gtp-binding proteins in membranes and the control of adenylate

THE JOURNAL OF Brouxrc~~ CHEMISTRY Vol. 252, No. 20, Issue of October 25, pp. 1224-1234, 1971

Prrnted ,n lJ S.A.

GTP-binding Proteins in Membranes and the Control of Adenylate Cyclase Activity*

(Received for publication, June 23, 1976, and in revised form, April 15, 1977)

THOMAS PFEUFFER

From the Department of Physiological Chemistry, University of Wiirzburg, School of Medicine, 8700 Wiirzburg, Federal Republic of Germany

To identify the GTP-binding site of catecholamine-stimulated adenylate cyclase (EC 4.6.1.1) from pigeon erythrocyte membranes photoreactive GTP derivatives have been synthesized. One of them, P”-(4-azidoanilido)-PI-5’-guanosine triphosphate, proved to be a potent activator of particulate and soluble adenylate cyclase which binds with high affinity (K,,ss 3.3 x lo-’ M) and competes effectively with guanyl-5’- yl imidodiphosphate (Gpp(NH)p) for the same binding site. Photoactivation of [“‘PIPJ-(4-azidoanilidoj-PI-5’-GTP-labeled membranes resulted in covalent incorporation of label into four major proteins with M, = 86,000, 52,000, 42,000, and 23,000, whereas in Lubrol PX-solubilized membranes only the GTP-binding proteins with M, = 42,000 and 23,000 were covalently labeled, although soluble adenylate cyclase preparations were stimulated by guanylnucleotides to about the same extent as membranous preparations. The bulk of the nucleotide binding sites, >95%, could be separated from adenylate cyclase without loss of activity by centrifugation of solubilized membranes through a sucrose density gradient, whereas most of the M, = 42,000 GTP-binding protein remained associated with adenylate cyclase activity.

Detergent-solubilized adenylate cyclase preparations were inactivated on contact with a GTP-Sepharose affinity matrix. Inactivation was due to dissociation of the adenylate cyclase complex into two protein fractions, one of which contained the guanylnucleotide-binding sites. Reacti- vation occurred on recombination of the fraction released from GTP-Sepharose with Gpp(NH)p or GTP with the fraction not adsorbed to GTP-Sepharose. Furthermore, guanylnucleotide-binding proteins from pigeon erythrocyte membranes reactivated rabbit myocardial adenylate cyclase preparations depleted of binding proteins. Reconstitution experiments with GTP-binding fractions obtained from isoproterenol-treated membranes suggested that the characteristic synergistic amplification of hormone action by guanylnucleotides is mediated via the guanyl nucleotide binding protein. Guanylnucleotide-binding proteins could also be

* This work was supported by Deutsche Forschtingsgemeinschaft Grant Pf 80/4 and by the Fonda der Chemischen Industrie. A report was given at the Titisee Conference on “Cyclic Nucleotides as Regu- lators of Proliferation and Differentiated Cell Function.” March 25 to 27, 1976, and at the 15th Symposium on Regulation of Enzyme Activity and Synthesis in Normal and Neoplastic Tissues. Indianap- olis, September 27 to 28, 1976. The costs of-publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

released from erythrocyte membranes by low ionic strength- EDTA solutions, which are known to solubilize peripheral contractile membrane proteins.

Purine nucleotides amplify the response of adenylate cyclase to hormones (cf. Ref. 1). Analogs of GTP, Gpp(CH,)p,’ Gpp(NH)p (2-6), and GTPr-S (6) are more effective than GTP. Because of their high affinity, guanylnucleotide analogs can be used to titrate binding sites in intact membranes or in detergent-solubilized membrane preparations (6, 7). Pigeon erythrocyte membranes, which we have mainly used, bind about 120 pmol of [3HJGpp(CH,)p/mg of protein (6). However, only 1 to 2 pmol of IgHlepinephrine and of the /3-adrenergic inhibitor [3H]propranolol are bound per mg of protein to turkey erythrocyte membranes (8). Subsequently the same amount was also found bound to pigeon erythrocyte membranes.* Thus, assuming hypothetically a 1:l relationship of hormone receptor to adenylate cyclase sites the GTP-binding sites would be in 50- to loo-fold excess. The possibility had to be considered therefore, that regulatory sites are in large excess over catalytic sites as seems to be the case with the regulatory Ca2+ binding protein of brain which stimulates adenylate cyclase and cyclic nucleotide phosphodiesterase (10). This assumption was in agreement with previous data which indicated that >90% of [W]Gpp(NH)p bound to a soluble preparation from pigeon erytbrocyte membranes could be separated from adenylate cyclase without significant loss of activity (6). Moreover, on the basis of these results we con- cluded at first that the guanylnucleotide binding protein is inhibitory, and that activation of adenylate cyclase results from its detachment by a mechanism analogous to that for the activation of CAMP-dependent protein kinase (6, 11, 12). An alternative possibility, namely that there exist several GTP- binding proteins in avian erythrocyte membranes, of which only one is involved in adenylate cyclase activation, was however not excluded.

It is the aim of this study to distinguish among these possibilities. Use was made of photoreactive GTP azides. Photolysis

’ The abbreviations used are: Gpp(CH,)p, guanyl-5’-yl-P,y-meth- ylene diphosphonate; App(NH)p and Gpp(NH)p, adenyl-5’-yl and guanyl-5’-yl imidcdiphosphate; GTPy-S, guanosine-5’-0-(3-thiotri- phosphate); GTP azidoanilide, P3-(I-azidoanilidoj-PI-guanosine triphosphate.

2 Brown et al. (9) found recently with turkey erythrocyte membranes and [‘2”Iliodohydroxybenzylpindolol even less binding sites, e.g. 0.2 to 0.3 pmol/mg of membrane protein.

7224

Regulation of Adenylate Cyclase 7225

of aliphatic and aromatic azides generates nitrenes which are

attacked by nucleophiles and are inserted into -C-H

bonds. Arylazides are more suitable, because they are less susceptible to rearrangements than alkyl- or acylazides (13). Moreover, aromatic azides absorb light at wavelengths >350 nm; hence, photolysis is not destructive to aromatic amino acid side chains. We have therefore synthesized photoreactive radioactive GTP arylazides with high affinity to membrane proteins. These compounds are potent activators of adenylate cyclase. With these radioactive GTP derivatives several binding proteins in intact and solubilized pigeon erythrocyte membranes were covalently labeled and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The experience gained with photoreactive GTP derivatives was applied to the synthesis of a novel GTP-Sepharose derivative. With this Sepharose derivative a protein fraction could be removed from detergent-solubilized adenylate cyclase preparations resulting in the loss of guanylnucleotide activation. In contrast to ear- lier experiments (6) with a GTPy-S-Sepharose derivative F- activation was likewise lost on treating adenylate cyclase with the new affinity matrix. Moreover, with the GTP-Sepharose derivative described in this paper the proteins adsorbed to the matrix could be removed with GTP or Gpp(NH)p. This was not possible with the previously described GTPy-S-Sepharose (6). I&addition of these proteins to the depleted adenylate cyclase preparations restored nucleotide and fluoride activation. Both separated components of the adenylate cyclase system were shown to be proteins sensitive to N-ethylmaleimide.

MATERIALS AND METHODS

Materials

Guanosine, 5’-GMP, GTP, Gpp(NH)p, ATP, imidodiphosphate, creatine phosphate, creatine kinase, alkaline phosphatase, snake venom (Crotalus) phosphodiesterase (EC 3.1.4.1), cytochromec, chy- motrypsinogen A, ovalhumin, bovine serum albumin, trypsin, and soybean trypsin inhibitor were obtained from Boehringer Mann- heim, and neutral A1203, N-ethyl-N’-(3-dimethylaminopropyl)- carbodiimide.HCl, triethanolamine. trihutylamine, 4-aminohenzoic acid, 4-aminoacetanilide, phenylenediamine, polyethyleneimine cellulose F thin layer plates were purchased from Merck, Darmstadt. DEAE-cellulose (DE52) and GF/C class fiber discs were from What- man, and Dowex 50-X8-H+ and pol&hylene glycol (M, = 6,000) from Serva, Heidelberg. Lubrol PX was a generous gift from ICI, Frank- furt, Germany. All other chemicals were of thehighest purity com- mercially available. Double-distilled water was used throughout.

[~Y-~*PIATP (0.5 to 2 Ci/mmol), inorganic [32Plphosphate (1550 to 4400 Cilmmol), and [VHlGpp(NH)p (2 to 8 Cilmmol) were purchased from The Radiochemical Centre, Amersham, England. The purity of the radioactive nucleotides was checked by thin layer chromatography on polvethyleneimine F thin layer plates using 0.5 to 1 M trikthylammonium bicarbonate buffer, pH 7.5, as developing solvent. Scintillator fluids were from Packard Instrument, Inc.

Methods

Preparations- Erythrocytes were collected from the blood of pi- geons obtained from a local source. Erythrocyte membranes were prepared according to 0ve and Sutherland (14) with the minor modifications described in Ref. 15. Membranes were suspended in Buffer A, which contains 20 mosm phosphate. 0.15 rnM NaCl, 1 rnM EDTA. 1 rnM dithioervthritol. O.Ol~mM~nhenvlmethvlsulfonvl fluo-

1 *

ride, and 3 mM MgCl,, pH 7.4. Adenylate cyclase acticity was solubilized with 20 mM Lubrol PX as described in Ref. 6. Soluble adenvlate cyclase from cardiac muscle of New Zealand rabbits was prepared according to Levey et al. (16).

Adenylate cyclase activity in particulate and soluble preparations was measured in a final volume of 200 pl ofN-morpholino-3-propane- sulfonic acid buffer, pH 7.4, described previously (6). [3’PlcAMP

formed from 1a”‘PlATP (10 to 15 Ci/moll was determined according to Ramachandran (17). The “‘P-nucleotide formed was routinely identified as 95 to 98% cyclic AMP by thin layer chromatography 0; by conversion to 5’-AMP after incubation with bovine heart phosphodiesterase.

Binding of lYHIGpp(NH)p or ly-3’PlP”-(4-azidoanilido)-P’-guanosine triphosphate to particulate or solubilized membrane preparations was determined as described (6). Protein concentrations were measured by the procedure of Lowry et al. (18) with bovine serum albumin as standard.

Analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed according to Neville and Glossmann (19). For greater amounts of protein (up to 1 mg/gel), larger gel tubes (1.2 x 10 cm) were used; 7.5 mA were applied per gel and protein bands were visualized with 0.25% Coomassie blue G-250 in 50% methanol containing 10% acetic acid.

The gels were destained electrophoretically. Radioactively labeled proteins were localized in gels frozen in dry ice. The frozen gels were sliced to l-mm discs with an automatic gel slicer (The Mickle Labo- ratory Engineering Co., Gomshall, England). The gel slices were not dissolved and were counted directly in Bray’s solution (20). Molecu- lar weights were estimated by comparing R,,, values with those of proteins of known molecular weights, i.e. cytochrome c, chymotryp- sinogen A, ovalbumin, and bovine serum albumin. R,,, values were plotted against log M, and the molecular weights were read from the graph.

Sucrose Gradient SeDaration - For density aradient centrifueation a linear sucrose gradient (5 to 30%) in 10 rnk Tris/HCl, 1 mM EDTA, 1 rnM MgCl,, 50 FM GMP buffer, pH 7.4, was employed. This buffer mixture is referred to as Buffer B.

Synthetic Procedures

Preparation of N2-(4-Azidobenzoyl)Gpp(NH)p- 4-Azidobenzoic acid was synthesized from 4-aminobenzoic acid according to Gold (21); m.p. 158” (literature, 160”). 4-Azidobenzoic acid was converted to 4-azidobenzoylchloride by reaction under reflux with a 1.5-fold molar excess of thionvlchloride. Unreacted thionvlchloride was removed by distillation in uacuo. 4-Azidobenzoylchloride was used without further purification. N-2’, 3’-Tris (I-azidobenzoylfGMP was synthesized in analogy with the synthesis of N2-2’, 3’-tris(benzoyl)- GMP from 0.5 mmol of 5’-GMP, 1 mmol of I’-morpholino-N,N’- dicvclohexvlcarhoxamidine. and 10 mmol of 4-azidobenzovlchloride in “dry pyiidine. N2-2’) 3’:Tris(4-azidobenzoyl)GMP was” partially saponified to N*-(I-azidobenzoyl)GMP bv the procedure of Postemak for the synthesis of NZ-(benzoyl)GMP (22). Yield was 70% based on 5’-GMP. N*-(I-AzidobenzoyljGMP (0.5 mmol) was converted to the free acid by treating the aqueous solution with Dowex 50-X8-H+. After removal of the Dowex heads 0.15 ml of tri-n-hutylamine was added and the solution evaporated to dryness. The residue was taken up in dimethylformamide (5 ml) and the solvent was removed under reduced pressure. This was repeated four times. The tributylammon- ium salt was dissolved in 5 ml of dry dimethylformamide and a solution of 3 mmol of carbonyldiimidazole in 4 ml of dimethylformamide was added. After 5 h. 4 mmol of ethanol were added and after standing for 50 min a solution of 3 mmol of trihutylammonium imidodiphosphate in 40 ml of dimethvlformamide was stirred in. The mixture was allowed to stand for 48-h protected against moisture. It was centrifuged and the precipitate was washed with dimethylformamide. The supernatanta were collected, combined, and evaporated to dryness. The residue was dissolved in 30 ml of water, applied on top of a DEAE-cellulose (DE521 column (2.5 x 20 cm. carbonate form,, and chromatographed using a linear gradient (2 liters, 0 to 0.5 M) of triethylammonium bicarbonate buffer, pH 7.5. The fractions containing N’-(l-azidobenzoyl)Gpp(NH)p were combined and evaporated. Yield was 40% based on N2-(4-azidobenzoyl)5’-GMP. All operations were carried out in the dark.

Preparation of P3-(4-Azidwnilido)-P’-5’-GTP- GTP.Na, (0.1 mmol) and N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide . HCl (0.4 mmol) were dissolved in 5 ml of 0.1 M triethanolamine buffer, pH 7.2. 4-Azidoaniline (0.8 mmol) was prepared from p-aminoacetanilide according to Silberrad and Smart (23) and mixed with 2 ml of peroxide-free 1,4-dioxane. The mixture was kept for 16 h at 20” in the dark. Unreacted 4-azidoaniline was extracted with three nortions of 10 ml each of diethyl ether. The water phase containing t-he nucleotide was diluted to 20 ml with distilled water and applied to a DEAE- cellulose (DE52) column (2.5 x 30 cm) in the carbonate form. GTP azidoanilide was eluted by a linear gradient, 0 to 0.5 M, of 2.4 liters of triethylammonium bicarbonate, pH7.5, at 4”. The fractions contain-

7226 Regulation of Adenylate Cyclase

ing the GTP azidoanilide were evaporated with methanol under reduced pressure. The compound was stored as a concentrated aqueous solution at -20”. Yield was 60% based on GTP; E czeo nm, = 30,600 [M-l X cm-‘] in 0.1 M phosphate buffer, pH 7.0. All operations were carried out in the dark.

Preparation of [yTLP]GTP Azidoanilide - [ySZPIGTP was prepared with minor modifications according to Glynn and Chappell (24) and used without further purification for the synthesis of [y- 32P]GTP azidoanilide by the above procedure. The labeled compound, 5 to 50 Cilmmol, was stored in aqueous solution at -20”. It was >95% radioactively pure. The synthesized compound was identified as GTP azidoanilide by the following criteria. It was photoreactive. On chromatography on polyethyleneimine cellulose in 0.8 M triethylammonium bicarbonate buffer, pH 7.5, reaction with polyethyleneimine cellulose occurred on exposure to ultraviolet light and a dark spot formed which did not move on rechromatography. GTP azidoanilide has a strong band in the infrared at 2150 cm-i characteristic for the -N, group (25). GTP azidoanilide has about the same mobility as GDP on chromatography on polyethyleneimine cellulose sheets in 0.8 M triethylammonium bicarbonate buffer, pH 7.5, R, for GDP = 0.46; and RF for GTP azidoanilide = 0.5, suggesting the presence of three negative charges.

[y-“*P]GTP azidoanilide had the same specific radioactivity as [y- 92PlGTP from which it was synthesized. The y-phosphate group was not liberated by treatment with alkaline phosphatase, but treatment with snake venom phosphodiesterase yielded a spot which moved with 5’-GMP on thin layer chromatography on polyethyleneimine cellulose.

Photoreaction - Suspensions of protein-boundly-3’PlGTP azidoanilide were irradiated at 4” in quartz cuvettes (0.5 to 2 cm light path) under gentle stirring with a 200 watt mercury lamp equipped with a cut off filter at 345 nm. Covalent incorporation after photolysis was shown by treating the material retained on Whatman GF/A glass fiber discs with 10% ice-cold trichloroacetic acid. The filters were washed extensively with 10% trichloroacetic acid, dried, and counted in Bray’s solution (201. With solubilized membranes 200 to 500 pg of bovine serum albumin were added as carrier.

Preparation of GTP-Sepharose- Carboxypropylamino-Sepharose 4B was prepared by coupling y-aminobutyric acid (4 mmol) in 50 ml of 0.1 M Na,CO,, pH 10, to 50 ml of packed Sepharose 4B activated with 10 g of CNBr for 16 h at 4” according to Cuatrecasas (26). GTP’Na, (33 pmol) and N-ethyl-iV’-(3-dimethylaminopropyl)- carbodiimide’HC1 (200 pmol) dissolved in 1 ml of distilled water were added to a solution ofp-phenylenediamine (600 pmol) in 1 ml of 1,4-dioxane and left for 3 h at 22” in the dark. The reaction mixture was poured under stirring into 50 ml of ice-cold ethanol and after 15 min at 4” collected by centrifugation at 10,000 x g for 5 min. The precipitate, washed twice with 10 ml of ice-cold ethanol, was finally dissolved in 4 ml of distilled water, mixed with 4 ml of packed carboxypropylamino-Sepharose 4B, and adjusted to pH 4.7. After adding N-ethyl-N’-f3-dimethylaminopropyl)carbodiimide . HCl (200 pmol) the mixture was stirred for 12 h at 22” in the dark with occasionally adjusting the pH to 4.7. The GTP-Sepharose derivative was washed with 1 M NaCl and finally stored at -20” in 125 mM NaCl solution containing 50% glycerol (w/w) and 0.02% NaN, (v/v). Prior to use, the Sepharose derivative was thoroughly washed with 250 mM NaCl at 4”, packed by centrifugation, and resuspended in 1.5 times its volume with 250 mM NaCl. The amount of GTP bound was I to 2 pmoliml of packed Sepharose as estimated by absorbance measurements at 256 nm after hydrolysis with 1 N HCl for 2 h at 37”. The corresponding P”-(4-aminoanilidol-P’-5’-adenosine triphosphate and ATP-Sepharose were prepared accordingly.

The buffer system used for affinity chromatography was 83 mM NaCl, 0.6 mM Lubrol PX, 0.7 mM Mg*+/EDTA, 7 mM Tris’HCl, pH 7.4, and is referred to as Buffer C.

RESULTS

Reaction of Membranes with GTP Azidoanilide

y-Substituted amidates and esters of GTP are potent activators of catecholamine-stimulated adenylate cyclase in pigeon erythrocyte membranes. Binding affinity and the ability to activate increase with increasing hydrophobicity of the y sub- stituent (27). In Fig. 1A are compared the concentration- dependent activation of pigeon erythrocyte membrane adenylate cyclase by NZ-(4azidobenzoyl)Gpp(NH)p and GTP azi-

FIG. 1. A, activation of membranous adenylate cyclase by photoreactive GTP analogs. Pigeon erythrocyte membranes, 230 pg of protein, were incubated in Buffer A with 50 pM m-isoproterenol and with the GTP analogs at the concentrations indicated. The final volume was 100 pl and incubation was for 20 min at 37”; 5 KM DL- propranolol was then added to displace m.-isoproterenol and adenylate cyclase activity was measured in 200 al as described under “Methods.” B, time course of the photoreaction of GTP azidoanilide with pigeon erythrocyte membranes. Membranes (4.8 mg/ml) were incubated in Buffer A with 7 x lo-’ M ly”2PlGTP azidoanilide (specific activity 5 Ci/mmol) for 40 min at 37”. When only binding of guanylnucleotides was measured incubations were carried out without nn-isoproterenol. The membranes were washed with Buffer A to remove excess nucleotide and 200-~1 aliquots were incubated in the presence and absence of 0.1 mM Gpp(NH)p for 20 min at 37” and photolyzed for the time indicated. The conditions for the photoreaction are given under “Methods.” The values in B represent the amount of analog covalently incorporated in the absence of Gpp(NH)p minus that in its presence. Control samples were kept in the dark. Radioactive covalently bound nucleotide was determined in 30-~1 aliquots of the membrane suspensions as described under “Methods.”

doanilide. The data demonstrate the greater efficacy of the latter compound. A K,,,,, value of 3.3 x lo-’ M was estimated for [y-“‘PIGTP azidoanilide and pigeon erythrocyte membranes. The amount bound was 120 pmollmg of protein. Com- parable affinity and number of binding sites were estimated for [3H]Gpp(CH,)p (6). The greater part of the photoreactive GTP analog apparently binds to the same sites to which Gpp(CHJp or Gpp(NH)p bind because all these analogs displace each other. On radiation, radioactivity from [yJ’P]GTP azidoanilide was incorporated covalently into membranes (Fig. 1B). The reaction was essentially completed after 60 min. In the dark or when [y-“‘PIGTP azidoanilide was photolyzed before it was added, little radioactivity was incorporated (Fig. 1B). Please note that the values in these and subsequent experiments give the difference between the nucleotide incor-


porated in the absence and presence of Gpp(NH)p. Thus, only those binding sites which are shared by GTP azidoanilide and Gpp(NH)p were considered. The data in Table I show that besides membrane proteins lipids also reacted with GTP azidoanilide. Interestingly, Gpp(NHlp also inhibited partially the covalent modification of lipids. This could open attractive possibilities to search for membrane lipids intimately associated with guanylnucleotide-binding sites. One estimates from the concentration and the specific radioactivity added that about 10 to 15% of the specifically bound photoaffinity label,

TABLE I Photoaffinity labeling of membranes with [y-32P]GTP azidoanilide

Membranes (5.1 mglml) were incubated with 3 pM [Y-~PIGTP azidoanilide (5 Ci/mmoll with or without 0.1 mM Gpp(NHlp for 30 min at 37” in Buffer A (see legend to Fig. 1B). The membranes were washed several times with the same buffer and 200-~1 aliquots were irradiated for 60 min at 4”. The membrane suspension with covalently bound nucleotide was precipitated with trichloroacetic acid on glass fiber discs (see “Methods”). Covalently labeled lipids were extracted by gently swirling the discs for 1 h with 2 ml of a 2:l (v/u) mixture of CHCl,/MeOH and 0.01 M HCl. Contaminating proteins were removed from the organic phase with 0.5 ml of 0.9% NaCl and the organic phase (1 ml) was counted for radioactivity in Bray’s solution (20). Control experiments were carried out with ly-32P]GTP azidoanilide photolyzed for 60 min prior to addition to membranes. The values represent the difference in incorporated radioactivity in the absence and presence of 0.1 mM Gpp(NH)p.

Compounds Iy-32Pl incorporated into

Proteins Lipids pmollmg

[y-“‘PIGTP azidoanilide 3.2 1.4 [y3’PlGTP azidoanilide (photolyzed prior 0.15 N.D.”

to addition)

n Not determined.

which is that portion that is displaced by Gpp(NH)p, were covalently incorporated into membranes after photolysis. This is comparable with the incorporation ofp-azidophenacyl-tRNA into ribosomes reported by Schwartz and Ofengand (28). Al- though the amount of nucleotide bound covalently is small, the photoreaction partially inactivates adenylate cyclase irreversibly. For example, from the total GTP azidoanilide bound specifically (but noncovalently) to membrane protein 14% was bound covalently after photoactivation. Activity measurements before and after photolysis indicated a 15% decrease in adenylate cyclase activity.

Guanylnucleotide Binding Proteins in Membranes

Pigeon erythrocyte membranes and soluble adenylate cyclase preparations were labeled covalently with GTP azidoanilide after incubation without and with 0.1 mM Gpp(NH)p and the labeled proteins were separated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As shown in Fig. 2A four membrane proteins with M, = 86,000, 52,000, 42,000, and 23,000 had reacted. The photoaffinity reagent was also incorporated inm a membrane protein of M, about 250,000 which corn&rates with spectrin but high concentrations of Gpp(NH)p did not prevent incorporation into that protein, hence suggesting that labeling was unspecific. In Lubrol PX- solubilized preparations lacking the spectrin complex, only proteins with M, = 42,000 and 23,000 were specifically labeled (Fig. 2B). Since soluble adenylate cyclase is fully responsive to nucleotide stimulation only the binding proteins with M, of 23,000 and 42,000 are therefore likely candidates for a role in adenylate cyclase activation. All radioactive bands in the sodium dodecyl sulfate-polyacrylamide gels were shown to be proteins because on digestion with trypsin the radioactivity shifted to new bands with greater R,,, values.

The larger portion of the nucleotide-binding proteins in

A c-4 8 ;

MEMeRANES

P

SLICE NUMBER

FIG. 2. Photoaffinity labeling of membranous and Lubrol PX- solubilized proteins. A, membranes, 2.5 mg/ml, were incubated with 3 PM [yJ*PlGTP azidoanilide (5 Cilmmol) in Buffer A for 30 min at 37” (see legend to Fig. 1B). After three washes with Buffer A each with 5 times the volume of the membrane suspension, the membranes were incubated for another 25 min at 37” without (O---O) and with (60) 0.1 mM Gpp(NHlp and photolyzed for 60 min at 4” (see “Methods”). The irradiated samples (250 pg of protein) were treated with 1% sodium dodecyl sulfate and 10 mM 2-mercaptoethanol for 15 min at 37” and electrophoresed in 12.5% polyacrylamide gels contain-

SLICE NUMBER

ing 0.1% sodium dodecyl sulfate. Phospholipids have about the same mobility as the tracking dye (TD). B, membranes were first incubated with GTP azidoanilide and washed as described for A. The membranes were then solubilized with 20 mM Lubrol PX for 30 min at 4” as described previously (61. The soluble material was incubated for 25 min at 37” in the absence (C---O) and presence (O---O) of 0.1 mM Gpp(NH)p and then photolyzed and separated electrophoreti- tally on polyacrylamide gels as described above and under “Meth- ods.”


solubilized preparations can be separated from adenylate cyclase activity without substantial inactivation. This can be achieved by chromatography on Sepharose 4B (61, by ion ex- change chromatography on DEAE-cellulose,” and by sucrose density centrifugation. We had previously observed that pretreatment of pigeon erythrocyte membranes with high (10m3 M)

concentrations of GMP yielded detergent-solubilized adenylate cyclase preparations which were stimulated nearly as effectively as membranous adenylate cyclase by Gpp(NH)p and other analogs (cf. Ref. 6). GMP is not or only weakly stimulatory and does not bind strong enough as to interfere with the binding and activation by Gpp(NH)p or GTP azidoanilide. GMP might protect the binding site in the course of solubilization. From the distribution of the bound radioactivity on centrifugation through a linear sucrose gradient one estimates that more than 95% of the nucleotide binding proteins sediment slower than adenylate cyclase and only about 5% of the nucleotide binding sites co-sediment with adenylate cyclase (Fig. 3). The guanylnucleotide-binding fractions separated by sucrose gradient centrifugation were labeled with lr- %P]GTP azidoanilide and analyzed by sodium dodecyl sulfate acrylamide gelelectrophoresis. The slowly sedimenting nucleotide binding peak was exclusively the 23,000 M, protein. The binding protein associated with adenylate cyclase was mainly the 42,000 M, protein. The bulk of the lipids and detergent does not sediment. It is therefore necessary to readd Lubrol PX to measure adenylate cyclase activity. Reactivation of soluble adenylate cyclase depleted of detergent by guanylnucleotides and F- was critically dependent on detergent concentration (see Fig. 4). Maximal adenylato cyclase activity in the presence of 0.1 mM Gpp(NH)p was obtained at a 3:l ratio (w/w) of Lubrol PX to protein.

Affinity Chromatography with GTP-Sepharose

Assuming a i& of 100,000 and the same concentration of active sites as there are catecholamine receptor sites, 1 pmol/ mg of adenylate cyclase would constitute only a small portion (0.01%) of the proteins in pigeon erythrocyte membranes (81. For the purification of a protein present in such small amounts procedures making use of characteristic ligand-binding properties are attractive. Detergent-solubilized pigeon erythrocyte membrane adenylate cyclase no longer responds to catecholamine but is fully responsive to guanylnucleotides. This prop- erty was therefore exploited and a GTP affinity column was synthesized making use of the high affinity of y-substituted aromatic amidates of GTP for membrane proteins. The affinity matrix used was Sepharose 4B-NH-(CH,),-CO-NHONH- pppG which is referred to as GTP-Sepharose.

The flowsheet (Fig. 5) illustrates the procedure. Adenylate cyclase was solubilized with Lubrol PX from membranes pretreated for 30 min at 37” with 1 mM GMP and 50 pM DL-

isoproterenol (cf. Ref. 6). Step A -The soluble preparation (1 ml) was added to 0.5 ml

of a GTP-Sepharose suspension and incubated with gentle shaking. The mixture was separated by centrifugation in the supernatant fraction I and the matrix bound material (II). Supernatant I contains the material not retained by the matrix.

Step B-The matrix-bound material II was washed three times with 1 ml of Buffer C and finally brought to the original volume (1.5 ml) with the same buffer containing 0.1 mM Gpp(NH)p or 0.2 mM GTP. The adsorbed proteins were re-

B T. Pfeuffer, unpublished results.

FIG. 3. Separation of solubilized membrane proteins by sucrose density gradient centrifugation. Membranes (8.8 mg of protein) were incubated in 1.5 ml of Buffer A with 1 rnM GMP and 50 pM DL- isoproterenol for 30 min at 37”. After three washes with 4 ml of Buffer B, the membranes were solubilized with 20 mM Lubrol PX as described in Ref. 6. The solubilized material was concentrated to a volume of 200 ~1 by centrifugation at 1000 x g in a “centrifilo Filter cone” device (CF 25-Amicon) and layered on top of 5 ml of a 5 to 30% linear sucrose gradient in Buffer B and centrifuged in a SW 50 rotor (Beckman) at 48,000 rpm for 14 h at 4”. Fractions of 0.25 ml were collected and adenylate cyclase activity was measured in 200 ~1 in the presence of 0.1 mM Gpp(NH)p and 0.5 mM Lubrol PX as described under “Methods.” Binding of 13HlGpp(NH)p was determined in lOO- ~1 aliquots according to Pfeuffer and Helmreich (6) and protein was measured as described under “Methods.”

T ‘c E

1 I I I I ti 3 5 10

RATIO LUBROL PX/PROTEIN (w/w I

FIG. 4. Dependence of soluble adenylate cyclase activity on detergent. Soluble adenylate cyclase (30 rg) depleted of Lubrol PX by sucrose density centrifugation as described in the legend to Fig. 3 was assayed in 200 ~1 with 0.1 mM Gpp(NH)p and in the presence of varying amounts (0 to 300 pg) of Lubrol PX. The ratio (w/w) of Lubrol:protein is given on the abscissa.

leased by incubation for 60 min under gentle shaking and the mixture separated by centrifugation thus yielding supernatant III. Since guanylnucleotides stabilize adenylate cyclase activity, supernatant I was likewise incubated for 60 min with either 0.1 mM Gpp(NH)p or 0.2 mM GTP.

Step C - For reactivation 50 ~1 of supernatant I and 50 ~1 of supernatant III were mixed and adenylate cyclase activity was measured immediately after mixing. To standardize the procedure the volumes were made equal. Since Step C results in a 2- fold dilution, aliquots (50 $1 of supernatants I and III were


GTP - Sepharose Centrifugation

10.000 x g ; 10 min; L’C

Supernatant I Matrix Proteins II

0 C Measure Actwity

t

Supernatant I Incubation

Recombwwd Measure

Actwity - I l 111 -

I

Supernatant 111

- +GpplriFp or GTP 1 W3a;h BufferC

60 min; 22’ C

Centri - 10,000 xg

0 B fugdlon L°C

Resuspension .

1.5ml in Buffer C

Release +Gpp(NH)p or GTP

6Omin; 22’C

Measure Activity

FIG. 5. Affmity chromatography of Lubrol PX-solubilized adenylate cyclase (see text).

TABLE II Resolution and reconstitution ofadenylate cyclase

The rationale of the procedure is outlined under “Methods” and in mation was linear throughout the assay. Protein concentrations are Fig. 5. The starting material was about 1.5 mg of soluble membrane given in microgram/200 ~1. Proteolysis with trypsin (2 pg/50 ~1) was protein. In Experiment D, Buffer C contained 0.1 rnM Gpp(NH)p and for 20 min at 22” and was terminated by incubation with soybean in Experiment K 0.2 rnM GTP. Fraction B was combined with the trypsin inhibitor (6 pg/50 ~1) for another 20 min. Reaction with N- binding proteins in Preparations D and K giving Preparations E and ethylmaleimide (3 mM) was for 20 min at 22” in Buffer C and was L, respectively. Since the protein concentration of Preparations D terminated by incubation with 6 mM 2-mercaptoethanol for another and K was very low, Buffer C without Lubrol was used. Adenylate 10 min. The values represent the mean f S.E. Each set of experi- cyclase activity was measured in 200 ~1 containing either 0.1 mM ments was carried out with a different preparation. The percentage Gpp(NH)p or 0.2 mM GTP and 10 mM NaF. Activities are given in of activity is given in parentheses and related to the activity of the picomoles of CAMP formed/min under assay conditions. CAMP for- contrnl (Preparation A)

Preparations (number of experiments) Adenylate cyclase activity with

GPP(NWP Fluoride

pm01 X min-’ X [ZOO plcll’

Protein concentration

/l&T x 1.200 PP A. Adenylate cyclase treated with Sepharose 4B (15) B. Adenylate cyclase treated with the GTP-Sepharose deriv-

ative (15) C. As in B but in the presence of 0.1 mM Gpp(NH)p (3) D. Guanylnucleotide-binding protein removed from the GTP-

Sepharose with 0.1 mM Gpp(NH)p (IO) E. B and D combined (10) F. Trypsin-treated B combined with D (2) G. B combined with trypsin-treated D (3) H. N-Ethylmaleimide-treated B combined with D (5) I. B combined with N-ethylmaleimide-treated D (5) K. Guanylnucleotide binding protein removed from the GTP-

Sepharose with 0.2 mM GTP (7) L. B and K combined (7) M. Trypsin-treated B combined with K (2) N. B combined with trypsin-treated K (4) 0. N-Ethylmaleimide-treated B combined with K (2)

65 c-t31 [lOO%l 10 (21) [15%1

61 (k6) [93%1 2 (to.31 [30/o]

35 (k1.3) WI%1 50 [lOO%l 17 (kO.8) [48%1 48 (kO.5) [97%1

49 (kl) 199%1 1.5 (kO.3) [3%1

39 (k2.2) [60%1 2.5 (50.7) [4%1

10 (k1.7) [15%1 3 (LO.5) [5%1 9.5 (kO.9) [lS%l

0.7 (kO.2) [2%1

29 (k2) [83%1 0.8 (r0.3) [2.5%1

18 (r2) [51%1 1.0 (kO.4) [3%1

P. B combined with N-ethylmaleimide-treated K (4) 19 (k2) [54%1


also diluted 2-fold by adding 50 ,nl of Buffer C containing 0.1 mM Gpp(NH)p or 0.2 mM GTP, respectively, and adenylate cyclase activity was measured. Since the protein concentration in supernatant III was very low, no Lubrol was added to Buffer C. When guanylnucleotide activation was measured Gpp(NH)p was added, whereas GTP was added when F- activation was measured. Control experiments were carried out with underivatized Sepharose 4B. The Sepharose protein mixture was treated like the experimental samples and carried through the same steps under identical conditions omitting only washing and the releasing step. Since it was shown in separate experiments that treatment of soluble adenylate cyclase with underivatized Sepharose 4B did not lower adenylate cyclase activity, regardless whether or not guanylnucleotides were present, the control supernatants I were treated like the experimental samples and were incubated likewise with Gpp(NH)p or GTP. The rationale of the whole procedure was to create equivalent conditions for measuring adenylate cyclase activities in supernatants I and III and in a mixture of both in order to make the measurements quantitatively comparable.

Resolution and Reconstitution of Adenylate Cyclase

Table II shows that treatment of detergent-solubilized adenylate cyclase with GTP-Sepharose results in a 85% loss of nucleotide stimulation (Experiment B). With underivatized Sepharose as control no activity was lost (Experiment A). After the experiments reported here were completed and the paper was in review it was found that the loss of guanylnucleotide activation was overestimated. The reasons for that are contaminating hydrolases in the preparation which were found to liberate GTP (or GDP) from GTP-Sepharose. These guanosine phosphates interfere with Gpp(NH)p (Experiment Bl thus lowering activation by the analog. GTP or GDP were ineffective when added after activation with Gpp(NH)p (Ex- periment C). Hydrolase activity in the soluble preparation can be inhibited by 0.5 mM GMP (or 0.5 mM PS-(4-aminoanilido)- PI-adenosine 5’-triphosphate) which bind only weakly to guanylnucleotide sites and do not interfere with Gpp(NH)p activation. Accordingly, when affinity chromatography is carried out in the presence and absence of these nucleotides, the F-- stimulated activity of supernatant I remained unchanged, whereas the Gpp(NH)p-stimulated activity did rise from about 15 to 30% of the original activity (see Experiment B). It should be emphasized, however, that the change in Gpp(NH)p-stimu-

TABLE III GTP enhancement of ~-stimulated adenylate cyclase activity

Membranes were treated with GMP and isoproterenol and solubilized with Lubrol PX as described. Untreated membranes were solubilized by the same procedure but omitting the preincubation step. Soluble protein (30 to 40 pg) was assayed for adenylate cyclase activity with and without 0.2 mM GTP in the presence of 10 mM NaF for 20 min at 37” in a final volume of 200 ~1. NaCl (40 mM) was added to establish the same conditions as in Table II. The values represent the mean of five experiments + SE.

Soluble reparations P ATP Fe-stimulated activity ram Ratio 2 GTP

With GTP Without GTP mhf pm01 x min x [ZOO p.&’

GMP/isoprotere- 0.1 47.1 (k3.5) 31 (k2.5) 1.5 (kO.11 nol-treated mem- 1.0 96 (k7.5) 87 (k7) 1.1 (-to.011 branes

“Untreated” mem- 0.1 37.8 (22.5) 37.2 (k2.5) 1.05 (kO.01) branes

lated activity in preparation B does not change the relative activity gain on recombination, since the activity in preparation E also increased (from 60 to 72%).

I@ activation of soluble adenylate cyclase prepared from GMP and isoproterenol-treated membranes is enhanced by GTP. This is shown in Table III which moreover demonstrates that GTP activation is dependent on the ATP concentration. The GTP activation of FO-stimulated adenylate cyclase activity assayed to 0.1 mM ATP concentrations was variable from preparation to preparation. The loss of FO-stimulated activity on treatment with GTP-Sepharose appeared considerably smaller when adenylate cyclase activity was measured with 0.1 mM ATP, but without GTP (29). The reason is that the effect of GTP on Fe activation of a preparation largely depleted of guanylnucleotide-binding proteins is much less than with the unfractionated preparation, because the binding proteins are needed for GTP activation (Experiment B). Thus the differences in the Fe-stimulated activities of control and experimental samples depend strongly on the extent of GTP activation of the control (compare Experiments A and B).

Binding of guanylnucleotides is time- and temperature-dependent (2). Considerably less binding protein was bound and recovered from the GTP affinity column at 0”. Activation of particulate and soluble adenylate cyclase preparations by guanylnucleotides is also a time- and temperature-dependent process. Catecholamines accelerate the conversion of adenylate cyclase in membranes to a quasi irreversibly activated form which is also formed, albeit more slowly, by guanylnucleotides alone (6, 30). Once formed, the active state with- stands solubilization and lasts for many hours. Since adenylate cyclase solubilized from membranes pretreated with GMP instead of Gpp(NH)p is unstable at higher temperatures (37”), the various steps involved in the affinity chromato- graphic procedure were carried out at room temperature and at the times stated.

A pretreatment of 20 min with 0.1 mM Gpp(NH)p was sufficiently long to convert adenylate cyclase to the maximally active state which was stable enough to give linear rates in the adenylate cyclase assay for at least 20 min at 37”.

The specificity of GTP-Sepharose for guanylnucleotide-binding sites is apparent from the effect of 0.1 mM Gpp(NH)p which prevented the attachment of binding proteins (Experiment C). Moreover, in other controls not shown here, the corresponding ATP-Sepharose derivative did not remove significant amounts of proteins required for adenylate cyclase activity and neither were proteins bound to GTP-Sepharose released by App(NH)p.

Detachment of protein from the affinity matrix by Gpp(NH)p (and GTP) requires neutral salt (about 0.1 M NaCl) and detergent (0.6 mM Lubrol) but once Gpp(NH)p is bound, the detergent is no longer needed for adenylate cyclase activity (cf. Ref. 6). Neither NaCl nor Lubrol alone or combined released proteins from the GTP-Sepharose. It is conceivable that high concentrations of neutral salt are needed to dissociate the adenylate cyclase complex. Since the activity of soluble adenylate cyclase is dependent on the ratio of detergent to protein and lipids (see Fig. 41, the concentration of detergent was reduced to 0.15 mM in Experiments D and K (4.5 mM in all other experiments), because the protein concentration of this material was less than 3% of the starting material (Table II). The starting soluble material (Preparation A, Table II) contained 220 pmol of Gpp(NH)p/mg of protein, whereas Prepara- tion D binds 1000 to 1600 pmol of Gpp(NHlp/mg, hence affinity chromatography results in a 5- to El-fold enrichment of sites. Recombination gives an activity gain of about 40% (compare


Experiments B with E in Table II). Assuming completely nonselective binding, one would have expected from the activity gain about 3300 pmol/mg in preparation D. Thus, the higher activity of the fewer sites (1000 to 1600 pmol) actually found in Preparation D suggests some enrichment of functional sites by affinity chromatography.

The reconstitution Experiments E and L clearly indicate that the fraction bound to GTP-Sepharose must be an essential component of the adenylate cyclase system because it is required for reactivation. Trypsin or N-ethylmaleimide abol- ished reactivation; hence Experiments F to I and M to P identify the matrix-bound fractions and the material not retained by the Sepharose derivative as proteins sensitive to- ward --SH reagents. Fe stimulation and guanylnucleotide binding and activation of adenylate cyclase have been reported to be sensitive to SH reagents (14, 31, 32). Reactivation did not occur when Fraction B was exposed for 1 h to temperatures above 45” or when the matrix-bound Fraction D was combined with bovine serum albumin, or Lubrol PX-solubilized membrane proteins from rat erythrocytes which are devoid of adenylate cyclase activity (33).y Some activity gain occurred, when the matrix-bound Fraction D was combined with the unfractionated Preparation A. This suggests an equilibrium between holoenzyme and its components and/or might mean that the binding protein concentration is limiting.

Preparation D from Table II was centrifuged through a linear sucrose density gradient (see Fig. 6). A fast sedimenting peak could be separated from slower sedimenting material

FRACTIONS

FIG. 6. Distribution of binding proteins in sucrose density gradient centrifugation. Pigeon erythrocyte membranes in 2 ml of Buffer A (9.4 mg of protein) were preincubated with m-isoproterenol and GMP and solubilized as described. Soluble adenylate cyclase (2.2 mg of protein in 1.6 ml) was treated with 0.8 ml of packed GTP- Sepharose for 30 min at 22”. Proteins attached to the affinity support were released with Gpp(NH)p as described in Fig. 5 and in the legend to Table II. The released material was concentrated to about 0.2 ml and layered on top of a linear sucrose gradient in Buffer B and centrifuged as described in legend to Fig. 3. Fractions of 0.33 ml were collected and 50-~1 aliquots were combined with either 56 ~1 of Buffer C (without Lubrol) or with 50 ~1 of the fraction not adsorbed to GTP-Sepharose (see Fig. 51. The activity of the fraction released with 0.1 rnsr Gpp(NH)p (Supernatant III, Fig. 5) is referred to as “intrinsic activity” (O-O). The volume of the fraction (50 ~11 not adsorbed to GTP-Sepharose (Supernatant I, Fig. 5) was made equal by addition of 50 ~1 of Buffer B. The activity of Supernatant I in the presence of 0.1 rnre Gpp(NH)p was 0.21 nmol x ml-’ x mini. Activity gain on recombination of Supernatants I and III (O-01 refers to activity with 0.1 mM Gpp(NHlp after recombination minus the sum of the activities of the isolated components. Protein is indicated by - - -.

TABLE IV Interaction of components of the adenylate cyclase system from pigeon

erythrocyte and rabbit cardial muscle membranes Rabbit myocardial membranes were solubilized with Lubrol PX

according to (Ref. 16). Solubilized myocardial and erythrocyte adenylate cyclase (1 mg/ml each) were treated with GTP-Sepharose as described in Fig. 5 and in the legend to Table II. GTP-binding fractions (50 ~~11 from pigeon erythrocyte adenylate cyclase (Prepara- tion C) were combined with 50 ~1 of either Buffer C (without Lubroll or with 50-~1 fractions from myocardial adenylate cyclase (not adsorbed to GTPSepharosel (Preparation Bl. Unfractionated Lubrol PX-solubilized myocardial adenylate cyclase served as control (Prep- aration Al and also received 50 ~1 of Buffer C. The concentration of Gpp(NHlp in the adenylate cyclase assay was 0.1 mM. Activity is expressed as picomoleslminlassay mixture under assay conditions. The values represent the mean f S.E. Percentage of activity is given in parentheses.

Preparations uuuaber of experiments) Gpp(NH) -stimulated ade- nv P ate cvclase

A. Myocardial adenylate cyclase 62 treated with Sepharose 4B (4)

(251 100%

B. Myocardial adenylate cyclase- 17 treated with GTP-Sepharose (41

(22) 27%

C. Guanylnucleotide-binding proteins 1.5 (zO.3) 2.5% from pigeon erythrocyte adenylate cyclase (3)

D. B and C combined (3) 36 (241 58%

devoid of activity. The latter contained the proteins required for reactivation as shown by the activity gain on recombination with the material not adsorbed to GTP-Sepharose. Com- parison of Fig. 6 with Fig. 3 shows that the intrinsic activity of the material detached from the matrix (Experiments D and K in Table II) can be ascribed to a small amount of unresolved holoenzyme. Separate experiments showed that the 23,000 M, fraction prepared from Gpp(NH)p and isoproterenol-treated membranes and separated by sucrose density gradient centrifugation (Fig. 3) did not reactivate. Thus, the experiments in Fig. 6 in conjunction with those in Fig. 3 again point to the 42,000 M, fraction as a likely candidate for a site of guanylnucleotide binding and action. The experimental verification of this suggestion will however depend on the purification of that protein, which is underway.

In Table IV is shown that guanylnucleotide-stimulated activity was regained on recombining binding fractions from pigeon erythrocyte membranes with a fraction prepared from myocardial adenylate cyclase and depleted of binding proteins (Table IV, Preparation D). Thus, adenylate cyclases from eukaryotes which are phylogenetically quite apart share similar control properties.

Synergistic Activation by Hormones and Guanylnucleotides

Guanylnucleotides and hormones activate synergistically adenylate cyclase from a variety of sources. The activity of adenylate cyclase solubilized from membranes preincubated with hormone and nucleotide is greater than the sum of the activities with either one of the effecters (3-61. We now found that exposure of intact membranes to nL-isoproterenol im- proves considerably the ability to reactivate of guanylnucleotide binding fractions prepared from such membranes (Table V). Reconstitution with a guanylnucleotide binding fraction from membranes not exposed to isoproterenol yielded little activity even when the other fraction came from hormonally activated membranes (Preparation IV in Table V). Con- versely, the guanylnucleotide binding fraction derived from


TABLE V

Effect of Dksoproterenol on reconstitution of adenylate cyclase activity

Adenylate cyclase was solubilized from pigeon erythrocyte membranes preincubated with GMP and with or without 50 PM DL- isoproterenol. The solubilized material was treated with GTP-Seph- arose by the procedures given in Fig. 5 and in the legend to Table II. Preparations I and II refer to unfractionated complex and III and IV to adenylate cyclase fractions depleted of GTP-binding proteins by treatment with GTP-Sepharose. The GTP-binding fractions represent material released from the affinity matrix with 0.1 mM Gpp(NH)p. Aliquots (50 ~1) of the adenylate cyclase preparations and fractions, respectively, were combined with 50 ~1 of Buffer C or with 50 ~1 of the binding fractions. Activity was measured in the presence of 0.1 rnM Gpp(NH)p and is expressed as picomobminlassay mixture under assay conditions. Adenylate cyclase activity of the GTP-binding fractions (k isoproterenol) was 1.5 pmol x min-’ x [200 ~I]-‘. The values represent in each case the mean of four experiments f S.E.

Gpp(NH)pactivated adenylate cyclase following addition of

Preparations B&For

GTP-binding GTP-binding fraction (- fraction (+ isoprotere- isoprotere-

n0l) 001)

pm01 x min-’ X 1200 @’ I. Adenylate cyclase corn- 19 (k2.5)

plex ( - isoproterenol) II. Adenylate cyclase corn- 60 (k5)

plex (+ isoproterenol) III. Adenylate cyclase frac- 4 (20.5) 11 (k2) 41 (k-4)

tion (- isoproterenol) IV. Adenylate cyclase frac- 7 (kO.51 18 (k1.5) 38 (k51

tion (+ isoproterenoll

TABLE VI

Release of GTP-binding proteins from membranes with EDTA solutions

Membranes (5 mg/ml) in 2 ml of Buffer A were incubated with or without 0.1 mM Gpp(NH)p and 50 FM m-isoproterenol for 20 min at 37” and dialyzed against 500 volumes of 0.1 mM EDTA solution, pH 8.0, for 16 h at 4” with one change of the solution. The dialyzed material was centrifuged for 10 min at 17,000 x g at 4” and the supernatant solution collected. This is the EDTA extract. The pellet was washed twice with 10 ml of Buffer A, adjusted to the original volume (2 ml), and assayed for adenylate cyclase activity with 0.1 mM Gpp(NH)p. It contained 50 to 70% of the original adenylate cyclase activity of the membranes. Preparation A represents Lubrol- solubilized adenylate cyclase depleted of GTP-binding proteins by GTP-Sepharose treatment (see Fig. 5 and legend to Table II). Ali- quota (50 ~11 of Preparation A were combined with 50 ~1 of the EDTA extracts (Preparations B or C) and adenylate cyclase activity was measured in the presence of 0.1 mM Gpp(NH)p and expressed as picomoles/min/assay mixture under assay conditions. Volumes and conditions were made equal by addition of 50 ~1 of 0.1 mM EDTA to Preparation A and conversely by addition of 50 ~1 of Buffer C without Lubrol PX to Preparations B and C. The values represent in each case the mean of five experiments k SE.

Preparations Adenylate cyclase activity

pm01 X min-’ x 1.200 @lull’

A. Soluble fraction from GTP-Sepharose treat- 10 (k1.4) ment

B. EDTA extract from untreated membranes 0.5 (50.1)

C. EDTA extract from Gpp(NH)p/nL-isoprotere- 0.7 (k0.15) no1 treated membranes

D. A+B 11 (21.4) E. A+C 31 (k2.7)

hormonally stimulated membranes reconstituted a more active adenylate cyclase even when the fraction depleted of binding proteins was derived from membranes not activated with isoproterenol. This points to an interesting relationship of the hormone receptor to the guanylnucleotide binding fraction which needs to be defined.

It may be seen from Table VI that guanylnucleotide binding proteins can also be solubilized from pigeon erythrocyte membranes with low ionic strength solutions, pH 8.0, containing 0.1 mM EDTA. This treatment reduced the guanylnucleotide- stimulated adenylate cyclase activity in the membranes by 30 to 50%. The solubilized material amounted to about 10 to 15% of the total membrane protein. Preparations B and C were practically inactive without or with various concentrations of Lubrol. Preparation C in contrast to B restored activity when added to Preparation A, which was passed through GTP- Sepharose. Therefore, reconstitution requires material extracted with EDTA from membranes pretreated with Gpp(NHlp (or Gpp(CH,)p) and nL-isoproterenol (compare Preparations D with E in Table VI). Other experiments indicated that the nucleotide treatment of the membranes was obligatory for reactivation whereas m-isoproterenol treatment greatly stimulated reactivation. These experiments were not included in Table VI because they merely support the conclu- sions drawn from the experiments shown in Table V.

DISCUSSION

We could show that adenylate cyclase from pigeon erythrocyte membranes can be reversibly inactivated by treatment with a GTP-Sepharose derivative. Loss of guanylnucleotide and fluoride activity was shown to result from dissociation of the adenylate cyclase complex into a protein fraction which is adsorbed to the GTP affinity matrix and bears the guanylnucleotide binding sites and a protein fraction whose function is not yet defined. Both nucleotide- and fluoride-stimulated activity was regained on recombining both fractions. Diagram 1 summarizes schematically two models. Model I assumes that dissociation of the active complex by affinity chromatography leaves the catalytic component (Cl in an inactive conforma- tion and that activity is regained on interaction of regulatory and catalytic components. Model II assumes that the material with the guanylnucleotide binding site (R) which is bound to the GTP-Sepharose, also contains the catalytic site (C). But in this case, reactivation would require interaction with another membrane protein X. Although we cannot yet decide on the basis of the available information between models I and II, the following findings favor model I. GTP (and possibly ATP) enhance Fe activation of adenylate cyclase (see Table III). Thus, the decrease in Fe-stimulated activity is at least in part

DIAGRAM 1


a consequence of the removal of the guanylnucleotide binding protein. Moreover, washing of the matrix-bound material II (cf. Fig. 5) with Buffer C (but without GTP) did not substan- tially increase Fe activity on addition of the wash fluid to supernatant I, thus making adsorption of undissociated ho- loadenylate cyclase to GTP-Sepharose an unlikely cause for the loss of Fe activation. Furthermore, adenylate cyclase solubilized with Lubrol PX from untreated membranes (not treated with GMP) was only poorly activated by Gpp(NHlp, although the preparation retained nearly all of the fluoride- stimulated activity which was, as to be expected, not stimulated by GTP. Consequently, treatment of this preparation with GTP-Sepharose lowered F’ stimulation only by about 6%. While these findings indicated that guanylnucleotide and fluoride activations can be separated, it is not clear whether these reflect different stabilities of the guanylnucleotide and the fluoride-activated enzyme or whether separation is due to an uncoupling of subunits with different functions. The problem is further complicated because one neither knows why and how Fe activates adenylate cyclase.

In this context it should be emphasized that one estimates from the results of this study that no more than 5% of the guanylnucleotide binding sites function in adenylate cyclase activation, although all the sites have similar affinity and specificity for guanylnucleotides and are sensitive to -SH reagents. But what other functions in membranes might guanylnucleotide-binding proteins have? One of the possibilities which we have considered is the presence of tubulin (which binds GTP) in pigeon erythrocyte membranes. Al- though we could recently identify tubulin as a constituent of the pigeon erythrocyte membrane (34), neither nucleotide activation, hormonal stimulation, or NaF activation of adenylate cyclase were affected by an antibody against ox brain tubulin. Another observation, namely that EDTA buffer treatment solubilized GTP-binding proteins from pigeon erythrocyte membranes deserves attention (Table VI). Low ionic strength EDTA buffer has been reported to solubilize contractile proteins, e.g. the spectrin complex from erythrocyte membranes (35). Thus, the similar solubility characteristics of the GTP- binding proteins involved in adenylate cyclase activation and of contractile membrane proteins may hint at an as yet ob- scure relationship of guanylnucleotide binding proteins to contractile elements in red blood cell membranes.

A problem which needs clarification concerns the interaction of hormone and guanylnucleotide whose functional consequence is the synergistic amplification of the action of both effecmrs. We now found that treatment of intact membranes with isoproterenol yields a guanylnucleotide-binding protein, which is more effective in reactivation of adenylate cyclase (see Table V). Thus, the hormone acts via the proteins containing the binding sites for guanylnucleotides. Recent findings of Mukherjee and Lefkowitz (36) point likewise to a relationship between hormone receptor and guanylnucleotides. Desensitized P-adrenergic receptors in frog erythrocyte membranes were rapidly and completely resensitized by exposure of membranes to Gpp(NH)p and other guanine nucleotides. The efficacy of nucleotides for resensitization was Gpp(NHlp > GTP > GDP > ATP > GMP and hence resem- bled their potency for activation of adenylate cyclase. Un- doubtedly, further progress will depend on the isolation and characterization of the components of the adenylate cyclase system including the hormone receptor. Coupling of hormone receptor to adenylate cyclase may however crucially depend on the lipid environment. Therefore, a better understanding of

the role of lipids in hormonal regulation of adenylate cyclase activity may be a prerequisite for the reconstitution of a hormonally sensitive complex.

The isolation and purification of the regulatory guanylnucleotide-binding protein might also clarify its hypothetically postulated GTPase activity (6). We have previously explained the greater efficacy of GTP analogs compared with the natural nucleotide GTP by assuming that the latter is hydrolyzed by a GTPase which is part of the adenylate cyclase complex (cf. Ref. 6). This proposal has recently received support from the discovery of a catecholamine-stimulated GTPase in turkey erythrocyte membranes (37). In this context, an interesting difference between GTP and GTP-y-S is noteworthy. Both guanylnucleotides bind to the same number of sites and are hydrolyzed by pigeon erythrocyte membranes at comparable rates. But, whereas only about 10% of the guanylnucleotide bound to membranes was recovered as GTP after 15 min of incubation with 1 &LM concentrations at 37”, 80% of the membrane bound GTPr-S was still unchanged (38). This suggests that GTP is hydrolyzed at additional sites and may explain why GTPr-S is a much better activator than GTP (6).

Acknowledgments -1 am indebted to Dr. Ernst J. M. Helm- reich, in whose laboratory this work was carried out, for his encouragement and support and for many valuable discus- sions. I also wish to thank Mr. Bolf Thomas who synthesized some of the photoaffinity reagents in the course of work carried out in partial fulfillment of the requirements for the Ph.D. thesis in the Science Faculty of the University of Wurz- burg. To Mrs. Elke Pfeuffer I am indebted for excellent techni- cal assistance.

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