochloride (33 Ci/mmol) were obtained sensitive fluorometric method. Under these conditions, the ,B- from New England Nuclear Corp. Cyclic [8-3H]AMP (13 Ci/ adrenergic receptor was purified approximately 2000-fold in mmol) was from the Commissariat a l'Energie Atomique (Sa- a single step with retention of all its pharmacological properties. clay, France). All other reagents were of the highest grade These experiments establish that the f-adrenergic receptor and commercially available. the adenylate cyclase are independent entities which may be on a functional basis. Preparations. Synthesis of bromo-alprenolol. Alprenolol separated contains an olefin moiety which can readily react with N- The mechanism of adenylate cyclase [ATP pyrophosphate-lyase bromosuccinimide in water to yield the corresponding brom- (cyclizing); EC 4.6.1.1] activation by polypeptide hormones or ohydrin (5). N-Bromosuccinimide (178 mg) was dissolved in catecholamines is generally thought to require several steps: the 100 ml of 10 mM (+)-alprenolol in water. The mixture was specific binding of the hormone to a membrane receptor, the stirred for 2 days at 40 in the dark. Physicochemical data -transmission of a signal to the enzyme, and finally an increase (thin-layer chromatography, ultraviolet spectra, mass spec- in 3':5'-cyclic AMP production (1). The elucidation of the trometry, and nuclear magnetic resonance spectra, unpub- molecular basis of hormonal activation requires an analytical lished) indicated the quantitative transformation of alprenolol approach, involving the sequential solubilization of the various to its corresponding bromohydrin. components, i.e., receptor, transmitter, and catalytic unit, and Preparation ofaffinity adsorbent. The alprenolol-agarose the reconstitution of a complete and functional cyclase system derivative is depicted in Fig. 1. Sepharose-4B activated with in vitro. Indirect evidence that the receptor and the catalytic cyanogen bromide (6) and linked to a spacer arm (7), was unit might be separable was reported recently (2-4), but the reacted with N-acetylhomocysteine thiolactone and di- existence of the transmitter is still speculative and the molecular thiothreitol (8), to yield the corresponding sulfhydryl-spacer properties of the adenylate cyclase-coupled receptors remain arm-agarose derivative. Bromo-alprenolol was coupled at 40, ill defined. in the dark. The agarose derivative (10 g) was suspended in 40 In this paper we report the digitonin solubilization of both ml of a solution of 6 mM bromo-alprenolol in 0.5 M sodium the ,B-adrenergic receptor and the adenylate cyclase from carbonate, pH 8. The suspension was gently stirred under a turkey erythrocytes. The fl-adrenergic receptor has been pu- nitrogen atmosphere for 3-5 days. The gel was successively rified by affinity chromatography on an alprenolol-agarose washed on a sintered-glass funnel with 1 liter of distilled water, adsorbent. We demonstrate that the solubilized adenylate cy- and on a small column with 4 liters of 10 mM HCl and 2 liters clase and f-adrenergic receptor are indeed discrete and inde- of 145 mM NaCl. The gel was stored in 0.1 M ethylenedi- pendent entities separable on the basis of their functional amine-tetraacetic acid (EDTA) containing 0.5% (vol/vol) n- properties. butanol, pH 4.8. The amount of alprenolol covalently linked per g of gel was estimated to be 1 ,umol. For affinity chroma- MATERIALS AND METHODS tography, 2 g of gel were packed into a small column and washed continuously with 2 liters of 10 mM Tris-HCI (pH 7.4) Materials. The following were obtained as gifts: (±)-al- containing 90 mM NaCl (buffer A) prior to use. prenolol hydrochloride (Geigy Laboratories); (-) and (+)- Preparation of turkey erythrocyte membranes. Erythrocyte propranolol (ICI); (+)-epinephrine bitartrate (Sterling-Win- membranes were prepared according to 0ye and Sutherland throp); protokylol (Lakeside Laboratory); butoxamine hydro- (9). The purified membranes were suspended in buffer A The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Abbreviations: Gpp(NH)p, 5'-guanylylimidodiphosphate; EGTA, "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate ethylene glycol-bis(fl-aminoethyl ether)-N,N'-tetraacetic acid; EDTA, this fact. ethylenediaminetetraacetic acid. 3710 Downloaded by guest on October 1, 2021 Biochemistry: Vauquelin et al. Proc. Natl. Acad. Sci. USA 74 (1977) 3711

SPACER ARM ALPRENOLOL 30° in buffer A in a final volume of 400 ,ul. At the end of the I v incubation, the assay mixture was chilled on ice and 200 Al of cold 24% (wt/vol) polyethylene glycol-6000 was added. The tubes were mixed and placed in ice for 6 min. Triplicate 100-Iul aliquots were then diluted in 4 ml of buffer B containing 8% (wt/vol) polyethylene glycol and filtered. The gel filtration technique allowed more accurate determinations of specific binding at high ligand concentrations. The FIG. 1. Structure of the alprenolol-agarose adsorbent used in antagonist-receptor complex was separated from free tracer affinity chromatography. by filtration of a 100-,al sample on a Sephadex G-50 column (0.5 X 12 cm) eluted by gravity with buffer A containing 0.05% containing 2 mM MgCI2, 1 mM ethylene glycol bis(Q-ami- (wt/vol) bovine gamma globulin. Fractions of 0.2 ml were noethyl ether)NN'-tetraacetic acid (EGTA), and 10% (vol/vol) collected. Fractions 9-12 contained the void volume of the glycerol, at a protein concentration of 10-14 mg/ml. Mem- column and the bound (R)-(-)-[3H]dihydr~Iprenolol. Free branes were stored under liquid nitrogen until use. ligand started with fraction 14. Specific binding was deter- Solubilization of (3-adrenergic receptor and adenylate cy- mined from the difference between total binding and binding clase. Ghosts were suspended in 5-7.5 volumes of buffer A in the presence of a 200-fold excess of (b)-alprenolol. containing 2 mM MgCl2, 1 mM EGTA, and 10 mM NaF, or 50 tiM Gpp(NH)p, or 50 ,uM isoproterenol, or isoproterenol plus RESULTS Gpp(NH)p, or no effector, and preincubated for 30 min at 300. Solubilization of 8-adrenergic receptor and adenylate Membranes were washed by centrifugation at 30,000 X g for 15 min and resuspended in buffer A containing 0.5% (wt/vol) cyclase suspended digitonin (3-4 mg of membrane protein per ml). The Basal adenylate cyclase activity from plasma membranes of mixture was allowed to stand in ice for 25-30 min with occa- turkey erythrocytes was increased from 0-30 to 200, 900, 1000, sional stirring and was centrifuged at 226,000 X g for 1 hr. The and 1700 pmol of cyclic AMP per mg per 30 min by clear supernatant constituted the solubilized preparation of Gpp(NH)p, NaF, isoproterenol, and isoproterenol plus fl-adrenergic binding sites and adenylate cyclase. Protein was Gpp(NH]p, respectively. Gpp(NH)p brought about a 10-fold determined by the method of Lowry et al. (10) and by a more increase in the affinity of the adenylate cyclase system for the sensitive method using fluorescamine (11) after affinity chro- catecholamines, whereas it did not modify the affinity of the matography. Crystalline bovine serum albumin was used as the binding sites. The order of potency of various agonists was standard; blanks containing buffer and digitonin were in- typical of a fll-adrenergic system: isoproterenol > protokylol cluded. > norepinephrine > epinephrine. Treatment with digitonin Methods. Adenylate cyclase assay. Adenylate cyclase ac- (15) solubilized 20-30% of the adenylate cyclase activity pro- tivity was measured as described (12, 13). Unless otherwise vided that the membranes were preincubated with Gpp(NH)p, stated, the assay medium contained 2 mM [a-32P]ATP (106 or isoproterenol plus Gpp(NH)p, or, preferably, NaF prior to cpm), 7 mM MgCI2, 0.5 mM EGTA, 1 mM cyclic AMP, 50 mM solubilization (Table 1). Under these conditions, the solubilized Tris-HCI (pH 7.6), an ATP-regenerating system (consisting of adenylate cyclase had a specific activity comparable to that of 25 mM phosphocreatine and creatine phosphokinase, 1 mg/ml), the membrane fraction, but no longer responded to catechol- and 10-100 ,ug of protein in a final volume of 60,ul. Incubation amines, NaF, or guanine nucleotides. was initiated by the addition of enzyme and performed for 30 The same treatment also solubilized 30% of the membrane- min at 30°. One enzyme unit is defined as the amount of en- bound (R-(-)[3H]dihydalprenolol binding sites. Despite the zyme that generates 1 pmol of cyclic AMP per 30 min at relatively low yield of sbiilization, those sites retained all the 300. essential characteristics of the membrane-bound receptors, i.e., Binding assays. Binding of (R)-(-)-[3H]dihydroalprenolol a dissociation constant of 3.5 nM, a binding capacity of 0.30- to membrane fractions was assayed by filtration on glass fiber 0.45 pmol per mg of protein, and a Hill coefficient of 0.91, in filters (Whatman GF/F; 25 mm in diameter). Unless otherwise contrast to 6 nM, 0.25 pmol/mg, and 0.92, respectively, in the stated, 0.4 mg of membrane protein was incubated with 10 nM original membrane fraction. In addition, solubilized receptors (R)-(-)-[3H1dihydroalprenolol for 10 min at 300 in 75 mM conserved similar affinities for the ,-blockers but displayed an Tris-HCI (pH 7.4)/25 mM MgCl2 (buffer B) in a final volume apparent 20- to 50-fold increase in the affinity for the agonists. of 200 Ml. At the end of the incubation, triplicate 50-,ul aliquots The order of potency remained typical of a fl1-adrenergic were diluted at 00 in 4 ml of buffer B, and filtered under re- system; the marked stereospecificity for the (-)-isomers was duced pressure through the filter discs. Filters were washed with not altered (Table 2). The observed shift in affinity might be 6 ml of ice-cold buffer B and placed in scintillation vials with due to a different conformational state of the receptor after 1 ml of 1 M HCI and 10 ml of Triton/toluene-base scintillation solubilization. fluid; radioactivity was determined. Under these conditions, Affinity chromatography of adrenergic receptor 90-95% of the membrane-bound tracer was displaced by 25,uM (+-alprenolol. The remaining counts were subtracted from the Specificity of Adsorbent. Batch experiments were first total bound radioactivity to determine the extent of specific performed to assess the affinity and the specificity of our af- binding to the fl-adrenergic receptors. finity gel. Fig. 2 shows that adsorption of solubilized receptor Binding of (R)-(-)-[3Hjdihydroalprenolol to solubilized re- increased with the amount of gel, while very little protein was ceptors was assayed by either one of the two following tech- bound. Addition of 10 nM tracer to the incubation mixture led niques. The polyethylene glycol precipitation technique (14) to a rightward shift of the adsorption curve (Fig. 2), thus indi- was most suitable for assaying a large number of samples with cating that the free ligand and the alprenolol-agarose were tracer concentrations below 20 nM. Soluble extracts (120-200 competing for the same sites. Moreover unsubstituted spacer gg of protein) were incubated with 10 nM tracer for 10 min at arm-agarose did not adsorb the receptor. Downloaded by guest on October 1, 2021 3712 Biochemistry: Vauquelin et al. Proc. Natl. Acad. Sci. USA 74 (1977)

Table 1. Solubilization of fl-adrenergic binding sites and Table 2. Apparent dissociation constants (Kapp) of adrenergic adenylate cyclase from turkey erythrocyte membranes agents for fl-adrenergic receptors Preincubation Yield, (R)-(-)-[3H]Dihydroalprenolol binding in conditions Membrane*t Solubilized*t % (R)-(-)-[3H]Dihydroalprenolol binding Adenylate Mem- Digitonin No effector 0.22 0.37 25 cyclasein brane, extract, Purified Gpp(NH)p 0.25 0.27 20.3 membrane, Kapp Kapp receptor,* NaF 0.20 0.40 29.2 Compound Kapp (MM) (MM) (MM) IC50 (MM) (-)-Isoproterenol ND ND ND Agonists (-)-Isoproterenol + (+)-Epinephrine 20 21 0.88 630 Gpp(NH)p ND ND ND (-)-Epinephrine 1.6 1.2 0.061 100 (-)-Norepineph- Adenylate cyclase rine 0.50 0.78 0.017 74 No effector 12 15 30 (±)-Protokylol 0.18 0.17 0.0095 6.3 Gpp(NH)p 131 180 28.2 (-)-Isoproterenol 0.10 0.12 0.0033 7.9 NaF 1310 1483 21.5 Antagonists (-)-Isoproterenol 1553 292 3.4 (±)-Butoxamine 3.2 9.3 13 ND (-)-Isoproterenol + (+)-Propranolol 0.24 0.60 0.82 37 Gpp(NH)p 1611 2234 34 (-)-Propranolol 0.0045 0.0050 0.0037 0.20 (±)-Alprenolol 0.015 0.015 0.011 0.17 Turkey erythrocyte membranes were preincubated with the indicated effector and solubilized by digitonin. (R)-(-)-[3HjDihydroal- (±)-Hydroxy- prenolol binding to solubilized preparations was assessed by the benzylpindolol 0.00020 0.00067 0.00087 ND polyethylene glycol precipitation technique. Concentrations of The affinity of agonists for the adenylate cyclase was determined Gpp(NH)p, NaF, and (-)-isoproterenol for the preincubation and from dose-response curves for stimulation of enzyme activity. The the adenylate cyclase assay were 50 ,M, 10 mM, and 50 gM, respec- affinity of antagonists was deduced from the IC50 obtained from the tively. At least three identical experiments were performed for each dose-response curves for inhibition of 0.5 MM (-)-isoproterenol- preincubation condition with different membrane preparations and stimulated adenylate cyclase activity. The affinities of agonists for reproducible results. Yield is expressed as percentage of activity the receptor sites were deduced from the IC50 obtained from the solubilized from the membranes. ND, not determined. (R)-(-)-[3HJdihydroalprenolol binding displacement curves. Kapp * Values are pmol/mg. values were calculated from the IC50 by the method ofLevitzki et al. t Values are specific activity (enzyme units per mg). (16). (R)-(-)-[3HJDihydroalprenolol binding in membrane and solubilized fractions was assayed by the precipitation technique. Binding to purified material was assayed by the gel filtration technique. As Purification of P-Adrenergic Receptor. The receptor was a control, the (R)-(-)-_3HJdihydroalprenolol binding displacement purified by column chromatography (Fig. 3). Ten milliliters curve by (-)-epinephrine was performed in the adenylate cyclase of digitonin extract containing 4-5 mg of protein were loaded assay conditions, i.e., in the presence of 2 mM ATP,1 mM cyclic AMP, on top of the affinity column (0.75 ml of packed gel). Most of 0.5 mM EDTA, and the ATP-regenerating system, with no significant the applied protein and 80-90% of the adenylate cyclase ac- change in the Kapp value of (-)-epinephrine. The values shown in this table represent the mean value of at least two separate experiments. tivity were found in the eluate that did not contain receptor ND, not determined. activity. The column was extensively washed and then eluted * The concentration of (R)-(-)-[3HJdihydroalprenolol to be displaced with 10 mM Tris-HCl (pH 7.4), containing 1 M NaCl, 40 nM in this experiment was 0.2 ,M. IC50 is the concentration of com- (R)-(-)-[3H]dihydroalprenolol, 720 nM (+)-alprenolol, and pound that causes 50% displacement. 0.1% bovine gamma globulin. Release of 25-30% of the adsorbed receptor was achieved. This binding activity was entirely displaceable by 8 ,uM (+)-alprenolol. No adenylate cyclase was synthesized by coupling the potent adrenergic antagonist activity coeluted with the receptor. Boiling of the membranes alprenolol to a spacer arm on agarose via a thio-ether bond (Fig. prior to solubilization and affinity chromatography yielded no 1). This gel exhibits major advantages over compounds de- alprenolol binding activity in the column eluate. Addition of scribed previously (17, 18). In contrast with norepinephrine 8 ,uM (+)-alprenolol to the solubilized receptors prevented the linked to agarose through an amido bond, no catechol group - specific retention of the receptor by the column. When the projects from the gel, thus avoiding retention of catechol ac- gamma globulin was omitted, the protein content of the al- ceptors; the amino group of the ethanolamine side chain, which prenolol eluate was too low to be detected by Lowry's tech- is important for receptor recognition, remains free. Unlike nique; it was estimated on pooled, concentrated elution samples catecholamines linked to agarose through diazotized hydro- obtained from three identical experiments by a method using phobic spacer arms, a hydrophilic arm was used in the present fluorescamine (11). The alprenolol eluate contained 0.013% of gel, thus minimizing nonspecific adsorption of solubilized the total applied protein; therefore the f3-adrenergic receptor membrane proteins. was purified by about 2000-fold in a single step, considering Batch and column experiments confirmed the ability of the the above-mentioned 30% yield. Its pharmacological properties alprenolol-agarose gel to adsorb the solubilized f,-adrenergic with respect to various agonists and antagonists were studied receptor from turkey erythrocytes in a biospecific manner. by displacement of bound tritiated alprenolol. As indicated in Adsorption of the receptor was prevented by addition of free Table 2, the purified binding sites retained all the essential alprenolol, indicating that the immobilized ligand binds to the characteristics of a ,B1-adrenergic receptor. receptor site. Buffers used to wash the affinity adsorbent, either in batch or in column, did not inhibit the binding of alprenolol DISCUSSION to fresh receptor, thus indicating that no ligand leached from A novel approach to the affinity chromatography of the cate- the gel. Binding activity, eluted from the column, was actually cholamine receptor is presented in this work. A new adsorbent due to the receptor itself as binding of tracer to the eluted Downloaded by guest on October 1, 2021 Biochemistry: Vauquelin et al. Proc. Natil. Acad. Sci. USA 74 (1977) 3713

oz z I- ~~~~~~~~~~~~~~~75- CL~~~~~~~~~~~~~~~~~~~~~~~~~~C 0 1 2 0 N.W50 50 S o z C-I-- U

0 1*2 LogE GEL J (mg/ml) FIG. 2. Adsorption of f-adrenergic binding sites and protein on the alprenolol-agarose derivative. Digitonin-solubilized turkey erythrocyte membranes (0.9 ml) were incubated for 10 min at 300 with the alprenolol-agarose derivative in a final volume of 1 ml, either in the absence (e) or in the presence of 10 nM free (R)-(-)-[3Hjdihydroalprenolol (0). After centrifugation (1000 X g, 4 min), residual specific binding of (R)- (-)-P3H]dihydroalprenolol was determined in the supernatant by the polyethylene glycol precipitation technique. Results are expressed in percent of receptor sites adsorbed by the gel. Protein content adsorbed by the gel (- --- *) was estimated by Lowry's technique and expressed in percent of total protein. fractions was completely inhibited by addition of 8 ,uM unla- raphy of this extract, the eluates could not bind the ligand. beled alprenolol; gamma globulin, used in the column proce- Therefore, in the normal procedure, the eluted binding activity dure as carrier protein to avoid nonspecific losses of the receptor was not due to digitonin micelles. (19), did not bind alprenolol. The digitonin extract of boiled The amount of receptor eluted from the column was in- membranes was devoid of binding activity; after chromatog- creased by elevating both the alprenolol concentration and the zO -Jo _coaD0 > 400. 0 5-

uJ I. I

-i u " 200. (-)'5C uJ

z U r" 0 0 (a

ELUATE (m ) FIG. 3. Affinity chromatography of detergent-solubilized ,l-adrenergic receptor from turkey erythrocyte membranes. Membranes were preincubated with 10 mM NaF and solubilized. Ten milliliters of original digitonin extract (i.e., approximately 5 mg of protein, corresponding to 1.4-1.5 pmol ofreceptor sites and 6500 enzyme units) were chromatographed at a flow rate of 10 ml/hr, at 300 over a column 0.5 X 2 cm, containing 0.75 ml of packed gel that had been equilibrated in 10mM Tris-HCl (pH 7.4), 90 mM in NaCl (buffer A). The column was then rapidly washed with 4 ml of buffer A, containing 0.1% (wt/vol) bovine gamma globulin (arrow 1), and 2 ml of 10 mM Tris-HCl (pH 7.4), containing 1 M NaCl and 0.1% (wt/vol) bovine gamma globulin (arrow 2). The receptor was eluted with 6 ml of the preceding buffer containing 40 nM (R)-(-)- [3HJdihydroalprenolol and 720 nM (+)-alprenolol (arrow 3). Fractions of 1 ml were collected at room temperature and transferred in an ice-water bath. Adenylate cyclase activities (X --- X) were determined in the presence of 10 mM NaF. Results are expressed in pmol of cyclic AMP formed per 20 min/ml. Specific (R)-(-)-[3Hldihydroalprenolol binding (0) was assayed by the gel filtration technique. Specific binding is expressed in fmol/ml. Bovine gamma globulin was omitted in the buffers for protein determinations by Lowry's technique (0); protein concentrations are expressed in mg/ml. When membranes were preincubated with 50 1tM Gpp(NH)p instead of NaF, no qualitative change was observed in the elution profile of the enzymatic activities and no qualitative or quantitative change was observed in the adsorption and elution of the f3- adrenergic binding sites. Downloaded by guest on October 1, 2021 3714 Biochemistry: Vauquelin et al. Proc. Natt. Acad. Sci. USA 74 (1977)

ionic strength of the medium. The need for a high ionic strength This work was supported by grants from the Belgian Nationaal in addition to an elevated alprenolol concentration to elute the Fonds voor Wetenschappelijk Onderzoek (NFWO), Fonds voor receptor from the column suggests that electrostatic interactions Geneeskundig Wetenschappelijk Onderzoek, and Algemene Spaar-en also occur between the receptor and the spacer arm (20). Al- Lijfrentekas-Kankerfonds, and the French Institut National de la Sante though these interactions are not potent enough to cause ad- et de la Recherche Medicale, Delegation G6nerale a la Recherche sorption of the receptor to the unsubstituted spacer arm-agarose Scientifique et Technique, and Fondation pour la Recherche Medicale. gel, they may increase the overall affinity of the gel for the G.V. is a recipient of the NFWO of Belgium. receptor. This type of synergistic action between spacer arm and ligand has been defined as "compound affinity" (7). Under the conditions described in this paper, the fl-adrenergic receptor could be eluted from the affinity column with 1. Helmreich, E. J. M., Zenner, H. P., Pfeuffer, T. & Cori, C. F. a 25-30% yield; the purification was estimated to be approxi- (1976) in Current Topics in Cellular Regulation, eds. Horecker, mately 200Q-fold. The release of only 30% of the binding ma- B. L. & Stadman, E. R. (Academic Press, New York), Vol. 10, pp. terial from the affinity column in the presence of alprenolol was 41-87. probably due to degradation of the receptor after solubilization. 2. Orly, J. & Schramm, M. (1976) Proc. Nati. Acad. Sci. USA 73, That the binding characteristics of the purified receptor eluted 4410-4414. 3. Insel, P. A., Maguire, M. E., Gilman, A. G., Bourne, H. R., Cof- from the column are identical to those of the membrane-bound fino, P. & Melmon, K. L. (1976) Mol. Pharmacol. 12, 1062- sites (Table 2) suggests that the purified receptor is fully rep- 1069. resentative of the original material. 4. Limbird, L. E. & Lefkowitz, R. J. (1977) J. Biol. Chem. 252, The direct demonstration that the f3-adrenergic receptor and 799-802. the adenylate cyclase are two distinct molecular entities con- 5. Guss, C. 0. & Rosenthal, R. (1955) J. Am. Chem. Soc. 77, stitutes a salient feature of the present study. Individualization 2549. of catalytic and hormone- or guanine nucleotide-binding ac- 6. Vauquelin, G., Lacombe, M. L., Hanoune, J. & Strosberg, A. D. tivities was an early, but incomplete, indication that each (1975) Biochem. Blophys. Res. Commun. 64,1076-1082. function was supported by different components, since each 7. O'Carra, P., Barry, S. & Griffin, T. (1974) in Methods in Enzy- of these activities could still be contaminated the others in mology, eds. Jacoby, W. B. & Wilchek, M. (Academic Press, New by York), Vol. 34, Part B, pp. 108-126. an active or inactive form. Further progress in that direction 8. Cuatrecasas, P. (1970) J. Biol. Chem. 245,3059-3065. was achieved when the two activities could be separated (2-4). 9. 0ye, I. & Sutherland, E. W. (1966) Blochim. Blophys. Acta 127, The elegant cell fusion experiments of Orly and Schramm (2) 347-354. have shown that the catecholamine receptor of turkey eryth- 10. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. rocyte ghosts could be coupled to the catalytic unit of Friend (1951) J. Blol. Chem. 193,265-275. erythroleukemia cells. This indicated that the receptor and the 11. B6hlen, P., Stein, S., Dairman, W. & Udenfriend, S. (1973) Arch. enzyme might be distinct molecules capable of independent Biochem. Biophys. 155,213-220. migration in the fluid bilayer of the biological membrane. On 12. Hanoune, J., Lacombe, M. L. & Pecker, F. (1975) J. Biol. Chem. the other hand, the existence of receptor activity 250,4569-4574. f3-adrenergic 13. Hanoune, J., Stengel, D., Lacombe, M. L., Coudrier, E. & Feld- in mouse lymphoma cells that appear to lack adenylate cyclase mann, G. (1977) J. Biol. Chem. 252,2039-2045. (3) suggested that the receptor is a product of a gene different 14. Cuatrecasas, P. (1972) Proc. Natl. Acad. Sci. USA 69, 318- from that coding for the enzyme. A partial separation of the 322. receptor and the enzyme from frog erythrocytes, based on their 15. Caron, M. G. & Lefkowitz, R. J. (1976) J. Biol. Chem. 251, apparent molecular weights, was reported by Limbird and 2374-2384. Lefkowitz (4). Our results demonstrate that the catalytic 16. Levitzki, A., Sevilla, N., Atlas, D. & Steer, M. L. (1975) J. Mol. component and the fl-adrenergic receptor from turkey eryth- Biol. 97, 35-53. rocytes are completely separable on the basis of their functional 17. Haber, E. & Wrenn, S. (1976) Physiol. Rev. 56,317-38. properties. In the present affinity chromatographic procedure, 18. Lefkowitz, R. J., Limbird, L. E., Mukherjee, C. & Caron, M. G. was without (1976) Biochim. Biophys. Acta 457,1-39. the adenylate cyclase eluted receptor activity and 19. Cuatrecasas, P. & Parikh, I. (1974) in Methods in Enzymology, the latter was purified free of enzyme activity. Consequently, eds. Jacoby, W. B. & Wilchek, M. (Academic Press, New York), the existence of a A-receptor macromolecule that is distinct and Vol. 34, Part B, pp. 653-670. separable from the adenylate cyclase enzyme has now been 20. Wilchek, M. & Miron, T. (1976) Biochem. Blophys. Res. Com- established. mun. 72, 108-113. Downloaded by guest on October 1, 2021