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Assay of the acetylcholine receptor by affinity labeling
ANALYTICAL
BIOCHEMISTRY
76,
442-45 1 (1976)
Assay of the Acetylcholine
Receptor
by Affinity
A. COWBURN~
ARTHURKARLIN,MARKG.MCNAMEE,'ANDDAVID Department
of Neurology.
College of Physicians and Surgeons, New York, New York 10032
Labeling
Columbia
University,
Received March 19. 1976; accepted June 22. 1976 An accurate and sensitive assay for nicotinic acetylcholine receptor binding sites is described which is based on the specificities of receptor both for an affinity label, 4-t N-maleimido)benzyltrimethylammonium iodide (MBTA). and for o-neurotoxins from Naja venoms. It has been demonstrated that MBTA reacts exclusively with one type of subunit of the acetylcholine receptors isolated from the electric tissue of Electrophonts ekcfricas and Torpedo californicc! and that this reaction is blocked in the presence of Naja naja siamensis a-neurotoxin and of other ligands of the acetylcholine binding site. Thus. in this assay the difference in the extent of labeling by MBTA in the absence and presence of N. n. siamensis toxin is considered the specific labeling of receptor. Although this assay is more complicated than direct c*-neurotoxin binding, it is justified by the wellestablished site specificity of the labeling. The specific activities of several different receptor preparations determined using this assay are one-half of those determined using toxin binding. It is possible to assay accurately as little as 0.25 pg of receptor in the presence of IOO-fold as much other protein.
The isolation. purification, and characterization of receptors depend first of all on the availability of suitable assays. For nicotinic-type acetylcholine receptors (AChR), three types of assays have been used (for reviews, see Ref. (l)-(3)): the reversible binding of acetylcholine and congeners (4), the very slowly reversible binding of polypeptide a-neurotoxins of elapid and hydrophid snake venoms (5), and the assay described here which involves the reduction and affinity alkylation of the AChR. It was inferred from physiological experiments with intact electroplax from electric tissue of Electrophortrs electricus that a disulfide bond at the periphery of the ACh binding site on the AChR is susceptible to reduction by low concentrations of dithiothreitol and that, following reduction, one of the SH groups formed reacts at an enhanced rate with various alkylating agents bearing a quaternary ammonium group compared with analogous reagents without a quaternary ammonium group (6). For example, 4(N-maleimido)benzyltrimethylammonium iodide (MBTA) was found to alkylate the reduced AChR about 5000-fold faster than does N-ethylmaleimide. The former reagent has considerable affinity for the unreduced ’ Present address: University of California, Davis, Calif. ” Present address: The Rockefeller University. New York. N.Y.
442 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form resewed.
ASSAY
OF THE
ACETYLCHOLINE
RECEPTOR
443
(i.e., native) ACh bindmg site (I(; = 8 x IO-"M); moreover, its reaction with the reduced AChR is retarded in the presence of specific reversible ligands. By these criteria (7), MBTA is an affinity label for the reduced AChR. Tritiated MBTA has been used to label and to quantitate ACh binding sites in intact cells from EIectrophorus electric tissue (S), in membrane fragments from the same tissue (9). and in solubilized and purified preparations of receptor from Electr-ophorrrs ( 10,ll) and from Torpedo califomica t 12.13). In all cases, a polypeptide of about 40,000 daltons is specifically labeled. The labeling of this component is blocked by the presence of low molecular weight ligands of the AChR and of a-neurotoxins or by affinity reoxidation of the AChR by dithiobischoline. For purposes of labeling a substantial quantity of receptor, conditions similar to those used in the electrophysiological studies are desirable and convenient. The preparation is reacted with 0.2 mM dithiothreitol at pH 8.0 for 10 to 20 min. the dithiothreitol is removed by gel filtration. the preparation is reacted with about lo-” M [“HIMBTA for 2 to 10 min. and finally excess alkylating agent is removed (10). This procedure is cumbersome, however, for the routine assay of multiple samples, and conditions were sought that would permit the sequential addition to AChR preparations of dithiothreitol and [“HIMBTA without the intervening removal of the dithiothreitol. Dithiothreitol is present in great excess over MBTA, with which it reacts quite rapidly. The reduced AChR, however, reacts even more rapidly with MBTA; thus. it was possible to find conditions which result in complete and specific reaction of MBTA with the reduced AChR before the label is consumed by reaction with dithiothreitol. METHODS
[3H]MBTA of specific activity of about 2 ciimmol was synthesized as previously described (8) and was stored in acetonitrile solution in sealed glass vials under liquid nitrogen. After a vial was opened, the contents were stored at -20°C. Label is prepared for use as follows: A small aliquot t -0.2 ml) is taken and dried in V(ICUO, and the residue is redissolved in -4 ml of 0.1 mivr HCl to give an approximate’concentration of label of 30 PM. The exact concentration of label is determined spectrophotometrically as follows: Present in the solution are MBTA and principally one hydrolysis product. 4-(N-maleamido)benzyltrimethy1ammonium iodide. The concentrations in moles per liter of MBTA (0) and its hydrolysis product (h). are determined from their molar extinction coefficients and the absorbances of the solution at 224.236.7 (isosbestic point), 260. and 290 nm (8). (Actually, the absorbances at any two wavelengths suffice to determine cl and h .) The following equations are used: u = (4930 A2.,I - 20.700 A ,<,,,)i(1.5 I x 1Ox)
[I1
h = (32,500 A,,,, - 45 1A,,,)/( 1.5 1 x IO”)
[?I
444
KARLIN.
MCNAMEE
AND
COWBURN
a = (8990 Azz4 - 20,700 A,,,)/(2.73 b = (32,500 Azso - 942 A&/(2.73 u+b=(A
23d15,900
x 108) x 108)
[31 [41
[51
The values of a and b obtained from the two pairs of equations, [I], [2] and [3], [4], are averaged. Also, the quantity (a + b), the total concentration of labeled species, is the average of the values obtained from the first pair, the second pair, and Eq. [5]. The solution of label in 0.1 M HCl can be stored at -20°C for several days. The following hydrolysis rates of MBTA are taken into account in storing and diluting the label: at pH 4, O.OOWday at -2o”C, O.Ol/hr at 0°C. and O.O8/hr at 25°C; and at pH 7.1,0.016/min at 25°C. Because of the rapidity of hydrolysis at pH 7.0, the label is kept at a low pH until it is added to AChR. Reduced, carboxamidomethylated, succinylated lysozyme (10,14) was prepared as follows: One hundred milligrams of hen egg white lysozyme is dissolved in 10 ml of saturated guanidine hydrochloride solution. The pH is adjusted to 8.5 with concentrated NaOH. The solution is continuously stirred on a magnetic stirrer, and 46 mg of dithiothreitol is added. After 20 min at room temperature, 555 mg of iodoacetamide is added over a few minutes. The pH is adjusted back to 8.5 with 3 to 5 M NaOH and maintained at 8.5 while adding 400 mg of succinic anhydride over about 10 min. After the pH no longer changes, the solution is dialyzed against about 7 vol of water and changed every 2 hr, for 8 hr total. A precipitate which eventually redissolves may form in the bag. The outside solution is changed to 10 mrvr Na,CO, for 2 hr and then changed to water again for two additional 2-hr periods. Any precipitate remaining is removed by centrifugation. The protein solution is directly lyophilized. or the protein is precipitated with 9 vol of acetone, washed with acetone, and dried in a vacuum desiccator. The principal a-neurotoxin of Nuju nuju siumensis (siamensis toxin) was purified (15) from lyophilized venom obtained from Miami Serpentarium. All other chemicals were obtained commercially and were the best grade available. Receptor was purified from the electric tissues of E. electricus and of T. culifornicu by affinity chromatography and sucrose density gradient centrifugation ( 11- 13). The labeling assay is as follows: In 15ml screw-top plastic tubes (Becton-Dickinson), quadruplicate 50-~1 samples containing 1 to 50 pg of protein in a buffer called TNPSO and containing 0.2% Triton X-100,50 mM NaCl, 10 mM NaPO,, 1 mM EDTA, and 3 mM NaN, (pH 7.0) are mixed with 50 ~1 of 0.4 mM dithiothreitol in 0.2% Triton X- 100, 150 mM NaCl, 20 mM Tris-Cl, 1 mM EDTA, and 3 mM NaN, (pH 8.3) to give a final buffer composition of 0.2% Triton X-100, 100 mM NaCl, 5 mM NaPO,, 10 mM
ASSAY OF THE ACETYLCHOLINE
0
0.2
D/THIOTHRE/TOL
04
06
RECEPTOR
0.8
445
1.0
CONCENTRATION ImMl
FIG. 1. The extent of specific alkylation of receptor as a function of the initial concentration of dithiothreitol. The assay mixture contained 2.4 pg of AChR purified from Torpedo californica of specific activity of 3.3 pmol of sitesiwgof protein. The receptor was reduced for 20 min with the indicated concentrations of dithiothreitol. and thereafter the assay was continued as described in Methods. In the case of the zero point, dithiothreitol was added to a final concentration of 2.5 PM just before addition of label.
Tris-Cl, 1 mM EDTA, and 3 mM NaN, (pH 8.0). (The concentrations of the components of the medium may be varied somewhat, but the final pH should be 8.0.) All incubations are at 25°C. After 20 min, 10 ~1 of 0.53 M NaPO, (pH 6.7) is added to bring the pH to 7.0. To two of the samples (set A) 400 ~1 of TNPISO (a buffer like TNPSO except the NaCl concentration is 150 mM) is added, and to the other two samples (set B) 400 ~1 of TNPl50 containing 3 pg of siamensis toxin/ml is added. After 15 to 20 min. 500 ~1 of 2 WM [3H]MBTA in 0.2% Triton X-100- 150 mM NaCl (unbuffered) is added to each sample. After a fixed interval, which can be from 0.5 to 20 min, 25 ~1 of approximately 0.1 M /?-mercaptoethanol is added. Finally, about 100 pg of reduced, carboxamidomethylated, succinylated lysozyme is added as carrier, and the tubes are capped and put in ice. The tubes may be put in the freezer for later processing, or the procedure may be carried forward immediately. It is possible to combine the 0.53 M NaPO, and TNP150 into one solution, and also the P-mercaptoethanol and carrier, to reduce the number of separate additions. Forty tubes can be conveniently carried through all the steps by one person making the appropriate additions at 30-set intervals. The filtration to isolate labeled protein is carried out as follows: To each tube is added 14 ml of saturated ammonium sulfate solution, the top is replaced, and the tube inverted. The contents are filtered under suction through a 24-mm glass-fiber filter (Whatman GF/A) held in a polypropylene filter holder (Gelman). The tubes, including the top, are washed twice with 15 ml of 50% saturated ammonium sulfate solution and the washings filtered. Each filter is washed three times with about 25 ml of 50% saturated
446
KARLIN.
MCNAMEE
REDUCTlON
AND
TIME
COWBURN
Imin)
FIG. 2. The extent of specific alkylation as a function of the time of reduction with 0.2 mM dithiothreitol. The conditions were the same as in Fig. 1. The abscissa represents the time from the addition of dithiothreitol to the first dilution and adjustment of the pH of the incubation mixture to 7.0.
ammonium sulfate solution, twice with 25 ml of ice-cold 1 N HCl, and twice with 15 ml of acetone; the vacuum is turned off while the funnel is being filled at each of these washings steps. The dry filters are placed in counting vials and treated with 0.1 ml of water and 0.6 ml of NCS solubilizer (AmershamSearle) for 1 hr at 50°C. Finally, 10 ml of a toluene-based scintillant (5 g of PPO and 0.3 g of dimethyl-POPOP/liter of toluene) is added. Typically, 1000 to 10,000 cpm was the tritium activity obtained per sample. The extent of site-specific labeling is the difference between the means of set A (fully labeled) and set B (protected with toxin). The factor converting counts per minute to moles of label is obtained by dividing the total moles of labeled species (a + b) by counts per minute in a counting standard; i.e., it is assumed that the counting efficiencies of 13HlMBTA and of its hydrolysis product are the same. A standard is prepared by spotting an aliquot of diluted label onto a glass filter in a vial. Such standards in duplicate are digested and counted identically to the assay samples. RESULTS
Optimal concentrations of the reactants in the assay are those that maximize the completeness of the labeling of the ACh binding site on the AChR (specific labeling) and minimize the labeling of other sites on the AChR and on other proteins that may be present (nonspecific labeling). Initially, the ACh binding site disulfide bond must be completely reduced. At 0.2 mM dithiothreitol (pH 8.0) (Fig. 1) and a reaction time of 20 min (Fig. 2), a plateau is reached in the extent of specific labeling. Higher concentrations of dithiothreitol or longer times of reduction result in at most a few percent greater specific reaction. The concentration of dithiothreitol during
ASSAY
OF THE
LABEL FIG. 3. The [3]MBTA. The about 3 pmol of The error bars
ACETYLCHOLINE
CONCENTRATION
extent of specific alkylation as assay mixture contained 2 pg of sitesipgof protein. Two different represent the average deviations
RECEPTOR
447
IpM)
a function of the initial concentration of AChR from Tovprdo of specific activity of receptor preparations (0.0) were assayed. of duplicate determinations.
the alkylation step in the procedure adopted is one-tenth of the initial concentration. In a few experiments, however, an initial concentration of 0.4 mM dithiothreitol (in 50 ~1) diluted during the alkylation to 0.02 mM resulted in the same specific labeling as did the usual initial concentration of 0.2 mM dithiothreitol (in 100 ~1) diluted during the alkylation to 0.02 mM. Therefore, the lack of an effect on specific labeling of higher dithiothreitol concentrations initially is not due to a cancelling of effects due to a higher dithiothreitol concentration during the alkylation. The concentration of siamensis toxin present in the control tubes (set B) during the second step is 2.4 pg/ml (0.3 PM), and, during the subsequent alkylation step. the concentration is 0.15 FM. Neither doubling nor quadrupling the siamensis toxin concentration decreases the extent of labeling in the controls (set B), which is about 5% of the total labeling (A) for highly purified AChR. The specific labeling (A - B) reaches a plateau as a function of 13HlMBTA concentration at 1 PM (Fig. 3). At much higher concentrations (>5 PM) there is a tendency towards higher values for A - B (see Discussion). Experimentally, the errors in A - B are large at label concentrations greater than 4 PM because both the background retention of label by the filter and the extent of nonspecific labeling become comparable to the specific labeling. At 1 pM MBTA, the average deviation in A - B in duplicate determinations is about 10%. The time course of the reaction of MBTA with AChR is very rapid and is essentially complete within 10 set under assay conditions (Fig. 4). in agreement with numerical solutions of the kinetic equations (to appear else-
448
KARLIN.
MCNAMEE
0
20
AND COWBURN
40
ALKYLATlON
60 TIME
180 600 (S)
FIG. 4. The extent of specific alkylation as a function of the time interval between the addition of [SH]MBTA and the addition of /Smercaptoethanol. The incubation mixture contained 2.4 pg of AChR from Torpedo. The zero-time point was obtained by adding P-mercaptoethanol before adding label. All other conditions were as described in Methods.
where). The extent of specific labeling is the same whether the P-mercaptoethanol is added 10 set or 10 min after addition of label. The addition of ,&mercaptoethanol in large excess over all reactants suppresses the reactions of protein SH with labeled species. The addition of P-mercaptoethanol has little effect on the extent of labeling when the samples are precipitated and filtered immediately, but it prevents an increase in nonspecific labeling which results when samples are stored overnight or longer at -20°C prior to filtering. Since the primary reactions of label with dithiothreitol and with all proteins are complete within 30 set, the slow reaction leading to the increase in nonspecific labeling is probably due to the formation of mixed disulfides between proteins and the dithiothreitol[3H]MBTA adduct. This reaction is likely to be suppressed by the presence of an approximately 2500-fold higher concentration of /3-mercaptoethanol, i.e., less than 1 PM monolabeled dithiothreitol compared with 2.5 mM ,&mercaptoethanol. The filtration procedure results in the recovery of at least 90% of the receptor in the assay mixture. as determined by using prelabeled receptor. Addition of carrier is crucial for good recovery. Reduced, carboxamidomethylated, succinylated lysozyme is the best carrier for this purpose found so far. If the recovery of receptor with the usual addition of 100 pg of carboxamidomethylated. succinylated lysozyme is taken as 100%. then the following recoveries are obtained with other amounts and proteins: 500 Kg of carboxamidomethylated, succinylated lysozyme, 100%; 100 pg of lysozyme, 57%; 100 pg of bovine serum albumin, 26%: 200 pg of gelatin, 75%; 200 pg of bovine y-globulin, 8%. The extensive wash procedure is required to obtain low blanks of the order of 500 cpm (0.05-o. 1% of the total radioactivity in the sample filtered). The filter at the conclusion of the washes is dry and free of ammonium sulfate and HCl. The assay is linear up to 5 pg of pure AChR: thereafter. there is a
ASSAY
OF THE
ACETYLCHOLINE
RECEPTOR
449
.I
RECEPTOR
l/~gl
FIG. 5. The extent of specific (0) and nonspecific (0) alkylation as a function of the quantity of receptor assayed. AChR from Torpedo of a specific activity of 2.4 pmol/Fg of protein was assayed according to the procedure described in Methods. The specific alkylation is that blocked by n-toxin and the nonspecific is that not blocked by a-toxin.
slight but increasing deviation from linearity (Fig. 5). The optimal range is 2 to 20 pm01 of sites per sample. The specificity of the reaction. defined as (A - B)/A, varies with the purity of the receptor sample from 12% for 1%pure AChR to about 95% for presumably pure AChR (Fig. 6). DISCUSSION The conditions for this assay were arrived at by our consideration of the rates of the reactions of MBTA with the different classes of SH groups present, namely the SH formed by reduction at the ACh binding site, the SH preexisting or formed by reduction elsewhere on the AChR or on other proteins present, and the SH of dithiothreitol. The first reaction is characterized by a rate for AChR from Elrctrophorus of 6 x lo5 M-‘set-’ (10) and for AChR from T. culifornica of 8 x lo5 MP’sec-‘. The second class of reactions, which result in nonspecific labeling, can be estimated to proceed with an average rate of the order of 600 M-‘set-‘. The rate of reaction of MBTA with the SH of dithiothreitol is about 5000 M-‘set-‘, determined spectrophotometrically as described previously for the reaction of MBTA with cysteine (6). The two SH groups of dithiothreitol appear to be alkylated independently. The calculated time course of these reactions shows completion of the alkylation of specific receptor SH groups in about 14 set (cf. Fig. 4). The increase in the concentration of the product of dithiothreitol and of MBTA almost exactly mirrors the fall in the concentration of MBTA; both changes lag behind the formation of specifically labeled AChR. The formation of labeled nonspecific SH is relatively slow. but since even in purified receptor we estimate that there are about I nonspecific SH groups per reacting ACh binding site SH. the nonspecific reaction is not insignificant.
450
KARLIN.
MCNAMEE
0 062 SPECINC
AND COWBURN
0.25 ACTlVlTY
I
4
(nmo/es/mgl
FIG. 6. The ratio of the specific alkylation of receptor to the total atkylation as a function of the purity of the receptor preparation. Purified receptor from Torpedo was mixed with a protein fraction obtained during the affinity chromatographic purification of receptor, which contained no receptor activity. Samples contained from 0 to 1.5 pg of receptor and from 0 to 14 Kg of nonreceptor protein. Assays were conducted according to the standard procedure as given in Methods. The specificity, defined as (A - B)/A, is plotted as a function of specific activity as determined by the assay (0). The line represents the values calculated by numerical solution of the kinetic equations (to appear elsewhere) taking as equivalent weights 240.000 g of receptor/mot of specific sites and 15.000 g of protein/m01 of nonspecific reactive sulfhydryl groups.
The experimental results are in accord with the calculations (to appear elsewhere) that, within a few percent, all specific sites are labeled at 1 PM MBTA (Fig. 3). The apparent increase in specific labeling above 5 PM label, if significant, may be due to the alkylation of the second thiol formed by disulfide reduction at the ACh binding site. When the saturation of the specific labeling of reduced receptor was investigated by the alternative procedure in which the reduced AChR is separated from dithiothreitol by gel filtration before the addition of MBTA, it was found that half-saturation occurs in 2 min at 9.6 nM MBTA in the case of Electrophonts AChR (10) and at 7.2 nM in the case of Torpedo AChR (unpublished results). With a given AChR preparation, the saturation values of the specific labeling obtained under these conditions at 100 to 500 nM MBTA, moreover, are the same as the values obtained with the presently described assay at 1 to 4 /JM MBTA. The specific activity of pure AChR determined by the binding of tritiated siamensis toxin has, in our hands, given a value exactly twice that obtained by the assay with MBTA (1 I- 13). Furthermore, although all labeling of purified AChR by MBTA is blocked by siamensis toxin, less than half of the binding of toxin is blocked by prelabeling of AChR with MBTA (11). The lower values obtained by affinity labeling could be due either to half-of-thesites reactivity of AChR towards MBTA or to the additional binding of (Ytoxins to a second site different from the ACh binding site. Not only is the specificity of the labeling by MBTA operationally well defined by the
ASSAY
OF THE
ACETYLCHOLINE
RECEPTOR
451
block of the labeling by a-toxin, but the location of this labeling has been shown to be exclusively on a unique subunit of the receptor ( 10,12,13). The specificity of this assay is therefore reliably established. ACKNOWLEDGMENTS The support provided by research grants NS-07065 from the National Institute of Neurological and Communicative Disorders and Stroke and BMS75-03026 from the National Science Foundation and by a grant-in-aid from The New York Heart Association, Inc.. is gratefully acknowledged.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. 1 I. 12. 13. 14. 15.
Karlin. A. (1974) L+r Sci. 14. 138% 1415. Rang, H. P. (1974) Quart. Ret,. Biophys. 7, X3-399. Changeux. J. -P., and Cohen. J. B. (1975) Annu. Ret,. Phtrrmucol. 15, 83- 103. O’Brien. R. D.. and Gilmour, L. P. (1969) Proc. Nat. Acud. SC;. USA 63, 496-503. Lee. C. Y. (1971) Annu. Rev. Phormucol. 12, 265-286. Karlin. A. ( 1969) J. Grn. P17~3siol. 54, 245s-264s. Singer. S. J. (1967) Advan. Prvteir7 Chrm. 22, I-54. Karlin. A.. Prives, J.. Deal. W.. and Winnik. M. (1971)5. Moi. B&i. 61, 175-188. Karlin. A.. and Cowburn. D. A. (1974) in Neurochemistry of Cholinergic Receptors (DeRobertis. E.. and Schacht. J., eds.). pp. 37-48. Raven Press. New York. Karlin. A.. and Cowburn. D. A. (1973) Proc. Not. Acad. Sci. USA 70. 3636-3640. McNamee. M. G., Weill. C. L.. and Karlin. A. (1975) in Protein-Ligand Interactions (Sund. H.. and Blauer. G.. eds.). pp. 316-327. de Gruyter. Berlin. Weill, C. L.. McNamee. M. G.. and Karlin. A. ( 1974) Biot,ltem. Biop/7ys. Rcs. Conrm1ci7. 61, 997- 1003. Karlin. A.. Weill. C. L., and McNamee. M. G. (1975) Cold Spring Hm+~r Syn7p. Quunr. Bid. 40, 203. Habeeb, A. F. S. A.. Cassidy. H. G., and Singer. S. J. ( 1958) Biochim. Ei0phy.s. AC~LI 29, 587-593. Karlsson. E.. Arnberg. H.. and Eaker. D. (1971) Eur. .I. Biochrm. 21. I-16.
BIOCHEMISTRY
76,
442-45 1 (1976)
Assay of the Acetylcholine
Receptor
by Affinity
A. COWBURN~
ARTHURKARLIN,MARKG.MCNAMEE,'ANDDAVID Department
of Neurology.
College of Physicians and Surgeons, New York, New York 10032
Labeling
Columbia
University,
Received March 19. 1976; accepted June 22. 1976 An accurate and sensitive assay for nicotinic acetylcholine receptor binding sites is described which is based on the specificities of receptor both for an affinity label, 4-t N-maleimido)benzyltrimethylammonium iodide (MBTA). and for o-neurotoxins from Naja venoms. It has been demonstrated that MBTA reacts exclusively with one type of subunit of the acetylcholine receptors isolated from the electric tissue of Electrophonts ekcfricas and Torpedo californicc! and that this reaction is blocked in the presence of Naja naja siamensis a-neurotoxin and of other ligands of the acetylcholine binding site. Thus. in this assay the difference in the extent of labeling by MBTA in the absence and presence of N. n. siamensis toxin is considered the specific labeling of receptor. Although this assay is more complicated than direct c*-neurotoxin binding, it is justified by the wellestablished site specificity of the labeling. The specific activities of several different receptor preparations determined using this assay are one-half of those determined using toxin binding. It is possible to assay accurately as little as 0.25 pg of receptor in the presence of IOO-fold as much other protein.
The isolation. purification, and characterization of receptors depend first of all on the availability of suitable assays. For nicotinic-type acetylcholine receptors (AChR), three types of assays have been used (for reviews, see Ref. (l)-(3)): the reversible binding of acetylcholine and congeners (4), the very slowly reversible binding of polypeptide a-neurotoxins of elapid and hydrophid snake venoms (5), and the assay described here which involves the reduction and affinity alkylation of the AChR. It was inferred from physiological experiments with intact electroplax from electric tissue of Electrophortrs electricus that a disulfide bond at the periphery of the ACh binding site on the AChR is susceptible to reduction by low concentrations of dithiothreitol and that, following reduction, one of the SH groups formed reacts at an enhanced rate with various alkylating agents bearing a quaternary ammonium group compared with analogous reagents without a quaternary ammonium group (6). For example, 4(N-maleimido)benzyltrimethylammonium iodide (MBTA) was found to alkylate the reduced AChR about 5000-fold faster than does N-ethylmaleimide. The former reagent has considerable affinity for the unreduced ’ Present address: University of California, Davis, Calif. ” Present address: The Rockefeller University. New York. N.Y.
442 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form resewed.
ASSAY
OF THE
ACETYLCHOLINE
RECEPTOR
443
(i.e., native) ACh bindmg site (I(; = 8 x IO-"M); moreover, its reaction with the reduced AChR is retarded in the presence of specific reversible ligands. By these criteria (7), MBTA is an affinity label for the reduced AChR. Tritiated MBTA has been used to label and to quantitate ACh binding sites in intact cells from EIectrophorus electric tissue (S), in membrane fragments from the same tissue (9). and in solubilized and purified preparations of receptor from Electr-ophorrrs ( 10,ll) and from Torpedo califomica t 12.13). In all cases, a polypeptide of about 40,000 daltons is specifically labeled. The labeling of this component is blocked by the presence of low molecular weight ligands of the AChR and of a-neurotoxins or by affinity reoxidation of the AChR by dithiobischoline. For purposes of labeling a substantial quantity of receptor, conditions similar to those used in the electrophysiological studies are desirable and convenient. The preparation is reacted with 0.2 mM dithiothreitol at pH 8.0 for 10 to 20 min. the dithiothreitol is removed by gel filtration. the preparation is reacted with about lo-” M [“HIMBTA for 2 to 10 min. and finally excess alkylating agent is removed (10). This procedure is cumbersome, however, for the routine assay of multiple samples, and conditions were sought that would permit the sequential addition to AChR preparations of dithiothreitol and [“HIMBTA without the intervening removal of the dithiothreitol. Dithiothreitol is present in great excess over MBTA, with which it reacts quite rapidly. The reduced AChR, however, reacts even more rapidly with MBTA; thus. it was possible to find conditions which result in complete and specific reaction of MBTA with the reduced AChR before the label is consumed by reaction with dithiothreitol. METHODS
[3H]MBTA of specific activity of about 2 ciimmol was synthesized as previously described (8) and was stored in acetonitrile solution in sealed glass vials under liquid nitrogen. After a vial was opened, the contents were stored at -20°C. Label is prepared for use as follows: A small aliquot t -0.2 ml) is taken and dried in V(ICUO, and the residue is redissolved in -4 ml of 0.1 mivr HCl to give an approximate’concentration of label of 30 PM. The exact concentration of label is determined spectrophotometrically as follows: Present in the solution are MBTA and principally one hydrolysis product. 4-(N-maleamido)benzyltrimethy1ammonium iodide. The concentrations in moles per liter of MBTA (0) and its hydrolysis product (h). are determined from their molar extinction coefficients and the absorbances of the solution at 224.236.7 (isosbestic point), 260. and 290 nm (8). (Actually, the absorbances at any two wavelengths suffice to determine cl and h .) The following equations are used: u = (4930 A2.,I - 20.700 A ,<,,,)i(1.5 I x 1Ox)
[I1
h = (32,500 A,,,, - 45 1A,,,)/( 1.5 1 x IO”)
[?I
444
KARLIN.
MCNAMEE
AND
COWBURN
a = (8990 Azz4 - 20,700 A,,,)/(2.73 b = (32,500 Azso - 942 A&/(2.73 u+b=(A
23d15,900
x 108) x 108)
[31 [41
[51
The values of a and b obtained from the two pairs of equations, [I], [2] and [3], [4], are averaged. Also, the quantity (a + b), the total concentration of labeled species, is the average of the values obtained from the first pair, the second pair, and Eq. [5]. The solution of label in 0.1 M HCl can be stored at -20°C for several days. The following hydrolysis rates of MBTA are taken into account in storing and diluting the label: at pH 4, O.OOWday at -2o”C, O.Ol/hr at 0°C. and O.O8/hr at 25°C; and at pH 7.1,0.016/min at 25°C. Because of the rapidity of hydrolysis at pH 7.0, the label is kept at a low pH until it is added to AChR. Reduced, carboxamidomethylated, succinylated lysozyme (10,14) was prepared as follows: One hundred milligrams of hen egg white lysozyme is dissolved in 10 ml of saturated guanidine hydrochloride solution. The pH is adjusted to 8.5 with concentrated NaOH. The solution is continuously stirred on a magnetic stirrer, and 46 mg of dithiothreitol is added. After 20 min at room temperature, 555 mg of iodoacetamide is added over a few minutes. The pH is adjusted back to 8.5 with 3 to 5 M NaOH and maintained at 8.5 while adding 400 mg of succinic anhydride over about 10 min. After the pH no longer changes, the solution is dialyzed against about 7 vol of water and changed every 2 hr, for 8 hr total. A precipitate which eventually redissolves may form in the bag. The outside solution is changed to 10 mrvr Na,CO, for 2 hr and then changed to water again for two additional 2-hr periods. Any precipitate remaining is removed by centrifugation. The protein solution is directly lyophilized. or the protein is precipitated with 9 vol of acetone, washed with acetone, and dried in a vacuum desiccator. The principal a-neurotoxin of Nuju nuju siumensis (siamensis toxin) was purified (15) from lyophilized venom obtained from Miami Serpentarium. All other chemicals were obtained commercially and were the best grade available. Receptor was purified from the electric tissues of E. electricus and of T. culifornicu by affinity chromatography and sucrose density gradient centrifugation ( 11- 13). The labeling assay is as follows: In 15ml screw-top plastic tubes (Becton-Dickinson), quadruplicate 50-~1 samples containing 1 to 50 pg of protein in a buffer called TNPSO and containing 0.2% Triton X-100,50 mM NaCl, 10 mM NaPO,, 1 mM EDTA, and 3 mM NaN, (pH 7.0) are mixed with 50 ~1 of 0.4 mM dithiothreitol in 0.2% Triton X- 100, 150 mM NaCl, 20 mM Tris-Cl, 1 mM EDTA, and 3 mM NaN, (pH 8.3) to give a final buffer composition of 0.2% Triton X-100, 100 mM NaCl, 5 mM NaPO,, 10 mM
ASSAY OF THE ACETYLCHOLINE
0
0.2
D/THIOTHRE/TOL
04
06
RECEPTOR
0.8
445
1.0
CONCENTRATION ImMl
FIG. 1. The extent of specific alkylation of receptor as a function of the initial concentration of dithiothreitol. The assay mixture contained 2.4 pg of AChR purified from Torpedo californica of specific activity of 3.3 pmol of sitesiwgof protein. The receptor was reduced for 20 min with the indicated concentrations of dithiothreitol. and thereafter the assay was continued as described in Methods. In the case of the zero point, dithiothreitol was added to a final concentration of 2.5 PM just before addition of label.
Tris-Cl, 1 mM EDTA, and 3 mM NaN, (pH 8.0). (The concentrations of the components of the medium may be varied somewhat, but the final pH should be 8.0.) All incubations are at 25°C. After 20 min, 10 ~1 of 0.53 M NaPO, (pH 6.7) is added to bring the pH to 7.0. To two of the samples (set A) 400 ~1 of TNPISO (a buffer like TNPSO except the NaCl concentration is 150 mM) is added, and to the other two samples (set B) 400 ~1 of TNPl50 containing 3 pg of siamensis toxin/ml is added. After 15 to 20 min. 500 ~1 of 2 WM [3H]MBTA in 0.2% Triton X-100- 150 mM NaCl (unbuffered) is added to each sample. After a fixed interval, which can be from 0.5 to 20 min, 25 ~1 of approximately 0.1 M /?-mercaptoethanol is added. Finally, about 100 pg of reduced, carboxamidomethylated, succinylated lysozyme is added as carrier, and the tubes are capped and put in ice. The tubes may be put in the freezer for later processing, or the procedure may be carried forward immediately. It is possible to combine the 0.53 M NaPO, and TNP150 into one solution, and also the P-mercaptoethanol and carrier, to reduce the number of separate additions. Forty tubes can be conveniently carried through all the steps by one person making the appropriate additions at 30-set intervals. The filtration to isolate labeled protein is carried out as follows: To each tube is added 14 ml of saturated ammonium sulfate solution, the top is replaced, and the tube inverted. The contents are filtered under suction through a 24-mm glass-fiber filter (Whatman GF/A) held in a polypropylene filter holder (Gelman). The tubes, including the top, are washed twice with 15 ml of 50% saturated ammonium sulfate solution and the washings filtered. Each filter is washed three times with about 25 ml of 50% saturated
446
KARLIN.
MCNAMEE
REDUCTlON
AND
TIME
COWBURN
Imin)
FIG. 2. The extent of specific alkylation as a function of the time of reduction with 0.2 mM dithiothreitol. The conditions were the same as in Fig. 1. The abscissa represents the time from the addition of dithiothreitol to the first dilution and adjustment of the pH of the incubation mixture to 7.0.
ammonium sulfate solution, twice with 25 ml of ice-cold 1 N HCl, and twice with 15 ml of acetone; the vacuum is turned off while the funnel is being filled at each of these washings steps. The dry filters are placed in counting vials and treated with 0.1 ml of water and 0.6 ml of NCS solubilizer (AmershamSearle) for 1 hr at 50°C. Finally, 10 ml of a toluene-based scintillant (5 g of PPO and 0.3 g of dimethyl-POPOP/liter of toluene) is added. Typically, 1000 to 10,000 cpm was the tritium activity obtained per sample. The extent of site-specific labeling is the difference between the means of set A (fully labeled) and set B (protected with toxin). The factor converting counts per minute to moles of label is obtained by dividing the total moles of labeled species (a + b) by counts per minute in a counting standard; i.e., it is assumed that the counting efficiencies of 13HlMBTA and of its hydrolysis product are the same. A standard is prepared by spotting an aliquot of diluted label onto a glass filter in a vial. Such standards in duplicate are digested and counted identically to the assay samples. RESULTS
Optimal concentrations of the reactants in the assay are those that maximize the completeness of the labeling of the ACh binding site on the AChR (specific labeling) and minimize the labeling of other sites on the AChR and on other proteins that may be present (nonspecific labeling). Initially, the ACh binding site disulfide bond must be completely reduced. At 0.2 mM dithiothreitol (pH 8.0) (Fig. 1) and a reaction time of 20 min (Fig. 2), a plateau is reached in the extent of specific labeling. Higher concentrations of dithiothreitol or longer times of reduction result in at most a few percent greater specific reaction. The concentration of dithiothreitol during
ASSAY
OF THE
LABEL FIG. 3. The [3]MBTA. The about 3 pmol of The error bars
ACETYLCHOLINE
CONCENTRATION
extent of specific alkylation as assay mixture contained 2 pg of sitesipgof protein. Two different represent the average deviations
RECEPTOR
447
IpM)
a function of the initial concentration of AChR from Tovprdo of specific activity of receptor preparations (0.0) were assayed. of duplicate determinations.
the alkylation step in the procedure adopted is one-tenth of the initial concentration. In a few experiments, however, an initial concentration of 0.4 mM dithiothreitol (in 50 ~1) diluted during the alkylation to 0.02 mM resulted in the same specific labeling as did the usual initial concentration of 0.2 mM dithiothreitol (in 100 ~1) diluted during the alkylation to 0.02 mM. Therefore, the lack of an effect on specific labeling of higher dithiothreitol concentrations initially is not due to a cancelling of effects due to a higher dithiothreitol concentration during the alkylation. The concentration of siamensis toxin present in the control tubes (set B) during the second step is 2.4 pg/ml (0.3 PM), and, during the subsequent alkylation step. the concentration is 0.15 FM. Neither doubling nor quadrupling the siamensis toxin concentration decreases the extent of labeling in the controls (set B), which is about 5% of the total labeling (A) for highly purified AChR. The specific labeling (A - B) reaches a plateau as a function of 13HlMBTA concentration at 1 PM (Fig. 3). At much higher concentrations (>5 PM) there is a tendency towards higher values for A - B (see Discussion). Experimentally, the errors in A - B are large at label concentrations greater than 4 PM because both the background retention of label by the filter and the extent of nonspecific labeling become comparable to the specific labeling. At 1 pM MBTA, the average deviation in A - B in duplicate determinations is about 10%. The time course of the reaction of MBTA with AChR is very rapid and is essentially complete within 10 set under assay conditions (Fig. 4). in agreement with numerical solutions of the kinetic equations (to appear else-
448
KARLIN.
MCNAMEE
0
20
AND COWBURN
40
ALKYLATlON
60 TIME
180 600 (S)
FIG. 4. The extent of specific alkylation as a function of the time interval between the addition of [SH]MBTA and the addition of /Smercaptoethanol. The incubation mixture contained 2.4 pg of AChR from Torpedo. The zero-time point was obtained by adding P-mercaptoethanol before adding label. All other conditions were as described in Methods.
where). The extent of specific labeling is the same whether the P-mercaptoethanol is added 10 set or 10 min after addition of label. The addition of ,&mercaptoethanol in large excess over all reactants suppresses the reactions of protein SH with labeled species. The addition of P-mercaptoethanol has little effect on the extent of labeling when the samples are precipitated and filtered immediately, but it prevents an increase in nonspecific labeling which results when samples are stored overnight or longer at -20°C prior to filtering. Since the primary reactions of label with dithiothreitol and with all proteins are complete within 30 set, the slow reaction leading to the increase in nonspecific labeling is probably due to the formation of mixed disulfides between proteins and the dithiothreitol[3H]MBTA adduct. This reaction is likely to be suppressed by the presence of an approximately 2500-fold higher concentration of /3-mercaptoethanol, i.e., less than 1 PM monolabeled dithiothreitol compared with 2.5 mM ,&mercaptoethanol. The filtration procedure results in the recovery of at least 90% of the receptor in the assay mixture. as determined by using prelabeled receptor. Addition of carrier is crucial for good recovery. Reduced, carboxamidomethylated, succinylated lysozyme is the best carrier for this purpose found so far. If the recovery of receptor with the usual addition of 100 pg of carboxamidomethylated. succinylated lysozyme is taken as 100%. then the following recoveries are obtained with other amounts and proteins: 500 Kg of carboxamidomethylated, succinylated lysozyme, 100%; 100 pg of lysozyme, 57%; 100 pg of bovine serum albumin, 26%: 200 pg of gelatin, 75%; 200 pg of bovine y-globulin, 8%. The extensive wash procedure is required to obtain low blanks of the order of 500 cpm (0.05-o. 1% of the total radioactivity in the sample filtered). The filter at the conclusion of the washes is dry and free of ammonium sulfate and HCl. The assay is linear up to 5 pg of pure AChR: thereafter. there is a
ASSAY
OF THE
ACETYLCHOLINE
RECEPTOR
449
.I
RECEPTOR
l/~gl
FIG. 5. The extent of specific (0) and nonspecific (0) alkylation as a function of the quantity of receptor assayed. AChR from Torpedo of a specific activity of 2.4 pmol/Fg of protein was assayed according to the procedure described in Methods. The specific alkylation is that blocked by n-toxin and the nonspecific is that not blocked by a-toxin.
slight but increasing deviation from linearity (Fig. 5). The optimal range is 2 to 20 pm01 of sites per sample. The specificity of the reaction. defined as (A - B)/A, varies with the purity of the receptor sample from 12% for 1%pure AChR to about 95% for presumably pure AChR (Fig. 6). DISCUSSION The conditions for this assay were arrived at by our consideration of the rates of the reactions of MBTA with the different classes of SH groups present, namely the SH formed by reduction at the ACh binding site, the SH preexisting or formed by reduction elsewhere on the AChR or on other proteins present, and the SH of dithiothreitol. The first reaction is characterized by a rate for AChR from Elrctrophorus of 6 x lo5 M-‘set-’ (10) and for AChR from T. culifornica of 8 x lo5 MP’sec-‘. The second class of reactions, which result in nonspecific labeling, can be estimated to proceed with an average rate of the order of 600 M-‘set-‘. The rate of reaction of MBTA with the SH of dithiothreitol is about 5000 M-‘set-‘, determined spectrophotometrically as described previously for the reaction of MBTA with cysteine (6). The two SH groups of dithiothreitol appear to be alkylated independently. The calculated time course of these reactions shows completion of the alkylation of specific receptor SH groups in about 14 set (cf. Fig. 4). The increase in the concentration of the product of dithiothreitol and of MBTA almost exactly mirrors the fall in the concentration of MBTA; both changes lag behind the formation of specifically labeled AChR. The formation of labeled nonspecific SH is relatively slow. but since even in purified receptor we estimate that there are about I nonspecific SH groups per reacting ACh binding site SH. the nonspecific reaction is not insignificant.
450
KARLIN.
MCNAMEE
0 062 SPECINC
AND COWBURN
0.25 ACTlVlTY
I
4
(nmo/es/mgl
FIG. 6. The ratio of the specific alkylation of receptor to the total atkylation as a function of the purity of the receptor preparation. Purified receptor from Torpedo was mixed with a protein fraction obtained during the affinity chromatographic purification of receptor, which contained no receptor activity. Samples contained from 0 to 1.5 pg of receptor and from 0 to 14 Kg of nonreceptor protein. Assays were conducted according to the standard procedure as given in Methods. The specificity, defined as (A - B)/A, is plotted as a function of specific activity as determined by the assay (0). The line represents the values calculated by numerical solution of the kinetic equations (to appear elsewhere) taking as equivalent weights 240.000 g of receptor/mot of specific sites and 15.000 g of protein/m01 of nonspecific reactive sulfhydryl groups.
The experimental results are in accord with the calculations (to appear elsewhere) that, within a few percent, all specific sites are labeled at 1 PM MBTA (Fig. 3). The apparent increase in specific labeling above 5 PM label, if significant, may be due to the alkylation of the second thiol formed by disulfide reduction at the ACh binding site. When the saturation of the specific labeling of reduced receptor was investigated by the alternative procedure in which the reduced AChR is separated from dithiothreitol by gel filtration before the addition of MBTA, it was found that half-saturation occurs in 2 min at 9.6 nM MBTA in the case of Electrophonts AChR (10) and at 7.2 nM in the case of Torpedo AChR (unpublished results). With a given AChR preparation, the saturation values of the specific labeling obtained under these conditions at 100 to 500 nM MBTA, moreover, are the same as the values obtained with the presently described assay at 1 to 4 /JM MBTA. The specific activity of pure AChR determined by the binding of tritiated siamensis toxin has, in our hands, given a value exactly twice that obtained by the assay with MBTA (1 I- 13). Furthermore, although all labeling of purified AChR by MBTA is blocked by siamensis toxin, less than half of the binding of toxin is blocked by prelabeling of AChR with MBTA (11). The lower values obtained by affinity labeling could be due either to half-of-thesites reactivity of AChR towards MBTA or to the additional binding of (Ytoxins to a second site different from the ACh binding site. Not only is the specificity of the labeling by MBTA operationally well defined by the
ASSAY
OF THE
ACETYLCHOLINE
RECEPTOR
451
block of the labeling by a-toxin, but the location of this labeling has been shown to be exclusively on a unique subunit of the receptor ( 10,12,13). The specificity of this assay is therefore reliably established. ACKNOWLEDGMENTS The support provided by research grants NS-07065 from the National Institute of Neurological and Communicative Disorders and Stroke and BMS75-03026 from the National Science Foundation and by a grant-in-aid from The New York Heart Association, Inc.. is gratefully acknowledged.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. 1 I. 12. 13. 14. 15.
Karlin. A. (1974) L+r Sci. 14. 138% 1415. Rang, H. P. (1974) Quart. Ret,. Biophys. 7, X3-399. Changeux. J. -P., and Cohen. J. B. (1975) Annu. Ret,. Phtrrmucol. 15, 83- 103. O’Brien. R. D.. and Gilmour, L. P. (1969) Proc. Nat. Acud. SC;. USA 63, 496-503. Lee. C. Y. (1971) Annu. Rev. Phormucol. 12, 265-286. Karlin. A. ( 1969) J. Grn. P17~3siol. 54, 245s-264s. Singer. S. J. (1967) Advan. Prvteir7 Chrm. 22, I-54. Karlin. A.. Prives, J.. Deal. W.. and Winnik. M. (1971)5. Moi. B&i. 61, 175-188. Karlin. A.. and Cowburn. D. A. (1974) in Neurochemistry of Cholinergic Receptors (DeRobertis. E.. and Schacht. J., eds.). pp. 37-48. Raven Press. New York. Karlin. A.. and Cowburn. D. A. (1973) Proc. Not. Acad. Sci. USA 70. 3636-3640. McNamee. M. G., Weill. C. L.. and Karlin. A. (1975) in Protein-Ligand Interactions (Sund. H.. and Blauer. G.. eds.). pp. 316-327. de Gruyter. Berlin. Weill, C. L.. McNamee. M. G.. and Karlin. A. ( 1974) Biot,ltem. Biop/7ys. Rcs. Conrm1ci7. 61, 997- 1003. Karlin. A.. Weill. C. L., and McNamee. M. G. (1975) Cold Spring Hm+~r Syn7p. Quunr. Bid. 40, 203. Habeeb, A. F. S. A.. Cassidy. H. G., and Singer. S. J. ( 1958) Biochim. Ei0phy.s. AC~LI 29, 587-593. Karlsson. E.. Arnberg. H.. and Eaker. D. (1971) Eur. .I. Biochrm. 21. I-16.