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Affinity labeling of the acetylcholine receptor in the electroplax
J. Mol. Biol. (1971) 61, 175-188
Affinity
Labeling of the Acetylcholine in the Electroplax
ARTHUR KARLIN,
JOAV
PRIVES,
WALTER
Receptor DEALT
AND
MITCHELL
WINNIK$
Departments of Neurology and Physiology College of Physicians and Surgeons Columbia University New York, N. Y. 10032, L7.S.A. (Received 22 March 1971) The receptor for acetylcholine in the electroplax of Electrophoru-s electricua is affinity labeled in. situ in a two-step process consisting of reduction by dithiothreitol followed by alkylation with tritiated 4-(N-maleimido)-or-benzyltrimethylammonium iodide. From the effect of these and other reagents on the response of the electroplax to cholinergic agents it has been inferred that dithiothreitol reduces an S-S group in the vicinity of the acetylcholine binding site on the receptor and that MBTA$ alkylates one of the SH-groups thus produced three orders of magnitude faster than other SH groups present. Furthermore, th(l alkylation by MBTA is specifically blocked either by prior affinity oxidation of t’he reduced receptor with dithiobischoline or by the simultaneous presence of hexamethonium. Consequently, the portion of the labeling of the dithiothreitoltreated electroplax by [3H]MBTA, which is eliminated by prior treatment with dithiobischoline and by the presence of hexamethonium. is considered to be specific for the receptor. This portion is 10 to 20% of the total labeling. The asympt,otic limit of the specific labeling yields an estimate of the quantity of receptor binding sit,es. The quantity of acetylcholinesterase catalytic sites is fo~lrfold t.o sevenfold as great. The average surface-density of receptor binding sites in the synaptic area of the electroplax membrane is approximately 3000 sitps pc’r ;Lm2.
1. Introduction Two general approaches to the assay of the receptor for acetylcholine, other than by t.he physiological response which it mediates, are currently in use or being developed. One approach is to determine the non-covalent binding by the material putatively containing the receptor of agents which either activate or inhibit the physiological response (O’Brien, Gilmour & Eldefrawi, 1970; La Ton-e, Lunt 6 De Robertis, 1970, Changeux, Kasai I% Lee, 1970; Miledi, Molinoff & Potter, 1971). The other is to react, the material covalently with affinity labels for the receptor (Gill & Rang, 1966; Changeux, Podleski & Wofsy, 1967; Karlin & Winnik, 1968; Karlin, 1969; Silman & Karlin, 1969: Rang & Ritter, 1969; Singer, 1970; Kiefer, Lindstrom, Lennox & i Present address: University of California, Riverside, Calif. 92502, U.S.A. $ Present address: Erindale College, University of Toronto, Toronto, Ontario. f Abbreviation used: MBTA, 4.(N-maleimido)-o-benzyltrimethylammonium iodide. 175
176
A. KARLIN,
J. PRIVES,
W. DEAL
AND
M. WINNIK
Singer, 1970). Problems of specificity abound, and convincing identification of the receptor in isolation will require the demonstration of reasonable interactions both with covalently and non-covalently reacting physiologically specific agents. The present work concerns the application of a two-step process for covalently labeling the acetylcholine receptor in the isolated living electroplax of Electrophorw electricus. Application of a dilute solution of dithiothreitol to the electroplax results in inhibition of the responseto acetylcholine (Karlin & Bartels, 1966). This inhibition can be completely reversed by subsequent application of an oxidizing agent such as 5,5’-dithiobis(2-nitrobenzoate). If a SH alkylating agent like N-ethylmaleimide, at a concentration otherwise without effect on the response,is added after dithiothreitol, then subsequent application of an oxidizing agent no longer results in reversal of the inhibition. These results suggest that dithiothreitol reduces an S-S group on a membrane component involved in the response to acetylcholine and that the SH groups formed either can be alkylated or can be re-oxidized to the original form. Further evidence supports the hypothesis that the S-S group whosereduction affects the responseis on the receptor and is close to those amino-acid residues which make contact with acetylcholine during binding. The salient points of the argument are: (1) application of dithiothreitol results in changes in the speciJicity of the responseof the electroplax to cholinergic agents; e.g. the response to carbamylcholine is inhibited (Karlin & Bartels, 1966) while that to decamethonium is enhanced (Karlin, 1969). All such changes are reversed following application of an oxidizing agent. Changesin the specificity of the responsepresumably reflect changesin the molecular organization of the binding site of the receptor. (2) Quaternary ammonium derivatives such as MBTA appear to alkylate the reduced receptor, as measuredby block of the reversibility of the effects of reduction by oxidizing agents, three orders of magnitude faster than do uncharged derivatives such as N-ethylmaleimide (Karlin & Winnik, 1968; Karlin, 1969). Furthermore, quaternary ammonium-oxidizing agents reverse the effects of reduction three orders of magnitude faster than do non-quaternary analogs(Bartels, Deal, Karlin & Mautner, 1970); e.g. O-5 PM-dithiobischoline applied for five minutes completely reverses the effects of dithiothreitol, whereas a lOOOfold higher concentration of cystine is without effect. (3) The reactions with the affinity-alkylating and affinity-oxidizing agents are retarded in the presence of 1 mMhexamethonium, a reversible ligand of the receptor (Karlin & Winnik, 1968; Bartels et al., 1970). (4) Following dithiothreitol, reactions with somequat,ernary ammonium maleimide derivatives and with two other quaternary ammonium derivatives, one acylating and one alkylating, result in a depolarizing response of the electroplax that is not reversed by washing (Karlin & Winnik, 1968; Silman & Karlin, 1969). The depolarization is reversed by high concentrations of reversible inhibitors such as d-tubocurarine but reappears after the inhibitor is washed out. Apparently, quaternary ammonium moieties covalently attached to an SH-group formed by reduction, interact non-covalently with the nearby acetylcholine binding site. Furthermore, the extent of activation increaseswith decreasinglength of the attached moiety (Karlin, 1969). MBTA appears to alkylate the reduced receptor 4700-fold faster than does N-ethylmaleimide. MBTA alkylates cysteine in solution 4*3-fold faster than does N-ethylmaleimide. Therefore, the enhancement in the rate of alkylation of the reduced receptor by MBTA over that of N-ethylmaleimide appears to be approximately llOO-fold due to the affinity of MBTA for the receptor and 4*3-fold due to t,he difference
LABELING
OF
ACETYLCHOLINE
177
RECEPTOR
in intrinsic reactivity (Karlin, 1969). Assuming that N-ethylmaleimide reacts witIll the SH group(s) in the vicinity of the binding site of the reduced receptor at approximately the same rate as it reacts with other SH groups elsewhere on the membrane, we expect that MBTA will react with the SH, in the vicinity of the binding site, approximately 1100-fold faster than with all other SH-groups. Despite this apparent’ high specificity, MBTA would also be expected to react with other SH groups. Criteria for specific reaction with the receptor can be developed from the physiological
observations cited above: (1) the specific reaction should be eliminated by prior treatment of the reduced receptor with the affinity-oxidizing agent, dithiobischoline, at a concentration shown to completely reverse the effects of reduction. (2) The specific reaction should be retarded in the presence of hexamethonium. (3) The extent of specific reaction as a function of concentration of MBTA should saturate, and half-saturation
should
occur
at approximately
t,he same concentration
as in
comparable physiological experiments. Using [3H]MBTA, electroplax, obtaining
we measure the extent of alkylation of the dithiothreitol-treated estimates of the quantity of specific and of non-specific
sites
of labeling. A comparison with the quantity of acetylcholinesterase is made. A preliminary report of some of these results has appeared (Karlin, Prives, Deal & Winnik,
1970).
2. Materials (a)
and Methods
4-(Il’-~naleimido)benz~~l-~-trimeth~l~~mmo~~~u~~~
iodide
S-(4-ol-dimetl~ylaminobenzyl)maleamic acid (I): a freshly prepared ether solution of p-amino-a-dimethylaminotoluene (Bennett & Willis, 1929), prepared by tin and HClreduction of P-nitro-a:-dimethylaminotoluene (Stedman, 1927), was added dropwise to a vigorously stirred solution of 1.2 equivalents of maleic anhydride in ether. After 1 hr at room temperature the solution was cooled in ice and filtered and the precipitate washed with ether to remove excess maleic anhydride. The combined yield for the reduction and acylation was 93%, giving white plates of m.p. 218 to 219°C after recrystallization twice from 80% aqueous ethanol. Molecular ion found at m/e 248; calculated, 248. Infrared maxima at 1680 cm-’ due to carboxyl, at 1,596 cm-l due to amide carbonyl and at 1540 cm-l due to amide II band. Analysis: talc. for C,,H,,O,X,: C, 62.89; H, 6.50; 5. 11.28; found: C, 62.60; H, 6.68; N, 11.13. 4-(N’-maleimido)-h’,N-dimethylbenzylamine (II). Cyclization of I to II was by the method of Cava, Deana, Muth & Mitchell (1961). A pasty mixture of 0.5 g of I, 88 mg sodium acetate (anhydrous) and 1 ml. acetic anhydrido was swirled over a steam bath for approximately 10 min until a yellow solution was formed. This was poured into icrwater and the mixture extracted twice with methylene chloride, adjusted to neutralitS with sodium bicarbonate, and extracted twice more with methylene chloride. The combined organic phase was dried over calcium sulfate. The solvent was removed under Induced pressure and 380 mg of bright yellow crystals (m.p. 86 to 87°C) were obtained from benzene-hexane solution. Molecular ion found at m/e 230; calculated 230. Infrared maximum (KBr) at 1710 cm-l due to the carbonyls. Nuclear magnetic reasonance peaks
(Ccl,)6 2.26 (singlet, 6H, (CH,),--), 3.48 (singlet 2H, -CH,N-), vinyl), 7.48 (AB quartet, 4H, aromat,ic). II polymerizes on standing.
6.86 (singlet It
2H,
is convcnicntl>
removed from polymer by dissolving in carbon tetrachloride and filtering. MBTA : 0.5 g of II was dissolved in 5 ml. methyl iodide and stirred overnight. MBTA was precipitated with ether and recrystallized from hot acetonitrile and benzene to yield 0.X g of yellow crystals, m.p. 204 to 205°C. Molecular ion, minus methyl iodide, found at m/e 230; calculated, 230. Infrared maximum at 1710 cm-l due to carbonyls. Nuclear magnetic resonance peak (D,O) 6 3.63 (singlet, 9H, (CH,),-), 4.75 (singlet, 2H, CH,N), 6.88 (singlet, 2H, vinyl), 7.68 (quartet, J=SHz dv 18 Hz, 4H aromatic). 1‘2
178
A.
KARLIN,
J.
PRIVES,
W.
DEAL
AND
M.
WINNIK
(b) [3H] 4-(N-maleimido)-ol-benz?/ltrinzethylam71~ iodide Into a vial containing 131 mg of II in O-5 ml. of methylene chloride was transferred 5.7 ~1. of tritiated methyl iodide (Tracerlab, 2.2 Ci/m-mole, 13 mg) in 0.5 ml. ether on 8 vacuum line. The vial w&8 sealed and stored at room temperature in the dark for 6 days. The vial W&S opened, 5 ml. ether added, and the sample was spun on a centrifuge and decanted. The precipitate wae washed with ether 4 times. The resulting bright yellow powder, dissolved in 0.8 ml. acetonitrile and 0.2 ml. methylene chloride, showed the proper nuclear magnetic resonance spectrum. The solution was added dropwise to 10 ml. ether with stirring. The precipitate was separated and dried in ~acuo to yield 39 mg of [3H]MBTA. This was dissolved in 100 ml. acetonitrile to yield a 9O-pM solution, samples of which were sealed into glass vials and placed in liquid nitrogen for storage. Solutions of [3H]MBTA for labeling experiments were prepared as follows. A vial containing [3H]MBTA in acetonitrile was removed from the liquid nitrogen and opened. The acetonitrile was removed under vacuum and the residue dissolved in O-1 mM-HCl to make approximately 0.25 mM-[3H]MBTA. A sample of this was taken for determination of concentration, and the remainder was divided into screwtop vials, frozen in liquid nitrogen, stored at -2O”C, and used over a period of 2 weeks. MBTA hydrolyzes to tho maleamic acid derivate 4.(N-maleamido)-or-benzyltrimethylammonium iodide (III). The concentration of MBTA and of III in the HCl solution was calculated from the extinction at 224, 236.7, 260 and 290 nm and the respective molar extinction coefficients for MBTA: 32,500, 15,900, 942, 451, and for III: 20,700, 15,900,8990, 4930. The extinction at the isosbestic point, 236.7 nm, yields the sum of MBTA and III. The extinction at the remaining 3 wavelengths were used pairwise to calculate the individual concentrations of MBTA and III, and the results, in all cases agreeing closely, were averaged. The actual solution of [3H]MBTA used in labeling was a dilution in eel Ringer solution (pH 7.1) of the stock solut,ion in HCl. The concentration of MBTA in the final solution was determined taking into account the following rates of hydrolysis of MBTA: 0.004/day in 0.1 mM-HCl at, -20°C; O*OlG/min in eel Ringer solution (pH 7.1) at 25°C. (c) Other reagents Dithiobischoline diiodide was synthesized according to Andrews, 1963. Other reagents were the best grade obtainable commercially.
Bergel
& Morrison,
(d) Labeling procedure Electroplax were dissected from the organ of Sachs of Electrophorus eleetricua as described by Schoffeniels (1957). In a typical experiment, 21 to 30 electroplax were divided into three groups of 7 to 10 electroplax each. The groups were treated simultaneously according to the following sequences of solutions: (A) (B) (C)
0.2 mM-dithiothreitol (10 min), R (5 min), R (5 min), R (20 min), 3[H]MBTA (10 min), R (5 min), R (5 min), R (5 min), R (6 min), R (5 min); 0.2 mM-dithiothreitol (10 min), R (5 min), R (5 min), R (5 min), 0.5 ran-dithiobischoline (5 min), R (5 min), R (5 min), [3H]MBTA (10 min), R (5 min), R (5 min), R (5 min), R (5 min), R (5 min); 0.2 mM-dithiothreitol (10 min), R (5 min), R (5 min), R (15 min), 1 mm-hexamethonium (5 min), 3[H]MBTA in 1 mM-hexamethonium (10 min), R (5 min), R (5 min), R (5 min), R (5 min), R (5 min),
where R is eel Ringer solution: 165 mm-N&l, 2.3 mM-KCl, 2 mM-C&l,, 2 mM-Mg&, 1.2 mM-K2HP04, 0.3 m&r-KH2P04, 10 mM-glucose (pH 7.1). All solutions were in R except for dithiothreitol which was dissolved in a Tris-Ringer solution (pH 8.0) identical with R except that Tris * HCl replaced phosphate and glucose wss omitted. The electroplax were transferred to 60 ml. of each solution and gently swirled for the times indicated. At the conclusion of the treatment sequence, the electroplax were blotted on filter paper and each placed in a tared vial and weighed. To each vial w&8 added 50 ~1. water and 0.5 ml. of NCS solubilizer (Amersham/Searle) and the vials were placed in a water bath at 6O’C for 2 hr. Ten-ml. scintillant (PPO and dimethylPOPOP in toluene) were added and the vials placed in a liquid-scintillation spectrometer for counting. The counting rate
LABELING
OF
ACETYLCHOLINE
RECEPTOR
was, in all cases, many-fold greater than background and the statistical were insignificant compared to other causes of variation.
179
errors in couming
(e) AcetyZchoZinesterase Single electroplax were blotted, weighed and homogenized in 1 ml. of 1 M-NaCl-1 mMI)otassium phosphate (pH 7.0)-0.01°h gelatin in a Ten Broek type all-glasshomogenizer. The homogenate was washed with the same medium into the titration vessel of an automatic titrator (Radiometer) operated as a pH-stat. The initial volume of the solution was 9 ml. Samples of 0.1 M-acetylcholine bromide were added successively to give 1 In&r, 3 1nM and 10 mM-acetylcholine. The steady-state rate of hydrolysis was determined at approximately pH 7.0. The titration was at 25°C under nitrogen with 0.01 M-NaOH as the titrant. (f) Electrophyaiologicul mea.wrevtwv& Electroplax were dissected as in (d) and the electrical potential differences across the ccl1 and across the innervated and the non-innervated membranes were measured as 1)reviously doscribod (Karlin, 1969).
3. Results (a) Physiological effects of 0.2 mM-dithiothreitol A lower concentration of dithiothreitol (O-2 mM) was used in the present work than that used previously (1 InM) (Karlin & Bartels, 1966). It was found that the lower concentration caused nearly aa much inhibition of the responseof the electroplax to carbamylcholine and resulted in less non-specific labeling by [3H]MBTA. The mean inhibition of the responseto 40 PM-carbamylcholine following a lo-minute application of 1 mM-dithiothreitol is 76% ; that following 0.2 mMdithiothreito1 is 65%. The inhibition due to 10 minutes of 1 mM-dithiothreitol is the maximum obtainable : 20 minutes of 5 maa-dithiothreitol results in the sameaverage inhibition. Given a linear relationship between the number of receptors reduced and the inhibition resulting, and a residual activity of the reduced receptor of 24%. it appears that
IO’x
Concn
(Ml
Fm. 1. The fraction of the inhibition of the response of the electroplax to carbamylcholine due to treatment with dithiothreitol followed by MBTA that is not reversed by subsequent treatment with 6,6’-dithiobis(2aitrobenzoate). Dithiothreitol w&9 applied for 10 min at concentrations of either 1 mu ( 0) or O-2 mu ( O,O). MBTA ~88 applied for 10 min at the oonoentration indicated on the absoissa either by itself (0, O), or in the presence of 1 mM-hexamethonium (0). Dithiobis(2-nitrobenzoate) wae applied for 10 min at 1 mu. The data for 1 mna-dithiothreitol, (o), are from Karlin (1969). The bars indicete standard errors of the mean.
180
A.
KARLIN,
J.
PRIVES,
W.
DEAL
AND
M.
WINNIK
approximately 85% (65 out of 76) of the receptors are reduced by 0.2 mM-dithiothreitol applied for 10 minutes. As with 1 m&r-dithiothreitol, the effects of O-2 mm-dithiothreitol are completely reversed by 10 minutes of 1 mM-5,5’-dithiobis(2-nitrobenzoate) (pH 8.0) or by 5 minutes of 0.5 ,uM-dithiobischoline. The extent to which alkylation of the reduced receptor by MBTA prevents the reversal by oxidizing agents is somewhat less after 0.2 mM than after 1 mM-dithiothreitol (Fig. 1). The concentration of MBTA, applied for 10 minutes, resulting in half-maximal inhibition of the reversal is approximately the same for the two concentrations of dithiothreitol, namely 2.5 x 10mg M. Hexamethonium partially protects the receptor reduced by 0.2 mMdithiothreito1 against alkylation by MBTA (Fig. 1). At 10m6 M-MBTA, the extent of inhibition in the presence of 1 mM-hexamethonium is 38% of that in the absence of hexamethonium. (b) The specific component of the labeling In the labeling of electroplax according to sequence (A), pre-existing SH groups and reduced S-S groups of both receptor and non-receptor proteins are labeled. In sequence (B), the reduced receptor is specifically reoxidized by dithiobischoline and consequently is not alkylated by [3H]MBTA. Sequence (B) is a measure of the nonspecific labeling. In sequence (C), the receptor is partially protected by hexamethonium; hence (C) is expected to measure a part of the specific labeling in addition to all of the non-specific labeling. Sequence (A) minus sequence (B), (A - B), and sequence (A) minus sequence (C), (A - C), are measures of the specific labeling, There is considerable scatter in the quantities measured using A - B and A - C expressed in moles/mg wet weight, especially at the higher concentrations of label (Figs 2 and 4). The scatter is significantly less when the data is expressed as mole/ power of the wet mg 2’3 (Figs 3 and 5). The justification for using the two-thirds weight is as follows: for identically treated cells, the quantity of label covalently attached varied on the average with the 0.58&O-12 power of the wet weight, as
4 I08xConcn
(M)
FIG. 2. The difference (A - B) between the mean quantity of C3H]MBTA bound by electroplex treated with 0.2 man-dithiothreitol (10 min) followed by [3H]MBTA (10 min) (A) and that bound by electroplex treated with dithiothreitol followed by 0.5 pM dithiobischoline (5 min) followed by [3H]MBTA (B). The concentration of [3H]MBTA is given on the abscissrt. The data are expressed aa molee/mg wet weight. The bars represent the standard error of the difference of the mean of A and B.
LABELING
OF
ACETYLCHOLINE
10*x
Fm. 3. The difference The curve is a weighted
(A - B) as in Fig. least-squares fit.
0
I
Concn
2, except
(M)
that
2 IO* x Concn
181
RECEPTOR
data
3
are
expressed
as molos/mg2’3.
4
(Ml
4. The difference (A - C) between the mean quantity of [3H]MBTA bound by electroplas with 0.2 mM-dithiothreitol (10 min) followed by [3H]MBTA (10 min) (A) and that bound by eloctroplax treated with dithiothreitol followed by [3H]MBTA in the presence of 1 mM-hcxamethonium (C). The concentration of [3H]MBTA is given on the abscissa. The data are oxpressod as moles/mg wet weight. FIG.
treated
by a parabolic least-squares tit of the data. The two-thirds power, which is within one standard deviation of the estimated parameter, makes sense dimensionally. Assuming equal densities and proportions for these cells, the two-thirds power of t,he wet weight is proportional to the surface area of the cells, with the same proportionality constant for all cells. It is reasonable to assume that the charged, quaternary ammonium MBTA does not penetrate the surface membrane in appreciable amounts during the lo-minute incubation period, and hence that almost all of the reaction takes place at the cell surface. Expressed as moles/mg 213, the quantities A - B and B - C appear to approach determined
182
A.
KARLIN,
J.
PRIVES,
W.
10*x
FIG. 5. The difbrence The curve is a weighted
(a - C) as in Fig. least-squares fit.
DEAL
Concn
4, except
AND
M.
WINNIK
(~1
that
data
are expressed
8s moles/mg2’3.
an upper limit with increasing concentration of [3H]MBTA (Figs 3 and 5). Estimates of this limit are obtained as follows. During the lo-minute incubation, the concentration of r3H]MBTA remains nearly constant, less than 1% being lost in alkylation reactions and approximately 16% being lost by hydrolysis. The reaction of the label with the receptor therefore, is probably well represented as pseudo-first order. The extent of reaction is given by Y = Y,,,(l
- ewktX)
where Y is moles of receptor alkylated; k is the second-order rate constant; t, is the duration of the reaction; and X is the concentration of [3H]MBTA. The equation may be written Y = Y,,,(l where X,., is the concentration time t). However,
- 2-x’xo,s)
of label at which
half-maximal
labeling occurs (at
z(x’xo~s) = 1 + (X/X,.,) approximately,
and therefore an approximation
for Y is the familiar form
yIn*xm + X0.,/X) which deviates from the exact expression for Y by, at most, 15% and is identical at X = Xo.,~Xo., and Y,,, may now be estimated by fitting a straight line to the points (l/X, l/Y). Estimates of these parameters and the standard errors of the estimates were obtained by a weighted least-squares analysis (Wilkinson, 1961) taking the inverse of the coefioient of variance as the weighting factor. Both the data expressed in moles per mg213 and those expressed in moles per mg were analyzed and the parameters estimated (Table 1). The two sets of parameters are consistent. X0 .s is approximately the same for both. Since in the range of electroplax weights obtained, the value in mg is approximately threefold the value in mg213,
LABELING
OF
ACETYLCHOLINE
The specific labeling by [3H]MBTA
of dithiothreitol-treated electroplux
(lW&.~
ww ynl,,
(moles/mg2’3)
(W
2.1 f 3.0 f
1.7 5 0.7 1.6 f 0.6
0.8 0.8
Extrapolat,ed 2.5 f 4.6 f
0.9 1.2
t,o complete
183
1
TABLE
(1W ylrmx
RECEPTOR
(moles/mg)
reduction
0.8 * 1.2 f
0.6 0.6
(lWXO.6 W) 2.1 f 1.6 2.3 & 1.1
and protection 0.9 f 1.8 f
0.7 0.9
Eleotroplax were treated with 0.2 mM-dithiothreitol followed by either [3H]MBTA (A), or 0.5 PM-dithiobischoline and then [3H]MBTA (B), or [3H]MBTA in the presence of 1 mM-hexamethonium (C). The differences, A - B and A - C, approach an asymptotic limit (Y,,,) with increasing concentration of [3H]MBTA. This limit, Y,,,, and the concentration (X,.,) applied for 10 min which results in half-maximal labeling were estimated by a weighted least-squares fit of t,he equation Y = Y,ax/(l + X,.,/X) 1 o the data. The data expressed in units of moles/mg and of molss/mga’3 were analyzed separately. The extrapolation to complete reduction of all receptors and complete protection by hexamethonium is based on the approximations that 0.2 man-dithiothreitol applied for 10 min reduces 85% of the receptors and that 1 mM-hexamcthonium protects against alkylation by MBTA to t,he extent of 77% (see text for details).
the values of Y,,, correspond s,swell. The relative errors of the estimates are considerably lessfor the data expressed as moles per mg2’3. Curves calculated using t,he estimated parameters are plotted in Figures 3 and 5. The values of Y,,, for A - B and A - C do not correspond to the total quantity of the receptor. It appears that 0.2 mM-dithiothreitol (10 min) reduces 85% of the total available receptors (seeabove). Dividing Y,,, by 0.85 gives an estimate of the t,otal quantity of receptor as determined by A - B (Table 1). From physiological experiments, hexamethonium protects the reduced receptor against alkylstion by MBTA 77% on the average over the concentration range tested (Fig. 1). Y,,, for A - C therefore corresponds to 0.85 x0.77=0*65 of the total quantity of receptor, and dividing Y,,, by 0.65 gives an estimate of the total (Table 1). These extrapolations increasethe spread between the limits of A - B and A - C. The difference hetween these two estimates for the total receptor is not statistically significant: however, A - C was, in most experiments, greater than A - B as are the corresponding values of Y,,,. The ratios of the specific labeling to the total labeling, (A - B) : A and (A - C) : A, decreasewith increasing concentrations of [3H]MBTA (Fig. 6). This decreasewould be expected if the specific component of the labeling were to saturate and the nonspecific component increase linearly with increasing concentration of label. This appears to be the case. (c) The non-speci$c componentof the labeling A considerable fraction of the total labeling is non-specific. This includes preexisting SH groups, non-receptor S-S groups reduced by dithiothreitol, and possibly other nucleophillic groups such as -NH, that might react with MBTA. That almost all of the groups that are labeled by [3H]MBTA are SH groups is demonstrated by
A. KARLIN,
184
J. PRIVES,
W. DEAL
AND
M. WINNIK
10Sx Concn(Ml Fm. 0. The fraction of the total labeling which is speoific. (A - B)/A, (0); The abscissa gives the concentration of [3H]MBTA. Data obtained in different slightly different conoentrations of r3H]MBTA are averaged here, the bars standard error of the mean.
(A - G)/A, experiments representing
(m). at the
the following: incubation of electroplax with 1 mM.-S&Y-dithiobis(2nitrobenzoate) (pH 8.0) for 10 minutes, either with or without prior incubation with dithiothreitol, followed by lo-* M-[~H]MBTA for 10 minutes, results in 3 to 5% of the labeling obtained in the absenceof incubation with 5,6’-dithiobis(2nitrobenzoate). Therefore, oxidation of SH groups eliminates nearly all of the groups that react with MBTA either before or after dithiothreitol. In contrast, the labeling of the pre-existing SH groups is not changed either by pretreatment with 0.5 ,uM-dithiobischoline for 5 minutes or by the presence of 1 mM-hexamethonium. This supports the assumption that at this low concentration dithiobischoline oxidizes only the SH groups of the reduced receptor and furthermore that hexamethonium protects only specific receptor SH groups against alkylation by MBTA. The labeling either before or after dithiothreitol was found to approach a limiting value at a concentration of approximately 1 mM-[3H]MBTA, applied for 10 minutes. Also, at a given concentration of [3H]MBTA, the quantity of labeling was approximately maximal following 5 mrvr-dithiothreitol applied for 20 minutes. Thus, the first TABLE
2
The quantity of group reacting with [3H]ikfBTA before reduction with dithiothreitol (designatedSH) and after reduction (designatedS-S) Labeling
procedure
1Ol4 x SH (moles/mgals)
10’4 x s-s (moles/mg2’s)
lOi xR (moles/mg2’s)
21,000
2-5
5 mM-dithiothreitol 0.8 mMj3H]MBTA
(20 min), (10 min),
6406
0.2 m&r-dithiothreitol 0.8 mM-[sH]MBTA
(10 min), (10 min),
6400
0.2 mM-dithiothreitol lOWa M-[3H]MBTA
(10 min), (10 min).
3000 3
The labeling procedure given is that for total labeling (SH obtained by omitting dithiothreitol. The quantity, S-S, was (SH + S-S). Inoluded in S-S is the quantity of receptor and elkylated, designated R. It is assumed that only one of the S-S is alkylated by MBTA.
2-4 3
l-2
+ S-S). The quantity, SH, was obtained by subtracting SH from disulfide whioh has been reduced two SH formed by reduction of an
LABELING
OF
ACETYLCHOLINE
RECEPTOR
185
line of Table 2 gives estimates of the total quantity of preexisting SH groups which can be alkylated by MBTA and of the quantity of S-S groups which can be reduced by dithiothreitol and alkylated by MBTA. Of the latter quantity, l/7 is actually reduced by 0.2 mMdithiothreito1. Of the quantity reduced, excluding receptor, at, most l/1500 is alkylated by lo-* M-MBTA. Of the quantity of susceptible preexisting SH groups, l/2100 is alkylated by lo-* M-MBTA. In contrast, of the t,obal quantity of receptor, approximately 85% is reduced by 0.2 mM-dithiothreitol and approximately 50% of that reduced is alkylated by MBTA. MBTA appears to alkylate SH groups of the reduced receptor approximately 1000-fold faster on the average than all other SH groups, as inferred from physiological experiments. It is clear from the data in Table 2 that the specificity of the labeling procedure. 0.2 mM-dithiothreitol followed by 10-O M-C3H]MBTA, would be doubled simply by t’he blocking of the pre-existing SH groups. An obvious approach is to alkylate these groups with a saturating concentration of non-radioactive MBTA, to reduce with dithiothreitol, and finally to alkylate with a low concentration of [3H]MBTA. This approach fails using either MBTA, N-ethylmaleimide, or iodoacetamide as the blocking agent, for the pre-existing SH groups. The resuhs of such an experiment are as f&on s : 10’4 ;< Label bountl (moles/mg2’3) 1O-8 M-[3H]MBTA 24 $0.2 10 - * M-MBTA, 10 - 8 M[~H]MBTA 0.4. j-o.1 O-2 m&r-dithiothreitol, 10e8 M-[3H]MBTA 3.8 $04 M-MBTA, 0.2 mm-dithiothreitol, 10e8 M-[3H]MBTA 4.0 10.2 Labeling sequence
lo-*
The difference between the labeling in A and B demonstrates that a considerable portion (83%) of the pre-existing SH groups is eliminated by pre-treatment with MBTA. The results of sequences C and D demonstrate that pretreatment with MBTA has no significant effect on the quantity of label bound after dithiothreitol. Similar results are obtained after pretreatment with 5 x 10m4 M-N-ethylmaleimide and wit.h 10 mM-iodoacetamide. One possible explanation is that dithiothreitol reverses the initial alkylation, restoring the SH groups. This possibility has been eliminated: the quantity of label bound by cells treated with 0.1 mrvr-[3H]MBTA is not decreased by subsequent treatment with 0.2 m&r-dithiothreitol. Also, the physiological inhibition due to alkylation by MBTA following dithiothreit,ol is not, changed by subsequent re-application of dithiothreitol. (d) Acetylcholinesteruse Electroplax were homogenized in 1 M-NaCl-091 y. gelatin-l mM-potassium phosphate (pH 7-O), ensuring that all the enzyme activity present, was being measured (Silman & Karlin, 1967). The rate was 5 to 10% higher at 3 mM-acetylcholinc than at either 1 mM or 10 mM, which is typical for this enzyme (Augustinsson & Nachmansohn, 1949). Based on the rate at 3 m&r-acetylcholine and on a molecular weight of 2.4 x lo5 and a specific activity of the pure enzyme of 660 m-mole acetylcholine hydrolyzed per mg enzyme per hour at pH 7.0 (Kremzner & Wilson, 1964), the average quantity of acetylcholinesterase is 3.4 ( f 0.5) x lo-l3 mole per cell, l-6 ( &O-2) x 10-l* mole per mg wet weight, or 4.5 ( f 0.4) x lo-l4 mole per mg213.
186
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KARLIN,
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PRIVES,
W.
DEAL
AND
M.
WINNIK
4. Discussion (a) Xpecifi labeling The first two criteria of specificity (see Introduction) are incorporated into the labeling procedure : only that portion of the total labeling that is eliminated by prior treatment with dithiobischoline (A - B) or by the presence of hexamethonium (A - C) is considered specific. In all experiments these differences were positive. However, the difference A - C was in most experiments greater than A - B, and the calculated asymptotic limit, Y,,,, is greater for A - C than for A - B (Table 1). This is unexpected since five minutes of 05 PM-dithiobischoline completely restores the response to carbamylcholine following inhibition by dithiothreitol, whereas 1 m&r-hexamethonium only partially protects against irreversible block by MBTA. One explanation would be that although five minutes of O-5 PM-dithiobischoline completely restores the response, it does not fully reoxidize all reduced receptor. Some reduced receptor then remains to be alkylated by [3H]MBTA in sequence B, decreasing the quantity A - B. Arguing against this explanation (and for the specificity of the oxidation) is the result that increasing the concentration of dithiobischoline fivefold to 2.5 pM failed to decrease significantly the quantity of label bound in sequence B. Another possible explanation, focusing on A - C, is that hexamethonium protects some other SH groups, as well as those proximal to the acetylcholine binding site, against alkylation by C3H]MBTA, causing C to be smaller than B. Hexamethonium does not, however, protect SH groups present before dithiothreitol-reduction against alkylation by [3H]MBTA. It appears that reaction of certain SH groups generated by reduction, present either on the receptor elsewhere than near the binding site or on some other component of the membrane, is retarded by the binding of hexamethonium. The apparent decrease in the rate of reaction of MBTA with the reduced receptor due to hexamethonium seen in physiological experiments does not appear to be simply competitive (Fig. 1). The third criterion of specificity, the saturation of the labeling, is clearly satisfied when the data are expressed as moles per mg 2’3 (Figs 3 and 5). The relative errors in the differences, A - B and A - C, increase with increasing concentration of label, as would be expected for the saturating difference between nearly linearly increasing quantities. The assumption that only one of the two SH groups generated by reduction of the S-S group near the acetylcholine binding site is labeled by [3H]MBTA, is based on the likelihood that the first molecule of MBTA added occupies the negative subsite so that reaction with the second molecule of MBTA would not be enhanced by binding; i.e. the rate of reaction with the second SH group would be lOOO-fold slower than with the first. The concentration of [3H]MBTA which in ten minutes results in half-saturation of A - B and A - C (approximately 1.6 x lo-* M, Table 1) is sixfold greater than the concentration of MBTA added for ten minutes which results in half-maximal irreversible block of the response of the electroplax (approximately 2.5 x IO- g M ; Fig. 1). This shift of the labeling curves (Figs 3 and 5) towards higher concentrations relative to the physiological inhibition curve (Fig. 1) can be accommodated within our ignorance of the exact relationship between the activation of the receptor and the physiological response. (b) Non-apec@c labeling That the specific labeling is only 10 to 20% of the total (Fig. 6), despite the lOOOfold enhancement of the rate of reaction of MBTA with the SH groups of the reduced
LABELING
OF
ACETYLCHOLINE
RECEPTOR
IS7
receptor, apparently results from a several thousand-fold greater quant’itg of nonspecific SH groups available after reduction by 0.2 mM-dithiothreitol comparctl with specific SH groups (Table 2). The failure of the blocking of the pre-exist8ing non-specific SH groups to improve the specificity of the labeling is difficult t)o undcrst,and. It appears that alkylating pre-existing SH groups with MBTA, N-ethylmaleimide or iodoacetamide increases the susceptibility of S-S groups to subsequent, reduction by dithiothreitol. It appears further that the number of additional nonspecific SH groups made available by dithiothreitol for alkylation by [3H]MB’TR approximately equals the number alkylated before dithiothreitol. Consequent’ly. blocking the pre-existing SH groups by alkylation does not result in an increase in t,he specificity of the labeling following dithiothreitol. (c) AcetyZchoZine.deruse Thu quantity of acetylcholinesterase in the electroplax det’ermined by us is approximately twice as large as that reported previously (Rosenberg & Dettbarn, 1963). This difference is ascribable to our homogenizing the cell in 1 M-NaCl, which has been previously shown to solubilize the membrane-bound enzyme completely, uncovering approximately twice as much activity as could be measured in an unt,reated membrane fraction from electric tissue (Silman t Karlin, 1967). With foul catalytic sites per molecule of acetylcholinesterase, the concentration of catalyt,ic sites is 18 X lo-l4 mole per mgz’3, or sevenfold greater than the maximum value of &4 - B and fourfold greater than the value of A - C. To the extent that the latter t’wo values are estimates of the quantity of acetylcholine binding sites, there is then a four to sevenfold greater number of catalytic sites of acetylcholinesterase than of receptor binding sites in the electroplax. Whether or not the enzyme is labeled 1)~. 13H]MBTA under the conditions used to label the electroplax is not known. The kinetic parameters of the enzyme, however, are unaffected by treatment with e&her dit8hiothreitol or by dithiothreitol followed by MBTA under the conditions used in the labeling experiments. (Karlin, 1967 ; Karlin & Silman, unpublished observations). (d) Surface deneity of receptors on the innervated membrane Electroplax from the organ of Sachs of Electrophorw are flat cells with t.ypical dimensions of 10 mm x 3 mm x 0.1 mm. One of the faces is innervated and the ot,hel is not. The innervated face alone is responsive to chemical and to electrical stimuli. The membrane on both faces protrudes in numerous papillae, and on the innervated face, the axons wrap around the papillae running in gutterlike folds of the membrane (Luft, 1957; Holtzman, unpublished work). Seen in section, virtually all axonal profiles in close apposition to the electroplax membrane contain synaptic vesicles (Holtzman, unpublished work), and thus the entire membrane area directly under t’he axons may function synaptically. We assume that most, if not all, receptor molecules are contained within this area. The size of this area for a typical 35 mg electroplax may be estimated as follows : the area of a plane of the same out line as the innervated face is 33 mm2. The length of the profile of the innervated membrane seen in phase and electron micrographs is approximately threefold the length of t,hc: section. Hence, the area of the membrane is approximately threefold that of tho subtended plane (for discussion of quantitative stereology see Loud, 1968). In addition, there are tubular invaginations of the membrane which increase the area further, approximately threefold. Finally, approximately 30% of the innervated membrane is directly under axons. The area of the innervated membrane therefore is 297 mmZ,
188
A. KARLIN,
J. PRIVES,
W. DEAL
AND
M. WINNIK
89 mm2 of which is subaxonal. A 35-mg electroplax contains 1.6 x 1011to 3-Ox 1011 acetylcholine binding sites per cell (Table 1; (35)213= 10.7). The average density of receptor sites is 1.8~ lo3 to 3.4~ lo3 sites per pm2 of subaxonal membrane. The receptor moleculesmay be oligomeric and may cluster, so that the actual distribution may differ markedly from the average. Assuming that the receptor protomer is a globular protein of molecular weight 40,OOOtand 5 nm diameter, close-packing on a plane would result in a density of approximately 4.7 x lo4 moleculesper pm2. Hence, a reasonable estimate is that the receptor protomer occupies of the order of 5% of the subaxonal membrane area, a result which suggeststhat our estimate for the number of receptors per electroplax is at least not spatially unreasonable. We thank Drs Hai Won Chang, David Cowburn, Eric Holtzman and Michael Reiter for their valuable assistance and suggestions, This work was supported in part by U.S. Public Health Service grants NS-07065 and NS-03304, by National Science Foundation grant GB-15906, and by a gift from the New York Heart Association, Inc. One of us (A. K.) is a career scientist of the Health Research Council of the City of New York. This work was presented in part at the Ciba Foundation Symposium on Molecular Properties of Drug Receptors, London, 1970. REFERENCES Andrews, K. J. M., Bergel, F. & Morrison, A. L. (1953). J. Chem. Sot. p. 3001. Augustinsson, K. B. & Nachmansohn, D. (1949). Science, 110, 98. Bartels, E., Deal, W., Karlin, A. & Mautner, H. (1970). Biochim. biophys. Acta, 203, 568. Bennett, G. M. & Willis, G. H. (1929). J. Chem. Sot. 264. Cava, M. P., Deana, A. A., Muth, K. & Mitchell, M. J. (1961). In Organic Synthesis, vol. 41, p. 93. New York: 5. Wiley & Sons. Changeux, J. P., Kasai, M. & Lee, C. Y. (1970). Proc. Nut. Acd Sci., Wash. 67, 1241. Changeux, J. P., Podleski, T. & Wofsy, L. (1967). Proc. Nut. Acad. Sk., Wash. 58, 2063. Gill, E. W. & Rang, H. P. (1966). Mol. PhwmueoZ. 2, 284. Karlin, A. (1967). Biochim. biophys. Acta, 139, 358. Karlin, A. (1969). J. aen. Physiol. 54, 245s. Karlin, A. & Bartels, E. (1966). Biochim. biophys. Acta, 126, 525. Karlin, A., Prives, J., Deal, W. & Winnik, M. (1970). In Cibu Foundation Symposium on Mole&or Properties of Drug Receptora, p. 247. London: J. & A. Churchill. Karlin, A. & Winnik, M. (1968). Proc. Nat. Acud. Sci., Wash. 60, 668. Kiefer, H., Lindstrom, J., Lennox, E. S. & Singer, S. J. (1970). Proc. Nat. Acad. Sci., Waivh. 67, 1688. Kremzner, L. T. & Wilson, I. B. (1964). Biochemistry, 3, 1902. La Torre, J. L., Lunt, G. S. & De Robertis, E. (1970). Proc. Nat. Acad. SC;., Wash. 65, 716. Loud, A. V. (1968). J. Cell Biol. 37, 27. Luft, J. H. (1957). J. Morphol. 100, 113. Miledi, R., Molinoff, P. & Potter, L. T. (1971). Nature, 229, 554. O’Brien, R. D., Gilmour, L. P. & Eldefrawi, M. E. (1970). Proc. Nat. Acd. Sci., Wash. 65, 438. Rang, H. P. $ Ritter, J. M. (1969). Mol. Pharmacol. 5, 394. Rosenberg, P. & Dettbarn, W. D. (1963). Biochim. biophys. Acta, 69, 103. Schoffeniels, E. (1957). Biochim. biophys. Acta, 26, 585. Silman, H. I. & Karlin, A. (1967). Proc. Nat. Acud. Sci., Wash. 58, 1664. Silman, H. I. & Karlin, A. (1969). Science,164, 1420. Singer, S. J. (1970). In Ciba Foundation Symposium on Molecukw Properties of Drug Receptora, p. 229. London: 5. & A. Churchill. Stedman, E. (1927). J. Chem. Sot. 1905. Wilkinson, G. N. (1961). Biochem. J. 80, 324. t Polyacrylamide gel electrophoresis of labeled receptor solubilized with 1% sodium dodecyl sulfate indicates a moleoular weight in 1% sodium dodecyl sulfate of 8pprOXtiatib’ 40,000 (Reiter, Cowburn, Prives & Karlin, manuscript in preparation).
Affinity
Labeling of the Acetylcholine in the Electroplax
ARTHUR KARLIN,
JOAV
PRIVES,
WALTER
Receptor DEALT
AND
MITCHELL
WINNIK$
Departments of Neurology and Physiology College of Physicians and Surgeons Columbia University New York, N. Y. 10032, L7.S.A. (Received 22 March 1971) The receptor for acetylcholine in the electroplax of Electrophoru-s electricua is affinity labeled in. situ in a two-step process consisting of reduction by dithiothreitol followed by alkylation with tritiated 4-(N-maleimido)-or-benzyltrimethylammonium iodide. From the effect of these and other reagents on the response of the electroplax to cholinergic agents it has been inferred that dithiothreitol reduces an S-S group in the vicinity of the acetylcholine binding site on the receptor and that MBTA$ alkylates one of the SH-groups thus produced three orders of magnitude faster than other SH groups present. Furthermore, th(l alkylation by MBTA is specifically blocked either by prior affinity oxidation of t’he reduced receptor with dithiobischoline or by the simultaneous presence of hexamethonium. Consequently, the portion of the labeling of the dithiothreitoltreated electroplax by [3H]MBTA, which is eliminated by prior treatment with dithiobischoline and by the presence of hexamethonium. is considered to be specific for the receptor. This portion is 10 to 20% of the total labeling. The asympt,otic limit of the specific labeling yields an estimate of the quantity of receptor binding sit,es. The quantity of acetylcholinesterase catalytic sites is fo~lrfold t.o sevenfold as great. The average surface-density of receptor binding sites in the synaptic area of the electroplax membrane is approximately 3000 sitps pc’r ;Lm2.
1. Introduction Two general approaches to the assay of the receptor for acetylcholine, other than by t.he physiological response which it mediates, are currently in use or being developed. One approach is to determine the non-covalent binding by the material putatively containing the receptor of agents which either activate or inhibit the physiological response (O’Brien, Gilmour & Eldefrawi, 1970; La Ton-e, Lunt 6 De Robertis, 1970, Changeux, Kasai I% Lee, 1970; Miledi, Molinoff & Potter, 1971). The other is to react, the material covalently with affinity labels for the receptor (Gill & Rang, 1966; Changeux, Podleski & Wofsy, 1967; Karlin & Winnik, 1968; Karlin, 1969; Silman & Karlin, 1969: Rang & Ritter, 1969; Singer, 1970; Kiefer, Lindstrom, Lennox & i Present address: University of California, Riverside, Calif. 92502, U.S.A. $ Present address: Erindale College, University of Toronto, Toronto, Ontario. f Abbreviation used: MBTA, 4.(N-maleimido)-o-benzyltrimethylammonium iodide. 175
176
A. KARLIN,
J. PRIVES,
W. DEAL
AND
M. WINNIK
Singer, 1970). Problems of specificity abound, and convincing identification of the receptor in isolation will require the demonstration of reasonable interactions both with covalently and non-covalently reacting physiologically specific agents. The present work concerns the application of a two-step process for covalently labeling the acetylcholine receptor in the isolated living electroplax of Electrophorw electricus. Application of a dilute solution of dithiothreitol to the electroplax results in inhibition of the responseto acetylcholine (Karlin & Bartels, 1966). This inhibition can be completely reversed by subsequent application of an oxidizing agent such as 5,5’-dithiobis(2-nitrobenzoate). If a SH alkylating agent like N-ethylmaleimide, at a concentration otherwise without effect on the response,is added after dithiothreitol, then subsequent application of an oxidizing agent no longer results in reversal of the inhibition. These results suggest that dithiothreitol reduces an S-S group on a membrane component involved in the response to acetylcholine and that the SH groups formed either can be alkylated or can be re-oxidized to the original form. Further evidence supports the hypothesis that the S-S group whosereduction affects the responseis on the receptor and is close to those amino-acid residues which make contact with acetylcholine during binding. The salient points of the argument are: (1) application of dithiothreitol results in changes in the speciJicity of the responseof the electroplax to cholinergic agents; e.g. the response to carbamylcholine is inhibited (Karlin & Bartels, 1966) while that to decamethonium is enhanced (Karlin, 1969). All such changes are reversed following application of an oxidizing agent. Changesin the specificity of the responsepresumably reflect changesin the molecular organization of the binding site of the receptor. (2) Quaternary ammonium derivatives such as MBTA appear to alkylate the reduced receptor, as measuredby block of the reversibility of the effects of reduction by oxidizing agents, three orders of magnitude faster than do uncharged derivatives such as N-ethylmaleimide (Karlin & Winnik, 1968; Karlin, 1969). Furthermore, quaternary ammonium-oxidizing agents reverse the effects of reduction three orders of magnitude faster than do non-quaternary analogs(Bartels, Deal, Karlin & Mautner, 1970); e.g. O-5 PM-dithiobischoline applied for five minutes completely reverses the effects of dithiothreitol, whereas a lOOOfold higher concentration of cystine is without effect. (3) The reactions with the affinity-alkylating and affinity-oxidizing agents are retarded in the presence of 1 mMhexamethonium, a reversible ligand of the receptor (Karlin & Winnik, 1968; Bartels et al., 1970). (4) Following dithiothreitol, reactions with somequat,ernary ammonium maleimide derivatives and with two other quaternary ammonium derivatives, one acylating and one alkylating, result in a depolarizing response of the electroplax that is not reversed by washing (Karlin & Winnik, 1968; Silman & Karlin, 1969). The depolarization is reversed by high concentrations of reversible inhibitors such as d-tubocurarine but reappears after the inhibitor is washed out. Apparently, quaternary ammonium moieties covalently attached to an SH-group formed by reduction, interact non-covalently with the nearby acetylcholine binding site. Furthermore, the extent of activation increaseswith decreasinglength of the attached moiety (Karlin, 1969). MBTA appears to alkylate the reduced receptor 4700-fold faster than does N-ethylmaleimide. MBTA alkylates cysteine in solution 4*3-fold faster than does N-ethylmaleimide. Therefore, the enhancement in the rate of alkylation of the reduced receptor by MBTA over that of N-ethylmaleimide appears to be approximately llOO-fold due to the affinity of MBTA for the receptor and 4*3-fold due to t,he difference
LABELING
OF
ACETYLCHOLINE
177
RECEPTOR
in intrinsic reactivity (Karlin, 1969). Assuming that N-ethylmaleimide reacts witIll the SH group(s) in the vicinity of the binding site of the reduced receptor at approximately the same rate as it reacts with other SH groups elsewhere on the membrane, we expect that MBTA will react with the SH, in the vicinity of the binding site, approximately 1100-fold faster than with all other SH-groups. Despite this apparent’ high specificity, MBTA would also be expected to react with other SH groups. Criteria for specific reaction with the receptor can be developed from the physiological
observations cited above: (1) the specific reaction should be eliminated by prior treatment of the reduced receptor with the affinity-oxidizing agent, dithiobischoline, at a concentration shown to completely reverse the effects of reduction. (2) The specific reaction should be retarded in the presence of hexamethonium. (3) The extent of specific reaction as a function of concentration of MBTA should saturate, and half-saturation
should
occur
at approximately
t,he same concentration
as in
comparable physiological experiments. Using [3H]MBTA, electroplax, obtaining
we measure the extent of alkylation of the dithiothreitol-treated estimates of the quantity of specific and of non-specific
sites
of labeling. A comparison with the quantity of acetylcholinesterase is made. A preliminary report of some of these results has appeared (Karlin, Prives, Deal & Winnik,
1970).
2. Materials (a)
and Methods
4-(Il’-~naleimido)benz~~l-~-trimeth~l~~mmo~~~u~~~
iodide
S-(4-ol-dimetl~ylaminobenzyl)maleamic acid (I): a freshly prepared ether solution of p-amino-a-dimethylaminotoluene (Bennett & Willis, 1929), prepared by tin and HClreduction of P-nitro-a:-dimethylaminotoluene (Stedman, 1927), was added dropwise to a vigorously stirred solution of 1.2 equivalents of maleic anhydride in ether. After 1 hr at room temperature the solution was cooled in ice and filtered and the precipitate washed with ether to remove excess maleic anhydride. The combined yield for the reduction and acylation was 93%, giving white plates of m.p. 218 to 219°C after recrystallization twice from 80% aqueous ethanol. Molecular ion found at m/e 248; calculated, 248. Infrared maxima at 1680 cm-’ due to carboxyl, at 1,596 cm-l due to amide carbonyl and at 1540 cm-l due to amide II band. Analysis: talc. for C,,H,,O,X,: C, 62.89; H, 6.50; 5. 11.28; found: C, 62.60; H, 6.68; N, 11.13. 4-(N’-maleimido)-h’,N-dimethylbenzylamine (II). Cyclization of I to II was by the method of Cava, Deana, Muth & Mitchell (1961). A pasty mixture of 0.5 g of I, 88 mg sodium acetate (anhydrous) and 1 ml. acetic anhydrido was swirled over a steam bath for approximately 10 min until a yellow solution was formed. This was poured into icrwater and the mixture extracted twice with methylene chloride, adjusted to neutralitS with sodium bicarbonate, and extracted twice more with methylene chloride. The combined organic phase was dried over calcium sulfate. The solvent was removed under Induced pressure and 380 mg of bright yellow crystals (m.p. 86 to 87°C) were obtained from benzene-hexane solution. Molecular ion found at m/e 230; calculated 230. Infrared maximum (KBr) at 1710 cm-l due to the carbonyls. Nuclear magnetic reasonance peaks
(Ccl,)6 2.26 (singlet, 6H, (CH,),--), 3.48 (singlet 2H, -CH,N-), vinyl), 7.48 (AB quartet, 4H, aromat,ic). II polymerizes on standing.
6.86 (singlet It
2H,
is convcnicntl>
removed from polymer by dissolving in carbon tetrachloride and filtering. MBTA : 0.5 g of II was dissolved in 5 ml. methyl iodide and stirred overnight. MBTA was precipitated with ether and recrystallized from hot acetonitrile and benzene to yield 0.X g of yellow crystals, m.p. 204 to 205°C. Molecular ion, minus methyl iodide, found at m/e 230; calculated, 230. Infrared maximum at 1710 cm-l due to carbonyls. Nuclear magnetic resonance peak (D,O) 6 3.63 (singlet, 9H, (CH,),-), 4.75 (singlet, 2H, CH,N), 6.88 (singlet, 2H, vinyl), 7.68 (quartet, J=SHz dv 18 Hz, 4H aromatic). 1‘2
178
A.
KARLIN,
J.
PRIVES,
W.
DEAL
AND
M.
WINNIK
(b) [3H] 4-(N-maleimido)-ol-benz?/ltrinzethylam71~ iodide Into a vial containing 131 mg of II in O-5 ml. of methylene chloride was transferred 5.7 ~1. of tritiated methyl iodide (Tracerlab, 2.2 Ci/m-mole, 13 mg) in 0.5 ml. ether on 8 vacuum line. The vial w&8 sealed and stored at room temperature in the dark for 6 days. The vial W&S opened, 5 ml. ether added, and the sample was spun on a centrifuge and decanted. The precipitate wae washed with ether 4 times. The resulting bright yellow powder, dissolved in 0.8 ml. acetonitrile and 0.2 ml. methylene chloride, showed the proper nuclear magnetic resonance spectrum. The solution was added dropwise to 10 ml. ether with stirring. The precipitate was separated and dried in ~acuo to yield 39 mg of [3H]MBTA. This was dissolved in 100 ml. acetonitrile to yield a 9O-pM solution, samples of which were sealed into glass vials and placed in liquid nitrogen for storage. Solutions of [3H]MBTA for labeling experiments were prepared as follows. A vial containing [3H]MBTA in acetonitrile was removed from the liquid nitrogen and opened. The acetonitrile was removed under vacuum and the residue dissolved in O-1 mM-HCl to make approximately 0.25 mM-[3H]MBTA. A sample of this was taken for determination of concentration, and the remainder was divided into screwtop vials, frozen in liquid nitrogen, stored at -2O”C, and used over a period of 2 weeks. MBTA hydrolyzes to tho maleamic acid derivate 4.(N-maleamido)-or-benzyltrimethylammonium iodide (III). The concentration of MBTA and of III in the HCl solution was calculated from the extinction at 224, 236.7, 260 and 290 nm and the respective molar extinction coefficients for MBTA: 32,500, 15,900, 942, 451, and for III: 20,700, 15,900,8990, 4930. The extinction at the isosbestic point, 236.7 nm, yields the sum of MBTA and III. The extinction at the remaining 3 wavelengths were used pairwise to calculate the individual concentrations of MBTA and III, and the results, in all cases agreeing closely, were averaged. The actual solution of [3H]MBTA used in labeling was a dilution in eel Ringer solution (pH 7.1) of the stock solut,ion in HCl. The concentration of MBTA in the final solution was determined taking into account the following rates of hydrolysis of MBTA: 0.004/day in 0.1 mM-HCl at, -20°C; O*OlG/min in eel Ringer solution (pH 7.1) at 25°C. (c) Other reagents Dithiobischoline diiodide was synthesized according to Andrews, 1963. Other reagents were the best grade obtainable commercially.
Bergel
& Morrison,
(d) Labeling procedure Electroplax were dissected from the organ of Sachs of Electrophorus eleetricua as described by Schoffeniels (1957). In a typical experiment, 21 to 30 electroplax were divided into three groups of 7 to 10 electroplax each. The groups were treated simultaneously according to the following sequences of solutions: (A) (B) (C)
0.2 mM-dithiothreitol (10 min), R (5 min), R (5 min), R (20 min), 3[H]MBTA (10 min), R (5 min), R (5 min), R (5 min), R (6 min), R (5 min); 0.2 mM-dithiothreitol (10 min), R (5 min), R (5 min), R (5 min), 0.5 ran-dithiobischoline (5 min), R (5 min), R (5 min), [3H]MBTA (10 min), R (5 min), R (5 min), R (5 min), R (5 min), R (5 min); 0.2 mM-dithiothreitol (10 min), R (5 min), R (5 min), R (15 min), 1 mm-hexamethonium (5 min), 3[H]MBTA in 1 mM-hexamethonium (10 min), R (5 min), R (5 min), R (5 min), R (5 min), R (5 min),
where R is eel Ringer solution: 165 mm-N&l, 2.3 mM-KCl, 2 mM-C&l,, 2 mM-Mg&, 1.2 mM-K2HP04, 0.3 m&r-KH2P04, 10 mM-glucose (pH 7.1). All solutions were in R except for dithiothreitol which was dissolved in a Tris-Ringer solution (pH 8.0) identical with R except that Tris * HCl replaced phosphate and glucose wss omitted. The electroplax were transferred to 60 ml. of each solution and gently swirled for the times indicated. At the conclusion of the treatment sequence, the electroplax were blotted on filter paper and each placed in a tared vial and weighed. To each vial w&8 added 50 ~1. water and 0.5 ml. of NCS solubilizer (Amersham/Searle) and the vials were placed in a water bath at 6O’C for 2 hr. Ten-ml. scintillant (PPO and dimethylPOPOP in toluene) were added and the vials placed in a liquid-scintillation spectrometer for counting. The counting rate
LABELING
OF
ACETYLCHOLINE
RECEPTOR
was, in all cases, many-fold greater than background and the statistical were insignificant compared to other causes of variation.
179
errors in couming
(e) AcetyZchoZinesterase Single electroplax were blotted, weighed and homogenized in 1 ml. of 1 M-NaCl-1 mMI)otassium phosphate (pH 7.0)-0.01°h gelatin in a Ten Broek type all-glasshomogenizer. The homogenate was washed with the same medium into the titration vessel of an automatic titrator (Radiometer) operated as a pH-stat. The initial volume of the solution was 9 ml. Samples of 0.1 M-acetylcholine bromide were added successively to give 1 In&r, 3 1nM and 10 mM-acetylcholine. The steady-state rate of hydrolysis was determined at approximately pH 7.0. The titration was at 25°C under nitrogen with 0.01 M-NaOH as the titrant. (f) Electrophyaiologicul mea.wrevtwv& Electroplax were dissected as in (d) and the electrical potential differences across the ccl1 and across the innervated and the non-innervated membranes were measured as 1)reviously doscribod (Karlin, 1969).
3. Results (a) Physiological effects of 0.2 mM-dithiothreitol A lower concentration of dithiothreitol (O-2 mM) was used in the present work than that used previously (1 InM) (Karlin & Bartels, 1966). It was found that the lower concentration caused nearly aa much inhibition of the responseof the electroplax to carbamylcholine and resulted in less non-specific labeling by [3H]MBTA. The mean inhibition of the responseto 40 PM-carbamylcholine following a lo-minute application of 1 mM-dithiothreitol is 76% ; that following 0.2 mMdithiothreito1 is 65%. The inhibition due to 10 minutes of 1 mM-dithiothreitol is the maximum obtainable : 20 minutes of 5 maa-dithiothreitol results in the sameaverage inhibition. Given a linear relationship between the number of receptors reduced and the inhibition resulting, and a residual activity of the reduced receptor of 24%. it appears that
IO’x
Concn
(Ml
Fm. 1. The fraction of the inhibition of the response of the electroplax to carbamylcholine due to treatment with dithiothreitol followed by MBTA that is not reversed by subsequent treatment with 6,6’-dithiobis(2aitrobenzoate). Dithiothreitol w&9 applied for 10 min at concentrations of either 1 mu ( 0) or O-2 mu ( O,O). MBTA ~88 applied for 10 min at the oonoentration indicated on the absoissa either by itself (0, O), or in the presence of 1 mM-hexamethonium (0). Dithiobis(2-nitrobenzoate) wae applied for 10 min at 1 mu. The data for 1 mna-dithiothreitol, (o), are from Karlin (1969). The bars indicete standard errors of the mean.
180
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WINNIK
approximately 85% (65 out of 76) of the receptors are reduced by 0.2 mM-dithiothreitol applied for 10 minutes. As with 1 m&r-dithiothreitol, the effects of O-2 mm-dithiothreitol are completely reversed by 10 minutes of 1 mM-5,5’-dithiobis(2-nitrobenzoate) (pH 8.0) or by 5 minutes of 0.5 ,uM-dithiobischoline. The extent to which alkylation of the reduced receptor by MBTA prevents the reversal by oxidizing agents is somewhat less after 0.2 mM than after 1 mM-dithiothreitol (Fig. 1). The concentration of MBTA, applied for 10 minutes, resulting in half-maximal inhibition of the reversal is approximately the same for the two concentrations of dithiothreitol, namely 2.5 x 10mg M. Hexamethonium partially protects the receptor reduced by 0.2 mMdithiothreito1 against alkylation by MBTA (Fig. 1). At 10m6 M-MBTA, the extent of inhibition in the presence of 1 mM-hexamethonium is 38% of that in the absence of hexamethonium. (b) The specific component of the labeling In the labeling of electroplax according to sequence (A), pre-existing SH groups and reduced S-S groups of both receptor and non-receptor proteins are labeled. In sequence (B), the reduced receptor is specifically reoxidized by dithiobischoline and consequently is not alkylated by [3H]MBTA. Sequence (B) is a measure of the nonspecific labeling. In sequence (C), the receptor is partially protected by hexamethonium; hence (C) is expected to measure a part of the specific labeling in addition to all of the non-specific labeling. Sequence (A) minus sequence (B), (A - B), and sequence (A) minus sequence (C), (A - C), are measures of the specific labeling, There is considerable scatter in the quantities measured using A - B and A - C expressed in moles/mg wet weight, especially at the higher concentrations of label (Figs 2 and 4). The scatter is significantly less when the data is expressed as mole/ power of the wet mg 2’3 (Figs 3 and 5). The justification for using the two-thirds weight is as follows: for identically treated cells, the quantity of label covalently attached varied on the average with the 0.58&O-12 power of the wet weight, as
4 I08xConcn
(M)
FIG. 2. The difference (A - B) between the mean quantity of C3H]MBTA bound by electroplex treated with 0.2 man-dithiothreitol (10 min) followed by [3H]MBTA (10 min) (A) and that bound by electroplex treated with dithiothreitol followed by 0.5 pM dithiobischoline (5 min) followed by [3H]MBTA (B). The concentration of [3H]MBTA is given on the abscissrt. The data are expressed aa molee/mg wet weight. The bars represent the standard error of the difference of the mean of A and B.
LABELING
OF
ACETYLCHOLINE
10*x
Fm. 3. The difference The curve is a weighted
(A - B) as in Fig. least-squares fit.
0
I
Concn
2, except
(M)
that
2 IO* x Concn
181
RECEPTOR
data
3
are
expressed
as molos/mg2’3.
4
(Ml
4. The difference (A - C) between the mean quantity of [3H]MBTA bound by electroplas with 0.2 mM-dithiothreitol (10 min) followed by [3H]MBTA (10 min) (A) and that bound by eloctroplax treated with dithiothreitol followed by [3H]MBTA in the presence of 1 mM-hcxamethonium (C). The concentration of [3H]MBTA is given on the abscissa. The data are oxpressod as moles/mg wet weight. FIG.
treated
by a parabolic least-squares tit of the data. The two-thirds power, which is within one standard deviation of the estimated parameter, makes sense dimensionally. Assuming equal densities and proportions for these cells, the two-thirds power of t,he wet weight is proportional to the surface area of the cells, with the same proportionality constant for all cells. It is reasonable to assume that the charged, quaternary ammonium MBTA does not penetrate the surface membrane in appreciable amounts during the lo-minute incubation period, and hence that almost all of the reaction takes place at the cell surface. Expressed as moles/mg 213, the quantities A - B and B - C appear to approach determined
182
A.
KARLIN,
J.
PRIVES,
W.
10*x
FIG. 5. The difbrence The curve is a weighted
(a - C) as in Fig. least-squares fit.
DEAL
Concn
4, except
AND
M.
WINNIK
(~1
that
data
are expressed
8s moles/mg2’3.
an upper limit with increasing concentration of [3H]MBTA (Figs 3 and 5). Estimates of this limit are obtained as follows. During the lo-minute incubation, the concentration of r3H]MBTA remains nearly constant, less than 1% being lost in alkylation reactions and approximately 16% being lost by hydrolysis. The reaction of the label with the receptor therefore, is probably well represented as pseudo-first order. The extent of reaction is given by Y = Y,,,(l
- ewktX)
where Y is moles of receptor alkylated; k is the second-order rate constant; t, is the duration of the reaction; and X is the concentration of [3H]MBTA. The equation may be written Y = Y,,,(l where X,., is the concentration time t). However,
- 2-x’xo,s)
of label at which
half-maximal
labeling occurs (at
z(x’xo~s) = 1 + (X/X,.,) approximately,
and therefore an approximation
for Y is the familiar form
yIn*xm + X0.,/X) which deviates from the exact expression for Y by, at most, 15% and is identical at X = Xo.,~Xo., and Y,,, may now be estimated by fitting a straight line to the points (l/X, l/Y). Estimates of these parameters and the standard errors of the estimates were obtained by a weighted least-squares analysis (Wilkinson, 1961) taking the inverse of the coefioient of variance as the weighting factor. Both the data expressed in moles per mg213 and those expressed in moles per mg were analyzed and the parameters estimated (Table 1). The two sets of parameters are consistent. X0 .s is approximately the same for both. Since in the range of electroplax weights obtained, the value in mg is approximately threefold the value in mg213,
LABELING
OF
ACETYLCHOLINE
The specific labeling by [3H]MBTA
of dithiothreitol-treated electroplux
(lW&.~
ww ynl,,
(moles/mg2’3)
(W
2.1 f 3.0 f
1.7 5 0.7 1.6 f 0.6
0.8 0.8
Extrapolat,ed 2.5 f 4.6 f
0.9 1.2
t,o complete
183
1
TABLE
(1W ylrmx
RECEPTOR
(moles/mg)
reduction
0.8 * 1.2 f
0.6 0.6
(lWXO.6 W) 2.1 f 1.6 2.3 & 1.1
and protection 0.9 f 1.8 f
0.7 0.9
Eleotroplax were treated with 0.2 mM-dithiothreitol followed by either [3H]MBTA (A), or 0.5 PM-dithiobischoline and then [3H]MBTA (B), or [3H]MBTA in the presence of 1 mM-hexamethonium (C). The differences, A - B and A - C, approach an asymptotic limit (Y,,,) with increasing concentration of [3H]MBTA. This limit, Y,,,, and the concentration (X,.,) applied for 10 min which results in half-maximal labeling were estimated by a weighted least-squares fit of t,he equation Y = Y,ax/(l + X,.,/X) 1 o the data. The data expressed in units of moles/mg and of molss/mga’3 were analyzed separately. The extrapolation to complete reduction of all receptors and complete protection by hexamethonium is based on the approximations that 0.2 man-dithiothreitol applied for 10 min reduces 85% of the receptors and that 1 mM-hexamcthonium protects against alkylation by MBTA to t,he extent of 77% (see text for details).
the values of Y,,, correspond s,swell. The relative errors of the estimates are considerably lessfor the data expressed as moles per mg2’3. Curves calculated using t,he estimated parameters are plotted in Figures 3 and 5. The values of Y,,, for A - B and A - C do not correspond to the total quantity of the receptor. It appears that 0.2 mM-dithiothreitol (10 min) reduces 85% of the total available receptors (seeabove). Dividing Y,,, by 0.85 gives an estimate of the t,otal quantity of receptor as determined by A - B (Table 1). From physiological experiments, hexamethonium protects the reduced receptor against alkylstion by MBTA 77% on the average over the concentration range tested (Fig. 1). Y,,, for A - C therefore corresponds to 0.85 x0.77=0*65 of the total quantity of receptor, and dividing Y,,, by 0.65 gives an estimate of the total (Table 1). These extrapolations increasethe spread between the limits of A - B and A - C. The difference hetween these two estimates for the total receptor is not statistically significant: however, A - C was, in most experiments, greater than A - B as are the corresponding values of Y,,,. The ratios of the specific labeling to the total labeling, (A - B) : A and (A - C) : A, decreasewith increasing concentrations of [3H]MBTA (Fig. 6). This decreasewould be expected if the specific component of the labeling were to saturate and the nonspecific component increase linearly with increasing concentration of label. This appears to be the case. (c) The non-speci$c componentof the labeling A considerable fraction of the total labeling is non-specific. This includes preexisting SH groups, non-receptor S-S groups reduced by dithiothreitol, and possibly other nucleophillic groups such as -NH, that might react with MBTA. That almost all of the groups that are labeled by [3H]MBTA are SH groups is demonstrated by
A. KARLIN,
184
J. PRIVES,
W. DEAL
AND
M. WINNIK
10Sx Concn(Ml Fm. 0. The fraction of the total labeling which is speoific. (A - B)/A, (0); The abscissa gives the concentration of [3H]MBTA. Data obtained in different slightly different conoentrations of r3H]MBTA are averaged here, the bars standard error of the mean.
(A - G)/A, experiments representing
(m). at the
the following: incubation of electroplax with 1 mM.-S&Y-dithiobis(2nitrobenzoate) (pH 8.0) for 10 minutes, either with or without prior incubation with dithiothreitol, followed by lo-* M-[~H]MBTA for 10 minutes, results in 3 to 5% of the labeling obtained in the absenceof incubation with 5,6’-dithiobis(2nitrobenzoate). Therefore, oxidation of SH groups eliminates nearly all of the groups that react with MBTA either before or after dithiothreitol. In contrast, the labeling of the pre-existing SH groups is not changed either by pretreatment with 0.5 ,uM-dithiobischoline for 5 minutes or by the presence of 1 mM-hexamethonium. This supports the assumption that at this low concentration dithiobischoline oxidizes only the SH groups of the reduced receptor and furthermore that hexamethonium protects only specific receptor SH groups against alkylation by MBTA. The labeling either before or after dithiothreitol was found to approach a limiting value at a concentration of approximately 1 mM-[3H]MBTA, applied for 10 minutes. Also, at a given concentration of [3H]MBTA, the quantity of labeling was approximately maximal following 5 mrvr-dithiothreitol applied for 20 minutes. Thus, the first TABLE
2
The quantity of group reacting with [3H]ikfBTA before reduction with dithiothreitol (designatedSH) and after reduction (designatedS-S) Labeling
procedure
1Ol4 x SH (moles/mgals)
10’4 x s-s (moles/mg2’s)
lOi xR (moles/mg2’s)
21,000
2-5
5 mM-dithiothreitol 0.8 mMj3H]MBTA
(20 min), (10 min),
6406
0.2 m&r-dithiothreitol 0.8 mM-[sH]MBTA
(10 min), (10 min),
6400
0.2 mM-dithiothreitol lOWa M-[3H]MBTA
(10 min), (10 min).
3000 3
The labeling procedure given is that for total labeling (SH obtained by omitting dithiothreitol. The quantity, S-S, was (SH + S-S). Inoluded in S-S is the quantity of receptor and elkylated, designated R. It is assumed that only one of the S-S is alkylated by MBTA.
2-4 3
l-2
+ S-S). The quantity, SH, was obtained by subtracting SH from disulfide whioh has been reduced two SH formed by reduction of an
LABELING
OF
ACETYLCHOLINE
RECEPTOR
185
line of Table 2 gives estimates of the total quantity of preexisting SH groups which can be alkylated by MBTA and of the quantity of S-S groups which can be reduced by dithiothreitol and alkylated by MBTA. Of the latter quantity, l/7 is actually reduced by 0.2 mMdithiothreito1. Of the quantity reduced, excluding receptor, at, most l/1500 is alkylated by lo-* M-MBTA. Of the quantity of susceptible preexisting SH groups, l/2100 is alkylated by lo-* M-MBTA. In contrast, of the t,obal quantity of receptor, approximately 85% is reduced by 0.2 mM-dithiothreitol and approximately 50% of that reduced is alkylated by MBTA. MBTA appears to alkylate SH groups of the reduced receptor approximately 1000-fold faster on the average than all other SH groups, as inferred from physiological experiments. It is clear from the data in Table 2 that the specificity of the labeling procedure. 0.2 mM-dithiothreitol followed by 10-O M-C3H]MBTA, would be doubled simply by t’he blocking of the pre-existing SH groups. An obvious approach is to alkylate these groups with a saturating concentration of non-radioactive MBTA, to reduce with dithiothreitol, and finally to alkylate with a low concentration of [3H]MBTA. This approach fails using either MBTA, N-ethylmaleimide, or iodoacetamide as the blocking agent, for the pre-existing SH groups. The resuhs of such an experiment are as f&on s : 10’4 ;< Label bountl (moles/mg2’3) 1O-8 M-[3H]MBTA 24 $0.2 10 - * M-MBTA, 10 - 8 M[~H]MBTA 0.4. j-o.1 O-2 m&r-dithiothreitol, 10e8 M-[3H]MBTA 3.8 $04 M-MBTA, 0.2 mm-dithiothreitol, 10e8 M-[3H]MBTA 4.0 10.2 Labeling sequence
lo-*
The difference between the labeling in A and B demonstrates that a considerable portion (83%) of the pre-existing SH groups is eliminated by pre-treatment with MBTA. The results of sequences C and D demonstrate that pretreatment with MBTA has no significant effect on the quantity of label bound after dithiothreitol. Similar results are obtained after pretreatment with 5 x 10m4 M-N-ethylmaleimide and wit.h 10 mM-iodoacetamide. One possible explanation is that dithiothreitol reverses the initial alkylation, restoring the SH groups. This possibility has been eliminated: the quantity of label bound by cells treated with 0.1 mrvr-[3H]MBTA is not decreased by subsequent treatment with 0.2 m&r-dithiothreitol. Also, the physiological inhibition due to alkylation by MBTA following dithiothreit,ol is not, changed by subsequent re-application of dithiothreitol. (d) Acetylcholinesteruse Electroplax were homogenized in 1 M-NaCl-091 y. gelatin-l mM-potassium phosphate (pH 7-O), ensuring that all the enzyme activity present, was being measured (Silman & Karlin, 1967). The rate was 5 to 10% higher at 3 mM-acetylcholinc than at either 1 mM or 10 mM, which is typical for this enzyme (Augustinsson & Nachmansohn, 1949). Based on the rate at 3 m&r-acetylcholine and on a molecular weight of 2.4 x lo5 and a specific activity of the pure enzyme of 660 m-mole acetylcholine hydrolyzed per mg enzyme per hour at pH 7.0 (Kremzner & Wilson, 1964), the average quantity of acetylcholinesterase is 3.4 ( f 0.5) x lo-l3 mole per cell, l-6 ( &O-2) x 10-l* mole per mg wet weight, or 4.5 ( f 0.4) x lo-l4 mole per mg213.
186
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KARLIN,
J.
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W.
DEAL
AND
M.
WINNIK
4. Discussion (a) Xpecifi labeling The first two criteria of specificity (see Introduction) are incorporated into the labeling procedure : only that portion of the total labeling that is eliminated by prior treatment with dithiobischoline (A - B) or by the presence of hexamethonium (A - C) is considered specific. In all experiments these differences were positive. However, the difference A - C was in most experiments greater than A - B, and the calculated asymptotic limit, Y,,,, is greater for A - C than for A - B (Table 1). This is unexpected since five minutes of 05 PM-dithiobischoline completely restores the response to carbamylcholine following inhibition by dithiothreitol, whereas 1 m&r-hexamethonium only partially protects against irreversible block by MBTA. One explanation would be that although five minutes of O-5 PM-dithiobischoline completely restores the response, it does not fully reoxidize all reduced receptor. Some reduced receptor then remains to be alkylated by [3H]MBTA in sequence B, decreasing the quantity A - B. Arguing against this explanation (and for the specificity of the oxidation) is the result that increasing the concentration of dithiobischoline fivefold to 2.5 pM failed to decrease significantly the quantity of label bound in sequence B. Another possible explanation, focusing on A - C, is that hexamethonium protects some other SH groups, as well as those proximal to the acetylcholine binding site, against alkylation by C3H]MBTA, causing C to be smaller than B. Hexamethonium does not, however, protect SH groups present before dithiothreitol-reduction against alkylation by [3H]MBTA. It appears that reaction of certain SH groups generated by reduction, present either on the receptor elsewhere than near the binding site or on some other component of the membrane, is retarded by the binding of hexamethonium. The apparent decrease in the rate of reaction of MBTA with the reduced receptor due to hexamethonium seen in physiological experiments does not appear to be simply competitive (Fig. 1). The third criterion of specificity, the saturation of the labeling, is clearly satisfied when the data are expressed as moles per mg 2’3 (Figs 3 and 5). The relative errors in the differences, A - B and A - C, increase with increasing concentration of label, as would be expected for the saturating difference between nearly linearly increasing quantities. The assumption that only one of the two SH groups generated by reduction of the S-S group near the acetylcholine binding site is labeled by [3H]MBTA, is based on the likelihood that the first molecule of MBTA added occupies the negative subsite so that reaction with the second molecule of MBTA would not be enhanced by binding; i.e. the rate of reaction with the second SH group would be lOOO-fold slower than with the first. The concentration of [3H]MBTA which in ten minutes results in half-saturation of A - B and A - C (approximately 1.6 x lo-* M, Table 1) is sixfold greater than the concentration of MBTA added for ten minutes which results in half-maximal irreversible block of the response of the electroplax (approximately 2.5 x IO- g M ; Fig. 1). This shift of the labeling curves (Figs 3 and 5) towards higher concentrations relative to the physiological inhibition curve (Fig. 1) can be accommodated within our ignorance of the exact relationship between the activation of the receptor and the physiological response. (b) Non-apec@c labeling That the specific labeling is only 10 to 20% of the total (Fig. 6), despite the lOOOfold enhancement of the rate of reaction of MBTA with the SH groups of the reduced
LABELING
OF
ACETYLCHOLINE
RECEPTOR
IS7
receptor, apparently results from a several thousand-fold greater quant’itg of nonspecific SH groups available after reduction by 0.2 mM-dithiothreitol comparctl with specific SH groups (Table 2). The failure of the blocking of the pre-exist8ing non-specific SH groups to improve the specificity of the labeling is difficult t)o undcrst,and. It appears that alkylating pre-existing SH groups with MBTA, N-ethylmaleimide or iodoacetamide increases the susceptibility of S-S groups to subsequent, reduction by dithiothreitol. It appears further that the number of additional nonspecific SH groups made available by dithiothreitol for alkylation by [3H]MB’TR approximately equals the number alkylated before dithiothreitol. Consequent’ly. blocking the pre-existing SH groups by alkylation does not result in an increase in t,he specificity of the labeling following dithiothreitol. (c) AcetyZchoZine.deruse Thu quantity of acetylcholinesterase in the electroplax det’ermined by us is approximately twice as large as that reported previously (Rosenberg & Dettbarn, 1963). This difference is ascribable to our homogenizing the cell in 1 M-NaCl, which has been previously shown to solubilize the membrane-bound enzyme completely, uncovering approximately twice as much activity as could be measured in an unt,reated membrane fraction from electric tissue (Silman t Karlin, 1967). With foul catalytic sites per molecule of acetylcholinesterase, the concentration of catalyt,ic sites is 18 X lo-l4 mole per mgz’3, or sevenfold greater than the maximum value of &4 - B and fourfold greater than the value of A - C. To the extent that the latter t’wo values are estimates of the quantity of acetylcholine binding sites, there is then a four to sevenfold greater number of catalytic sites of acetylcholinesterase than of receptor binding sites in the electroplax. Whether or not the enzyme is labeled 1)~. 13H]MBTA under the conditions used to label the electroplax is not known. The kinetic parameters of the enzyme, however, are unaffected by treatment with e&her dit8hiothreitol or by dithiothreitol followed by MBTA under the conditions used in the labeling experiments. (Karlin, 1967 ; Karlin & Silman, unpublished observations). (d) Surface deneity of receptors on the innervated membrane Electroplax from the organ of Sachs of Electrophorw are flat cells with t.ypical dimensions of 10 mm x 3 mm x 0.1 mm. One of the faces is innervated and the ot,hel is not. The innervated face alone is responsive to chemical and to electrical stimuli. The membrane on both faces protrudes in numerous papillae, and on the innervated face, the axons wrap around the papillae running in gutterlike folds of the membrane (Luft, 1957; Holtzman, unpublished work). Seen in section, virtually all axonal profiles in close apposition to the electroplax membrane contain synaptic vesicles (Holtzman, unpublished work), and thus the entire membrane area directly under t’he axons may function synaptically. We assume that most, if not all, receptor molecules are contained within this area. The size of this area for a typical 35 mg electroplax may be estimated as follows : the area of a plane of the same out line as the innervated face is 33 mm2. The length of the profile of the innervated membrane seen in phase and electron micrographs is approximately threefold the length of t,hc: section. Hence, the area of the membrane is approximately threefold that of tho subtended plane (for discussion of quantitative stereology see Loud, 1968). In addition, there are tubular invaginations of the membrane which increase the area further, approximately threefold. Finally, approximately 30% of the innervated membrane is directly under axons. The area of the innervated membrane therefore is 297 mmZ,
188
A. KARLIN,
J. PRIVES,
W. DEAL
AND
M. WINNIK
89 mm2 of which is subaxonal. A 35-mg electroplax contains 1.6 x 1011to 3-Ox 1011 acetylcholine binding sites per cell (Table 1; (35)213= 10.7). The average density of receptor sites is 1.8~ lo3 to 3.4~ lo3 sites per pm2 of subaxonal membrane. The receptor moleculesmay be oligomeric and may cluster, so that the actual distribution may differ markedly from the average. Assuming that the receptor protomer is a globular protein of molecular weight 40,OOOtand 5 nm diameter, close-packing on a plane would result in a density of approximately 4.7 x lo4 moleculesper pm2. Hence, a reasonable estimate is that the receptor protomer occupies of the order of 5% of the subaxonal membrane area, a result which suggeststhat our estimate for the number of receptors per electroplax is at least not spatially unreasonable. We thank Drs Hai Won Chang, David Cowburn, Eric Holtzman and Michael Reiter for their valuable assistance and suggestions, This work was supported in part by U.S. Public Health Service grants NS-07065 and NS-03304, by National Science Foundation grant GB-15906, and by a gift from the New York Heart Association, Inc. One of us (A. K.) is a career scientist of the Health Research Council of the City of New York. This work was presented in part at the Ciba Foundation Symposium on Molecular Properties of Drug Receptors, London, 1970. REFERENCES Andrews, K. J. M., Bergel, F. & Morrison, A. L. (1953). J. Chem. Sot. p. 3001. Augustinsson, K. B. & Nachmansohn, D. (1949). Science, 110, 98. Bartels, E., Deal, W., Karlin, A. & Mautner, H. (1970). Biochim. biophys. Acta, 203, 568. Bennett, G. M. & Willis, G. H. (1929). J. Chem. Sot. 264. Cava, M. P., Deana, A. A., Muth, K. & Mitchell, M. J. (1961). In Organic Synthesis, vol. 41, p. 93. New York: 5. Wiley & Sons. Changeux, J. P., Kasai, M. & Lee, C. Y. (1970). Proc. Nut. Acd Sci., Wash. 67, 1241. Changeux, J. P., Podleski, T. & Wofsy, L. (1967). Proc. Nut. Acad. Sk., Wash. 58, 2063. Gill, E. W. & Rang, H. P. (1966). Mol. PhwmueoZ. 2, 284. Karlin, A. (1967). Biochim. biophys. Acta, 139, 358. Karlin, A. (1969). J. aen. Physiol. 54, 245s. Karlin, A. & Bartels, E. (1966). Biochim. biophys. Acta, 126, 525. Karlin, A., Prives, J., Deal, W. & Winnik, M. (1970). In Cibu Foundation Symposium on Mole&or Properties of Drug Receptora, p. 247. London: J. & A. Churchill. Karlin, A. & Winnik, M. (1968). Proc. Nat. Acud. Sci., Wash. 60, 668. Kiefer, H., Lindstrom, J., Lennox, E. S. & Singer, S. J. (1970). Proc. Nat. Acad. Sci., Waivh. 67, 1688. Kremzner, L. T. & Wilson, I. B. (1964). Biochemistry, 3, 1902. La Torre, J. L., Lunt, G. S. & De Robertis, E. (1970). Proc. Nat. Acad. SC;., Wash. 65, 716. Loud, A. V. (1968). J. Cell Biol. 37, 27. Luft, J. H. (1957). J. Morphol. 100, 113. Miledi, R., Molinoff, P. & Potter, L. T. (1971). Nature, 229, 554. O’Brien, R. D., Gilmour, L. P. & Eldefrawi, M. E. (1970). Proc. Nat. Acd. Sci., Wash. 65, 438. Rang, H. P. $ Ritter, J. M. (1969). Mol. Pharmacol. 5, 394. Rosenberg, P. & Dettbarn, W. D. (1963). Biochim. biophys. Acta, 69, 103. Schoffeniels, E. (1957). Biochim. biophys. Acta, 26, 585. Silman, H. I. & Karlin, A. (1967). Proc. Nat. Acud. Sci., Wash. 58, 1664. Silman, H. I. & Karlin, A. (1969). Science,164, 1420. Singer, S. J. (1970). In Ciba Foundation Symposium on Molecukw Properties of Drug Receptora, p. 229. London: 5. & A. Churchill. Stedman, E. (1927). J. Chem. Sot. 1905. Wilkinson, G. N. (1961). Biochem. J. 80, 324. t Polyacrylamide gel electrophoresis of labeled receptor solubilized with 1% sodium dodecyl sulfate indicates a moleoular weight in 1% sodium dodecyl sulfate of 8pprOXtiatib’ 40,000 (Reiter, Cowburn, Prives & Karlin, manuscript in preparation).