Biochimicq et Bioptlysica Acta, 1034(1990) 29-38
29
Elsevier BBAGEN23278
An improved procedure for the isolation and purification of nicotinic acetylcholine receptor from Torpedofuscomaculata electric organ E.A. K a p p a n d C . G . W h i t e l e y Department of Chemistry and Biochemistry, Rhodes University, Grahamstown (Republic of South Africa)
(Received25 April 1989) (Revisedmanuscriptreceived7 December1989)
Key words: Receptorpurification;Chromatofocusing;Nicotiniccholinergicreceptor
The nicotinic acetyicholinergic receptor has been isolated and purified from extracts of the electric organ of the fish Torpedo fuscomaculata. The isolation procedure involves (a) a series of purification steps including preparation of membrane fragments, extraction of receptors with non-ionic detergents and chromatofocusing; (b) a novel fluorimetric titration assay. The purified receptor is isolated following a 9-fold purification with an overall yield of 12% and a specific activity of 4027 nM . g - !. Gel electrophoresis in the presence of sodium dodecylsulphate produced only one major band with molecular weight of 44 600 associated with the a-subunit. A comparison is made with other established procedures. Affinity chromatography on cobratoxin CNBr-Sepharose CIAB produced a 6.8-fold purification, 5% yield and 2900 n M . g - i specific activity, while in ion-exchange chromatography on DEAE Sepharose 613 gave a 4.7-fold purification, 3% yield and specific activity of 1988 nM • g - 1
Introduction The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion-channel protein [1,2], so elucidation of its molecular mechanism of function, therefore, requires an intimate understanding of its ligand binding properties. A prerequisite for obtaining most of the receptor preparations functioning both in ligand recognition and ion-channel gating is a purification under conditions which protect the receptor from proteolytic attack [3,4]. Homogenisation of the electroplax tissue in the presence of ethylene diamine tetracetic acid (EDTA) and sulphydryl blocking agents such as N-ethylmaleimide or iodoacetamide has been recommended [3]. While this procedure apparently preserves the chemical integrity of the receptor proteins, this may not necessarily be the case for its functional integrity [5]. Current techniques for the preparative isolation of proteins use the chromatographic recognition of a
Abbreviation: rtAChR,nicotinicacetylcholinereceptor. Correspondence: C.G. Whitely, Department of Chemistryand Biochemistry,RhodesUniversity,P.O. Box94, Grahamstown,6140 South Africa.
variety of physical parameters. The ideal separation method employs an easily identifiable physical parameter unique to the protein of interest [6]. Gel filtration, affinity chromatography with biospecific or group specific adsorbent, ion-exchange chromatography and preparative electrophoresis have proven to be versatile separation techniques. Seldom does any one of these procedures provide sufficient resolution or characterisation to obtain a homogeneous protein from a complex biological material. For this reason purification procedures usually combine some or all of these techniques to increase purity in a series of chromatographic/ electrophoretic steps. Each of these steps, however, suffer from several disadvantages. The resolving power of gel filtration is limited by chromatographic factors and cannot really yield fractions of discrete molecular size. Ion-exchange chromatography, a technique based on the nature and degree of available ionisable groups on the protein molecules [7], is not sufficiently characteristic and many proteins elute under similar conditions. The interaction between proteins and biospecific affinity adsorbents may be too strong, causing problems in desorption, or too weak making an inefficient adsorbent. Group specific affinity adsorbents have eliminated this problem [8,9], but in doing so they have decreased the highly specific recognition and purifica-
0304-4165/90/$03.50 © 1990 ElsevierSciencePublishersB.V. (BiomedicalDivision)
30 tion parameters of biological specificity and thus the degree of purification. The nicotinic acetylcholine receptor protein was the first receptor for a neurotransmitter to be isolated, purified and characterised as a protein [10]. In recent years, affinity chromatography has proven to be a successful technique for the purification of this membrane bound protein [11]. Elapid snake venoms contain polypeptide neurotoxins which possess the extraordinary properties of binding with very high affinity and with nearly total specificity to the nicotinic acetylcholine receptor found in excitable membranes at neuromuscular junctions [12]. Since interaction of toxin and receptor is not affected by the presence of detergent, suitably labelled neurotoxins can be used to assay for solubilised receptor molecules. Any such assay involves the separation of toxin-receptor complexes from excess toxin and procedures such as gel filtration [13], ammonium sulphate precipitation [14], ultra filtration, electrophoresis and ion-exchange chromatography may be used. With the advent of radioligand binding assays neurotransmitter receptors have now been well characterised [15,161. In all the protocols that follow an affinity chromatography step there have been complaints of rather low yields [10] and that modification of the properties of the purified protein occurs. Controversy has also been raised with the reversible nature between the cobratoxin (Naja naja) - acetylcholine receptor interaction and the known agonist carbamylcholine [17]. As part of a study in these laboratories on nicotinic acetylcholine receptor we required large amounts of the purified protein. The published procedures for isolation of this receptor proved inadequate and in view of the foregoing discussion a new and improved method for purification and assay was sought. Purification and fractionation of proteins from biological fluids is facilitated by their different isoelectric points. The possibility of producing a pH gradient using an ion-exchange column has provided techniques for separation with resolution and recovery comparable to those of electrophoretic systems. With ampholyte displacement column chromatography a pH gradient is produced either by mixing buffers of different pH or by employing the buffering action of an ion-exchanger and a running buffer initially adjusted to one pH through a column adjusted to another pH. The advantages of this system are that ordinary chromatographic equipment may be used; there is no restriction on the size of column; mixtures of ordinary buffers may be used and proteins are not subjected to more extreme pH values than their pI [18]. We report here a chromatofocusing procedure for the purification of nicotinic acetylcholine receptor and show that it is a superior technique from the usual ion-exchange or affinity chromatographic processes.
Materials and Methods
Materials The materials used in this work were obtained as follows. Live electric rays (Torpedo fuscomaculata) were caught in the Bushman's esturary off the South East coast of South Africa. Pure Naja naja venom (acobratoxin) was purchased from the Council for Scientific Industry and Research, Pretoria. Polybuffer exchanger (PBE 94), Polybuffer 74, Sephacryl S-400 and CNBr-Sepharose 4B were purchased from Pharmacia. Phenylmethylsulphonyl fluoride, molecular weight standards, bovine serum albumin, electroplax of electric eel (Electrophorus electricus), acetylthiocholine chloride, 5,5-dithiobis-(2-nitrobenzoic acid), carbamylcholine, acetylcholine and DEAE-Sepharose 6B were obtained from Sigma. Triton X-100 was obtained from Fischer Scientific Co.. EDTA and sodium azide were bought from Aldrich Chemical Co. Acrylamide, ammonium persulphate, N, N '-methylene bisacrylamide, sodium dodecylsulphate (SDS), and Coomassie brilliant blue 250 were from Bio-Rad. Dialysis membranes were obtained from Spectropor and all other inorganic and buffer materials were of reagent grade.
Methods Extraction of receptor. Electric organs were excised from a freshly killed Torpedo ray and stored at - 8 0 °C until required. Electric tissue (120 g) was homogenised (2 min) in imidazole buffer (25 mM, pH 7.4, 150 ml) containing sodium azide (0.01%), EDTA (10 mM) and phenylmethylsulphonyl fluoride (0.1 mM). The homogenate was then centrifuged (20 000 X g, 60 min, 4 ° C) and the supernatant discarded. The pellet was resuspended in the same buffer solution and Triton X-100 added to a final concentration of 1%. The mixture was stirred (18 h, 4°C) and then centrifuged (100000 x g, 60 min), the pellet was resuspended in the above buffer, centrifuged again and the supernatants were pooled. Chromatofocusing. The receptor extract (10 ml) was added to a PBE 94 column (11 x 1.6 cm) previously equilibrated and washed successively with imidazole buffer (25 mM, pH 7.4, 40 ml) and PB 74 (polybuffer) (5 ml, pH 4.0). The column was eluted with PB 74 (pH 4.0) at 54 ml .h -1, fractions (3.0 ml) collected and monitored for absorbance at 280 nm. Finally, the column was washed with sodium chloride (1 M, 50 ml) to elute the proteins not displaced by low ionic strength. Affinity chromatography. Cobratoxin (15 mg) is incubated (18 h 4 ° C) with CNBr-activated Sepharose 4B (40 ml), washed with Tris-HC1 (0.1 M pH 7.6), then poured into a column (7.5 x 2.6 cm). The partially purified receptor in Tris-HC1 buffer is filtered through the matrix at a rate of 80 ml- h - 1, fractions (3.0 ml) are collected and monitored for absorbance at 280 nm. After all the receptor solution has been applied, the
31 column is washed with adsorbing buffer until the A280 of effluent has dropped to baseline levels. Carbamylcholine (1 M, 20 ml) in Tris-HC1 buffer was then passed through the column and fractions (3.0 ml) are collected and monitored for absorbance at 280 nm and receptor activity. DEAE-Sepharose 6B chromatography. The resin is equilibrated in Tris-HC1 buffer (0.1 M, pH 7.6) and poured into a column (2.6 × 10 cm). The partially purified receptor in Tris-HC1 buffer (0.1 M) is filtered through the matrix at a rate of 80 ml- h-1 and fractions (3.0 ml) are collected and monitored for absorbance at 280 ran. After all the receptor solution has been applied, the column is washed with Tris-HC1 (0.1 M, pH 7.6) until the A2so of effluent has dropped to baseline levels. A linear gradient of sodium chloride is then started with a flow rate of 80 m l - h -1, fractions (3.0 ml) collected and monitored for absorbance at 280 nm and for receptor activity. Ammonium sulphate fractionation. Ammonium sulphate was added to the receptor active fractions from each of the chromatofocusing, affinity chromatography and DEAE-Sepharose 6B chromatography stages, the precipitate was collected by centrifugation 10000 × g, 10 rain redissolved in Tris-HC1 (0.1 M, pH 7.6, 1.0 ml) and dialysed against the same buffer (2 1; 4 h) and finally lyophilised.
Molecular weight determinations Disc polyacrylamide gel electrophoresis. Electrophoresis of fractions of receptor on SDS slab gels is used to monitor the purification of the receptor [19]. The acrylamide/bisacrylamide mixture (10% and 0.26%, respectively) is polymerised in 0.01 M Tris-HC1 ~ H 7.6) containing 0.025% N,N,N',N'-tetramethylenediamine. Polymerisation is catalysed by the addition of freshly prepared ammonium persulphate 1.5% (w/v). The upper and lower buffer compartments are filled with 0.1% SDS/Tris-HC1 buffer sample, (50/~1) is applied to the gel and a potential of 100 mV for 30 rain then 150 mV for 3 h is applied. Protein is stained with 0.2% solution of Coomassie blue in a mixture of methanol/water/glacial acetic acid. Destaining is achieved by extensive washings in methanol and acetic acid. Sephacryl S-400 chromatography. The molecular weight of the purified receptor was determined by chromatography on Sephacryl S-400 in Tris-HC1, (0.1 M, pH 7.6). The column (1.6 × 40 cm) was standardised with Blue dextran (M r = 2- 106), lactate dehydrogenase (M r = 140 000), DNase (M r = 63 000) and carboxypeptidase A (M r = 34 300). The receptor was eluted with the same buffer, at a rate of 33 ml. h-1 fractions (3.0 ml) collected and monitored for absorbance at 280 nm. Protein determination. Protein is measured by the modification of the Lowry [20] method using bovine serum albumin as the standard. The presence of Triton
X-100 caused the formation of precipitate during the reaction. It was shown that this precipitate and its subsequent removal by centrifugation had no effect on the final result.
Receptor assay Fluorimetry. A fluorescence titration assay is described here which allows a rapid and reproducible means for determining receptor site concentrations without many of the difficulties associated with the use of radiochemicals [21]. d-Tubocurarine, a specific competitive antagonist of nAChR, shows similar affinities for both membrane bound and solubilised receptor and, hence, includes its use as a fluorescence quenching ligand [22]. Since the intensity of excitation light decreases along the light path due to the absorption by d-tubocurarine and inner filter effects, the decrease in the tryptophanyl fluorescence is corrected by dividing the apparent intensity (Fa) by the light intensity in the sample cell Fc = Fa/lO e~o
(1)
KJ 1 - 0 = [ d T b c ] t / 0 - p ( A ) t
(2)
0 = AF/AFma x
(3)
where Fc is the corrected fluorescence intensity, E and c are molar extinction coefficient and molar concentration of d-tubocurarine, respectively, a is an instrumental constant which was found to be 0.588 [23], (A)t is the total concentration of receptor; p is the total number of binding sites; O is the fractional occupancy of total receptor sites by ligand; AF is the increase in fluorescence in the presence of known amount of ligand; AFm~x is the increase in fluorescence at full saturation with ligand; [dTbc]t is the total concentration of dtubocurarine. The fluorescence measurements were made with a Hitachi spectrofluorimeter with excitation light source from a Xenon 150 lamp, the fluorescence was measured through the cell at an angle of 90 o to the incident beam. Increasing concentrations of d-tubocurarine (0-60 /~M) were titrated against a fixed concentration of receptor (100 #1) in phosphate buffer (0.1 M, pH 7.2). An excitation wavelength of 295 nm and emission wavelength of 340 nm were used. The fluorescence data was used to determine total concentration of receptor (Eqns. 2 and 3). Spectrophotometry. In order to verify the fluorimetric assay of the receptor described above, the binding of d-tubocurarine to nicotinic acetylcholine receptor was also monitored spectrophotometrically using a Bausch and Lomb spectrophotometer. Reaction mixtures (3.0 cm3) contained purified receptor (100 #1) and d-tubocurarine (2-50 #M) in imidazole buffer (0.1 M, pH 7.4). The absorbance was monitored at 230 nm. The total
32
Theoretical Actual q74 74 72 737 7.0 716 6.8 7.04
concentration of receptor was determined according to the method of Scatchard [24] (Eqns. 4 and 5).
(dThc)f = (dTbc)t -- AAx/AAxmax" (A)t ~'/(dTbc)f
. =
p
- -
p/K d
t
(4)
(5)
where (dTbc)t is the total d-tubocurarine concentration; (A)t is the total receptor concentration; AA x is the difference in absorbance observed when d-tubocurarine is mixed with receptor (A); AAxmax is the m a x i m u m absorbance observed when all of the tubocurarine molecules are bound to the receptor; 1, is the real of bound tubocurarine; p is the number of binding sites; K a is the dissociation constant of d-tubocurarine-receptor complex; and (dTbc)r is the concentration of free tubocurarine determined using the absorption coefficient (A]%c,n) of 118 at 280 nm and Mr of 695. Results
Extraction of receptor The acetylcholine receptor is deeply integrated into the membrane and detergents are needed to release it into solution. The non-denaturing detergent Triton X100 was effective in solubilising the receptor, without loss of toxin or cholinergic ligand binding sites, after an incubation period of 18 h. The Triton had to be removed before any protein determinations could be made [25,26]. Due to serious problems created by proteolysis [27], maximum precautions were taken and all solutions contained certain proteinase inhibitors such as E D T A and phenylmethylsulphonyl fluoride. Sodium azide was also included to inhibit bacterial growth. F r o m 120 g electric organ this extraction procedure yielded receptor with a specific activity of 438- 3 n M . g-x (Table I). No noticeable differences were observed when organs which had been stored at - 20 o C even for m a n y months were used instead of fresh ones. A second detergent extraction carried out on the material which remained after the first detergent treatment yielded little receptor. Thus this extra step was regarded as being unnecessary. Lowering the concentration of detergent in crude extracts by dilution to 0.01% ( w / v ) or less resulted in aggregation of the receptor protein. Thus, in crude preparations the presence of detergent at 1% ( w / v ) is needed to keep the receptor protein in solution.
Chromatofocusing The results of the chromatographic separations are shown in Fig. 1. With the fact that Triton X-100 absorbs at 280 nm it is seen that the removal of this detergent and extraneous protein occurs rapidly. This offers a suitable alternative to sucrose gradient centrifugation [22] or the more harsh treatments such as adsorption to charcoal or prolonged dialysis.
90C
2.0 x'' I
o 8oc I
~ 7oc
{ 6oc
6,6
\\ 1.5
t
\\ \\ \
t
v
~ 5oc
1.0
~ 400 g 300
~2c~
\\
\.
O.5
m
100
t
60
6.24
5.6 5.6 5.4 5.2
5.78 5:75 5.51 540
5.0
5.26
t
\\
'5
6.94
6.4 6.73 6,2 6.57
\
t
pH
4.8 4.97 46 4 80 44
t
4.67
~xx 4.2 4,56 4.0 4 50 go ~+o 1~o ~ioo 250 Volume (cm 3 )
Fig. 1. Chromatofocusing of solubilised nicotinic acetylcholine receptor on polybuffer exchanger PBE 94. The absorbance is monitored at 280 nm and the pH range 7.4-4.0 is indicated ( . . . . . ). The arrow indicates elution by 1 M NaC1. Binding capacity (nM, O-- -- -- e) of receptor is also shown. Standard proteins eluted through the same column under the same conditions are superimposed (. . . . . . ) (a, cytochrome c, pl = 10.1) (b, carbonic anhydrase, pl = 5.4). Standard Triton X-100 is also indicated ( . . . . . . ).
Elution of the active receptor in a sharp peak is readily accomplished by a step increment in ionic strength to 1 M NaC1. The PBE-94 step gave a specific activity of 4026.6 n M . g - ] , a 9.2-fold purification with a 12% overall yield. The combined fractions from this elution (16 ml) was saturated with a m m o n i u m sulphate to improve the fold purification slightly. The estimated degree of purity based upon electrophoretic analysis before and after chromatofocusing was judged to be greater than 90%. Fig. 2a represents an electrophoretogram scan of S D S - P A G E analysis of the purified receptor. Table I summarises the results of all the various steps. After freeze drying, purified receptor was obtained in a 12.5-fold purification and 2.26% overall yield. N o acetylcholinesterase activity was detected in the main receptor fraction. This was not unusual as phenylmethylsulphonyl fluoride, present in the incubation mixture, is a known esterase inhibitor.
Affinity chromatography The affinity column used consisted of a-cobratoxin coupled to cyanogen bromide activated Sepharose 4B. Approx. 10% of the toxin residues retained the ability to bind soluble A C h R protein. The elution profile obtained by chromatography of solubilised cobratoxinA C h R complex is shown in Fig. 3. Extraneous protein material was removed by adding alternatively Tris-HCl buffer + 0.1% Triton X-100 and 1 M NaC1. Although several neuroactive agents efficiently displace the toxin
33 a (3 1.0 1.O
500
"6
400
E
8 L o.~
300
i
TD
29
Elsevier BBAGEN23278
An improved procedure for the isolation and purification of nicotinic acetylcholine receptor from Torpedofuscomaculata electric organ E.A. K a p p a n d C . G . W h i t e l e y Department of Chemistry and Biochemistry, Rhodes University, Grahamstown (Republic of South Africa)
(Received25 April 1989) (Revisedmanuscriptreceived7 December1989)
Key words: Receptorpurification;Chromatofocusing;Nicotiniccholinergicreceptor
The nicotinic acetyicholinergic receptor has been isolated and purified from extracts of the electric organ of the fish Torpedo fuscomaculata. The isolation procedure involves (a) a series of purification steps including preparation of membrane fragments, extraction of receptors with non-ionic detergents and chromatofocusing; (b) a novel fluorimetric titration assay. The purified receptor is isolated following a 9-fold purification with an overall yield of 12% and a specific activity of 4027 nM . g - !. Gel electrophoresis in the presence of sodium dodecylsulphate produced only one major band with molecular weight of 44 600 associated with the a-subunit. A comparison is made with other established procedures. Affinity chromatography on cobratoxin CNBr-Sepharose CIAB produced a 6.8-fold purification, 5% yield and 2900 n M . g - i specific activity, while in ion-exchange chromatography on DEAE Sepharose 613 gave a 4.7-fold purification, 3% yield and specific activity of 1988 nM • g - 1
Introduction The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion-channel protein [1,2], so elucidation of its molecular mechanism of function, therefore, requires an intimate understanding of its ligand binding properties. A prerequisite for obtaining most of the receptor preparations functioning both in ligand recognition and ion-channel gating is a purification under conditions which protect the receptor from proteolytic attack [3,4]. Homogenisation of the electroplax tissue in the presence of ethylene diamine tetracetic acid (EDTA) and sulphydryl blocking agents such as N-ethylmaleimide or iodoacetamide has been recommended [3]. While this procedure apparently preserves the chemical integrity of the receptor proteins, this may not necessarily be the case for its functional integrity [5]. Current techniques for the preparative isolation of proteins use the chromatographic recognition of a
Abbreviation: rtAChR,nicotinicacetylcholinereceptor. Correspondence: C.G. Whitely, Department of Chemistryand Biochemistry,RhodesUniversity,P.O. Box94, Grahamstown,6140 South Africa.
variety of physical parameters. The ideal separation method employs an easily identifiable physical parameter unique to the protein of interest [6]. Gel filtration, affinity chromatography with biospecific or group specific adsorbent, ion-exchange chromatography and preparative electrophoresis have proven to be versatile separation techniques. Seldom does any one of these procedures provide sufficient resolution or characterisation to obtain a homogeneous protein from a complex biological material. For this reason purification procedures usually combine some or all of these techniques to increase purity in a series of chromatographic/ electrophoretic steps. Each of these steps, however, suffer from several disadvantages. The resolving power of gel filtration is limited by chromatographic factors and cannot really yield fractions of discrete molecular size. Ion-exchange chromatography, a technique based on the nature and degree of available ionisable groups on the protein molecules [7], is not sufficiently characteristic and many proteins elute under similar conditions. The interaction between proteins and biospecific affinity adsorbents may be too strong, causing problems in desorption, or too weak making an inefficient adsorbent. Group specific affinity adsorbents have eliminated this problem [8,9], but in doing so they have decreased the highly specific recognition and purifica-
0304-4165/90/$03.50 © 1990 ElsevierSciencePublishersB.V. (BiomedicalDivision)
30 tion parameters of biological specificity and thus the degree of purification. The nicotinic acetylcholine receptor protein was the first receptor for a neurotransmitter to be isolated, purified and characterised as a protein [10]. In recent years, affinity chromatography has proven to be a successful technique for the purification of this membrane bound protein [11]. Elapid snake venoms contain polypeptide neurotoxins which possess the extraordinary properties of binding with very high affinity and with nearly total specificity to the nicotinic acetylcholine receptor found in excitable membranes at neuromuscular junctions [12]. Since interaction of toxin and receptor is not affected by the presence of detergent, suitably labelled neurotoxins can be used to assay for solubilised receptor molecules. Any such assay involves the separation of toxin-receptor complexes from excess toxin and procedures such as gel filtration [13], ammonium sulphate precipitation [14], ultra filtration, electrophoresis and ion-exchange chromatography may be used. With the advent of radioligand binding assays neurotransmitter receptors have now been well characterised [15,161. In all the protocols that follow an affinity chromatography step there have been complaints of rather low yields [10] and that modification of the properties of the purified protein occurs. Controversy has also been raised with the reversible nature between the cobratoxin (Naja naja) - acetylcholine receptor interaction and the known agonist carbamylcholine [17]. As part of a study in these laboratories on nicotinic acetylcholine receptor we required large amounts of the purified protein. The published procedures for isolation of this receptor proved inadequate and in view of the foregoing discussion a new and improved method for purification and assay was sought. Purification and fractionation of proteins from biological fluids is facilitated by their different isoelectric points. The possibility of producing a pH gradient using an ion-exchange column has provided techniques for separation with resolution and recovery comparable to those of electrophoretic systems. With ampholyte displacement column chromatography a pH gradient is produced either by mixing buffers of different pH or by employing the buffering action of an ion-exchanger and a running buffer initially adjusted to one pH through a column adjusted to another pH. The advantages of this system are that ordinary chromatographic equipment may be used; there is no restriction on the size of column; mixtures of ordinary buffers may be used and proteins are not subjected to more extreme pH values than their pI [18]. We report here a chromatofocusing procedure for the purification of nicotinic acetylcholine receptor and show that it is a superior technique from the usual ion-exchange or affinity chromatographic processes.
Materials and Methods
Materials The materials used in this work were obtained as follows. Live electric rays (Torpedo fuscomaculata) were caught in the Bushman's esturary off the South East coast of South Africa. Pure Naja naja venom (acobratoxin) was purchased from the Council for Scientific Industry and Research, Pretoria. Polybuffer exchanger (PBE 94), Polybuffer 74, Sephacryl S-400 and CNBr-Sepharose 4B were purchased from Pharmacia. Phenylmethylsulphonyl fluoride, molecular weight standards, bovine serum albumin, electroplax of electric eel (Electrophorus electricus), acetylthiocholine chloride, 5,5-dithiobis-(2-nitrobenzoic acid), carbamylcholine, acetylcholine and DEAE-Sepharose 6B were obtained from Sigma. Triton X-100 was obtained from Fischer Scientific Co.. EDTA and sodium azide were bought from Aldrich Chemical Co. Acrylamide, ammonium persulphate, N, N '-methylene bisacrylamide, sodium dodecylsulphate (SDS), and Coomassie brilliant blue 250 were from Bio-Rad. Dialysis membranes were obtained from Spectropor and all other inorganic and buffer materials were of reagent grade.
Methods Extraction of receptor. Electric organs were excised from a freshly killed Torpedo ray and stored at - 8 0 °C until required. Electric tissue (120 g) was homogenised (2 min) in imidazole buffer (25 mM, pH 7.4, 150 ml) containing sodium azide (0.01%), EDTA (10 mM) and phenylmethylsulphonyl fluoride (0.1 mM). The homogenate was then centrifuged (20 000 X g, 60 min, 4 ° C) and the supernatant discarded. The pellet was resuspended in the same buffer solution and Triton X-100 added to a final concentration of 1%. The mixture was stirred (18 h, 4°C) and then centrifuged (100000 x g, 60 min), the pellet was resuspended in the above buffer, centrifuged again and the supernatants were pooled. Chromatofocusing. The receptor extract (10 ml) was added to a PBE 94 column (11 x 1.6 cm) previously equilibrated and washed successively with imidazole buffer (25 mM, pH 7.4, 40 ml) and PB 74 (polybuffer) (5 ml, pH 4.0). The column was eluted with PB 74 (pH 4.0) at 54 ml .h -1, fractions (3.0 ml) collected and monitored for absorbance at 280 nm. Finally, the column was washed with sodium chloride (1 M, 50 ml) to elute the proteins not displaced by low ionic strength. Affinity chromatography. Cobratoxin (15 mg) is incubated (18 h 4 ° C) with CNBr-activated Sepharose 4B (40 ml), washed with Tris-HC1 (0.1 M pH 7.6), then poured into a column (7.5 x 2.6 cm). The partially purified receptor in Tris-HC1 buffer is filtered through the matrix at a rate of 80 ml- h - 1, fractions (3.0 ml) are collected and monitored for absorbance at 280 nm. After all the receptor solution has been applied, the
31 column is washed with adsorbing buffer until the A280 of effluent has dropped to baseline levels. Carbamylcholine (1 M, 20 ml) in Tris-HC1 buffer was then passed through the column and fractions (3.0 ml) are collected and monitored for absorbance at 280 nm and receptor activity. DEAE-Sepharose 6B chromatography. The resin is equilibrated in Tris-HC1 buffer (0.1 M, pH 7.6) and poured into a column (2.6 × 10 cm). The partially purified receptor in Tris-HC1 buffer (0.1 M) is filtered through the matrix at a rate of 80 ml- h-1 and fractions (3.0 ml) are collected and monitored for absorbance at 280 ran. After all the receptor solution has been applied, the column is washed with Tris-HC1 (0.1 M, pH 7.6) until the A2so of effluent has dropped to baseline levels. A linear gradient of sodium chloride is then started with a flow rate of 80 m l - h -1, fractions (3.0 ml) collected and monitored for absorbance at 280 nm and for receptor activity. Ammonium sulphate fractionation. Ammonium sulphate was added to the receptor active fractions from each of the chromatofocusing, affinity chromatography and DEAE-Sepharose 6B chromatography stages, the precipitate was collected by centrifugation 10000 × g, 10 rain redissolved in Tris-HC1 (0.1 M, pH 7.6, 1.0 ml) and dialysed against the same buffer (2 1; 4 h) and finally lyophilised.
Molecular weight determinations Disc polyacrylamide gel electrophoresis. Electrophoresis of fractions of receptor on SDS slab gels is used to monitor the purification of the receptor [19]. The acrylamide/bisacrylamide mixture (10% and 0.26%, respectively) is polymerised in 0.01 M Tris-HC1 ~ H 7.6) containing 0.025% N,N,N',N'-tetramethylenediamine. Polymerisation is catalysed by the addition of freshly prepared ammonium persulphate 1.5% (w/v). The upper and lower buffer compartments are filled with 0.1% SDS/Tris-HC1 buffer sample, (50/~1) is applied to the gel and a potential of 100 mV for 30 rain then 150 mV for 3 h is applied. Protein is stained with 0.2% solution of Coomassie blue in a mixture of methanol/water/glacial acetic acid. Destaining is achieved by extensive washings in methanol and acetic acid. Sephacryl S-400 chromatography. The molecular weight of the purified receptor was determined by chromatography on Sephacryl S-400 in Tris-HC1, (0.1 M, pH 7.6). The column (1.6 × 40 cm) was standardised with Blue dextran (M r = 2- 106), lactate dehydrogenase (M r = 140 000), DNase (M r = 63 000) and carboxypeptidase A (M r = 34 300). The receptor was eluted with the same buffer, at a rate of 33 ml. h-1 fractions (3.0 ml) collected and monitored for absorbance at 280 nm. Protein determination. Protein is measured by the modification of the Lowry [20] method using bovine serum albumin as the standard. The presence of Triton
X-100 caused the formation of precipitate during the reaction. It was shown that this precipitate and its subsequent removal by centrifugation had no effect on the final result.
Receptor assay Fluorimetry. A fluorescence titration assay is described here which allows a rapid and reproducible means for determining receptor site concentrations without many of the difficulties associated with the use of radiochemicals [21]. d-Tubocurarine, a specific competitive antagonist of nAChR, shows similar affinities for both membrane bound and solubilised receptor and, hence, includes its use as a fluorescence quenching ligand [22]. Since the intensity of excitation light decreases along the light path due to the absorption by d-tubocurarine and inner filter effects, the decrease in the tryptophanyl fluorescence is corrected by dividing the apparent intensity (Fa) by the light intensity in the sample cell Fc = Fa/lO e~o
(1)
KJ 1 - 0 = [ d T b c ] t / 0 - p ( A ) t
(2)
0 = AF/AFma x
(3)
where Fc is the corrected fluorescence intensity, E and c are molar extinction coefficient and molar concentration of d-tubocurarine, respectively, a is an instrumental constant which was found to be 0.588 [23], (A)t is the total concentration of receptor; p is the total number of binding sites; O is the fractional occupancy of total receptor sites by ligand; AF is the increase in fluorescence in the presence of known amount of ligand; AFm~x is the increase in fluorescence at full saturation with ligand; [dTbc]t is the total concentration of dtubocurarine. The fluorescence measurements were made with a Hitachi spectrofluorimeter with excitation light source from a Xenon 150 lamp, the fluorescence was measured through the cell at an angle of 90 o to the incident beam. Increasing concentrations of d-tubocurarine (0-60 /~M) were titrated against a fixed concentration of receptor (100 #1) in phosphate buffer (0.1 M, pH 7.2). An excitation wavelength of 295 nm and emission wavelength of 340 nm were used. The fluorescence data was used to determine total concentration of receptor (Eqns. 2 and 3). Spectrophotometry. In order to verify the fluorimetric assay of the receptor described above, the binding of d-tubocurarine to nicotinic acetylcholine receptor was also monitored spectrophotometrically using a Bausch and Lomb spectrophotometer. Reaction mixtures (3.0 cm3) contained purified receptor (100 #1) and d-tubocurarine (2-50 #M) in imidazole buffer (0.1 M, pH 7.4). The absorbance was monitored at 230 nm. The total
32
Theoretical Actual q74 74 72 737 7.0 716 6.8 7.04
concentration of receptor was determined according to the method of Scatchard [24] (Eqns. 4 and 5).
(dThc)f = (dTbc)t -- AAx/AAxmax" (A)t ~'/(dTbc)f
. =
p
- -
p/K d
t
(4)
(5)
where (dTbc)t is the total d-tubocurarine concentration; (A)t is the total receptor concentration; AA x is the difference in absorbance observed when d-tubocurarine is mixed with receptor (A); AAxmax is the m a x i m u m absorbance observed when all of the tubocurarine molecules are bound to the receptor; 1, is the real of bound tubocurarine; p is the number of binding sites; K a is the dissociation constant of d-tubocurarine-receptor complex; and (dTbc)r is the concentration of free tubocurarine determined using the absorption coefficient (A]%c,n) of 118 at 280 nm and Mr of 695. Results
Extraction of receptor The acetylcholine receptor is deeply integrated into the membrane and detergents are needed to release it into solution. The non-denaturing detergent Triton X100 was effective in solubilising the receptor, without loss of toxin or cholinergic ligand binding sites, after an incubation period of 18 h. The Triton had to be removed before any protein determinations could be made [25,26]. Due to serious problems created by proteolysis [27], maximum precautions were taken and all solutions contained certain proteinase inhibitors such as E D T A and phenylmethylsulphonyl fluoride. Sodium azide was also included to inhibit bacterial growth. F r o m 120 g electric organ this extraction procedure yielded receptor with a specific activity of 438- 3 n M . g-x (Table I). No noticeable differences were observed when organs which had been stored at - 20 o C even for m a n y months were used instead of fresh ones. A second detergent extraction carried out on the material which remained after the first detergent treatment yielded little receptor. Thus this extra step was regarded as being unnecessary. Lowering the concentration of detergent in crude extracts by dilution to 0.01% ( w / v ) or less resulted in aggregation of the receptor protein. Thus, in crude preparations the presence of detergent at 1% ( w / v ) is needed to keep the receptor protein in solution.
Chromatofocusing The results of the chromatographic separations are shown in Fig. 1. With the fact that Triton X-100 absorbs at 280 nm it is seen that the removal of this detergent and extraneous protein occurs rapidly. This offers a suitable alternative to sucrose gradient centrifugation [22] or the more harsh treatments such as adsorption to charcoal or prolonged dialysis.
90C
2.0 x'' I
o 8oc I
~ 7oc
{ 6oc
6,6
\\ 1.5
t
\\ \\ \
t
v
~ 5oc
1.0
~ 400 g 300
~2c~
\\
\.
O.5
m
100
t
60
6.24
5.6 5.6 5.4 5.2
5.78 5:75 5.51 540
5.0
5.26
t
\\
'5
6.94
6.4 6.73 6,2 6.57
\
t
pH
4.8 4.97 46 4 80 44
t
4.67
~xx 4.2 4,56 4.0 4 50 go ~+o 1~o ~ioo 250 Volume (cm 3 )
Fig. 1. Chromatofocusing of solubilised nicotinic acetylcholine receptor on polybuffer exchanger PBE 94. The absorbance is monitored at 280 nm and the pH range 7.4-4.0 is indicated ( . . . . . ). The arrow indicates elution by 1 M NaC1. Binding capacity (nM, O-- -- -- e) of receptor is also shown. Standard proteins eluted through the same column under the same conditions are superimposed (. . . . . . ) (a, cytochrome c, pl = 10.1) (b, carbonic anhydrase, pl = 5.4). Standard Triton X-100 is also indicated ( . . . . . . ).
Elution of the active receptor in a sharp peak is readily accomplished by a step increment in ionic strength to 1 M NaC1. The PBE-94 step gave a specific activity of 4026.6 n M . g - ] , a 9.2-fold purification with a 12% overall yield. The combined fractions from this elution (16 ml) was saturated with a m m o n i u m sulphate to improve the fold purification slightly. The estimated degree of purity based upon electrophoretic analysis before and after chromatofocusing was judged to be greater than 90%. Fig. 2a represents an electrophoretogram scan of S D S - P A G E analysis of the purified receptor. Table I summarises the results of all the various steps. After freeze drying, purified receptor was obtained in a 12.5-fold purification and 2.26% overall yield. N o acetylcholinesterase activity was detected in the main receptor fraction. This was not unusual as phenylmethylsulphonyl fluoride, present in the incubation mixture, is a known esterase inhibitor.
Affinity chromatography The affinity column used consisted of a-cobratoxin coupled to cyanogen bromide activated Sepharose 4B. Approx. 10% of the toxin residues retained the ability to bind soluble A C h R protein. The elution profile obtained by chromatography of solubilised cobratoxinA C h R complex is shown in Fig. 3. Extraneous protein material was removed by adding alternatively Tris-HCl buffer + 0.1% Triton X-100 and 1 M NaC1. Although several neuroactive agents efficiently displace the toxin
33 a (3 1.0 1.O
500
"6
400
E
8 L o.~
300
i
TD
