Proceeding8 of the National Academy of Science8

Vol. 65, No. 2, pp. 438-445, February 1970

A Muscarone-Binding Material in Electroplax and Its Relation to the Acetylcholine Receptor, II. Dialysis Assay
R. D. O'Brien, L. P. Gilmour, and M. E. Eldefrawi
SECTION OF NEUROBIOLOGY AND BEHAVIOR, CORNELL UNIVERSITY, ITHACA, NEW YORK

Communicated by F. A. Long, November 10, 1969

Abstract. An equilibrium dialysis technique, applied to lyophilized particulate fractions of Torpedo electroplax, gave data consistent with a single kind of macromolecular binding of muscarone, with binding constant, 7 X 10-v M and an amount of 1 nmole per gram original electroplax. The effects on muscarone binding of 38 drugs suggested that muscarone binding reflected acetylcholine receptor activity. Of 18 enzyme preparations, only trypsin, chymotrypsin, and phospholipase C reduced binding activity, suggesting that a phospholipoprotein was binding. Partial "solubilization" of the binding protein was achieved, but the "solubilized" activity did not migrate on electrophoresis. Additional evidence was provided that acetyicholinesterase was not responsible for this muscarone binding.

In the preceding paper1 we presented evidence that muscarone binding to a particulate fraction of Torpedo electroplax behaved in all the ways explored as one would expect from binding to acetylcholine receptor, e.g., in its high affinity for muscarone, acetylcholine, tubocurarine, and other cholinergic drugs; its insensitivity to noncholinergic drugs; its reversibility; and its amount. In the present paper we report data obtained following a change of assay procedure which permitted examination of soluble as well as particulate binding material, and gave greatly improved kinetic data.
Procedure. The original extraction procedure' was modified in several ways to permit preparation on a larger scale, and to permit lyophilization. Electric organ with its attached skin was homogenized in water at 20% with a Waring blender at maximum speed for 1 min, then filtered through cheesecloth to remove skin fragments. It was centrifuged at 12,000 X g for 90 min in the cold. The precipitate was either used directly or (where indicated below) lyophilized overnight, weighed, and stored frozen. For binding studies, suitable quantities representing 0.3-3 gm of original electroplax were used directly or (in the case of lyophilized material) rehomogenized in water. The 1-ml samples were placed in 1/4-in. dialysis tubing, tied at each end, and suspended in 100 vol of a solution of H3-muscarone (at 10-6 M, unless otherwise indicated) in a KrebsRinger solution2 containing 0.7 mM calcium. The system was shaken on a reciprocating or swirling shaker overnight at 40C, and 0.5-ml samples from the bath and the bag contents were counted as before. Analyses: Protein was measured by the method of Lowry3 with bovine serum albumin as standard, and organic matter by the method of Johnson4 with sucrose as
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standard. Values obtained were as follows (in mg/gm original tissue): whole electroplax: protein 21.3, organic matter 44.0. Precipitate at 27,000 X g (as used in centrifugal studies') protein 7.3, organic matter 17.9. Precipitate at 12,000 X g (as used in dialysis studies) protein 7.7, organic matter 16.2. The plax powder contained 0.68 mg protein per milligram of powder, corresponding to 9.3 mg protein per gram original tissue. Electrophoresis: The polyacrylamide disk electrophoresis technique as described by Davis5 was used, except that the sample in 20% sucrose was layered under the buffer surface. After electrophoresis the gel columns were frozen with freon and sliced into 0.5-mm slices with a tissue slicer, and each slice weighed and then placed in a small beaker containing 2 ml of 10-6 M H'-muscarone in Ringer's solution for 40 min at room temperature. The gel slices were solubilized with 30% H202 according to the method of Gray and Steffensen,6 and the samples counted in a Bray7 scintillation cocktail. Enzymes: The enzyme preparations were as follows: Hyaluronidase: Sigma Type I, bovine testes, 350 NF units/mg. Peptidase: Sigma, intestinal mucosa, 15 units/gm. Ribonuclease: Sigma, bovine pancreas, 5X crystallized. Deoxyribonuclease: Sigma, bovine pancreas, crude, 550 Kunitz units/mg. Pepsin, Sigma, 2650 units/mg. Pancreatin, Sigma, Grade II, porcine pancreas. Carboxypeptidase-A (DFP-treated), Sigma, recrystallized. Collagenase, Sigma, Cl. hystolyticum. Sulfatase, Sigma, limpet, 10 units/mg. Lysozyme, Nutritional Biochemicals. Papain, Nutritional Biochemicals. Neuraminidase, Sigma, Type V, Cl. perfringens. Phospholipase D, Sigma, Type II, cabbage. Crotalus adamanteus venom, Sigma. Bee venom, Sigma, Grade I. Chymotrypsin, a, Sigma, Type II, bovine pancreas, crystallized. Trypsin, Worthington, bovine pancreas, 180 units/mg. Phospholipase C, Worthington, Cl. perfringens, 1-2 units/mg. Results. The equilibrium dialysis technique proved more sensitive than the original centrifugal procedure, permitting the use of ten times less tissue per assay. At this stage we shifted to routine use of the optimal medium, the phosphate-Ringer solution described above, and employed 10-6 M muscarone. With the dialysis technique, it was possible to examine the additivity of activities obtained in different fractions. The means of two such runs showed the following activities: in whole homogenate, 0.86 nmoles per gram original plax; in precipitate from 12,000 X g for 90 minutes, 0.55 nmoles per gram; in supernatant, 0.19 nmoles per gram. The activity in the two fractions was, therefore, 86 per cent of that in the whole homogenate. We next stockpiled 36 kg of electroplax (from 264 Torpedo) frozen in 280 gm portions. The previous paper showed that binding activity was not lost after lyophilization. Homogenates at 20 per cent in water were prepared, and the precipitate at 12,000 X g (90-min centrifugation) was collected and lyophilized, to give a total of 493 gm of "plax powder." This powder was flown from Naples to Ithaca for the subsequent work, and was stored at -20C. The material gave an excellent linear relation between muscarone binding and quantity of tissue (Fig. 1) and a good Lineweaver-Burk plot relating muscarone binding to muscarone concentration (Fig. 2). From this plot the following parameters were computed by the weighted regression technique: binding constant, K = 7.19(0.59) X 10-7 M; maximal binding, R = 1.01(40.031)nmoles per gram fresh electroplax equivalent. As expected, elevated temperatures shortened the time-to-equilibrium, from ten hours at 4C to five hours, at 380C. In addition, there was a positive temperature dependence of binding, as judged by the equilibrium level. The nmoles bound per gram original plax were 0.45 at 40C, 0.58 at 220C, and 0.69 at 380C. Drug profile: One would expect that agents acting upon the receptor, whether

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2.0-

1.5 o
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agonists or antagonists (their physiological action being to stimulate or to block) would compete with muscarone for that receptor and therefore show blockade in this binding test. The data in Table 1, from experiments with plax powder and dialysis technique with

determinngduplicateinhibitionvalues on each of two different days, thus giving four values. For the compounds shown in Table 1, the error (defined as the maximal departure from the mean) never exceeded 9 per cent of the mean. The table shows that the 11 neuromuscular agents tested were potent blockers at 10-5 M(see Webb8 for description of the WIN compounds). The three cholinergic agents which are not good neuromuscular agents have rather low activity (21-51% blockade at 10-4 M). The following 12 miscellaneous neuroactive agents, which are regarded pharmacologically as ineffective on cholinergic receptors, were not effective blockers (as judged by the fact that muscarone binding in the presence of 10-4 M agent was between 90 and 110 per cent of the control): amphetamine, codeine, epi-

ing to freeze-dried 12,000 X g precipitate upon weight of plax from which that precipitate

10-6 M muscarone, confirm and extend the findings reported previously' with co fresh plax and the centrifugal technique, and assayed with 2.5 X 10-7 M 0 . muscarone. O/ The mean of 43 control results gathered over three months was 0.485 /, , , nmoles per gram original plax, with a 3 4 2 l standard deviation of 4 0.062 (=13%) gms TISSUE and a range of 0.638 to 0.380. However, the variance in the inhibition data FIG. 1.-Dependence of muscarone bind- was less. The usual procedure involved

was derived. Muscarone was 10-6M.

nephrine, eserine, glutamine, L-alanine, mephenesin, N-methyl-N-chloroethyl-2chloro-2-phenylethylamine, pilocarpine, psilocybin, serotonin, and tyramine. Three neuroactive agents gave evidence of a small activating effect upon muscarone binding, as judged by the following means (as percentage of control) and ranges of quadruplicate tests: 'y-aminobutyrate 125 (122-129); norepinephrine 116 (108-124); imipramine 127 (122-136). Four neuroactive compounds not usually considered cholinergic gave, at 10-4 M, measurable blockade. These were hordenine, bretylium, picrotoxin, and especially strychnine. The significance of these observations is discussed below. Three other agents were tested. N-ethyl maleimide (which is without effect on eel plax receptor9) was inactive at 10-4 M on muscarone binding. p-Chloromercuribenzoate at 0.5 m:\{, a concentration which blocks eel plax receptor9 was inhibitory (Table 1). The reducing agent dithiothreitol at 1 mMI, a concentration which blocks eel plax receptor9 was, however, without action on mus-

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FIG. 2.-Dependence upon muscarone concentrations (S) of binding (B) to freeze-dried 12,000 X g precipitate derived from 0.5 of plax (dialysis method). Three points at low values of S are omitted because they would be far off scale. The line shown is computed from all points by the Wilkinson method.22

carone binding. It was reported by Karlin10 that these two latter agents were inactive against eel acetylcholinesterase, and we have confirmed this inactivity using Torpedo plax powder for a source of acetylcholinesterase. Attempted solubilization: Attempts were made to solubilize with a variety of agents, by treating 26 mg of plax powder per milliliter for one hour at room temperature with shaking, then centrifuging at 100,000 X g, and assaying the supernatant. Two kinds of problems were encountered.
TABLE 1. Effective blockers of binding of 10-6 M muscarone to plax powder.

Agent

Curare, 10-5 Al Acetylcholine, 4 X 10- Al (after paraoxon 10-4 M) WIN 7758, 10-5 M WIN 7846, 10-r M WIN 7789, 10-5 M WIN 3317, 10-r M

(%) Agent Neuromuscular agents 79 WIN 13357, 10-5 M 82 Flaxedil, 10-6 M

Blockade

Blockade

(%)
31 58 59 82 86 77

Nicotine, 10-6 M
78 70 79 69
10-4 M

Succinylcholine 10-5 M Decamethonium 10-5 M

Tetraethylammonium Hexamethonium
Parachloromercuribenzoate 5 X 10-4M Hordenine Bretylium

Other cholinergic agents 32 Atropine 51 Noncholinergic agents 38 Picrotoxin

21

35

19 25

Strychnine

76

All agents at 10-4 M, unless otherwise stated. The following WIN compounds are the indicated analogs of benzoquinonium. 7758: bis-p-chlorobenzyl; 7846; bis-ethyl; 7789; bis-o-chlorobenzyl; 3317:bis-methvl. WIN 13357 is the bromide salt of ambenonium. See Webb.8

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Solubilization with sodium dodecyl sulfate (SDS) appeared to be very successful, but we suspected that an SDS-plax complex might be formed in the dialysis bag. Confirmation of this possibility was found when it was shown that bovine serum albumin (BSA) and human plasma ,8-lipoprotein, which show no binding by themselves, gave binding in the presence of SDS. For example, with 0.5 per cent SDS during assay, BSA gave 35.4 pmoles per milligram, and lipoprotein gave 36.8 pmoles per milligram. Under identical conditions, 0.5 per cent SDS alone accounted for only 22 per cent of this binding. Neither the SDS-treated plax nor the SDS-treated BSA or lipoprotein were strongly inhibited by 10-5 M curare, the maximum inhibition being 32 per cent for the lipoprotein-SDS preparation. We attribute the SDS artifact to the formation within the dialysis bag of SDSprotein micelles with a surrounding negative charge, which binds muscarone nonspecifically. With Triton-X100, a different problem was encountered. This nonionic detergent inhibited binding; thus 0.5 per cent Triton-X100 inhibited binding by whole plax powder 58 per cent. Inhibition increased with increasing Triton-X100 concentration. When plax powder was extracted with 2 per cent Triton-X100, then centrifuged, and the supernatant diluted four-fold for dialysis assay, 21 per cent of the activity was solubilized (when the data were corrected for the above inhibition). Other solubilizing agents included cholate, desoxycholate, taurocholate, urea, papain, lysozyme, pancreatin, Crotalus venom, and bee venom; none gave superior solubilization and all (except the enzymes and venoms) were at least 30 per cent inhibitory. Because of the difficulty of removing detergents, and of their interference in the assay, solubilization by sonication in the presence of various salt and urea concentrations was explored using a Bronwill Biosonik ultrasonic disintegrator at 50 per cent of maximum power, and ice-and-water cooling. Under optimal conditions (0.01 M NaCl and 0.5 M urea), typically for three periods of two minutes with two-minute intervals, solubilization of up to 23 per cent has been obtained. Enzymic degradation: In an attempt to characterize the muscarone-binding activity, the effect of treatment with various enzymes was explored, using (arbitrarily) 1 mg of enzyme and 6.8 mg of plax powder per milliliter of water, incubating one hour at room temperature (the pH, set by the powder, was 7.0), and assaying. No inhibition was given by: hyaluronidase, peptidase, ribonuclease, deoxyribonuclease, pepsin, pancreatin, carboxypeptidase-A (DFPtreated), collagenase, sulfatase, lysozyme, papain, neuraminidase, phospholipase D, lipase, Crotalus adamanteus venom (a source of phospholipase A), and bee venom. Chymotrypsin inhibited 80 per cent, trypsin inhibited 67 per cent, and phospholipase C inhibited 48 per cent. It should be noted that some of these enzymes, especially hyaluronidase and pepsin, have pH optima far removed from 7.0, so their inactivity is not conclusive. The data therefore suggest that the binding material is a phospholipoprotein, or perhaps a mixture of a protein and a phospholipid. Our findings do not cqn-

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firm the view of Thesleff and Albuquerque" that the effects of phospholipase C in blocking the electrical excitability of rat muscle are due to interference "with the action potential mechanism, leaving cholinergic receptors functionally intact." Electrophoresis: In disc gel electrophoresis of many different preparations, binding activity has only been observed in two zones: at the basal slice, i.e., the first slice taken from the stacking gel, and at the front, as marked by bromphenol blue. The front or fastest-running component was always found in 2 per cent SDS-solubilized plax powder, but it was also seen in 2 per cent SDStreated bovine serum albumin, and was only 7 per cent inhibited by 10-4 Al curare. We therefore believe this is artifactual binding, just as observed in dialysis assays. The basal slice, by contrast, showed binding with 2 per cent SDS-solubilized plax powder, and with sonicated material. The binding was from 73 to 100 per cent inhibited by 10-4 Al curare. It therefore seems that this is nonartifactual binding. It was absent from 2 per cent SDS-treated bovine serum albumin and from Triton-X100-solubilized plax powder; presumably in this latter case the high concentration of Triton-X100 was fully inhibitory. Thus SDS "solubilization" does not yield a curare-sensitive binding material that migrates electrophoretically even though it yields numerous migrating soluble proteins. Muscarone binding and acetylcholinesterase: In addition to evidence previously cited,' three findings show that muscarone binding, in the concentration range studied herein, was not due to acetylcholinesterase. (a) We studied the centrifugal behavior of the binding and of the enzyme in fresh electroplax homogenates and found them to differ in several ways. The most distinctive feature was that centrifuging at 100,000 X g for one hour gave a supernatant with zero binding activity, but with 9 per cent of the acetylcholinesterase. This supernatant acetylcholinesterase activity behaved identically with the particulate activity in the following respects (selected as features distinguishing acetylcholinesterase from butyrylcholinesterase). Both were inhibited 56-57 per cent by excess substrate (i.e., by 10'-1 Al as compared with 10-3 l acetylcholine); were little affected, i.e., 7-14 per cent inhibited, by 10-3 M MIIPA (N, N-bis (isopropyl)phosphoramidofluoridate) and did not hydrolyze benzoylcholine. In these ways, both behaved identically with purified bovine erythrocyte acetycholinesterase, and entirely unlike horse serum butyrylcholinesterase, which was found to be 23 per cent activated by excess acetylcholine; to be 100 per cent inhibited by 10-1 AlMIPA; and to hydrolyze benzoylcholine (10-3 M) at 50 per cent of the rate it hydrolyzed acetylcholine (10-3 M). It was concluded that the acetylcholinesterase of the particles and the supernatant were of the same type, and therefore that the centrifugal behavior of the enzyme and of the muscarone-binding activity differed. (b) The binding of muscarone to acetylcholinesterase was studied by the dialysis technique, using Winthrop's bovine erythrocyte or Worthington's highly purified (1000 units/mg) Electrophorus acetylcholinesterase. The amount of enzyme used per dialysis bag was that having as much acetyicholinesterase activity as the standard 6.9 mg of plax powder. Whereas that amount of plax

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powder bound 0.243 nmole of muscarone when the latter was 10-6 M, the enzyme showed zero binding. Thus in eight runs with Electrophorus enzyme the contents of the bag averaged 22 dpm over the value of 97,907 dpm for the same volume drawn from the bath. The finding is in accord with the observation' that muscarone's binding constant for acetylcholinesterase, as measured by enzyme inhibition, is 2.5 X 10-4 M, so that negligible binding would be seen at a muscarone concentration 250 times lower than this constant. (c) Plax powder was extracted with cold toluene for 24 hours, the supernatant discarded, and the residue was re-extracted as before. The residue was centrifuged down and then homogenized in buffer. It contained 74 per cent of the acetylcholinesterase activity of the original plax powder, but only 4 per cent of the muscarone-binding activity. One may roughly estimate if receptor and acetylcholinesterase are present in comparable amounts. The plax powder contained, as stated above, 1 nmole of receptor per gram of original plax. We measured acetylcholinesterase by the pH-stat method, and found an activity of 98 rmoles acetylcholine hydrolyzed per minute per gram original plax. If the turnover number is 3 X 10' min-','3 there are 0.3 nmole of acetylcholinesterase per gram of original plax. Discussion. It is clear that the dialysis assay technique as applied to plax powder gives kinetic data far superior to those reported previously, for a centrifugal assay applied to fresh plax precipitates.' The linearity of the data of Figure 1 and the small variance of the data of Figure 2 give us good confidence in the new estimates of the muscarone binding constant. The data on the "drug profile" go some way beyond (and fully confirm) those previously reported from centrifugal assay. All the highly potent neuromuscular agents are highly effective blockers of muscarone binding; most important are the data for curare, Flaxedil, succinylcholine, and four benzoquinoniums, all of which give 70 per cent or more blockade at 10-5 M. By contrast, the considerable list of noncholinergic drugs which are inactive at 10-4 Ml in blocking binding demonstrates that the blockade by neuromuscular agents is indeed a specific one. In short, the pharmacological and the binding data are in excellent harmony. Other than simply expanding the list of compounds, the only additional step can be the comparison of binding constants by physiological response of whole cells and by binding to homogenates, using identical tissue and a variety of drugs. Only one agent, DTT, has beei reported to be effective in blocking depolarization of Electrophorus plax cells, but is ineffective on blocking muscarone binding to Torpedo plax material. This could be due to a species difference, but more plausible explanations are that DTT affects a step in depolarization other than the binding step, or that the S-S group with which DTT presumably reacts is some 8-10 A away from the binding site. 14 The other anomaly to be resolved is why the four "noncholinergic" agents were moderately active blockers, i.e., were active at 10-4 J1. (a) The most potent was strychnine; although it is not normally thought of as a cholinergic agent, in fact it is one, for Lanari and Lucco"5 have shown it blocks the neuromuscular junction and autonomic ganglia, and Alving'16 has shown the blockade to be curare-like. (b) As for bretylium, it has been suggested that it acts by blocking the release by

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acetylcholine of noradrenaline at adrenergic nerve terminals;'7 18 this suggestion would account for the findings that bretylium blocks the sympathomimetric actions of acetylcholine in intact animals19 and tissues2 and inhibits acetylcholine-induced contractions of guinea pig ileum.2' (c) We cannot account for the activity of hordenine and picrotoxin. The activity was small. They appear not to have been tested on a physiological preparation of electroplax. In conclusion, the data of this and the preceding paper constitute rather strong evidence that the electroplax protein which binds muscarone with a binding constant of 7 X 10-7 M is either the acetylcholine receptor itself, or else is the binbing component of that receptor.
We are grateful to the Geigy Co. of Switzerland for donating nor-muscarone, to Mr. B. D. Hilton for synthesizing H3-muscarone, to Sterling-Winthrop Research Institute for the WIN compounds, to Dr. A. Toppozada for studying binding to acetylcholinesterase, and to Mr. Sam Rhine for computer analysis. Financial support is gratefully acknowledged from U.S. Public Health Service grants, GM07804 and training grant ES 98.
O'Brien, R. D., and L. P. Gilmour, these PROCEEDINGS, 63, 496 (1969). Dawson, R. M. C., D. C. Elliott, W. H. Elliott, and K. M. Jones, in Data for Biochemical Research, (Oxford: Oxford University Press, 1959), p. 208. 3 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 4 Johnson, M. J., J. Biol. Chem., 181, 707 (1949). 6 Davis, B. J., Ann. N.Y. Acad. Sci., 121, 404 (1963). 6 Gray, R. H., and D. M. Steffensen, Anal. Biochem., 24, 44 (1968). 7 Bray, A. G., Anal. Biochem., 1, 279 (1960). 8 Webb, G. D., Biochim. Biophys. Acta, 102, 172 (1965). 9 Karlin, A., and E. Bartels, Biochim. Biophys. Acta, 126, 525 (1966). 10 Karlin, A., Biochim. Biophys. Acta, 139, 358 (1967). 11 Thesleff, S., and E. X. Albuquerque, Ann. N.Y. Acad. Sci., 144, 534 (1967). 12 Witkop, B., R. C. Durant, and S. L. Fries, Experientia, 15, 300 (1959). 13 Cohen, J. A., and M. G. P. J. Warringa, Biochim, Biophys. Acta, 11, 52 (1953). 14 Karlin, A., and M. Winnik, these PROCEEDINGS, 60, 668 (1968). 15 Lanari, L., and J. V. Lucco, Amer. J. Physiol., 126, 277 (1939). 16 Alving, B. O., Arch. Int. Pharmacodyn., 131, 123 (1961). 17 Burn, J. H., in Adrenergic Mechanisms, ed. W. A. Bain (London, Churchill, 1960), p. 131. 18 Triggle, D. J., in Chemical Aspects of the Autonomic Nervous System, (New York: Academic Press, 1965), p. 206. 19 Boura, A. L. A., and Green, A. F., Brit. J. Pharmacol., 14, 536 (1959). 20 Hukovic, S., Brit. J. Pharmacol., 15, 117 (1960). 21 Kosterlitz, H. W. and G. M. Lees, Brit. J. Pharmacol., 17, 82 (1961). 22 Wilkinson, G. M., Biochem. J., 80, 324 (1961).
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