Large-Scale Purification of the Acetylcholine-Receptor Protein. from Torpedo marmorata Electric Organ

Transcription

1 Eur. J. Biochem. 80, (1977) Large-Scale Purification of the Acetylcholine-Receptor Protein in ts Membrane-Bound and Detergent -Ext ract ed Forms from Torpedo marmorata Electric Organ Andre SOBEL, Michel WEBER, and Jean-Pierre CHANGEUX Laboratoire de Neurobiologie Molkculaire, nstitut Pasteur, Paris (Received April 18, 1977) A method is described for the large-scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol a-toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor-rich membrane fragments is composed of only one class of membrane fragments covered with 8-nm rosettes identified as acetylcholine receptor molecules. This preparation is used as a starting material for the detergent solubilization and the large-scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X-100 extract of receptor-rich membranes done in the presence of 2-mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy. Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecyl sulfate reveals three bands of apparent molecular weights * 1 000, & and _ , the two heaviest ones being present in significantly lower amounts than the Mr one. The comparison of several samples of purified protein with different specific activities reveals a striking variability of the ratios of the Mr band to the and Mr ones. The acetylcholine (nicotinic) receptor protein from fish electric organ was the first receptor for a neurotransmitter to be isolated, purified and characterized as a protein (for review see [2-41). Moreover, the development of a fractionation method giving membrane fragments particularly rich in rechptor protein rendered it accessible to physicochemical investigations in a membrane-bound and still functional state [5]. All the protocols followed to purify the detergentextracted protein, except one [6], included a step of affinity chromatography. However, the yields from all these methods were often rather low [4] and it was also established that after affinity chromatography the binding properties of the purified protein are modified ~71. n this article we present a fractionation method of the electric organ from Torpedo marmorata, which A preliminary account of this work [] was presented at the Congress0 Nazionale della Societa taliana di Biochimica, Venice, October (1976). Abbreviations. Buffer : 100 mm NaCl, 100 mm Tris-HC1 ph 7.4,1 %Triton X-100; solution 1 : 0.1 mm phenylmethylsulfonyl fluoride, 0.02 % NaN, ; PhMeSO,F, phenylmethylsulfonyl fluoride. Enzyme. Acetylcholinesterase (EC 3.1,.7). yields large quantities of highly purified receptor-rich membrane fragments and which is based on improvements of the method of Cohen et al. [8]. These membrane fragments are used as a starting material for the purification of the detergent-extracted receptor protein without using affinity chromatography. The peptide chain composition of the highly purified protein appears simpler than currently accepted. MATERALS AND METHODS Assays for the Nicotinic Receptor Site, Acetylcholinesterase and Proteins The nicotinic receptor sites from both membranebound and detergent-extracted receptor protein were routinely assayed with Naja nigricollis 3H-labelled a-toxin (12-25 Ci/mmol) after complete dissolution by Triton X-100, precipitation by dilution in the presence of helper [9] and filtration on Millipore filters as described by Meunier et al. [9], except for the composition of the helper : 5 mg/ml bovine serum albumin, 5 mg/ml egg lecithin, 1 Triton X-100, 100

2 21 6 Purified Membrane-Bound and Detergent-Extracted Acetylcholine Receptors mm Tris-HC1 ph 7.4, 100 mm NaCl and 0.02% NaN,. With highly purified samples of receptor protein some irreproducibility in the results was noted, a significant fraction of the re~eptora-[~h]toxin complex passing through the filter. n these instances an assay based on the separation of free from bound ~-[~H]toxin by Sephadex G-50 filtration was used. Both free and bound ~-[~H]toxin were determined giving more reliable and reproducible results. The specific activity of the purified fractions were determined by this method. A 25-ml column of Sephadex G-50 was equilibrated with 1% Triton X-100, 100 mm NaCl, 100 mm Tris-HC1 ph 7.4 and 0.02% NaN, buffer. The sample preincubated with an excess of ~-[~H]toxin (1 pmol) in 100 pl buffer 1 for 1 h at room temperature, was layered on top of the column and fractions of 0.5 ml collected and counted with 5 ml Bray s solution in an ntertechnique SL 30 scintillation counter. The radioactivity in the excluded peak was taken as a measure of the a-[3h]toxin.acetylcholinereceptor complex. Acetylcholinesterase was assayed by the method of Ellman [lo]. Proteins were estimated by the method of Lowry et al. [ll], using bovine serum albumin as the standard. Detergents and buffers at appropriate concentrations were always included in the blank and standard assays. Throughout this paper the specific activity of the acetylcholine receptor protein is expressed in nmol or pmol of N. nigricollis a-[3h]toxin sites/g protein. Purijication of Acetylcholine- Receptor-Rich Membrane Fragments kg freshly dissected electric organ from Torpedo marmorata was minced into 5-g pieces, resuspended in its volume of cold (4 C) twice-distilled water containing 0.1 mm phenylmethylsulfonyl fluoride (PhMeS0,F) and 0.02% NaN, (solution 1) and homogenized by 150-g batches of tissue in a Virtis homogenizer for 2 x 1 min at maximal speed. The homogenate was then centrifuged at low speed in a Beckman JA 10 rotor at 5000 rev./min for 10 min. The supernantant (S,) was collected and the pellet (Pl) homogenized again in two volumes of solution 1 and centrifuged under the same conditions as the crude homogenate. The supernatants (S,) and (S,) were combined to yield (SJ, which was centrifuged in a Beckman JA 10 rotor at 7000 rev./min for 120 min. The supernatant was discarded and the pellet (P3) resuspended in a minimal volume of 2 M sucrose in solution 1 with a conical all-glass homogenizer, adjusted to 32% (w/w) sucrose with solution 1 and sonicated under nitrogen for 4 x 15 s at maximum power with a Siduse US-77-5 bath sonicator at a frequency of 1 MHz; 25 ml of the homogenized pellet (El) were then layered on top of a discontinuous sucrose gradient (25 ml at 41.5%, w/w, 15 ml at 37.5%, w/w, 10 ml at 35%, w/w) in a 75-ml tube, and centrifuged in a Beckmann 35 Ti rotor for 6 h at rev./min. After this first high-speed centrifugation, the 32 /, sucrose layer and the pellet (P4) were discarded and 15 ml of 36% sucrose injected between the 35% and 37.5% layers in order to collect the 35% (S,) and the 37.5% (E,) layers separately; (E,) was diluted with one volume of solution 1, centrifuged at rev./min in a Beckman 35 Ti rotor for 60 min and the resulting pellet resuspended, homogenized and sonicated as P, ; 10 ml of the resuspended pellet were layered on top of a 30-ml continuous /, sucrose gradient in solution 1 and centrifuged in a Beckmann SW 27 rotor at rev./min for 6 h. Fractions of 1.5 ml were collected and assayed for protein and a-toxin binding sites. 4- ( N-Maleimido) -phen~l-[~h] trimethylammonium Labelling of Acetylcholine-Receptor-Rich Membrane Fragments The acetylcholine-receptor-rich membrane fragments ( nmol ~-[~H]toxin sites/g protein were labelled with 4-(N-maleimid0)-phenyl-[~H]-trimethylammonium following the procedure of Karlin [12], as described by Barrantes et al. [13]. The suspension of receptor-rich membrane fragments was incubated for 10 min at room temperature with 1 mm dithiothreitol in buffer (O.1 M NaC1, 1 mm EDTA, 0.01 M Tris-HC1,pH 8). The reaction was stopped by adding 0.5 M sodium phosphate buffer, ph 6.7, and cooling to 0 C. After centrifugation at xg for 90 min, the pellet was resuspended in buffer 1 (0.15 m NaC, 1 mm EDTA, 0.01 M sodium phosphate buffer ph 7.0). An aliquot was incubated for 90 min at 20 C with an excess of a-toxin from Naja nigricollis, the remainder being stored for the same length of time at the same temperature. The two samples were then labelled in parallel with 4-(N-maleirnid0)-phenyl-[~H]- trimethylammonium (about 2 Cijmmol) for 2 min at 25 C. The labelling was stopped by adding an excess of 2-mercaptoethanol. Polyacrylamide Gel Electrophoresis Native gel electrophoresis in Triton X-100 was performed at ph 7.5 as described by Meunier et al. [9]. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed on 1.5-mmthick slab gels by the method of Laemmli [14], as modified by Anderson and Gesteland [15]. The running and the stacking gels contained respectively 10% and 5 % acrylamide, and 0.13 /, N, N -methylenebisacryl-

3 A. Sobel, M. Weber, and J.-P. Changeux 217 amide; pl samples containing pg proteins, 2% sodium dodecyl sulfate, M Tris-HC ph 6.8, 10% glycerol and 1 /, 2-mercaptoethanol were incubated for 5 min at 100 C and then layered on to the gel. The electrophoresis was carried out at a constant current of 30 ma ( V) for 3-4 h. n one case (Fig. 2B) a 5-15x linear gradient polyacrylamide gel has been used, following the procedure of Ames [16]. The gel was then fixed in 25% isopropanol/lo% acetic acid/65 /, water for 1 h, stained overnight with 0.25% Coomassie brilliant blue in the same medium, and destained using the fixation medium. Destained gels were scanned with a Vernon gel scanner and mobilities (RF) estimated from relative displacement to the tracking dye (bromophenol blue). Standard proteins and their subunit molecular weight were : rabbit muscle phosphorylase (94 000), bovine serum albumin (68 000), ovalbumin (45 000), chymotrypsinogen (25 000). 4-(N-Maleimido)-phenyl- [3H]trimethylammoniumlabelled peptides were revealed on the slab gel by fluorography, following the procedure described by Bonner and Laskey [17]. The developed film was scanned as the Coomassie-stained gel. Purification of the Acetylcholine Receptor Acetylcholine-receptor-rich membrane fragments were supplemented to a final concentration of about 15 mg protein/ml with 100 mm NaCl, 10 mm Tris- HC1 ph 7.4, 15 mm 2-mercaptoethanol and 4% detergent: Triton X-100, Berol 043 or sodium cholate. After incubation at room temperature for 30 min, ml of the mixture was layered on top of a 11-ml linear, 5-20% (w/v) sucrose gradient in 100 mm NaCl, 10 mm Tris-HC1 ph 7.4, 15 mm 2-mercaptoethanol, 0.1 % Triton X-100,0.06% BerolO43 or 0.01 % cholate and centrifuged for 16 h at rev./min in a Beckman SW 41 rotor. Fractions of approximately 0.5 ml were collected and their acetylcholine receptor content estimated either by direct counting of the radioactivity of an aliquot previously labelled with cr-[3h]toxin or 4-(Nmaleimido)-phenyl- [3H]trimethylammonium or by the standard filtration assay with ~-[~H]toxin. soelectric Focussing Liquid-phase isoelectric focussing was carried out in an 80-ml glass column similar to that described by Joseleau-Petit and Kepes [18]. The anode solution contained 0.2% H2S04 in 50% sucrose and the cathode solution was 0.15 M NaOH. The focussing was carried out in a 1 % solution of Ampholine ph in a /, sucrose gradient supplemented with various concentrations of urea dissolved immediately before use as specified in Results. After a 30-min prerun at 400 V, focussing was achieved in 15 h, with a final current of 0.6 ma and a final voltage of 900 V. Fractions of 1.5 ml were collected and the ph measured immediately in each fraction before neutralization with 10 p1 of 2 M Tris-HC1 ph 7.4. Finally, the acetylcholine receptor was assayed in each fraction. Chemical Sources Acrylamide bisacrylamide, N, N,N, -tetramethylethylenediamine, 2-mercaptoethanol were from Eastman-Kodak Ltd ; sodium dodecyl sulfate from Serlabo; Triton X-100 from Sigma; Berol 043 from Berolkemi AB ; sodium deoxycholate, sodium cholate, urea (analytical grade) from Merck. 4-(N-Maleimido)-phenyl- [3H]trimethylammonium was synthesized by Dr Pichat at the CEA (Saclay). Actin was a gift from Dr Whalen and tubulin was a gift of Dr Karsenti. RESULTS ACETYLCHOLNE-RECEPTOR-RCH MEMBRANE FRAGMENTS Cohen et al. [8] have described a fractionation method of the electric organ of Torpedo marmorata, which yields membrane fragments highly enriched in acetylcholine receptor protein with a maximal specific activity of 3000 nmol/g but which still present a significant heterogeneity. We present here several improvements of this method. An mproved Method of Purification Use of Anti-proteolytic Agents. As an improvement of the method of Cohen et al. [8] two protease inhibitors : phenylmethylsulfonyl fluoride (PhMeS0,F) and EDTA have been tested. n the presence of 0.1 mm PhMeSO,F, the acetylcholine-receptor-rich membranes migrate on a sucrose gradient exactly as they do in the absence of PhMeS0,F. On the other hand, in the presence of 1 mm EDTA many proteins, including the Na+-K ATPase and acetylcholinesterase, migrate with the acetylcholine-receptor-rich membranes on a sucrose gradient, resulting in a decrease of their specific activity. Also, it was noticed that, in the presence of 10 mm Ca2+, most of the membrane material migrates to the bottom of the tubes. n the final purification method, all the steps have been carried out in the presence of 0.1 mm PhMeS0,F to avoid proteolysis and 0.02 NaN, to inhibit bacterial growth.

4 218 Purified Membrane-Bound and Detergent-Extracted Acetylcholine Receptors - Fresh electric oraan from Toroedomormoraia Homogenization in 1 vol. soh 1 Centrifugation?OOOx g, 10 min p1 s1 Homogenization 1 vol.soln1 Centrifugation 7OOOxg, Omin p2 S2 s12 centrifugation 10000% g, 120 min 1 Resuspension in 32% sucrose Sonication Centrifugotion 1OOOOOx~, 5h, on discontinuous sucrose gradient (41.5%-37.5% - 34%) Receptor enriched membrane fraction ( 325% sucrose) Dilution - Centrifugation OOOOOxg. 60min Resuspension in 32% sucrose 2 Centrif ugot ion rg, 5 h on continuous 35% -43% sucrose gradient Pool of AChR- rich fractions M Triton X-100 solubilisation Centrifugation SW rev /rnin, 16h on continuous 5%- 20 % sucrose gradient with 0.01 M 2-rnercaptoethanol Purified AChR A Fig. 1. Scheme of the purijication procedure of the acetylcholinereceptor-rich membrane fragments and of the acetylcholine receptor protein. AChR, acetylcholine receptor Table 1. Large-scale purification of the acetylcholine-receptor-rich membrane fragments and of the acetylcholine receptor protein from the electric organ of Torpedo marmorata by successive sucrose gradient centrifugations Values are given for a purification starting from 1 kg fresh electric organ Fraction Proteins Acetyl- Specific choline activity receptor u-[~h]- toxin binding sites mg nmol nmol/g Low-speed supernatants (S12) Sample for the discontinuous sucrose gradient (El) Sample for the continuous sucrose gradient (E2) Purified acetylcholine-receptorrich membrane fragments (M Purified acetylcholine-receptor protein (R) Large-Scale Purijication of Acetylcholine-Receptor- Rich Membrane Fragments. To improve the yield and purity of acetylcholine-receptor-rich membrane fragments, several modifications of the method of Cohen et al. [8] have been adopted (see Methods). The homogenization was done in one instead of two volumes of buffer 2. After the first low-speed centrifugation, the pellet was rehomogenized and centrifuged in the same conditions, but the membrane fragments of the two pooled supernatants were concentrated and separated from soluble proteins by centrifugation before further purification. This gives concentrated samples, which were sonicated to prevent aggregation and then centrifuged on two consecutive sucrose gradients. A discontinuous gradient centrifugation first yielded membrane fragments with a specific activity of the same order as that of the Cohen et al. [8] preparation, but in larger amounts. Then, a centrifugation on a continuous sucrose gradient gives rise to a membrane preparation with the highest specific activity. From 1 kg electric organ, this purification procedure (Fig. 1) yields membrane fragments (M) containing 140mg of protein and 560 nmol of C- [3H]toxin sites (Table l), in other words with a specific activity of 4000 nmol/g for the acetylcholine receptor. The best specific activity obtained was 4500 nmol/g. This value is approximately one half of the maximal specific activities found in several laboratories for the purified acetylcholine receptor protein in its detergentsoluble form (see Discussion). Any attempt to further purify these membrane fragments has failed. Washing with 0.2 or 0.4 M KCl, as well as dialysis against distilled water, did not remove any significant amount of protein. Properties of the Highly Purified Acetylcholine-Receptor-Rich Membrane Fragments Polypeptide Composition. The highly purified acetylcholine-receptor-rich membrane fragments were submitted to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate following the procedure of Anderson et al. [15] (see Methods). Four main polypeptide chains of apparent molecular weights 40000~1000; ; and are, in general, present (Fig. 2A,B). f the ;etylcholine-receptor-rich membrane fragments have been previously labelled with 4-(N-maleimido)-phenyl- [3H]trimethylammonium, a covalent affinity reagent specific of the cholinergic receptor site [12], fluorography [71 of the gel reveals that, in agreement with the results of Karlin and collaborators [12], only the Mr band is radioactive (Fig. 2A). On the other hand, if the electrophoresis is carried out with the system of Ames [16] three bands are observed (Fig. 2B) ; this method does not give a separation of the nd M, chains.

5 A. Sobel, M. Weber, and J.-P. Changeux 219 from 1 :1 to 2:l. The high proportion of proteins in these membranes may explain their remarkably high density (1.17 g/cm3). E T.D. t Fig. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the acetylcholine receptor-rich membrane fragments. (A) Membrane fragments have been labelled with 4-(N- maleimido)-phenyl-[3h]trimethylammonium in the absence or presence of a-toxin, and 20 pg protein were submitted to electrophoresis according to Anderson et al. [15]. () Scan of the Coomassiebrilliant-blue-stained gel on a 10% acrylamide gel. T.D., tracking dye; A and T, location of actin and tubulin when added to the sample. (1 and 111) Scans of the films obtained after fluorography [17] of the gels obtained with 4-(N-maleimid0)-phenyl-[~Htrimethylammonium labelled membrane fragments in the absence (11) or the presence (111) of a-toxin to protect the receptor binding site. (B) A given fraction of acetylcholine-receptor-rich membrane fragments has been submitted to polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate () on 10% acrylamide gel according to Anderson et al. [15] and (11) on a 5-15% acrylamide gradient gel according to Ames [16]. T.D., tracking dye Regarding the nature of the other components, co-migration with actin reveals that only a minor band with an apparent M, of in the sodium dodecyl sulfate gel could be actin. On the other hand, tubulin did not co-migrate with the band of M, (Fig. 2A). Other Properties. By electron microscopy and after negative staining, the preparation of receptor-rich membranes appears to contain fragments of variable size (0.1-1 pm diameter), nearly all of them being covered with a high density of 8-nm rosettes similar to those observed with detergent-purified acetylcholine receptor protein [9,19] (and this paper). The protein : lipid ratio of the acetylcholine-receptor-rich membranes ranges, with increasing purity, T.D t DETERGENT-SOLUBLZED ACETYLCHOLNE RECEPTOR PROTEN Solubilization and Purijication of the Acetylcholine Receptor Protein Effect of 2-Mercaptoethanol. Solubilization of the acetylcholine receptor from the acetylcholine-receptorrich membranes can be achieved with neutral detergents, such as Triton X-100 and Berol 043, or with anionic detergents such as sodium cholate or sodium deoxycholate. t is known [6,20,21] that Torpedo acetylcholine receptor solubilized from crude homogenates, migrates on a sucrose gradient as at least two main peaks, with apparent sedimentation coefficients of 9 S and 12 S, and as even higher aggregates. n the absence of salt, acetylcholine receptor solubilized from the acetylcholine-receptor-rich membrane fraction appears to exist as very large aggregates, most of which pellet under the same conditions of sucrose gradient centrifugation (Fig. 3). High salt (1 M NaCl) or urea (1 or 2 M) partly dissociate these large aggregates but never give a single peak. On the other hand, in the presence of 0.01 M 2-mercaptoethanol, the acetylcholine receptor protein solubilized by either Triton X-100 or sodium cholate migrates as a single sharp peak on sucrose gradient with a standard sedimentation coefficient of 9 S (Fig. 3). No effect of 2-mercaptoethanol could be observed on the binding of acetylcholine to the acetylcholine receptor (Wollman F. A., unpublished results) ; however, at concentrations higher than 0.01 M 2-mercaptoethanol a significant loss of ~-[~H]toxin binding takes place. This latter effect might be caused by the reduction of some essential disulfide bridges of the toxin by 2-mercaptoethanol. PuriJication by Centrifugation on Sucrose Gradients. Acetylcholine-receptor-rich membrane fragments were therefore solubilized in the presence of 0.01 M 2-mercaptoethanol and 4% Triton X-100 or Berol 043 and centrifuged in a sucrose gradient in the presence of 2-mercaptoethanol (Fig. 1). Table 1 gives the results for a typical purification. The best preparation had a specific activity of 9000 nmol/g, which is in the range of the specific activities found in other laboratories for purified acetylcholine receptor protein (see Discussion). Criteria of Homogeneity After centrifugation on a sucrose gradient, the acetylcholine receptor appears as a single and symmetrical peak of standard sedimentation coefficient 9 S. The proteins were not measured along the gradient as 2-mercaptoethanol interferes with the protein assay.

6 220 Purified Membrane-Bound and Detergent-Extracted Acetylcholine Receptors Fraction number Fig. 3. Sucrose gradient centrifugation of detergent-solubilized acetylcholine receptor. (A) A crude membrane fraction was solubilized with 1 % Triton X-100 and centrifuged for 16 h at rev./min in a Beckman SW-41 rotor on a 5-20% (w/v) sucrose gradient containing 10 mm Tris-HC1 ph 7.4, 100 mm NaCl, 0.1 % Triton X-100,0.02% NaN, and 0.1 mm PhMeS0,F. Fractions were approx. 0.5 ml in volume. (B) and (C) Purified acetylcholine-receptor-rich membrane fractions were solubilized with 4 % BerolO43, preincubated with trace amounts of ~-[~H]toxin, and centrifuged for 16 h at rev./min in a Beckman SW-41 rotor on a 5-20% (w/v) sucrose gradient containing 10 mm Tris-HC1 ph 7.4, 0.06% BerolO43, 0.02% NaN,, 0.1 mm PhMeS0,F and (B) 1 M NaCl (0) or 1 M urea (0) or (C) 10 mm 2-mercaptoethanol By polyacrylamide gel electrophoresis in the presence of a non-denaturing detergent, the purified acetylcholine receptor migrates as a single band stained by Coomassie brilliant blue (Fig. 4). Electron microscopy after negative staining of the purified acetylcholine receptor preparation shows an homogeneous population of 8-nm rosette-like particles similar to those observed with highly purified acetylcholine-receptor-rich membranes and with purified acetylcholine receptor protein from Electrophorus electricus [5]. Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecyl sulfate gives 3 polypeptide bands stained with Coomassie blue of apparent molecular weights f 1000; f 2000 and 66000)2000 (Fig. 6). The M, band and a few minor ones have therefore been eliminated from the membrane extract by sucrose gradient centrifugation. n addition, in some preparations, the M, and M, bands appear markedly reduced. Finally, the purified acetylcholine receptor, partially labelled with 4-(N-maleirnid0)-phenyl-[~H]trimethylammonium, was submitted to liquid-phase isoelectric focussing as described in Methods. The position of the acetylcholine receptor in the fractions collected after 15 h of focussing was followed both by measuring the radioactivity of 4-(N-rnaleimid0)-phenyl-[~H]trimethylammonium and by assaying the free receptor sites with ~-[~H]toxin (cf. Methods). When 3 or 4 M urea were present during focussing only the 4-(N-maleimido)-phenyl-[3H]trimethylammonium could be followed as a significant loss of ~-[~H]toxin binding sites takes place. The isoelectric point of the acetylcholine receptor was 5.3 in 1 and 2 M urea, and shifted to 5.6 in 3 and 4 M urea. n all cases, the acetylcholine Fig. 4. Polyacrylamide gel electrophoresis of the purified acetylcholine receptor in non-denaturing conditions. 30 pg purified acetylcholine receptor protein has been run on a polyacrylamide gel at ph 7.5 and containing 1 /, Triton X-100 (see Methods). The proteins on the gel have been stained with Coomassie-blue and scanned at 550 nm. T.D., tracking dye receptor peak was rather broad, covering more than one ph unit (Fig. 5). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the fractions along the ph gradient showed only minor differences between themselves and the original sample (see next paragraph). SUBUNT COMPOSTON OF THE ACETYLCHOLNE RECEPTOR To test the possibility that the acetylcholine receptor protein purified by our method is still heterogene-

7 A. Sobel, M. Weber, and J.-P. Changeux Fraction number Fig. 5. soelectric focussing profile of the purified acetylcholine receptor. 4 nmol purified, partially 4-(N-maeimid0)-phenyl-[~H]trimethylammonium-labelled acetylcholine receptor have been applied on a 50-ml sucrose gradient containing 1 % Ampholine ph , 1 M urea, 1 % Triton X-100 and 10 mm 2-mercaptoethanol. Electrofocussing was carried out for 15 h at approx. constant power, with a final voltage of 900 V. Fractions of 1.5 ml were collected and the ph of each fraction was measured (0). Each fraction was then neutralized with 2 M Tris-HC1 buffer, ph 7.4. The radioactivity due to 4-(N-maleimido)-phenyl-[3H]trimethylammonium (0) was measured in aliquots and the acetylcholine receptor (A) was assayed according to Meunier et al. [9] i 40 m 40 ]ADAD, Gel Length m D c m - - Gel length Fig. 6. Densitometric scans of polyacrylamide gels obtained after electrophoresis in the presence of sodium dodecyl sulfate and Coomassie blue staining of the proteins of preparations of increasing purity (-) of (A) acetylcholine-receptor-rich membrane fragments and (B) detergent- solubilized andpuri$ed acetylcholine receptor. Specific activities in nmol/g: () 6000; (11) 7000; (111) T.D., tracking dye. Numbers above the bands indicate x apparent molecular weights of the corresponding polypeptide chains ous, the peptide composition of samples with different specific activities was investigated in a systematic manner by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Fig. 6 shows scans of several specimens of highly purified acetylcholine-receptor-rich membranes and acetylcholine receptor protein after Coomassie blue staining. t is clear that the ratios of the three major bands of the purified acetylcholine receptor preparation of apparent molecular weights 40000, and vary significantly among these samples in a way related to their specific activities. Quantitative comparison of these ratios from the scans appears valid, since the intensity of staining of a given band was shown to be proportional to the amount of protein present in that band. Under these conditions the ratios of the and M, bands to the M, one varied from 0.15 to A variation of these ratios was also noticed among the different fractions taken along the detergent-con-

8 222 Purified Membrane-Bound and Detergent-Extracted Acetylcholine Receptors taining sucrose gradient and the isoelectric focussing column. These observations indicate that the stoichiometry of the different polypeptide chains is not constant. As the M, component is the only one labelled by 4-(N-maleimido)-phenyl-[3H]trimethylammonium, the two others may not be integral parts of the acetylcholine receptor molecule. DSCUSSON n this paper we describe large-scale methods of purification of the acetylcholine receptor in its membrane-bound and detergent-solubilized forms without using affinity chromatography. Acetylcholine-receptor-rich membrane fragments have been purified in several laboratories [8, with maximal specific activities close to 3000 nmol/g. The procedure reported in this paper yields membrane preparations of specific activities as high as 4500 nmol/g, in which more than 99% of the fragments are densely covered with characteristic acetylcholine receptor particles. The method has also the advantage of being simple and giving very large quantities of membrane fragments (up to 140 mg in proteins from 1 kg of electric organ) with a high recovery of up to 50%. These highly purified membrane fragments respond in vitro [25,26] to cholinergic agonists by an increase of 22Na permeability and are therefore excitable (M. Briley & J. L. Popot, unpublished results). They also exhibit characteristic changes of extrinsic fluorescence in the presence of cholinergic ligands after labelling by quinacrine [27]. They constitute an excellent preparation to study the functional properties of the acetylcholine receptor in its membrane environment. The polypeptide chain composition of these membrane fragments revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is remarkably simple : only four polypeptide chains of apparent molecular weights _+ 1000; k 1000 ; k 2000 and n preparations [22,24] made from Torpedo calijornica five bands of M approximately 40000; 50000; 60000; and were found. With preparations of low purity we also observe small amounts of a M, band. The presence of the M, component in our membranes from T. marmorata and the presence of the M one in those from T. californica may be due to a species difference. However, the resolution of the gel system used may not have been good enough to separate polypeptides with apparent molecular weights of and (Fig. 2B). n all instances the M, band is labelled by Karlin s affinity reagent and therefore carries the acetylcholine receptor site. These membranes have a very low content in lipids, which explains their high density. The highly purified acetylcholine-receptor-rich membrane fragments observed by electron microscopy appear covered by 8 nm acetylcholine receptor molecules, with a surface density close to that of the 8-nm particles observed in situ in the subsynaptic membrane [28,30] and 3-5 times smaller than that of u-rh]- toxin binding sites measured under the nerve terminal [29,311. The acetylcholine-receptor-rich membranes therefore consist of pure subsynaptic membrane fragments. Since the acetylcholine receptor protein represents nearly 50% of the proteins in these membrane fragments, they have been used as a starting material for its solubilization and purification without using affinity chromatography. The method of purification described is simple and consists of a single ultracentrifugation step following detergent solubilization. The omission of affinity chromatography from the purification procedure is of importance, since this technique gives preparations of receptor protein with modified binding properties [7] as a consequence of either a change of conformation or a selection of a particular sub-population of receptor molecules. An interesting property of the acetylcholine receptor, which made this purification possible, concerns the effect of 2-mercaptoethanol. n its absence the acetylcholine receptor from Torpedo electric organ sediments as (at least) two peaks in a sucrose gradient [6,20,21]. n its presence, however, the acetylcholine receptor migrates as a single, sharp peak. The disaggregation by 2-mercaptoethanol or dithiothreitol may involve reduction of inter and/or intramolecular S-S bonds (see Note 1). The purified acetylcholine receptor fraction appears homogeneous by several criteria: (a) it gives a single and symmetrical peak by centrifugation on sucrose gradient; (b) it migrates as a single protein band by polyacrylamide gel electrophoresis under non-denaturing conditions; (c) by electron microscopy, it is composed of a single class of particles; (d) further conventional purification steps do not result in an increase of specific activity. The specific activity of the purest acetylcholine receptor fractions was 9000 nmol/g, a value close to the highest reported in the literature for acetylcholine receptor purified by affinity chromatography [cf. 6,19, The broadness of the acetylcholine receptor peak given by liquid-phase isoelectric focussing suggests that a micro-heterogeneity of the purified protein exists. The analysis of the polypeptide chain composition of several fractions leads to the same conclusion. The polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the purest fractions give three polypeptide components with apparent

9 A. Sobel, M. Weber, and J.-P. Changeux 223 molecular weights of ; 50000~2000 and f Several bands have also been found by various authors with their purified fractions of acetylcholine receptor from Torpedo marmorata [35-371, T. nobiliana [33], T. ocellata [34] and T. californica [19,23,32,38,39]. n several instances [32,38] a constant ratio between the four bands present in the preparation have been reported with stoichiometries of 4:2:1:1 calculated from the amino acid analysis of bands [32] or 1.0:0.3 :0.2:0.3 calculated from the ratio of the staining intensities divided by the apparent molecular weight of each component [38]. By this last method we find, for our best fraction of acetylcholine receptor from T. marmorata, three bands with a ratio of 11 : 2 : 1. Patrick et al. [40] have suggested that components of molecular weight smaller than arise from proteolytic digestion. This does not seem to be the case, since the whole purification is done in the presence of 0.1 mm PhMeSO,F, a potent protease inhibitor. n addition, Reiter et al. [41] have shown that a M chain is labelled in vivo with 4-(N-maleimido)-benzyl- [3H]trimethylammonium and the same apparent molecular weight is found for the 4-(N-maleimido)-benzyl- [3H]trimethylammonium or 4-(N-maleimido)-phenyl- [3H]trimethylammonium-labelled chain after purification of the acetylcholine receptor protein [12] (and this paper). n our case the ratio of the three bands has been shown to differ from one preparation to another of either acetylcholine-receptor-rich membrane fragments or purified acetylcholine receptor protein. n all instances the contribution of the M component increased with the specific activity of the preparation. Also the successive fractions given by sucrose gradient centrifugation or by isoelectric focussing showed a slight but significant variation of the ratios of the three bands. Most likely, the and M dalton components are contaminants of the acetylcholine receptor protein. The pure protein should then give only one band corresponding to a molecular weight of This M band may, however, be made up of a single or of several polypeptide chains with the same apparent M. n the case of the purified acetylcholine receptor from Electrophorus electricus two bands have been generally reported [9,12,42,43] with apparent molecular weights of about and ; a third band at M has been also observed [12] (Sobel, unpublished). Only the M carries the acetylcholine binding site. The role of the other chain is unknown: it might, for instance, be the ionophore or a contaminant. Suggestions in favor of the last possibility have been presented by Valderrama et al. [44]. These authors have shown that the immunological cross-reactivity between acetylcholine receptor from Electrophorus and Torpedo is due only to the M, chain. Studies done in parallel on the 35S-labelled acetylcholine receptor from calf myotubes in culture (Merlie, Changeux and Gros, unpublished results) also indicate that only the M, component is present in this purified receptor molecule (see Note 2). How then to explain that the M and the M chains copurify with the acetylcholine receptor? t is known with other membrane proteins, such as the proteins El, E, and E, of the Semliki Forest virus that stable complexes between different polypeptide chains form upon detergent solubilization [45]. A similar phenomenon may occur between the acetylcholine receptor and other proteins present in the membrane. The co-micelles once formed would be so difficult to dissociate that the contaminant peptide chain would behave as authentic subunits of the acetyb choline receptor molecule. We thank Dr P. Boquet for a gift of a-toxin from Najd nigricollis and Drs Menez, Morgat and Fromageot for its tritiation. We are indebted to Dr J. Cartaud for the electron micrographs of the acetylcholine receptor in its purified and membrane-bound forms. We are grateful to Drs M. Briley and J. P. Merlie for helpful discussions and to Dr R. Whalen for the gift of a sample of purified actin. This work was supported by funds from the Collkge de France, the Centre National de la Recherche Scientijique, the nstitut National de la Santl. et de la Recherche mhdicale, the DLlhgation Ghnhrale a la Recherche Scientijique et Technique and the Commissariat a Energie A tomique. REFERENCES 1. Sobel, A. & Changeux, J. P. (1977) Biochem. SOC. Trans. 5, Briley, M. & Changeux, J. P. (1977) nt. Rev. Meurobiol. in the press. 3. Changeux, J. P. (1975) Handb. Psychopharmacol. 6, Karlin, A. (1974) Life Sci. 14, Changeux, J. P., Benedetti, L., Bourgeois, J.-P., Brisson, A., Cartaud, J., Devaux, Ph., Grunhagen, H., Moreau, M., Popot, J.-L., Sobel, A. Weber, M. (1976) Cold Spring Harbor Symp. Quant. Biol. 40, Potter, L. 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10 224 A. Sobel, M. Weber, and J.-P. Changeux: Purified Membrane-Bound and Detergent-Extracted Acetylcholine Receptors 18. Joseleau-Petit, D. & Kepes, A. (1975) Biochim. Biophys. Acta, 406, Eldefrawi, M. E. & Eldefrawi, A. T. (1975) Ann. N.Y. Acad. Sci. 264, Gibson, R. E., O Brien, R., Edelstein, S. J. &Thompson, W. R. (1976) Biochemistry, 15, Raftery, M. A., Schmidt, J. & Clark, D. G. (1972) Arch. Bioehem. Biophys. 152, Flanagan, S. D., Barondes, S. H. & Taylor, P. (1976) J. Biol. Chem. 251, Nickel, E. & Potter, L. T. (1973) Brain Res. 57, Reed, K., Vandlen, R., Bode, J., Duguid, J. & Raftery, M. A. (1975) Arch. Biochem. Biophys. 167, Kasai, M. &Changeux, J. P. (1971) J. Membrane Biol. 6, Hazelbauer, G. L. & Changeux, J. P. (1974) Proc. Natl Acad. Sci. U.S.A. 71, Griinhagen, H. & Changeux, J. P. (1976) J. Mol. Biol. 106, Cartaud, J. (1975) Exp. Brain Res. 23, suppl Fertuck, H. C. & Salpeter, M. M. (1976) J. Cell Biol. 69, Rosenbludt, J. (1975) J. Neurocytology, 4, Bourgeois, J. P., Ryter, A,, Menez, A,, Fromageot, P., Boquet, P. & Changeux, J. P. (1972) FEBS Lett. 25, Raftery, M. A,, Vandlen, R. L., Reed, K. L. & Lee, T. (1976) Cold Spring Harbor Symp. Quant. Biol. 40, Ong, D. E. & Brady, R. N. (1974) Biochemistry, 13, Riibsamen, H., Montgomery, M., Hess, G. P., Eldefrawi, A. T. & Eldefrawi, M. E. (1976) Biochem. Biophys. Res. Commun. 70, Heilbronn, E. & Mattson, C. (1974) J. Neurochem. 22, Mattson, C. & Heilbronn, E. (1975) J. Neurochem. 25, Gordon, A., Bandini, G. & Hucho, F. (1974) FEBS Lett. 47, Karlin, A., Weill, C., McNamee, M. & Valderrama, R. (1976) Cold Spring Harbor Symp. Quant. Biol. 40, Hucho, F., Layer, P., Kiefer, H. R. & Bandini, G. (1976) Proc. Nail Acad. Sci. USA. 73, Patrick, J., Boulter, J. & O Brien, J. C. (1975) Bioehem. Biophys. Res. Commun. 64, Reiter, M. J., Cowburn, D. A,, Prives, J. M. & Karlin, A. (1972) Proc. Nail Acad. Sei. U.S.A. 69, Lindstrom, J. & Patrick, J. (1974) in Synaptic Transmission and Neuronal nteraction (Bennett, M. V. L., ed.) pp , Raven Press, New York. 43. Biesecker, G. (1973) Biochemistry, 12, Valderrama, R., Weill, C. L., McNamee, M. & Karlin, A. (1976) Ann. N.Y. Acad. Sci. 274, Simons, K., Helenius, A. & Garoff, H. (1973) J. Mol. Biol. 80, A. Sobel, M. Weber, and J.-P. Changeux, Laboratoire de Neurobiologie Moleculaire, nstitut Pasteur, 25/28 Rue du Docteur-Roux, F Paris-Cedex-15, France Notes Added in Prmc 1. A similar observation has been independently made by H. W. Chang and E. Bock (Biochemisrry, in press) with purified acetylcholine receptor protein from Torpedo ealifornica. 2. n the course of the present purification of the detergentextracted receptor protein from T. nmrmorata a membrane component made up almost exclusively of the M, chain can be recovered from the pellet of the last centrifugation step. Fluorescence studies done with quinacrine indicate that this protein carries a site for the fluorescent local anesthetic quinacrine and for the frog toxin histrionicotoxin. The M, chain. which is absent in the purified protein, might then be part of the acetylcholine ionophore (Sobel, A,, Heidmann, T., Hofler, J. & Changeux, J. P., Proc. Nut1 Acad. Sci. U.S.A., in the press).