Characterization of acetylcholine receptor-rich and acetylcholinesterase-rich membrane particles from Torpedo californica electroplax

ARCHIVES OF BlOCHEMlSl’BY AND BIOPHYSICS 167,138-144 (19%)’

Characterization of Acetylcholine Receptor-rich and

Acetylcholinesterase-Rich Membrane Particles

from Torpedo californica Electroplax

K. REED, R. VANDLEN, J. BODE, J. DUGUID, AND M. A. RAFTERY

Church Laboratory of Chemical Biology, Division of Chemistry and Chemical Engineering,’ California Institute of Technology, Pasadena, California 91109

Received September 4, 1974

Large-scale purification of acetylcbolinesterase-rich and acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax is described. Electron microscopy studies reveal structural differences in the two types of particles and the results are discussed in terms of structural aspects of the postsynaptic cleft. Polyacrylamide gel electrophoresis of receptor-rich fragments reveals that the fragments contain the same polypeptide components observed in receptor preparations purified from the same electroplax membranes, indicating that purified Torpedo receptor is not composed of species degraded by proteolysis. Results obtained from fluorescence studies of a cholinergic analog allow conclusions to be reached regarding species differences in electroplax acetylcholine receptor preparations.

The clear separation obtained between acetylcholine receptor-rich (AcChR2-rich) and acetylcholinesterase-rich (AcChE- rich) membrane particles from Torpedo californica by sucrose density-gradient centrifugation (1) prompted the large-scale isolation of such particles as described in this communication. Provided that such particles are enriched enough in the specific receptor and enzyme, respectively, they can be used for studies of ligand binding, the kinetics of such binding, electron microscopy, and X-ray analysis since they represent these important molecules in their native environments. In this communication we describe the large-scale isolation of these particles and their charac-

‘Contribution No. 4953. This research was sup- ported by UPHS-NS 10294. J. B. was the recipient of a fellowship from the Deutscheforschungsgemein- schaft and M.A.R. of a USPHS Research Career Development award.

2 Abbreviations used are: acetylcholine receptor, AcChR; acetylcholinesterase, AcChE; a-bungaro- toxin, a-Bgt; 1-(5-dimethylaminonaphthalene-l-sulfonamide)ethane-3trimethylammonium iodide (Dns- chol).

1 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

terization by electron microscopy, SDS gel electrophoresis and by the binding of a fluorescent cholinergic analog which has previously been used by Cohen et al. (2) to detect a specific agonist-induced conformation change in receptor-rich particles from the electroplax of Torpedo marmorata. The results presented here are discussed in terms of the structure of the synaptic cleft and of the individual membrane particles enriched in AcChR or AcChE. The AcChR particles are also compared to isolated AcChR (3, 4).

EXPERIMENTAL

Preparation of membrane fragments. The electric organs were excised from Torpedo californica and rapidly frozen to -90°C. Organs were thawed just

prior to use. Similar results were obtained from organs removed from live animals and not subjected to freezing. Approximately 500 g of tissue were minced with scissors and transferred to a commercial Waring Blendor with 250 ml of 1 M NaCl solution in buffer (10 mM sodium phosphate pH 7.4). This suspension was then homogenized for 3 min at full speed. All homoge- nization and centrifugation steps were carried out at 4°C. The homogenate was centrifuged at 5000 rpm for 10 min in a Sorval GSA rotor to remove large pieces of

38

ACETYLCHOLINE RECEPTOR AND ESTERASE IN MEMBRANE PARTICLES 139

connective tissue and other debris. The supernatant was filtered through cheesecloth and centrifuged at 100,OOOg for 45 min in a Beckman LZ-65 centrifuge. The supernatant, containing soluble proteins, was discarded and the pelleted membrane fragments were resuspended in ‘125 ml of buffered 0.4 M NaCl using a Virtis-23 homogenizer at 75% of maximum speed for 4 min. This homogenate was then centrifuged at 100,OOOg for 45 min. The pellet was resuspended in 125 ml of buffer solution for 4 min as above and the membrane suspension was recentrifuged at 100,000g for 45 min. The final pellet was resuspended in 150 ml of buffer solution. Fifty-five milliliters of 60% sucrose

(wt/vol) buffered with 10 mM sodium phosphate was added to make the homogenate 16% in sucrose and to give a final volume of approximately 200 ml. Best reproducibility of results was obtained when this membrane suspension was allowed to sit at 4°C for 12-24 h before sucrose gradient centrifugation.

Sucrose density gradient. Further purification of the crude membrane suspension was carried out using a Beckman Ti-15 Zonal rotor. A discontinuous sucrose gradient was routinely used in these experiments. The gradient consisted of 20 layers of sucrose from 25%-55% (wt/vol) with a total volume of 1100 ml. All sucrose solutions were buffered with 10 mM sodium phosphate, pH 7.4. The gradient and sample were centrifuged at 30,000 rpm for 12 h at 4°C. Fractions of 25 ml were then collected and analyzed for protein, acetylcholine receptor, acetylcholinesterase, and ATPase activity.

Assays. Protein concentration was estimated by the method of Lowry (5) with bovine serum albumin as a standard. Acetylcholinesterase activity was determined by the method of Ellman (6) using acetylthiocholine as the substrate. The activity is expressed as the observed increase in optical density at 412 rim/s/ml of sample analyzed. The acetylcholine receptor present in the membrane fragments was determined by the binding of [‘Y]a-BgT using the method of Schmidt and Raftery (7). The binding is expressed as pg of toxin bound/ml sample. The ATPase activity was determined by the method of Post and Sen (8) with activity expressed in terms of rmoles ATP hydrolyzed/h per ml of sample.

SDS gel electrophoresis. SDS gel electrophoresis was performed essentially as described by Fairbanks et al. (9). Samples (50 pg) were denatured with 3% SDS, 3 mM E:DTA, 30 mM Tris, pH 7.4 at 100°C for 3 min. The protein sample was applied to gels containing 7.5’% acrylamide, 0.2% methylene bisacrylamide, 0.2% SDS, and run at a constant current of 7 mA/gel for 2.5 h. Bromophenol blue was used as the tracking dye. After electrophoresis the gels were removed from the gel tubes, stained with Coomassie Brilliant Blue and destained as described (9). Gels were scanned at 550 nm by a Gilford Spectrophotome- ter with a Linear Transport unit. Determination of

molecular weights was made by comparison with gels containing the molecular weight standards bovine serum albumin, aldolase, &galactosidase, and myo- globin. Nondenaturing gel electrophoresis was performed with gels containing 4% acrylamide, 0.2% methylene bisacrylamide in buffer containing 100 mM Tris, pH 7.4, 200 mM acetate, 0.1% Na cholate, or 0.1% Triton. Protein samples (lo-20 pg) were applied to the gels and electrophoresed at 6 mA/tube for 4 h. Staining and destaining was accomplished in the man- ner described above.

RESULTS

Centrifugation of Torpedo californica electroplax membrane particles prepared as described in the Experimental section on a large scale, results in an improved separation of particle types over that previously presented (1). As shown in Fig. 1, three main fractions are observed in the gradient; the lightest fraction contains a small amount of protein and has considera- ble AcChE activity, but has essentially no [1251]a-Bgt binding indicating the absence of AcChR molecules in these particles. The middle fractions (16-34) have a low protein content and contain approximately 60% of the total [1251]a-Bgt binding sites; these particles are, therefore, highly enriched in AcChR. The specific activity of the pooled fractions (M-28), compared with the specific activity of isolated purified AcChR (3, 4), is such that of the total protein in these fractions the AcChR content is approxi-

FIG. 1. Sucrose density gradient separation of Torpedo electroplax membranes. The AcChR-rich

fragments band at a sucrose density of 1.17 g/cc.

140 REED ET AL.

mately 20 to 50%, depending on the preparation. The most dense fraction found in the gradient, peaking at about fraction number 40, contains all of the ATPase activity in the preparation, approximately 30% of the [*251]a-Bgt binding sites and the majority of the AcChE activity (70-80s). Because of this complexity the most dense fraction was not studied further.

Negative staining of the AcChR-rich particles reveals closed structures (Fig. 2a) at least 80% of which show masses of ap- parently circular structures in the membrane. .These structures are approximately 80-90 A in diameter, and contain a central pit as shown in Fig. 2b. These results are similar to those of Nichol and Potter (10 and Cartaud et al. (11). Similar 80- to 90- d structures have been observed by Meunier et al. (12) for isolated purified AcChR from Electrophorus electricus; thus, it is reason- able to consider the circular structures as AcChR molecules embedded in the membrane.

Figure 2c shows a thin section of the AcChE-rich particles stained for AcChE activity by the method of Karnofsky and Root (13). When examined by negative staining procedures the AcChE-rich fragments are quite different from the AcChR- rich particles. Each fragment is sur- rounded by a halo of small particles and these are presumed to be AcChE molecules which are associated with a basic membrane structure (Fig. 2d).

We have previously shown (1) that SDS gel electrophoresis of AcChE-rich and AcChR-rich particles clearly shows great differences in the polypeptide chains which are present in each type of particle. In the studies reported here we compare the SDS gel electrophoresis pattern of the AcChR- rich particles with a similar pattern of AcChR purified from the same particles by affinity chromatography methods (3) (Fig. 3). The obvious difference between these samples is the presence in the membrane fragments but not in the purified preparation of a 105,000-M, component and of a 40,000-M, component which overlaps the AcChR subunit, as judged by a comparison of the relative ratios of the protein bands of the purified AcChR material. The larger

molecular-weight component has previously been shown (1) to be the most abun- dant polypeptide chain in whole unfrac- tionated electroplax membranes. These patterns further emphasize the enrichment of acetylcholine receptor molecules in these particles.

The AcChR-rich particles have been used to study cholinergic ligand binding (14, 15). We have also studied the binding of the fluorescent dye l-(5-dimethylaminonaphthalene-l-sulfonamide) ethane-3-trimethylammonium iodide (Dns-chol) origi- nally synthesized by Weber et al. (16) and first used to study binding to a proteo- lipid isolated from electroplax (17). More recently, Dns-chol has been used by Co- hen and Changeux (2) and by Cohen et al. (18) to determine its ability to bind to AcChR-rich fragments isolated from the electric organ of Torpedo marmorata. We have studied the binding of the Dns-chol to AcChE-rich fragments and to AcChR-rich fragments from the Torpedo californica and the results of these studies are shown in Fig. 4. For AcChE-rich fragments excitation at 287 nm leads to emission with a maximum at 527 nm, as shown on the left side of Fig. 4. Addition of a-Bgt does not result in any decrease in the emission intensity as expected, since the AcChE- rich fragments do not bind cy-Bgt. On the other hand, addition of carbamylcholine at various concentrations reduced the fluorescence intensity, without any shift in the wavelength of maximum emission. The maximum emission with AcChR-rich fragments is 516 nm. As shown on the right- hand side of Fig. 4, addition of a-Bgt to saturation causes a reduction in the intensity at this wavelength. Additionally, carbamylcholine at saturating concentrations, reduces the emission intensity to almost the same level as a-Bgt at saturation. There is no change in the wavelength of maximum emission upon addition of the agonist carbamylcholine to AcChR-rich fragments from Torpedo californica. Also, when isolated purified AcChR was first denatured by heating, the same amount of fluorescence intensity was observed at 516 nm as was found with AcChR poisoned with a-Bgt. Therefore, we conclude that

the Act Tor

C elec


FIG. 2. Electron microscopy of AcChR-rich and AcChE-rich membrane fragments.

binding of Dns-chol to isolated purified crose density gradients in zonal rotors pro- :hR or to AcChR-rich fragments from vides a preparative method for obtaining pedo cal’ifornica is largely nonspecific. large quantities of AcChE-rich and espe-

DISCUSSION cially AcChR-rich membrane fragments. Such quantities of enriched receptor frag-

:entrifugation of Torpedo californica ments allow study at various physical lev- ztroplax membrane fragments in su- els of the acetylcholine receptor in its

142 REED ET AL.

40 MEMBRANE FRAGMENTS

40 PURIFIED AcChR FROM FRAGMENTS

FIG. 3. SDS-gel electrophoresis of AcChR-rich and purified AcChR, according to Fairbanks et al. (9).

470 510 550 5!30nm 4x) 510 550 590nm Emission

FIG. 4. The binding of Dns-chol to AcChE-rich and AcChR-rich membrane fragments. Excitation of bound dye at 287 nm leads to emission as shown. Displacement of Dns-chol by carbamylcholine results in reduction of emission intensity. Dashed line depicts effects of o-B@.

native environment. This is especially important, since it has been shown (14) that the properties of the isolated purified receptor differ in some respect from the same receptor in its native membrane environment. Although this environment can be reproduced by reconstitution of the isolated purified receptor with Torpedo phos- pholipids (19, 201, it is nevertheless impor-

tant to be able to correlate these results with those obtained from the membrane fragments themselves.

Our electron microscopic investigations of AcChE-rich and AcChR-rich fragments allow certain conclusions about the rela- tionship of these two important macromolecules in postsynaptic depolarization mechanisms. The first and most obvious conclusion is that the two macromolecules are not identical. Furthermore, they do not reside in or on the same membrane structure. For instance, they do not present a mosaic on the same membrane since they are associated with different particulate structures. It is clear that the AcChR is an integral membrane protein which cannot be extracted by salt solutions, but only by detergents. On the other hand, AcChE can be extracted by high concentrations of salt. This observation suggests that the esterase may be associated with a particulate structure by means of electrostatic interactions. Since we observe a halo of macromolecules associated with the AcChE-rich particles, it is tempting to conclude that these molecules are attached to membranes via the 150-A taillike structures which have been recently observed (21, 22). The possibility exists that the esterase is associated with a membrane entirely different from that of the receptor or that the receptor and esterase molecules are perhaps associated with different regions of the same membrane in the synaptic cleft, i.e., they are localized in different regions of the cleft. Another point which comes from the electron microscopy results is that receptor molecules do not appear to be organized in any obvious pattern in these fragments. Cartaud et al. (11) have reported the presence of hexago- nal lattice structures in AcChR-rich fragments from Torpedo marmorata. We have never observed such lattice arrangements in any of our preparations. This discrep- ancy may be due to differences between the two species of Torpedo or may be due to differences in the preparation of these fragments. Preliminary evidence suggests that the exact conditions experienced by the fragments results in more or less order- ing of the receptor molecules within such fragments.


The SDS gel patterns show that the AcChR-rich fragments contain the same polypeptide chains as those observed after purification of the AcChR from these same fragments by affinity chromatography procedures. The suggestion has been made (12) that the isolated receptor from Elec- trophorus electricus may have undergone proteolytic degradation and the subunit molecular weights observed may, therefore, be low. Our observation is that if the isolated receptor subunits observed in our studies are proteolytic degradation prod- ucts, then proteolysis has occurred prior to extraction of the receptor from the membranes and has occurred in a quantitative fashion. We think this highly unlikely and believe that the receptor subunit molecular weights observed represent the true subunit structures of the receptor molecule. In addition to a major 40,000 molecular- weight species observed on SDS gels of purified receptor, polypeptide components of A!, 50,000 and 65,000 are also consist- ently observed. It is not clear at the present time whether these higher molecular- weight components are actually an integral part of the receptor macromolecule of whether they are components which strongly adhere to the receptor and are not separated from it during the purification procedure.

Our studies of the binding of Dns-chol to AcChR-rich membrane fragments are at variance with the results of Cohen et al. (2) in which they observe an agonist-specific conformation change, monitored by a blue shift in the fluorescence of Dns-chol bound to specific sites other than the cholinergic binding site. We have not observed such a change in AcChR-rich fragments, in AcChE-rich fragments, or in purified AcChR; in fact, our observation that the same amount, representing approximately 80% of the total of Dns-chol bound by AcChR in the purified state, can also be bound by a boiled denatured receptor suggests that in the receptor from Torpedo californica there is no specific second class of binding sites. There are obvious differences between AcChR in species as closely related as Torpedo californica and Torpedo marmorata. For example, in Torpedo

marmoruta (24), the number of cholinergic ligand binding sites equals the number of Naja nigricollis a-toxin binding sites. How- ever, the binding of acetylcholine to AcChR-rich fragments from Torpedo californica is not cooperative (14), and the number of cholinergic ligand binding sites is always less than the number of a-Bgt binding sites. Furthermore, it has been shown (14, 25) that in contrast to the situation with Electrophorus electricus AcChR, the receptor from Torpedo californica binds cholinergic agonists con- siderably more strongly in the AcChR-rich fragments than in the isolated state. It, therefore, appears that there are species differences in the AcChR molecules that have been studied to date.

Although we have not been able to detect conformation changes by the method of Cohen et al. (2) for Torpedo californica AcChR-rich fragments we nevertheless are led to the conclusion that conformation changes do occur in this cholinergic receptor (15). This notion is based on the idea that, although there are fewer cholinergic ligand binding sites than a-Bgt sites, addition of cholinergic ligand to the receptor from Torpedo californica reduces the rate of combination of [1251]a-Bgt with all of its binding sites on the cholinergic receptor. Therefore, it would appear that all cholinergic ligands can cause some conformation change which is propagated throughout the receptor molecule and affects all toxin- binding sites. Therefore, we contend that detection of a given conformation change is not necessarily important to events such as the ion-translocation mechanism. Such a mechanism is an integral part of the isolated receptor since the isolated macromolecule in combination with phospho- lipids has both the cholinergic ligand rec- ognition site and the ion-translocation ap- paratus necessary to effect depolarization (19, 20).

REFERENCES

1. DUGUID, J. R., AND RAFTERY, M. A. (1973) Bio- chemistry 12, 3593.

2. COHEN, J. B., AND CHANGEUX, J.-P. (1973) Bio- chemistry 12, 4855.

3. SCHMIDT, J., AND RAFTERY, M. A. (1973) Biochem- istry 12, 852.

144 REED ET AL.

4. VANDLEN, R. L., SCHMIDT, J., AND RAFTERY, M. A. (1974) J. Macromol. Chem., in press.

5. LOWRY, 0. H., R~SEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265.

6. ELLMAN, G. L., COUXTNEY, K. D., ANDRES, V., AND FEATHERSTONE, R. M. (1961) Biochem. Pharma- col. 7, 68.

7. SCHMIDT, J., AND RAFTERY, M. A. (1973) Anal. Biochem. 52, 349.

8. POST, R. L., AND SEN, A. K. (1967) Methods Enzymol. 10. 762.

9. FAIRBANKS, G., STECK, T., AND WALLACH, F. (1971) Biochemistry 10, 2606.

10. NICHOL, A., AND POWER, L. T. (1973) Brain Res. 57, 508.

11. CARTAUD, J., BENEDE~I, E. L., COHEN, J., MEUNIER, J.-C., AND CHANGEUX, J.-P. (1973) Far. Eur. Biochem. Sot. Lett. 33, 109.

12. MUENIER, J.-C., SEALOCK, R., OLSEN, R., AND CHANGEUX, J.-P. (1974) Eur. J. Biochem. 45, 371.

13. KAFKNOFSKY, M., AND ROOT, L. (1964) J. Histo- them. Cytochem. 12, 219.

14. VANDLEN, R. L., CHAO, Y., CLAUDIO, T., REED, K.,

AND RAFIXRY, M. A. (1974) Biochemistry, submitted.

15. DEUTSCH, J., AND RAFTERY, M. A. (1974) Biochem- istry, submitted.

16. WEBER, G., BORRIS, D., DEROBERTIS, E., BARRANTES, F., LA TORRE, J., AND DE CARLIN, M. (1971) Mol. Pharmacol. 7, 530.

17. DE ROBERTIS, E. (1971) Science 171, 963. 18. COHEN, J. B., WEBER, M., AND CHANGEUX, J.-P.

(1974) C. R. H. Acad. Sci. Ser. D. 278, 1269. 19. MICHAELSON, D., VANDLEN, R., BODE, J., MOODY,

T., SCHMIDT, J., AND RAFTERY, M. A. (1974) Arch. Biochem. Biophys., submitted.

20. MICHAELSON, D., AND RAFTERY, M. A. (1974) Proc. Nat. Acad. Sci. USA, submitted.

21. DUDAI, Y., HERZBERG, M., AND SILMAN, I. (1973) Proc. Nat. Acad. Sci. USA, submitted.

22. RIEGEFI, F., BON, S., MASSOULIC, J., AND CARTLAND, J. (1973) Eur. J. Biochem. 34, 539.

23. STROUD, R. M., Ross, M., AND RAFTERY, M. A. (1974). unpublished results.

24. WEBER, M., AND CHANGEUX, J.-P. (1974) Mol. Pharmacol. 10, 15.

25. BODE, J., AND RAFTERY, M. A. (1974) Biochem. Biophys. Res. Commun., submitted.

Documents

Characterization of acetylcholine receptor-rich and acetylcholinesterase-rich membrane particles from Torpedo californica electroplax