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Biochemical characterization of solubilized muscarinic acetylcholine receptors
Brain Research
Bulletin,
Vol. 5,
pp. 703-709.
Printed
in the U.S.A.
Biochemical Characterization Muscarinic Acetylcholine
of Solubilized Receptors
H. REPKE AND H. MATTHIES Institute of Neurobiology and Brain Research, Academy of Sciences of GDR 301 Magdeburg, Leipziger Strasse 44, German Democratic Republic Received
8 August 1980
REPKE, H. AND H. MATTHIES. Biochemical characterization of solubilized muscarinic acetylcholine receptors. BRAIN RES. BULL. 5(6) 70%709, 1980.-Several in part new methods (salt extraction, phospholipase treatment, transfer to serumlipoproteins, nonionic detergens mixtures) have been extensively examined in order to obtain a solubilized muscarinic acetylcholine receptor with properties suitable for further purification. An optimal mixture of digitonin and gitonin has been found which forms a tightly bound detergent wall around the receptor protein as indicated by ion exchange and monolayer studies and which allows to solubilize the receptor from enriched synaptosomal membranes with a high yield and stability. The specific 3H-QNB binding of this preparation is nearly unchanged between pH 7.5 and 10. The isoelectric point of the muscarinic acetylcholine receptor has been determined by isoelectric focusing to be between pH 4.5 and 4.6. Solubilization
Muscarinic receptor Isoelectric point
Phospholipases
SEVERAL attempts have been undertaken to solubilize muscarinic acetylcholine receptors (mAchR) which have been shown to be more sensitive to the commonly used detergents than the nicotinic receptor [l, 3, 6, 131. Due to the relatively low concentration of the mAchR in neuronal tissues a high stability of the receptor in the solubilized state is required to enable the subsequent purification procedures. Furthermore the masking influence of a strong detergent wall around the protein should be avoided because it leads to difficulties during ion exchange and affinity chromatography ([ 171 Repke and Matthies, submitted for publication). In an extensive search we studied a number of partially new solubilization procedures in respect to these requirements. Similar to others [2, 7, 91 we found digitonin preparations to be most effective in mAchR solubilization and characterized the role of the main components digitonin and gitonin. The solubilized mAchRs were furthermore characterized in respect to its temperature and pH stability, isoelectric point and the intluence of the detergent on its ionic properties. METHOD Chemicals
All salts were used in p.a. purity. NaCl and KC1 were additionally recrystallized once, (NH&SO, twice from water. LiBr (suprapure) has been obtained from Merck. Digitonin from different lots was supplied from VEB Ysat and Merck. Na-desoxycholate and Na-cholate have been obtained from Serva and recrystallized twice from ethanol. Ampholine carrier ampholytes (pH ranges from 4-6 to 3-10) were supplied from LKB. The enzymes were obtained from the following sources: phospholipase A2 from snake venom (Boehringer); phospholipase C from Cl. perfrigens (&lb&hem); bungarotoxin from B. multicinctus (Boehringer); lipase from wheat germ
Copyright
o 1980 ANKHO
International
Digitonin
Gitonin
Monolayer
(Ferak); lipase from Rhizopus (Fe&); lipase from pankreas (Serva) and human serum albumin (Serva). Human serum lipoproteins were prepared from the blood of a selected donor and prepared as follows. The detibrillated serum was adjusted with KBr to 1,064 g/ml and centrifuged at 125,000 g for 25 hours at 4°C. The pellet was resuspended and adjusted to 1,125 g/ml with KBr. The supematant after the subsequent centrifugation for 44 hours at 125,000 g/4”C was dialysed against 0.9% NaCl(l6 pM/EDTA) and stored at 4°C until use. 3H-QNB (16 and 12 WmMol) were supplied from Amersham Radiochemical Centre and 3H-atropine (1 Ci/mMol) from I.R.E., Belgium. The stock solutions were checked for radiochemical purity before use. Extraction
Procedures
Enriched synaptosomal membranes @fold from homogenate [ 161) were used for starting material in all preparations as suspensions containing about 10 mg protein per ml. If not indicated the membranes were suspended in 50 mM phosphate buffer according to Sorensen (pH 7.3). After the subsequent extraction procedures the membranes were pelleted at 100,000 g during 1.5 hours. The proteins in the corresponding supematants were considered to be solubilized. Extraction methods, time and temperature are listed in detail in Table 1. (a) Salt extraction. Membrane suspensions and salt solutions were mixed in an ice bath to give the final salt concentrations which are listed in Table 1. After extraction and suspensions were diluted with distilled water to avoid the persistence of nonsolubilized material in the 100,000 g supematant. No precipitation of the solubilized proteins has been observed during equilibrium dialysis in 0.1 M NaCV50 mM phosphate buffer pH 7.3 which was employed for binding studies.
Inc.-0361-9230/80/060703-07$01.20/O
REPKE AND MATTHIES
704 TABLE
Detergens
Optimal concentration (examined range in paranthesis)
1
pMol mAchR per ml extract at the optimal conditions
Extraction procedures (optimal parameters) extraction time (hours)
NaCl Nd
KC1 LiBr Phospholipase
A
Phospholipase
A,
P-Bungarotoxin Phospholipase C Wheat germ lipase Pig pankreas lipase Phizopus lipase Human serum lipoproteins Digitoningitonin (recrystallized from ethanol) Digitonimgitonin (3:2) Na-cholate Na-desoxycholate
2.1 M (2.b2.4 M) (l&2.5 M) 2.0 M (1.5-2.5 M) (1.0-2.0 M) 3.3 U/ml (1.4-6.7 U/ml) 3.3 U/ml (0.6-6.7 U/ml) (0.3-0.7 U/ml) 3.3 U/ml (0.7-3.3 U/ml) (0.06-0.3 U/ml) (0.6-2.4 U/ml) (66267 &ml) 3.3 vol% (1.6-6.6 vol %) 0.07 % wlv (0.05-0.1 % w/v)
0.48
4
extraction method and temperature
Number of experiments
stirring at 4°C ,, I,
11
3 10
2 2
0.0
5
0.16
4
0.0
0.40
4 0.5
0.74
0.5
,I incubation at 31.5”C ,,
0.0
0.5 0.5
,I I,
2 2
0.82
0.75 0.75 0.75 0.5
I, I, 8, !,
3 3 3 8
0.98
9.5
1 % wlv
4.36
1.5
1 % wlv 1 % wlv
0.0
1.5 1.5
(b) O.M-O.l% Digitoninigitonin. For these experiments enriched synaptosomal membranes were prepared according to [ 161 from rat brains which were incubated in 0.74 sucrose at 4°C for 24-45 hours. This results in a partial degradation of the membranes and allows integral membrane proteins to be extracted by using very low detergent concentrations. Digitonin has been recrystallized twice from % ethanol and did not precipitate after solubilization. (c) Lipases. Membrane suspensions (according to b) were incubated at 315°C (50 mM Tris/HCl pH 7.3; 3 mM Ca*+) with different amounts of the enzymes for 30 min. After addition of EDTA (4 mM) the suspensions were transferred in an ice bath and pelleted. During the extraction experiments in the presence of digitonin the suspensions were shaken for 5 hours at 4°C. (d) Serumlipoproteins. The serumlipoproteins as obtained after dialysis have been added up to a concentration of 6.6 ~01% to the membrane suspensions (according to b) which results in a large increase in unspecific binding of the labelled ligands to the supematants after the corresponding extraction procedures. The addition of phospholipase A2 (l-5 U/ml) results in the precipitation of the protein part of the lipoproteins after incubation at 4°C during 24 hours in the presence of 3 mM Caz+. (e) Phospholipases. The membranes (according to b) were resuspended with 50 mM Tris/HCl buffer (3 mM Ca2+;
0.68 0.0 0.0 0.0
0.29
shaking at 4°C I, I, ,I
7
28 20 3 3
pH 7.3) and 3-16 mg human serum albumin were added per ml of the suspension to absorb the arising lysolipids. Human serum albumin has been selected from other serum albumins to have the lowest unspecific binding under different experimental conditions. The reaction was started by addition of the enzyme and stopped after 30 min at 31.5”C by EDTA addition (10 mM) and cooling. cf) 1% Digitoninlgitonin. Enriched synaptosomal membranes from freshly prepared rat brains were lyophilized and stored dry at -20°C. There is no decline in the number of detectable mAchRs for weeks. These membranes were resuspended at 0°C in the extraction buffer (0.1 M NaCl, 2.5 mM NaH,PO,, 2 mM KC1 adjusted to pH 7.4 with NaOH; 0.01 M Tris/HCl pH 7.4). A digitonimgitonin mixture (see below) was added to give a 1% solution and the membranes were extracted at 4°C by gentle shaking for 60-90 min. DigitoninlGitonin
Preparation
Commercially available “digitonin” preparations were used for starting material for the separation of the main components digitonin and gitonin (which occur in nearly equal portions in the mixture) according to [ 153. The pure components as well as their mixtures have been tested in extraction experiments as 1% solutions. A digitoninlgitonin mixture (about 3:2) with nearly optimal properties could be prepared
SOLUBILIZED
MUSCARINIC
705
RECEPTORS
as follows. Crude digitonin was completely solubilized at 70°C for 10-20 min as a 2% solution in distilled water. During one week at 4°C a gitonin enriched mixture of both components precipitates which represents about one half of the solubilized material. The supematant was freeze dried and stored dry at room temperature. The composition of the detergent mixtures was examined by thin layer chromatography [15]. Binding to Whatman GFIC Glass Filter Membianes Aliquots of rat brain homogenates (0.12 mg protein/ml) and digitonin/gitonin extracts of enriched synaptosomal membranes (0.5 mg protein/ml) were incubated in the corresponding buffers (Tris/HCl and phosphate, pH 7.4 and 9.2) during 60 min at room temperature with 3H-QNB (0.1-5.5 nM). 5x 10m7M QNB sulfate were added to determine unspecific binding. The samples were filtered through Whatman GF/C glass filter membranes under mild suction and washed 4 times with 2 ml aliquots of the corresponding ice cold buffer and placed into scintillation vials. Isoelectrofocusing Preparative isoelectrofocusing has been carried out by using the LKB 8101 equipment (110 ml column) with ampholine carrier ampholytes. The columns were cooled during the whole run to 4°C while 400-1000 V were applied during 15-24 hours. The zones, which are in part precipitated, were stabilized with a continuous sucrose gradient. Extracts (1% digitonin/gitonin) were freshly prepared and passed through a Sephadex G 15 column (eluted with 1% digitonin/gitonin in distilled water) to remove residual buffer substances. Then we added 3H-QNB to the extract up to a concentration of 10 nM and incubated for 30 min at room temperature and 4 hours at 4°C. The columns were eluted and fractionated by means of a LKB uvichord equipment. The ampholyte concentration was 1% in the runs with a pH range from 3-10 and 4-6 and 0.3% in pH gradients from 3.8 to 5.3. The latter pH range was prepared by collecting the corresponding fractions from a gradient (pH 3-6). Binding studies were carried out at pH 8.4. Binding Measurements Specific 3H-QNB and 3H-atropine binding at saturation concentrations (2x 10w9M and 10m8 respectively) has been used for mAchR detection. The binding was measured by equilibrium dialysis of 1 ml aliquots against 7 ml of the extraction buffer at 4°C for 20-24 hours under gentle shaking at pH 7.4 if not indicated. Nonspecific binding was determined in the presence of 2x 10m7QNB sulfate respectively 10w6M atropine sulfate. 3H-QNB has been used for routine experiments. The pH dependency of 3H-QNB binding was measured by using a 1% digitoninlgitonin extract. During the equilibrium dialysis of these samples we used 50-100 mM phosphate and glycin/NaOH buffers of the corresponding pH for outer medium. It could be shown, that due to the high amount and buffer capacity of the outer medium the pH within the dialysis bag changes slowly to the value of the outer medium. Radioactivity measurements were carried out with an Intertechnique scintillationspectrometer where the corresponding quenching curves were used for automated dpm calculation.
RESULTS
Methods for Solubilization
of mAchR
In Table 1 all procedures are summarized, which have been tested in order to develop receptor preparations with properties suitable for further purification. NaCl has already been described to solubilize mAchRs [ 1,3]. We found relatively small amounts of the mAchR in the 100,000 g supernatant, which could not be substantially increased by changes in salt concentration, extraction time and procedure. We could not reproduce the results of Carson et al. [6] using (NH&SO, precipitation of the NaCl extract, which resulted in our experiments in a marked decrease of the yield and specific binding per mg protein. The use of the NaCl extraction method is limited by the frequent loss of activity, which occurs at 4°C during 18-24 hours. We were not able to overcome this denaturation by the addition of phosphatidylcholine liposomes, protease inhibitors, sucrose and Ca*+. The low yield could not be improved by the use of different extraction techniques, the preextraction of lipids at -70°C or the combination of NaCl with the action of phospholipase A* (unpublished results). Further chaotropic agents as NaJ, KC1 and LiBr which already have been used in membrane protein solubilization were tested without success. Phospholipases and lipases were used for partial degradation of the membrane structure which leads to the liberation of some membrane proteins. The incubation of enriched synaptic membranes from brains (which were kept for 24-45 hours at 4°C) with three of the phospholipases (listed in Table 1) led to the appearance of mAchRs in the 100,000 g supematant. By this method a higher yield and stability can be reached compared with the salt extractions. Nevertheless the disadvantages of this method as the need for purified enzymes as well as the albumin contamination (added for absorption of lysolipids) led to the conclusion that this procedure is not suitable for routine preparations. The treatment under similar conditions of enriched synaptosomal membranes with lipases from different sources did not lead to mAchR solubilization, but it improved up to an optimal enzyme activity the mAchR extraction with 0.07% digitoninl gitonin (unpublished results). This might argue against possible protease contaminations to be responsible for the negative results. Human serum lipoproteins consisting of a hydrophobic protein and a surrounding lipid layer do not sediment at 100,000 g/90 min and seem to allow the transfer of some proteins from the membrane to the lipid core of the lipoprotein at 31.5”C. As shown by electrophoretic experiments the protein portion of the lipoproteins precipitates selectively from a protein solution after application of phospholipase AZ. This may lead to the liberation of absorbed proteins. We found that it is in fact possible to transfer certain quantities of mAchR into the lipoprotein containing supematant during an incubation at 3 1.5”C with an enriched synaptosomal membrane suspension from brains incubated for 24-45 hours at 4°C whereas shaking at 4°C has nearly no effect. Digitonin (commercially available preparations, recrystallized from ethanol) has been used in concentrations below 0.1% to develop a method which might fail to have some of the disadvantages which occur at higher detergent concentrations as masking of functional groups and binding sites. mAchRs could be only extracted by this method after previous incubation of the whole brains at 4°C in 0.74 M sucrose. During 24-45 hours a partial degradation of the
REPKE
706
AND MATTHIES
dpm x ICC0 mg protein
20 c 1L
-
12
-
8
-
L
-
--I
w
0.8
0.6
0,4
0,2
0
0
0,2
0.4
0.6
08
0 Na-chotate
Na-desoxycholate
( % w/v 1 - OOOQ0000
o
o
o
o
0
Q
GITONIN DIGITONIN
FIG. 2. Dependence of specific :‘H-QNB binding to protein extracts from enriched synaptosomal membranes on the composition of Nadesoxycholate/Na-cholate mixtures.
FIG. 1. Dependence of specific “H-QNB binding to protein extracts from enriched synaptosomal membranes on the composition of digitonin/gitonin mixtures which were used as 1% (w/v) solutions (see methods). Digitonin and gitonin were separated by thin layer chromatography on silica gel according to [ 151.
% \
membranes takes place which does not lead to a remarkable decrease of detectable mAchRs. This method has been extensively examined in order to optimize all parameters such as digitonin concentration, pH, Ca*+, protein-detergent proportion, time of preincubation, extraction time and additives (enzymes, lipoproteins, glycerin) so that the present yield may represent on optimum. This is considerable less compared with the receptor amounts which can be extracted with 1% solutions of digitonin/gitonin (3:2) mixtures (see below) which have been found to be the optimal detergent for mAchR solubilization. mAchR
Solubilization
with Detergent
Mixtures
By thin layer chromatography we found that commercially available digitonin consists from nearly equal portions of digitonin and gitonin and some minor components which have not been identified. It is only partially water soluble and differs remarkable in its detergent properties. We separated digitonin and gitonin by preparative adsorption chromatography [ 151and tested them alone as well as mixtures containing both components at different portions for their capability to solubilize the mAchR. The results shown in Fig. 1 demonstrate that the highest concentrations of extracted receptors within the 100,000 g supernatant is reached with a mixture of both components at a proportion of about 3:2 (digitonin:gitonin). The yield of the solubilized receptors ranges between 40 and 60%. The variation of the concentration of pure digitonin between 0.1 and 1% did not improve the yield of extracted mAchR. This suggests that the capability of pure digitonin to solubilize mAchRs can be improved by the addition of gitonin (which itself is much less water soluble) up to a certain concentration. The chemical heterogeneity of the detergent micelles influences not only the stability and yield of the extracted mAchR but also the average amount of extracted membrane proteins. With digitonin only 76.4-+5.6% (gitonin 52.6?8.3%) of the protein amount which was extracted with the optimal mixture could be extracted.
.\
l\. \ 0
I
I
12
1
4
I
7
1
1
9
11
days
FIG. 3. Time dependent decline of the specific “H-QNB binding to protein extracts from enriched synaptosomal membranes at 4°C. Detergens: Digitoninigitonin (3:2) 1% (w/v)).
We tested whether similar phenomena can be observed by using mixtures of Na-cholate and Na-desoxycholate. As shown in Fig. 2 we found a small amount of mAchR which could be extracted with Na-desoxycholate and which declines with increasing portions of Na-cholate. On the other hand only a small decrease in the specific “H-QNB binding has been observed if the digitonitigitonin is exchanged from the corresponding extracts against Nadesoxycholate. Stability
The stability of the solubilized mAchR is a crucial point for all further purification steps. We tested extracts obtained by the extraction with digitonin/gitonin mixtures (1%) during storage at 4°C. The specific 3H-QNB binding declines during 9 days to 50% of the original value (Fig. 3). For long time storage the extract can be frozen at -20°C which results in a loss of 3126% of the initial specific binding after thawing. pH Dependency Figure
of Specific
:‘H-QNB Binding
4 shows the specific 3H-QNB binding
of digito-
SOLUBILIZED
MUSCARINIC
RECEPTORS
707
ninlgitonin solubilized mAchR at different pH values. There is a range of nearly unchanged specific binding between pH 7.5 and 10. Additionally we found that the pH can be varied within this range in the extraction buffer without remarkable influence on the yield of extracted 3H-QNB binding sites. This has been shown to be a prerequisite for the use of several purification techniques such as ion exchange chromatography because digitonin/gitonin extracted mAchRs do not bind to DEAE sephadex at pH values around 7 as it should be expected according to its isoelectric point (see below). We found an optimal reversible binding to the gel at pH 9.2 (unpublished results). Binding to Whatman GFIC Glass Filter Membranes According to the isoelectric point of the digitoninlgitonin solubilized mAchR its binding to the charged surface of glass should be expected. This could simplify the method for binding measurements compared to the equilibrium dialysis. We could not observe any solubilized mAchR to be absorbed to the filter using extracts containing 3 pM mAchR per mg protein. The same type of glass filter membranes was employed for binding measurements with rat brain homogenates which resulted in a quantitative absorption up to 1 mg protein per filter (8 1.8 cm). The binding of solubilized mAchRs could not be increased at pH 9.2 or by the exchange of buffer systems. Spreading of DigitoninlGitonin Extracts as Protein Monolayers on the Air/Water Interphase Digitonin/gitonin extracts from enriched synaptosomal membranes can be spread out as a monolayer film on the surface of tridistilled water (total 500 cm*) at constant pressure (5 dyn/cm’) using an equipment as described by Szundi
L
5
6
7
8
9
10
12
PH
FIG. 4. Dependency on pH of specific 3H-QNB binding to digitonin/gitonin (3:2)solubilized mAcbRs. [14]. The surface tension was measured by the Wilhelmy plate method. Changes in the surface potential were followed with an air ionizing electrode. The comparison of the surface pressure-area curves of the unmodified extract and that of an extract which was filtered 2 times through sephadex G-25 show a shift to higher slopes of the surface tension and surface potential curves during compression of the film (Fig. 5). This change can be reversed by the addition of digitonin to the water phase. The exchange of the water phase beneath the film (at 20 dyn/cm2) led to a further increase in the slope of both surface tension and potential curves to its final shape which cannot be changed by further washing or compression/relaxation cycles. This indicates that there is a considerable amount of digitonin molecules which remain bound to the extracted
mV boo
dyn /cm2
100
11
200
300
cm’
FIG. 5. Changes in the surface pressure (solid lines) and surface potential (broken lines) during compression and relaxation of protein monolayer films from digitoningitonin extracts at the air/water interphase. Pretreatment of the extracted proteins: (1) Sephadex G-25 filtration (2 times); exchange of the water subphase after spreading and compression of the film. (2) Sephadex G-25 filtration (2 times). (3) untreated extract.
708
REPKE AND MATTHIES PH 6
20
fractions
FIG. 6. Is~l~t~focusing of digitooj~gito~u solubilized proteins from enriched synaptic membranes. Specific 3H-QNB binding to the fractions has been measured in the equilibrium dialysis at pH 8.4.
proteins also after repeated gel filtration or dialysis and which can be removed only if the digitonin has to compete for binding to presumably hydrophobic binding sites of the proteins with those from other proteins in the compressed film. The shape of the curves of the digitonin free film resembles to that obtained from total brain lipids. The extract film can be sucked off at constant pressure (10 dynlcm*) quantitatively as shown by the use of lz5J labelled proteins and has been characterized to consist at least mainly from proteins. The introduction of 5 M urea into the water subphase results in a long lasting expansion of the film at constant pressure (5 dyn/cm*) which can be interpreted as the result of protein unfolding. Isoelectric Point of the nAchR Preparative isoelectrofocusing was carried out using three different pH gradients (3-10,4-6 and 3%5.3). Only lO-30% of the specific 3H-QNB binding sites applied to the column could be recovered in the equilibrium dialysis at pH 8.4. Using the gradient from pH 3 to 10 the mAchR could be observed between pH 4 and 5; in the gradient from 4 to 6 between 4.5 and 5 and in the gradient from 3.8 to 5.3 it was detected in the fractions with a pH of 4.5-4.6. An example for the latter experiment is shown in Fig. 6. The result suggests a large difference between the isoelectric point and the pH optimum for antagonist binding and argues for a single population of mAchRs in respect to their IP. It must be concluded, that the loss of the binding capacity at low pH values as shown in the above experiments does not represent an irreversible denaturation of the mAchR. DISCUSSION
In order to find suitable methods for mAchR solubilization which allow its further purification we tested a number of in part new procedures and their combinations with each other. Summ~zing it can be concluded, that the use of di~to~~~tonin mixtures for mAchR solubilization might be
optimal in respect to the stability and binding properties of mAchR which have been already characterized by Hurko 191. Aronstam [2] and Gorissen [7f. Disadvantages result from the apparently strong detergent wall which must be assumed to surround the receptor molecule. This is indicated by the studies of mAchR binding to DEAE sephadex and glass filter membranes, the behaviour of the extract at the water-air interphase and furthermore underlined by affinity chromatography experiments (Repke and Matthies, submitted for pubiication). We have found that digitonin preparations contain considerable amounts of gitonin which itseif does not solubilize mAchRs but increases remarkably the efficiency of the pure digitonin up to a certain concentration. This phenomenon could not be observed after the addition of Na-cholate to Na-desoxycholate solutions which itself are slightly effective in mAchR solubiIization. It must be concluded, that the observed properties of digitoni~gitonin mixtures do not represent a common property of mixed detergent micelles of structural related compounds. According to the observation, that some digitonin lots fail to extract the mAchR [2] it can be assumed, that these preparations contain a higher gitonin portion than others. Despite some of the advantages, such as the lack of a masking detergent wail, we did not succeed in establishing a procedure to obtain stable salt extracted receptors. It can not be excluded that different results are in part due to small membrane fragments which have been observed to remain in the supernatant as well as to the difficulties to detect specific muscarinic binding sites with a Iigand of much lower specific activity and affinity to the mAchR such as atropine in comparison to QNB. According to our results, it seems that the mAchR can be partially liberated from the membranes by use of phospholipases without destruction of its binding site. This might be partly due to a lipid annulus surrounding the protein which is protected against the action of the enzyme as observed for a Ca2+ ATPase [4]. We could not observe a total loss of binding sites after phospholipase A treatment as re-
SOLUBILIZED MUSCARINIC RECEPTORS
709
cently described by Jiann-Wu Wei and Sulakhe [Ill for mAchRs from rat atrium. This could be due to different expe~ment~ conditions as the preincubatio~ of the brains at 4°C before membrane preparation as well as lower enzyme activities. The use of human serum lipoproteins for protein extraction according to our method might represent a useful tool also for other solubilization problems because it might be assumed, that the transfer of proteins exhibits a certain selectivity, which has not been examined in detail. The success of the enzyme and lipoprotein treatments might generally depend from a certain degree of membrane degradation as obtained after incubation at 4°C because we failed to succeed in mAchR sofubilization if we used membranes from freshly prepared brains as starting material for these procedures. The digitoni~gitonin solubilized mAehR exhibits a remarkable stability of the specific JH-QNB binding between pH 7.5 and 10. Very different results have been obtained using NaCl extracted mAchR by Alberts and Bartfai [I], who found an optimum around pH 7. As we have shown by the monolayer studies digitoninlgitonin cannot be completely removed from the extracted proteins by gel filtration or dialysis so it cannot be decided whether this difference is due
to the remaining detergent wail or whether it reflects differences between the complex molecule and the subunit presumably solubilized by NaCl. The observed value for the isoelectric point of the mAchR is very similar to that of one of the two forms of the nicotinic acetylcholine receptor [IS] but this does not allow conclusions about further similarities between these proteins. Furthermore these results suggest the existence of only one molecular species of the mAchR similar as it has been indicated by electrophoretic experiments 151.The observed heterogeneity of its binding properties might be rather due to isomerization or ditTerent molecular environments as indicated by others [8, 10, 121. On the other hand it cannot be excluded that during the equilibrium dialysis of the fractions from the pH gradient other forms of the mAchR might not be able to reconstitute their 3H-QNB binding capacity which is lost at the pH of its isoelectric point as shown by our results. ACKNOWLEDGEMENTS
We thank Drs. I. Szundi and A. B&c& Institute of Biophysics, BRC, Szeged, Hungary for performing the monolayer studies and Mrs. M. CoDbau for skillful technical assistance. This study was supported by the Mi~st~ of Science and Technolo~ of G.D.R.
REFERENCES 1, Alberts, P. and T. Bartfai. Muscarinic acetylcholine receptor from rat brain. Partial purification and characterization. J. biol. Chem. 251: 15431547, 1976. 2. Aronstam, R. S., D. C. Schuessler and M. E. Eldefrawi, Solubilization of muscarinic acetylcholine receptors of bovine brain. Life Sci. 23: 1377-1382, 1978. 3. Bartfai, T., J. Anner, M. Schultzberg and J. Montelius. Partial pu~~cation and characterization of a muscarinic acetylcholine receptor from rat cerebral cortex. Biochem. biophys. Res. Commun. 59: 72S-733, 1974. 4. Bennet, J. P., K. A. McGill and G. B. Warren. Transbilayer disposition of the phospholipid annulus surrounding a calcium transvort protein. Nature 274: 823-825. 1978. 5. Bird&l, N. J. M., A. S. V. Burgen and B. C. Hulme. A study of the muscarinic receptor by gel electrophoresis. Br. J. Pharmac. 66: 337-342, 1979. 6. Carson, S., S. Godwin, M. Massonlie and G. Kato. Solubilization of atropine binding material from brain. Nature 266: 176178, 1977. 7. Gorissen, H., G. Aerts and P. Laduron. Characterization of digitonin solubilized muscarinic receptor from rat brain. FEBS let!. %: 64-68, 1978. 8. Hammer, R., C. P. Berrie, N. J. M. Birdsall, A. S. V. Burgen
and E. C. Hulme. Pirenzepine distinguishes between different subclasses of muscarinic receptors. gature 283: 90-91, 1980. 9. Hurko. 0. Soecific $H-GNB bind&z activitv in diaitonin solubilized preparaiions from
10. J&r, J., B. Heldung and T. Bartfai. Isomerization of the muscarinic receptor antagonist komplex. J. biol. Chem. 254: SSQS5589, 1979. Il. Jiann-Wu Wei and P. V. Sulakhe. Properties of the muscarinic cholinergic receptors in rat brain. Naunyn-Schmiedebergs Arch. Pharmac.
309: 259-269.
1979.
12. Kloog, Y., Y. Egozi and M. -Sokolovsky. Characterization of muscarinic acetylcholine receptors from mouse brain: Evidence for regional heterogeneity and isomerization. &fotec. Pharmac, 15: 541-558,
1979.
13. Saraceno, H. and E. deRobertis. Isolation by affinity chromatography of a cholinergic proteolipid from nucleus caudatus. Biochem.
bioohvs. Res. Commun.
69: SSS-561.
1976.
14. Szundi, I. The interaction of lipids with iodine in monomolecular films. Chem. Phys. Lip, 22: 153-161, 1978. IS. Repke, H. and H. Matthies. Preparative separation of digitonin and gitonin and characterization of its detergent properties. Pharmazie
35: 233-234,
1980.
16. Repke, H., H. Nitsch, W. Thiemann and H. Matthies. A simple method for purification of synaptic membranes. Z. med. Labordiagn., in press. 17. Repke, H., H. Matthies, G. Dittrich and H. G. Werchan. Synthesis of affinity gels for the isolation of the muscarinic acetylcholine receptor. 2. Chem. 20: 60-61, 1980. 18. Teichberg, V. 1. and J. P. Changeux. Presence of two forms of acetylcholine receptor with different isoelectric points in the electric organ of electrophorus electricus and their catalytic interconversion in vitro. FEBS lett. 67: 264-268, 1976.
Bulletin,
Vol. 5,
pp. 703-709.
Printed
in the U.S.A.
Biochemical Characterization Muscarinic Acetylcholine
of Solubilized Receptors
H. REPKE AND H. MATTHIES Institute of Neurobiology and Brain Research, Academy of Sciences of GDR 301 Magdeburg, Leipziger Strasse 44, German Democratic Republic Received
8 August 1980
REPKE, H. AND H. MATTHIES. Biochemical characterization of solubilized muscarinic acetylcholine receptors. BRAIN RES. BULL. 5(6) 70%709, 1980.-Several in part new methods (salt extraction, phospholipase treatment, transfer to serumlipoproteins, nonionic detergens mixtures) have been extensively examined in order to obtain a solubilized muscarinic acetylcholine receptor with properties suitable for further purification. An optimal mixture of digitonin and gitonin has been found which forms a tightly bound detergent wall around the receptor protein as indicated by ion exchange and monolayer studies and which allows to solubilize the receptor from enriched synaptosomal membranes with a high yield and stability. The specific 3H-QNB binding of this preparation is nearly unchanged between pH 7.5 and 10. The isoelectric point of the muscarinic acetylcholine receptor has been determined by isoelectric focusing to be between pH 4.5 and 4.6. Solubilization
Muscarinic receptor Isoelectric point
Phospholipases
SEVERAL attempts have been undertaken to solubilize muscarinic acetylcholine receptors (mAchR) which have been shown to be more sensitive to the commonly used detergents than the nicotinic receptor [l, 3, 6, 131. Due to the relatively low concentration of the mAchR in neuronal tissues a high stability of the receptor in the solubilized state is required to enable the subsequent purification procedures. Furthermore the masking influence of a strong detergent wall around the protein should be avoided because it leads to difficulties during ion exchange and affinity chromatography ([ 171 Repke and Matthies, submitted for publication). In an extensive search we studied a number of partially new solubilization procedures in respect to these requirements. Similar to others [2, 7, 91 we found digitonin preparations to be most effective in mAchR solubilization and characterized the role of the main components digitonin and gitonin. The solubilized mAchRs were furthermore characterized in respect to its temperature and pH stability, isoelectric point and the intluence of the detergent on its ionic properties. METHOD Chemicals
All salts were used in p.a. purity. NaCl and KC1 were additionally recrystallized once, (NH&SO, twice from water. LiBr (suprapure) has been obtained from Merck. Digitonin from different lots was supplied from VEB Ysat and Merck. Na-desoxycholate and Na-cholate have been obtained from Serva and recrystallized twice from ethanol. Ampholine carrier ampholytes (pH ranges from 4-6 to 3-10) were supplied from LKB. The enzymes were obtained from the following sources: phospholipase A2 from snake venom (Boehringer); phospholipase C from Cl. perfrigens (&lb&hem); bungarotoxin from B. multicinctus (Boehringer); lipase from wheat germ
Copyright
o 1980 ANKHO
International
Digitonin
Gitonin
Monolayer
(Ferak); lipase from Rhizopus (Fe&); lipase from pankreas (Serva) and human serum albumin (Serva). Human serum lipoproteins were prepared from the blood of a selected donor and prepared as follows. The detibrillated serum was adjusted with KBr to 1,064 g/ml and centrifuged at 125,000 g for 25 hours at 4°C. The pellet was resuspended and adjusted to 1,125 g/ml with KBr. The supematant after the subsequent centrifugation for 44 hours at 125,000 g/4”C was dialysed against 0.9% NaCl(l6 pM/EDTA) and stored at 4°C until use. 3H-QNB (16 and 12 WmMol) were supplied from Amersham Radiochemical Centre and 3H-atropine (1 Ci/mMol) from I.R.E., Belgium. The stock solutions were checked for radiochemical purity before use. Extraction
Procedures
Enriched synaptosomal membranes @fold from homogenate [ 161) were used for starting material in all preparations as suspensions containing about 10 mg protein per ml. If not indicated the membranes were suspended in 50 mM phosphate buffer according to Sorensen (pH 7.3). After the subsequent extraction procedures the membranes were pelleted at 100,000 g during 1.5 hours. The proteins in the corresponding supematants were considered to be solubilized. Extraction methods, time and temperature are listed in detail in Table 1. (a) Salt extraction. Membrane suspensions and salt solutions were mixed in an ice bath to give the final salt concentrations which are listed in Table 1. After extraction and suspensions were diluted with distilled water to avoid the persistence of nonsolubilized material in the 100,000 g supematant. No precipitation of the solubilized proteins has been observed during equilibrium dialysis in 0.1 M NaCV50 mM phosphate buffer pH 7.3 which was employed for binding studies.
Inc.-0361-9230/80/060703-07$01.20/O
REPKE AND MATTHIES
704 TABLE
Detergens
Optimal concentration (examined range in paranthesis)
1
pMol mAchR per ml extract at the optimal conditions
Extraction procedures (optimal parameters) extraction time (hours)
NaCl Nd
KC1 LiBr Phospholipase
A
Phospholipase
A,
P-Bungarotoxin Phospholipase C Wheat germ lipase Pig pankreas lipase Phizopus lipase Human serum lipoproteins Digitoningitonin (recrystallized from ethanol) Digitonimgitonin (3:2) Na-cholate Na-desoxycholate
2.1 M (2.b2.4 M) (l&2.5 M) 2.0 M (1.5-2.5 M) (1.0-2.0 M) 3.3 U/ml (1.4-6.7 U/ml) 3.3 U/ml (0.6-6.7 U/ml) (0.3-0.7 U/ml) 3.3 U/ml (0.7-3.3 U/ml) (0.06-0.3 U/ml) (0.6-2.4 U/ml) (66267 &ml) 3.3 vol% (1.6-6.6 vol %) 0.07 % wlv (0.05-0.1 % w/v)
0.48
4
extraction method and temperature
Number of experiments
stirring at 4°C ,, I,
11
3 10
2 2
0.0
5
0.16
4
0.0
0.40
4 0.5
0.74
0.5
,I incubation at 31.5”C ,,
0.0
0.5 0.5
,I I,
2 2
0.82
0.75 0.75 0.75 0.5
I, I, 8, !,
3 3 3 8
0.98
9.5
1 % wlv
4.36
1.5
1 % wlv 1 % wlv
0.0
1.5 1.5
(b) O.M-O.l% Digitoninigitonin. For these experiments enriched synaptosomal membranes were prepared according to [ 161 from rat brains which were incubated in 0.74 sucrose at 4°C for 24-45 hours. This results in a partial degradation of the membranes and allows integral membrane proteins to be extracted by using very low detergent concentrations. Digitonin has been recrystallized twice from % ethanol and did not precipitate after solubilization. (c) Lipases. Membrane suspensions (according to b) were incubated at 315°C (50 mM Tris/HCl pH 7.3; 3 mM Ca*+) with different amounts of the enzymes for 30 min. After addition of EDTA (4 mM) the suspensions were transferred in an ice bath and pelleted. During the extraction experiments in the presence of digitonin the suspensions were shaken for 5 hours at 4°C. (d) Serumlipoproteins. The serumlipoproteins as obtained after dialysis have been added up to a concentration of 6.6 ~01% to the membrane suspensions (according to b) which results in a large increase in unspecific binding of the labelled ligands to the supematants after the corresponding extraction procedures. The addition of phospholipase A2 (l-5 U/ml) results in the precipitation of the protein part of the lipoproteins after incubation at 4°C during 24 hours in the presence of 3 mM Caz+. (e) Phospholipases. The membranes (according to b) were resuspended with 50 mM Tris/HCl buffer (3 mM Ca2+;
0.68 0.0 0.0 0.0
0.29
shaking at 4°C I, I, ,I
7
28 20 3 3
pH 7.3) and 3-16 mg human serum albumin were added per ml of the suspension to absorb the arising lysolipids. Human serum albumin has been selected from other serum albumins to have the lowest unspecific binding under different experimental conditions. The reaction was started by addition of the enzyme and stopped after 30 min at 31.5”C by EDTA addition (10 mM) and cooling. cf) 1% Digitoninlgitonin. Enriched synaptosomal membranes from freshly prepared rat brains were lyophilized and stored dry at -20°C. There is no decline in the number of detectable mAchRs for weeks. These membranes were resuspended at 0°C in the extraction buffer (0.1 M NaCl, 2.5 mM NaH,PO,, 2 mM KC1 adjusted to pH 7.4 with NaOH; 0.01 M Tris/HCl pH 7.4). A digitonimgitonin mixture (see below) was added to give a 1% solution and the membranes were extracted at 4°C by gentle shaking for 60-90 min. DigitoninlGitonin
Preparation
Commercially available “digitonin” preparations were used for starting material for the separation of the main components digitonin and gitonin (which occur in nearly equal portions in the mixture) according to [ 153. The pure components as well as their mixtures have been tested in extraction experiments as 1% solutions. A digitoninlgitonin mixture (about 3:2) with nearly optimal properties could be prepared
SOLUBILIZED
MUSCARINIC
705
RECEPTORS
as follows. Crude digitonin was completely solubilized at 70°C for 10-20 min as a 2% solution in distilled water. During one week at 4°C a gitonin enriched mixture of both components precipitates which represents about one half of the solubilized material. The supematant was freeze dried and stored dry at room temperature. The composition of the detergent mixtures was examined by thin layer chromatography [15]. Binding to Whatman GFIC Glass Filter Membianes Aliquots of rat brain homogenates (0.12 mg protein/ml) and digitonin/gitonin extracts of enriched synaptosomal membranes (0.5 mg protein/ml) were incubated in the corresponding buffers (Tris/HCl and phosphate, pH 7.4 and 9.2) during 60 min at room temperature with 3H-QNB (0.1-5.5 nM). 5x 10m7M QNB sulfate were added to determine unspecific binding. The samples were filtered through Whatman GF/C glass filter membranes under mild suction and washed 4 times with 2 ml aliquots of the corresponding ice cold buffer and placed into scintillation vials. Isoelectrofocusing Preparative isoelectrofocusing has been carried out by using the LKB 8101 equipment (110 ml column) with ampholine carrier ampholytes. The columns were cooled during the whole run to 4°C while 400-1000 V were applied during 15-24 hours. The zones, which are in part precipitated, were stabilized with a continuous sucrose gradient. Extracts (1% digitonin/gitonin) were freshly prepared and passed through a Sephadex G 15 column (eluted with 1% digitonin/gitonin in distilled water) to remove residual buffer substances. Then we added 3H-QNB to the extract up to a concentration of 10 nM and incubated for 30 min at room temperature and 4 hours at 4°C. The columns were eluted and fractionated by means of a LKB uvichord equipment. The ampholyte concentration was 1% in the runs with a pH range from 3-10 and 4-6 and 0.3% in pH gradients from 3.8 to 5.3. The latter pH range was prepared by collecting the corresponding fractions from a gradient (pH 3-6). Binding studies were carried out at pH 8.4. Binding Measurements Specific 3H-QNB and 3H-atropine binding at saturation concentrations (2x 10w9M and 10m8 respectively) has been used for mAchR detection. The binding was measured by equilibrium dialysis of 1 ml aliquots against 7 ml of the extraction buffer at 4°C for 20-24 hours under gentle shaking at pH 7.4 if not indicated. Nonspecific binding was determined in the presence of 2x 10m7QNB sulfate respectively 10w6M atropine sulfate. 3H-QNB has been used for routine experiments. The pH dependency of 3H-QNB binding was measured by using a 1% digitoninlgitonin extract. During the equilibrium dialysis of these samples we used 50-100 mM phosphate and glycin/NaOH buffers of the corresponding pH for outer medium. It could be shown, that due to the high amount and buffer capacity of the outer medium the pH within the dialysis bag changes slowly to the value of the outer medium. Radioactivity measurements were carried out with an Intertechnique scintillationspectrometer where the corresponding quenching curves were used for automated dpm calculation.
RESULTS
Methods for Solubilization
of mAchR
In Table 1 all procedures are summarized, which have been tested in order to develop receptor preparations with properties suitable for further purification. NaCl has already been described to solubilize mAchRs [ 1,3]. We found relatively small amounts of the mAchR in the 100,000 g supernatant, which could not be substantially increased by changes in salt concentration, extraction time and procedure. We could not reproduce the results of Carson et al. [6] using (NH&SO, precipitation of the NaCl extract, which resulted in our experiments in a marked decrease of the yield and specific binding per mg protein. The use of the NaCl extraction method is limited by the frequent loss of activity, which occurs at 4°C during 18-24 hours. We were not able to overcome this denaturation by the addition of phosphatidylcholine liposomes, protease inhibitors, sucrose and Ca*+. The low yield could not be improved by the use of different extraction techniques, the preextraction of lipids at -70°C or the combination of NaCl with the action of phospholipase A* (unpublished results). Further chaotropic agents as NaJ, KC1 and LiBr which already have been used in membrane protein solubilization were tested without success. Phospholipases and lipases were used for partial degradation of the membrane structure which leads to the liberation of some membrane proteins. The incubation of enriched synaptic membranes from brains (which were kept for 24-45 hours at 4°C) with three of the phospholipases (listed in Table 1) led to the appearance of mAchRs in the 100,000 g supematant. By this method a higher yield and stability can be reached compared with the salt extractions. Nevertheless the disadvantages of this method as the need for purified enzymes as well as the albumin contamination (added for absorption of lysolipids) led to the conclusion that this procedure is not suitable for routine preparations. The treatment under similar conditions of enriched synaptosomal membranes with lipases from different sources did not lead to mAchR solubilization, but it improved up to an optimal enzyme activity the mAchR extraction with 0.07% digitoninl gitonin (unpublished results). This might argue against possible protease contaminations to be responsible for the negative results. Human serum lipoproteins consisting of a hydrophobic protein and a surrounding lipid layer do not sediment at 100,000 g/90 min and seem to allow the transfer of some proteins from the membrane to the lipid core of the lipoprotein at 31.5”C. As shown by electrophoretic experiments the protein portion of the lipoproteins precipitates selectively from a protein solution after application of phospholipase AZ. This may lead to the liberation of absorbed proteins. We found that it is in fact possible to transfer certain quantities of mAchR into the lipoprotein containing supematant during an incubation at 3 1.5”C with an enriched synaptosomal membrane suspension from brains incubated for 24-45 hours at 4°C whereas shaking at 4°C has nearly no effect. Digitonin (commercially available preparations, recrystallized from ethanol) has been used in concentrations below 0.1% to develop a method which might fail to have some of the disadvantages which occur at higher detergent concentrations as masking of functional groups and binding sites. mAchRs could be only extracted by this method after previous incubation of the whole brains at 4°C in 0.74 M sucrose. During 24-45 hours a partial degradation of the
REPKE
706
AND MATTHIES
dpm x ICC0 mg protein
20 c 1L
-
12
-
8
-
L
-
--I
w
0.8
0.6
0,4
0,2
0
0
0,2
0.4
0.6
08
0 Na-chotate
Na-desoxycholate
( % w/v 1 - OOOQ0000
o
o
o
o
0
Q
GITONIN DIGITONIN
FIG. 2. Dependence of specific :‘H-QNB binding to protein extracts from enriched synaptosomal membranes on the composition of Nadesoxycholate/Na-cholate mixtures.
FIG. 1. Dependence of specific “H-QNB binding to protein extracts from enriched synaptosomal membranes on the composition of digitonin/gitonin mixtures which were used as 1% (w/v) solutions (see methods). Digitonin and gitonin were separated by thin layer chromatography on silica gel according to [ 151.
% \
membranes takes place which does not lead to a remarkable decrease of detectable mAchRs. This method has been extensively examined in order to optimize all parameters such as digitonin concentration, pH, Ca*+, protein-detergent proportion, time of preincubation, extraction time and additives (enzymes, lipoproteins, glycerin) so that the present yield may represent on optimum. This is considerable less compared with the receptor amounts which can be extracted with 1% solutions of digitonin/gitonin (3:2) mixtures (see below) which have been found to be the optimal detergent for mAchR solubilization. mAchR
Solubilization
with Detergent
Mixtures
By thin layer chromatography we found that commercially available digitonin consists from nearly equal portions of digitonin and gitonin and some minor components which have not been identified. It is only partially water soluble and differs remarkable in its detergent properties. We separated digitonin and gitonin by preparative adsorption chromatography [ 151and tested them alone as well as mixtures containing both components at different portions for their capability to solubilize the mAchR. The results shown in Fig. 1 demonstrate that the highest concentrations of extracted receptors within the 100,000 g supernatant is reached with a mixture of both components at a proportion of about 3:2 (digitonin:gitonin). The yield of the solubilized receptors ranges between 40 and 60%. The variation of the concentration of pure digitonin between 0.1 and 1% did not improve the yield of extracted mAchR. This suggests that the capability of pure digitonin to solubilize mAchRs can be improved by the addition of gitonin (which itself is much less water soluble) up to a certain concentration. The chemical heterogeneity of the detergent micelles influences not only the stability and yield of the extracted mAchR but also the average amount of extracted membrane proteins. With digitonin only 76.4-+5.6% (gitonin 52.6?8.3%) of the protein amount which was extracted with the optimal mixture could be extracted.
.\
l\. \ 0
I
I
12
1
4
I
7
1
1
9
11
days
FIG. 3. Time dependent decline of the specific “H-QNB binding to protein extracts from enriched synaptosomal membranes at 4°C. Detergens: Digitoninigitonin (3:2) 1% (w/v)).
We tested whether similar phenomena can be observed by using mixtures of Na-cholate and Na-desoxycholate. As shown in Fig. 2 we found a small amount of mAchR which could be extracted with Na-desoxycholate and which declines with increasing portions of Na-cholate. On the other hand only a small decrease in the specific “H-QNB binding has been observed if the digitonitigitonin is exchanged from the corresponding extracts against Nadesoxycholate. Stability
The stability of the solubilized mAchR is a crucial point for all further purification steps. We tested extracts obtained by the extraction with digitonin/gitonin mixtures (1%) during storage at 4°C. The specific 3H-QNB binding declines during 9 days to 50% of the original value (Fig. 3). For long time storage the extract can be frozen at -20°C which results in a loss of 3126% of the initial specific binding after thawing. pH Dependency Figure
of Specific
:‘H-QNB Binding
4 shows the specific 3H-QNB binding
of digito-
SOLUBILIZED
MUSCARINIC
RECEPTORS
707
ninlgitonin solubilized mAchR at different pH values. There is a range of nearly unchanged specific binding between pH 7.5 and 10. Additionally we found that the pH can be varied within this range in the extraction buffer without remarkable influence on the yield of extracted 3H-QNB binding sites. This has been shown to be a prerequisite for the use of several purification techniques such as ion exchange chromatography because digitonin/gitonin extracted mAchRs do not bind to DEAE sephadex at pH values around 7 as it should be expected according to its isoelectric point (see below). We found an optimal reversible binding to the gel at pH 9.2 (unpublished results). Binding to Whatman GFIC Glass Filter Membranes According to the isoelectric point of the digitoninlgitonin solubilized mAchR its binding to the charged surface of glass should be expected. This could simplify the method for binding measurements compared to the equilibrium dialysis. We could not observe any solubilized mAchR to be absorbed to the filter using extracts containing 3 pM mAchR per mg protein. The same type of glass filter membranes was employed for binding measurements with rat brain homogenates which resulted in a quantitative absorption up to 1 mg protein per filter (8 1.8 cm). The binding of solubilized mAchRs could not be increased at pH 9.2 or by the exchange of buffer systems. Spreading of DigitoninlGitonin Extracts as Protein Monolayers on the Air/Water Interphase Digitonin/gitonin extracts from enriched synaptosomal membranes can be spread out as a monolayer film on the surface of tridistilled water (total 500 cm*) at constant pressure (5 dyn/cm’) using an equipment as described by Szundi
L
5
6
7
8
9
10
12
PH
FIG. 4. Dependency on pH of specific 3H-QNB binding to digitonin/gitonin (3:2)solubilized mAcbRs. [14]. The surface tension was measured by the Wilhelmy plate method. Changes in the surface potential were followed with an air ionizing electrode. The comparison of the surface pressure-area curves of the unmodified extract and that of an extract which was filtered 2 times through sephadex G-25 show a shift to higher slopes of the surface tension and surface potential curves during compression of the film (Fig. 5). This change can be reversed by the addition of digitonin to the water phase. The exchange of the water phase beneath the film (at 20 dyn/cm2) led to a further increase in the slope of both surface tension and potential curves to its final shape which cannot be changed by further washing or compression/relaxation cycles. This indicates that there is a considerable amount of digitonin molecules which remain bound to the extracted
mV boo
dyn /cm2
100
11
200
300
cm’
FIG. 5. Changes in the surface pressure (solid lines) and surface potential (broken lines) during compression and relaxation of protein monolayer films from digitoningitonin extracts at the air/water interphase. Pretreatment of the extracted proteins: (1) Sephadex G-25 filtration (2 times); exchange of the water subphase after spreading and compression of the film. (2) Sephadex G-25 filtration (2 times). (3) untreated extract.
708
REPKE AND MATTHIES PH 6
20
fractions
FIG. 6. Is~l~t~focusing of digitooj~gito~u solubilized proteins from enriched synaptic membranes. Specific 3H-QNB binding to the fractions has been measured in the equilibrium dialysis at pH 8.4.
proteins also after repeated gel filtration or dialysis and which can be removed only if the digitonin has to compete for binding to presumably hydrophobic binding sites of the proteins with those from other proteins in the compressed film. The shape of the curves of the digitonin free film resembles to that obtained from total brain lipids. The extract film can be sucked off at constant pressure (10 dynlcm*) quantitatively as shown by the use of lz5J labelled proteins and has been characterized to consist at least mainly from proteins. The introduction of 5 M urea into the water subphase results in a long lasting expansion of the film at constant pressure (5 dyn/cm*) which can be interpreted as the result of protein unfolding. Isoelectric Point of the nAchR Preparative isoelectrofocusing was carried out using three different pH gradients (3-10,4-6 and 3%5.3). Only lO-30% of the specific 3H-QNB binding sites applied to the column could be recovered in the equilibrium dialysis at pH 8.4. Using the gradient from pH 3 to 10 the mAchR could be observed between pH 4 and 5; in the gradient from 4 to 6 between 4.5 and 5 and in the gradient from 3.8 to 5.3 it was detected in the fractions with a pH of 4.5-4.6. An example for the latter experiment is shown in Fig. 6. The result suggests a large difference between the isoelectric point and the pH optimum for antagonist binding and argues for a single population of mAchRs in respect to their IP. It must be concluded, that the loss of the binding capacity at low pH values as shown in the above experiments does not represent an irreversible denaturation of the mAchR. DISCUSSION
In order to find suitable methods for mAchR solubilization which allow its further purification we tested a number of in part new procedures and their combinations with each other. Summ~zing it can be concluded, that the use of di~to~~~tonin mixtures for mAchR solubilization might be
optimal in respect to the stability and binding properties of mAchR which have been already characterized by Hurko 191. Aronstam [2] and Gorissen [7f. Disadvantages result from the apparently strong detergent wall which must be assumed to surround the receptor molecule. This is indicated by the studies of mAchR binding to DEAE sephadex and glass filter membranes, the behaviour of the extract at the water-air interphase and furthermore underlined by affinity chromatography experiments (Repke and Matthies, submitted for pubiication). We have found that digitonin preparations contain considerable amounts of gitonin which itseif does not solubilize mAchRs but increases remarkably the efficiency of the pure digitonin up to a certain concentration. This phenomenon could not be observed after the addition of Na-cholate to Na-desoxycholate solutions which itself are slightly effective in mAchR solubiIization. It must be concluded, that the observed properties of digitoni~gitonin mixtures do not represent a common property of mixed detergent micelles of structural related compounds. According to the observation, that some digitonin lots fail to extract the mAchR [2] it can be assumed, that these preparations contain a higher gitonin portion than others. Despite some of the advantages, such as the lack of a masking detergent wail, we did not succeed in establishing a procedure to obtain stable salt extracted receptors. It can not be excluded that different results are in part due to small membrane fragments which have been observed to remain in the supernatant as well as to the difficulties to detect specific muscarinic binding sites with a Iigand of much lower specific activity and affinity to the mAchR such as atropine in comparison to QNB. According to our results, it seems that the mAchR can be partially liberated from the membranes by use of phospholipases without destruction of its binding site. This might be partly due to a lipid annulus surrounding the protein which is protected against the action of the enzyme as observed for a Ca2+ ATPase [4]. We could not observe a total loss of binding sites after phospholipase A treatment as re-
SOLUBILIZED MUSCARINIC RECEPTORS
709
cently described by Jiann-Wu Wei and Sulakhe [Ill for mAchRs from rat atrium. This could be due to different expe~ment~ conditions as the preincubatio~ of the brains at 4°C before membrane preparation as well as lower enzyme activities. The use of human serum lipoproteins for protein extraction according to our method might represent a useful tool also for other solubilization problems because it might be assumed, that the transfer of proteins exhibits a certain selectivity, which has not been examined in detail. The success of the enzyme and lipoprotein treatments might generally depend from a certain degree of membrane degradation as obtained after incubation at 4°C because we failed to succeed in mAchR sofubilization if we used membranes from freshly prepared brains as starting material for these procedures. The digitoni~gitonin solubilized mAehR exhibits a remarkable stability of the specific JH-QNB binding between pH 7.5 and 10. Very different results have been obtained using NaCl extracted mAchR by Alberts and Bartfai [I], who found an optimum around pH 7. As we have shown by the monolayer studies digitoninlgitonin cannot be completely removed from the extracted proteins by gel filtration or dialysis so it cannot be decided whether this difference is due
to the remaining detergent wail or whether it reflects differences between the complex molecule and the subunit presumably solubilized by NaCl. The observed value for the isoelectric point of the mAchR is very similar to that of one of the two forms of the nicotinic acetylcholine receptor [IS] but this does not allow conclusions about further similarities between these proteins. Furthermore these results suggest the existence of only one molecular species of the mAchR similar as it has been indicated by electrophoretic experiments 151.The observed heterogeneity of its binding properties might be rather due to isomerization or ditTerent molecular environments as indicated by others [8, 10, 121. On the other hand it cannot be excluded that during the equilibrium dialysis of the fractions from the pH gradient other forms of the mAchR might not be able to reconstitute their 3H-QNB binding capacity which is lost at the pH of its isoelectric point as shown by our results. ACKNOWLEDGEMENTS
We thank Drs. I. Szundi and A. B&c& Institute of Biophysics, BRC, Szeged, Hungary for performing the monolayer studies and Mrs. M. CoDbau for skillful technical assistance. This study was supported by the Mi~st~ of Science and Technolo~ of G.D.R.
REFERENCES 1, Alberts, P. and T. Bartfai. Muscarinic acetylcholine receptor from rat brain. Partial purification and characterization. J. biol. Chem. 251: 15431547, 1976. 2. Aronstam, R. S., D. C. Schuessler and M. E. Eldefrawi, Solubilization of muscarinic acetylcholine receptors of bovine brain. Life Sci. 23: 1377-1382, 1978. 3. Bartfai, T., J. Anner, M. Schultzberg and J. Montelius. Partial pu~~cation and characterization of a muscarinic acetylcholine receptor from rat cerebral cortex. Biochem. biophys. Res. Commun. 59: 72S-733, 1974. 4. Bennet, J. P., K. A. McGill and G. B. Warren. Transbilayer disposition of the phospholipid annulus surrounding a calcium transvort protein. Nature 274: 823-825. 1978. 5. Bird&l, N. J. M., A. S. V. Burgen and B. C. Hulme. A study of the muscarinic receptor by gel electrophoresis. Br. J. Pharmac. 66: 337-342, 1979. 6. Carson, S., S. Godwin, M. Massonlie and G. Kato. Solubilization of atropine binding material from brain. Nature 266: 176178, 1977. 7. Gorissen, H., G. Aerts and P. Laduron. Characterization of digitonin solubilized muscarinic receptor from rat brain. FEBS let!. %: 64-68, 1978. 8. Hammer, R., C. P. Berrie, N. J. M. Birdsall, A. S. V. Burgen
and E. C. Hulme. Pirenzepine distinguishes between different subclasses of muscarinic receptors. gature 283: 90-91, 1980. 9. Hurko. 0. Soecific $H-GNB bind&z activitv in diaitonin solubilized preparaiions from
10. J&r, J., B. Heldung and T. Bartfai. Isomerization of the muscarinic receptor antagonist komplex. J. biol. Chem. 254: SSQS5589, 1979. Il. Jiann-Wu Wei and P. V. Sulakhe. Properties of the muscarinic cholinergic receptors in rat brain. Naunyn-Schmiedebergs Arch. Pharmac.
309: 259-269.
1979.
12. Kloog, Y., Y. Egozi and M. -Sokolovsky. Characterization of muscarinic acetylcholine receptors from mouse brain: Evidence for regional heterogeneity and isomerization. &fotec. Pharmac, 15: 541-558,
1979.
13. Saraceno, H. and E. deRobertis. Isolation by affinity chromatography of a cholinergic proteolipid from nucleus caudatus. Biochem.
bioohvs. Res. Commun.
69: SSS-561.
1976.
14. Szundi, I. The interaction of lipids with iodine in monomolecular films. Chem. Phys. Lip, 22: 153-161, 1978. IS. Repke, H. and H. Matthies. Preparative separation of digitonin and gitonin and characterization of its detergent properties. Pharmazie
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