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Conversion of high affinity acetylcholine receptor from Torpedo californica electroplax to an altered form
ARCHIVES
OF BIOCHEMISTRY
Conversion Torpedo
AND
BIOPHYSICS
169, 458-463 (1975)
of High Affinity californica
Acetylcholine
Electroplax
R. D. O’BRIEN
AND
Receptor
to an Altered
from
Form
R. E. GIBSON
Section of Neurobiology and Behaoior, Cornet/ University, Ithaca, Neu York i&53 Received April
16, 1975
Most Triton extracts of fresh electroplax membranes showed a single kind of acetylcholine binding, of high affinity, with Kd = 17 no. Fresh membrane preparations without Triton also showed only high-affinity binding (K, = 14 nM), but membrane preparations from electroplax stored for 5 months in liquid nitrogen gave equal amounts of low-affinity binding material (K, = 0.2 FM). Heating Triton extracts at 40°C for 40 min or treating with 10 ~’ M p-chloromercuribenzoate or 5,5’-dithiobis(2-nitrobenzoic acid) converted the preparation to more than 90%’ low-affinity binding, It is suggested that the high-affinity is the native form and the low-affinity is an oxidation product.
When acetylcholine binding to Torpedo marmorata electroplax was first described (l), it was noted that the Scatchard plot of binding was curvilinear, and was compatible with the existence of mixed low-affinity and high-affinity binding in the ratio of 8: 1 in particulate preparations or 2: 1 in a crude Lubrol-solubilized preparation (2) or a highly purified Triton-solubilized preparation (11). An alternative possible explanation for the curved plots was that they reflected similar sites interacting with negative cooperativity (3). In unpurified Triton-solubilized preparations of T. marmorata, we found a ratio of 5.4:1, with a high-affinity binding having Kd = 11.3 nM and a low-affinity having Kd = .562 FM; we provided evidence that the two kinds of binding were to two kinds of receptor site (designated A and B), based on differing responses to drugs, heat, pH, and phospholipase A (4). All of the studies described in this paragraph made use of a large batch of lyophilized T. marmorata plax membrane harvested (5) in 1968 and stored at -25°C. By contrast with the above, two laboratories reported at first only a single kind of binding; but since their two reported dissociation constants differ 600-fold, it was possible that each had one of the forms we
had described. Thus Weber and Changeux (6) studied the displacement of [3H]-~toxin of Naja nigricollis by cholinergic ligands and found only one acetylcholine binding site in membrane fragments of T. marmorata, with Kd = 8 nM (close to our high-affinity value of 11.3 nM). In Raftery’s laboratory a fluorescent displacement technique with a purified preparation from T. californica (7) showed only one acetylcholine binding site, with Kd = 5 PM. Equilibrium dialysis against [3H]acetylcholine (8) gave Kd = 2.3 PM (fairly close to our low-affinity value of 0.56 PM). Recently Cohen, Weber and Changeux (9) found two sites in sodium cholate solubilized preparations from T. marmorata, with approximate Kd values of 55 nM and 0.8 /IM, quite close to our values. We report herein that fresh unpurified extracts of electroplax membranes from T. californica typically show only high-affinity binding, whether they are soluble or particulate, but that aging or treatment with heat or 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) or p-chloromercuribenzoate (PCMB)’ always convert this form to a ’ Abbreviations used: DTNB, 5,5’-dithiobis(2nitrobenzoic acid); and PCMB, p-chloromercuribenzoate.
458 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
CONVERSION
OF ACETYLCHOLINE
predominantly low-affinity binding form. We therefore propose that the low-affinity binding of acetylcholine, which is found in aged, crude and purified preparations, is a consequence of oxidation of the native form of the receptor. METHODS Torpedo californica was shipped alive from Venice, CA or as N,-frozen plax where indicated. The procedure of Miledi et al. (10) was used to harvest the heavy-particle fraction, which was then lyophilized. The resulting material was stored at -25°C. then homogenized at 10 mg/ml in a Krebs-Ringer solution containing 0.7 rnM calcium and 1% Triton X-100 (4). The homogenate was centrifuged for 1.25 h at 90,OOOg at 4°C. Seven-milliliter samples of the supernatant were then set aside on ice (for control or later treatment with PCMB or DTNB) or heated at 40°C for 40 min, then cooled on ice: then 0.07 ml of lo-” M PCMB or DTNB or water was added. After 30 min at room temperature, 0.07 ml of 10m2M Tetram [oxalate of 0-0-diethyl S-(2-diethylaminoethyl) phosphorothiolate] was added in order to inhibit acetylcholinesterase. 45 min later, samples of 0.5 ml each were placed in dialysis bags in a 4°C bath of the above Ringer solution containing lo-’ M Tetram and acetylcholine at concentrations from 5 x 10m9 M to 1Om6M. Acetylcholine binding was then assayed precisely as described previously (4). When a protective action of agents upon the heat transformation was explored, the agent at the indicated concentration was added just prior to heating. Thus its concentration during dialysis would be much reduced by diffusional loss. Binding constants for control acetylcholine-binding curves which showed only one component (as judged by Scatchard plot) were obtained from a computerized version of the Wilkinson weighted regression analysis (Lang 1). When the curve showed positive cooperativity the dissociation constant was obtained from the linear portion of the curve by the above method. When two-component systems (defined below) were observed, a program (Lang 2) was used based upon the Langmuir isotherm (i.e., a plot of binding as a function of ligand concentration). Linear weighting of the data points was based upon a histoFam analysis (programs Calu and Calr) of 40 random data sets. A detailed description of these programs with listings may be obtained from the authors. RESULTS
In complete contrast to our earlier studies with aged T. marmorata electroplax, Triton-solubilized crude preparations from fresh T. californica electroplax typically
RECEPTOR
459
displayed Scatchard plots (Fig. la) which were linear at acetylcholine concentrations above 5 KIM, with none of the curvilinearity previously present at high concentrations above 100 nM. In 17 such preparations, the average dissociation constant for acetylcholine was 17.0 i 11.1 nM and the amount was 0.97 i 0.33 nmol per g of original plax. This kind of binding appears to be the same as the high-affinity form we previously reported as one of the components of the aged T. marmorata preparations, with Kd = 11.3 * 2.2 nM in the same TritonRinger (4). Eldefrawi and Eldefrawi (11) have shown with highly purified T. marmorata receptor from aged electroplax that positive cooperativity is seen at acetylcholine concentrations below 10 nM. The lowest acetylcholine concentration normally used in the present study was 5 nM. At this concentration, evidence of positive cooperativity was frequently seen, i.e., the value of B (the amount bound; see Fig. 1) was lower than expected for simple noncooperative binding, resulting in a Scatchard plot with a maximum (Fig. lb). There was considerable variability in the concentration at which it was seen, a phenomenon which is discussed at length elsewhere (12). In several solubilized preparations of as well as fresh T. californica, low-affinity high-affinity binding was seen (Fig. lc); for eight of these, the average constants were 4.09 f 2.83 nM for the high and 0.509 * 0.332 PM for the low affinity; the high-affinity binding averaged 28’% of the activity (ranging between 6 and 65%). We were unable to discover what slight procedural changes may have produced this result. In runs performed with fresh particulate preparations without Triton X-100 (as in Methods but without solubilizing with Triton X-100), excellent linearity, implying one high-affinity component was always seen; the average in four runs was Kd = 13.9 f 7.7 nM. In particulates from material stored 5 months in liquid nitrogen, twocomponent systems were again seen; in three runs the Kd values averaged 1.77 nM and 0.198 pM, and the high affinity represented 46%. In purified preparations of fresh T. californica receptor, prepared ac-
460
O’BRIEN
AND GIBSON
a
LL,,
1
,
2 3
,
.L .5
1
J
6 .7 6 .9 10
b
6 Lt
FIG. 1. Acetylcholine binding to unpurified extracts of fresh electroplax membranes. B = binding (nmol/g original plax). L = acetylcholine concentration (FM); lowest L = 0.0005. (a) Commonest form: one component, no positive cooperativity. (b) Example in which positive cooperativity is seen even with lowest L = 0.5 nM. (c) Frequent form: two components, no positive cooperativity (high-affinity = 33%).
cording to the procedure developed for T. marmorata (9) and with only traces of residual detergent, low-affinity binding was always the predominant form. In three runs, the Kd values averaged 17.2 nM and 0.48 PM, with the high affinity representing 32% (the Scatchard plots have been published elsewhere (13) but without numerical analysis). When crude preparations from T. californica electroplax were heated at 40°C for 40 min, or treated with 10m4M PCMB or DTNB, they were converted to a material containing a mixture of high- and lowaffinity binding components (Fig. 2a, b, cl, with the latter much in excess. In seven
cases of heat treatment the average value for the high-affinity dissociation constant, KH, was 9.34 f 7.65 nM, and for the low-affinity constant, KL, it was 0.848 f 0.44 FM; the high-affinity binding contributed 4% of the total binding. For PCMBtreated electroplax (four experiments) the binding constants were KH = 4.96 * 2.47 nM and K, = 1.026 & 0.64 PM and the high-affinity form contributed 6%. The values of K, and K, (from two separate experiments) for DTNB-treated receptor were 6.81 nM and 0.561 PM, and the highaffinity form represented 8% of the observed binding. The values of KH and KL obtained for the three different treatments
CONVERSION
OF ACETYLCHOLINE
02
OL
06
06
10
12
461
RECEPTOR
1L
16
16
b
OL I
,,
OL
1,.
08
12
16
20
2L
.2t3
32
36
08
12
16
20
2L
B
FIG. 2. Effect of treatments upon single-component preparations of Triton extracts of fresh electroplax membranes. B, L as in Fig. 1. (a) Heating, 40°C for 40 min. (b) 1 mM PCMB. (c) 1 mM DTNB. The B of high-affinity in these examples was 3, 13, and 5%.
did not differ significantly from each other or the estimates of K, and K, obtained from two-component control curves. These constants are also in good agreement with KH and K, determined for aged T. marmorata. With heat treatment, there was, in addition to conversion of high- and low-affinity forms, a net loss of activity averaging 71%, a finding which is capatible with our previous report (14) for T. marmorata that the half-life at 40°C in Triton X-100 is about 45 min for low and 4 min for high-affinity binding. We would have wished to show that highly purified receptor was also convertible from all-high-affinity to mostly-lowaffinity by heat. Unfortunately, as indicated above, all purified receptor contains much low-affinity binding. Furthermore, Triton itself reduces binding strongly so that heating under conditions strictly comparable to work with crude preparations (i.e., in 1% Triton) yielded very low activities (Fig. 3b). When pure receptor was heated without detergent, the preparation was far less heat-sensitive than in the
presence of detergent (Fig. 3a), an observation which we shall elaborate upon in a future paper. Particulate preparations of T. californica were also relatively insensitive to heat, a phenomenon which might reflect innate stability, or perhaps only the lack of detergent. Other variables, covering procedures used in other laboratories or publications, were explored: one-component binding was found with (a) Triton-soluble and particulate preparations from freshly killed T. californica prepared (5) in the way used for the T. marmorata studies referred to in the first paragraph of the present paper, i.e., homogenization of the plax in water instead of 0.4 M NaCl, and centrifugation for 90 min instead of 15 min (b) treatment of fresh T. californica before and during dialysis with lo-” M diisopropyl phosphorofluoridate instead of lo-“ M Tetram as an anticholinesterase. In view of the fact that a mercurial SH-agent (PCMB) and an SH-oxidizing agent (DTNB) could mimic the effect of heating, it seemed plausible to suppose that SH-oxidation was responsible for the
462
O’BRIEN
AND GIBSON b
FIG. 3. Effect of heat, 40°C for 40 min, on highly purified receptor. B = binding (in nmol/mg (PM). (a) In the absence of added detergent. Note protein). L = acetylcholine concentration relative insensitivity. (b) In the presence of 1% Triton X-100. That detergent greatly reduces control binding, and increases heat-sensitivity. Note different ordinate scales for (a) and (b).
heating effect. However, not all SH-agents Form B has a binding constant of 0.85, have an effect upon receptor. Sodium arse- 1.03, or 0.56 PM for these three treatments, nite, N-ethyl maleimide and iodoacetavery similar to (a) that with Kd = 5 or 2.3 mide at 10m3 M had little or no effect. PM reported (by different procedures) for We explored the possibility that appro- purified T. californica (7, 8); (b) the lowpriate reducing conditions during heating affinity component of T. marmorata recepmight lessen the heat denaturation. We tor, reported as 1.97 FM for highly purified hoped that such experiments would also material without excess detergent or as 0.6 enable us to find conditions for preparation PM for unpurified Triton extracts of T. of controls which always had the highmarmorata (4); (c) the low-affinity compoaffinity binding only. But concentrations of nent of cholate extracts of T. marmorata l-100 mM of mercaptoethanol, ascorbate, reported (9) as 3 PM. dithiothreitol, sodium bisulfite, sodium It is a plausible hypothesis that the sulfite, or reduced glutathione did not native form of receptor is Form A, found in lessen the production of low-affinity bindfresh particulates or fresh detergent exing, as judged by the relative amounts of tracts of T. californica. This form is oxidizbinding assayed at lOmEand 1Om6M acetylable under fairly mild conditions, and choline. presumably the purified preparations described above are partially or fully oxidized to the B form, as is the long-stored T. DISCUSSION marmorata described in the opening paraTriton extracts of fresh T. californica graph. SH groups are probably involved in plax usually display only one form (Form this oxidation. It has been shown (15) that A) of high-affinity binding for acetylchoour aged T. marmorata has half of the free line. Its dissociation constant (17 nM) sug- SH groups per mole of acetylcholine-bindgests that it is the same as (a) that with Kd ing sites as does fresh T. californica pre= 8 nM observed (6) in T. californica plax pared under identical conditions, perhaps particulate using either Naja toxin dis- accounting for the differences in their conplacement or acetylcholine dialysis; (b) the tent of A and B forms. Furthermore T. high-affinity component of aged T. marmorata receptor purified under deoxmarmorata (4), with Kd = 11.3 nM in 1% ygenating conditions had 6.4 SH groups Triton X-100 or of highly purified Tritonper mole, as compared with 2.4 for that free T. marmorata (11) with Kd = 20 nM. purified under normal conditions. It should be stressed that cu-bunHeating, or treatment with PCMB or DTNB, converts most of the receptor (i.e., garotoxin binds strongly to both A and B 8596%) to a form (Form B) with a drastiforms so that the toxin cannot detect their cally reduced affinity for acetylcholine. interconversion; acetylcholine is the rea-
CONVERSION
OF ACETYLCHOLINE
gent of choice to distinguish the forms, because it is the ligand for which the difference in affinity is maximal (4). Our proposal that Form A is the native form of the receptor, and is typically present alone in fresh particulate and detergent extracts, runs counter to the proposal of Cohen, Weber and Changeux (9). They conclude that (a) the occurrence of Form A is related to the removal of the receptor from the membrane environment by detergent. But we find that such removal usually gives preparation containing only A; and that the fresh membrane preparations without detergent invariably contain only A; (b) the physiologically significant form is B, because of certain time constraints associated with binding in the nM range; but our findings suggest that B is simply a denatured form of A; (c) that the A form loses affinity on solubilization; they had earlier found (6) Kd = 8 nM in particulate preparations, and later (9) found Kd = 40-70 nM for cholate-solubilized receptor. But it should be stressed that the variance is high in all these binding studies, and also different techniques give somewhat different values, e.g., for the B form, Kd = 0.8 FM and 3 PM in two different procedures (9). We find no significant changes in solubilization, e.g., from Kd = 14 nM in an all-A particulate to 17 nM in an all-A soluble. The B form had Kd = 0.2 PM in particulates and 0.5 PM in soluble preparations. Detergents are well known to modify protein properties, and Triton X-100 inhibits acetylcholine-binding to purified receptor (Fig. 3 and Ref. 16). Yet paradoxically the crude fresh soluble receptor in 1% Triton X-100 is a better model of fresh membrane-bound receptor (i.e., both giving only-A preparations) than the highly purified receptor, which is mostly-B in spite of its small detergent content.
RECEPTOR
463
ACKNOWLEDGMENTS We are grateful to the National Institutes of Health Grant NS 09144, which funded this work in large part, to National Institutes of Health Post-doctoral Award GM 53317 to R. E. G., and to Nancy Baston and Carol Timpone for excellent technical assistance. Drs. M. E. and A. T. Eldefrawi kindly provided the purified receptor. REFERENCES 1. ELDEFRAWI, M. E., BRITTEN, A: G., AND ELDEFRAWI, A. T. (1971) Science 173, 338-340. 2. ELDEFRAWI, M. E., ELDEFRAWI, A. T., SEIFERT, S., AND O’BRIEN, R. D. (1972) Arch. Biochem. Biophys. 150, 210-218. 3. ELDEFRAWI, M. E., AND ELDEFRAWI, A. T. (1973) Biochem. Pharmacol. 22, 31453150. 4. O’BRIEN, R. D., AND GIBSON, R. E. (1974) Arch. Biochem. Biophys. 165, 681-690. 5. O’BRIEN, R. D., GILMOUR, L. P., AND ELDEFRAWI, M. E. (1970) Proc. Nat. Acad. Sci. USA 65, 438-445. 6. WEBER, M., AND CHANGEUX, J.-P. (1974) Mol. Pharmacol. 10, 15-24. 7. MARTINEZ-CARRION, M., AND RAFTERY, M. A. (1973) Biochem. Biophys. Res. Commun. 55, 1156-1164. 8. MOODY, T., SCHMIDT, J., AND RAFTERY, M. A. (1973) Biochem. Biophys. Res. Commun. 53, 761-772. 9. COHEN, J. B., WEBER, M., AND CHANGEUX, J.-P. (1974) Mol. Pharmacol. 10, 904-932. 10. MILEDI, R., MOLISOFF, P., AND POTTER, L. T. (1971) Nature (London) 229, 554-557. 11. ELDEFRAWI, M. E., AND ELDEFRAWI, A. T. (1973) Arch. Biochem. Biophys. 159, 362-373. 12. GIBSON, R. E., in preparation. 13. ELDEFRAU’I, M. E., ELDEFRAWI, A. T., PENFIELD, L. A., O’BRIEN, R. D., AND VAN CAMPEN, D. (1975) Life Sci. 16, 925-936. 14. O’BRIEN, R. D., THOMPSON, W. R., ANDGIBSON, R. E. (1974) in Neurochemistry of Cholinergic Receptors (De Robertis, E.. and Schacht, J., eds.), Raven Press, New York. 15. ELDEFRAWI, M. E., ELDEFRA~I, A. T., AND WILSON, D. (1975) Biochemistry (submitted). 16. EDELSTEIN. S. J., BEYER, W. B., ELDEFRAWI, A. T., AND ELDEFRAWI, M. E. (1975) J. Biol. Chem., in press.
OF BIOCHEMISTRY
Conversion Torpedo
AND
BIOPHYSICS
169, 458-463 (1975)
of High Affinity californica
Acetylcholine
Electroplax
R. D. O’BRIEN
AND
Receptor
to an Altered
from
Form
R. E. GIBSON
Section of Neurobiology and Behaoior, Cornet/ University, Ithaca, Neu York i&53 Received April
16, 1975
Most Triton extracts of fresh electroplax membranes showed a single kind of acetylcholine binding, of high affinity, with Kd = 17 no. Fresh membrane preparations without Triton also showed only high-affinity binding (K, = 14 nM), but membrane preparations from electroplax stored for 5 months in liquid nitrogen gave equal amounts of low-affinity binding material (K, = 0.2 FM). Heating Triton extracts at 40°C for 40 min or treating with 10 ~’ M p-chloromercuribenzoate or 5,5’-dithiobis(2-nitrobenzoic acid) converted the preparation to more than 90%’ low-affinity binding, It is suggested that the high-affinity is the native form and the low-affinity is an oxidation product.
When acetylcholine binding to Torpedo marmorata electroplax was first described (l), it was noted that the Scatchard plot of binding was curvilinear, and was compatible with the existence of mixed low-affinity and high-affinity binding in the ratio of 8: 1 in particulate preparations or 2: 1 in a crude Lubrol-solubilized preparation (2) or a highly purified Triton-solubilized preparation (11). An alternative possible explanation for the curved plots was that they reflected similar sites interacting with negative cooperativity (3). In unpurified Triton-solubilized preparations of T. marmorata, we found a ratio of 5.4:1, with a high-affinity binding having Kd = 11.3 nM and a low-affinity having Kd = .562 FM; we provided evidence that the two kinds of binding were to two kinds of receptor site (designated A and B), based on differing responses to drugs, heat, pH, and phospholipase A (4). All of the studies described in this paragraph made use of a large batch of lyophilized T. marmorata plax membrane harvested (5) in 1968 and stored at -25°C. By contrast with the above, two laboratories reported at first only a single kind of binding; but since their two reported dissociation constants differ 600-fold, it was possible that each had one of the forms we
had described. Thus Weber and Changeux (6) studied the displacement of [3H]-~toxin of Naja nigricollis by cholinergic ligands and found only one acetylcholine binding site in membrane fragments of T. marmorata, with Kd = 8 nM (close to our high-affinity value of 11.3 nM). In Raftery’s laboratory a fluorescent displacement technique with a purified preparation from T. californica (7) showed only one acetylcholine binding site, with Kd = 5 PM. Equilibrium dialysis against [3H]acetylcholine (8) gave Kd = 2.3 PM (fairly close to our low-affinity value of 0.56 PM). Recently Cohen, Weber and Changeux (9) found two sites in sodium cholate solubilized preparations from T. marmorata, with approximate Kd values of 55 nM and 0.8 /IM, quite close to our values. We report herein that fresh unpurified extracts of electroplax membranes from T. californica typically show only high-affinity binding, whether they are soluble or particulate, but that aging or treatment with heat or 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) or p-chloromercuribenzoate (PCMB)’ always convert this form to a ’ Abbreviations used: DTNB, 5,5’-dithiobis(2nitrobenzoic acid); and PCMB, p-chloromercuribenzoate.
458 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
CONVERSION
OF ACETYLCHOLINE
predominantly low-affinity binding form. We therefore propose that the low-affinity binding of acetylcholine, which is found in aged, crude and purified preparations, is a consequence of oxidation of the native form of the receptor. METHODS Torpedo californica was shipped alive from Venice, CA or as N,-frozen plax where indicated. The procedure of Miledi et al. (10) was used to harvest the heavy-particle fraction, which was then lyophilized. The resulting material was stored at -25°C. then homogenized at 10 mg/ml in a Krebs-Ringer solution containing 0.7 rnM calcium and 1% Triton X-100 (4). The homogenate was centrifuged for 1.25 h at 90,OOOg at 4°C. Seven-milliliter samples of the supernatant were then set aside on ice (for control or later treatment with PCMB or DTNB) or heated at 40°C for 40 min, then cooled on ice: then 0.07 ml of lo-” M PCMB or DTNB or water was added. After 30 min at room temperature, 0.07 ml of 10m2M Tetram [oxalate of 0-0-diethyl S-(2-diethylaminoethyl) phosphorothiolate] was added in order to inhibit acetylcholinesterase. 45 min later, samples of 0.5 ml each were placed in dialysis bags in a 4°C bath of the above Ringer solution containing lo-’ M Tetram and acetylcholine at concentrations from 5 x 10m9 M to 1Om6M. Acetylcholine binding was then assayed precisely as described previously (4). When a protective action of agents upon the heat transformation was explored, the agent at the indicated concentration was added just prior to heating. Thus its concentration during dialysis would be much reduced by diffusional loss. Binding constants for control acetylcholine-binding curves which showed only one component (as judged by Scatchard plot) were obtained from a computerized version of the Wilkinson weighted regression analysis (Lang 1). When the curve showed positive cooperativity the dissociation constant was obtained from the linear portion of the curve by the above method. When two-component systems (defined below) were observed, a program (Lang 2) was used based upon the Langmuir isotherm (i.e., a plot of binding as a function of ligand concentration). Linear weighting of the data points was based upon a histoFam analysis (programs Calu and Calr) of 40 random data sets. A detailed description of these programs with listings may be obtained from the authors. RESULTS
In complete contrast to our earlier studies with aged T. marmorata electroplax, Triton-solubilized crude preparations from fresh T. californica electroplax typically
RECEPTOR
459
displayed Scatchard plots (Fig. la) which were linear at acetylcholine concentrations above 5 KIM, with none of the curvilinearity previously present at high concentrations above 100 nM. In 17 such preparations, the average dissociation constant for acetylcholine was 17.0 i 11.1 nM and the amount was 0.97 i 0.33 nmol per g of original plax. This kind of binding appears to be the same as the high-affinity form we previously reported as one of the components of the aged T. marmorata preparations, with Kd = 11.3 * 2.2 nM in the same TritonRinger (4). Eldefrawi and Eldefrawi (11) have shown with highly purified T. marmorata receptor from aged electroplax that positive cooperativity is seen at acetylcholine concentrations below 10 nM. The lowest acetylcholine concentration normally used in the present study was 5 nM. At this concentration, evidence of positive cooperativity was frequently seen, i.e., the value of B (the amount bound; see Fig. 1) was lower than expected for simple noncooperative binding, resulting in a Scatchard plot with a maximum (Fig. lb). There was considerable variability in the concentration at which it was seen, a phenomenon which is discussed at length elsewhere (12). In several solubilized preparations of as well as fresh T. californica, low-affinity high-affinity binding was seen (Fig. lc); for eight of these, the average constants were 4.09 f 2.83 nM for the high and 0.509 * 0.332 PM for the low affinity; the high-affinity binding averaged 28’% of the activity (ranging between 6 and 65%). We were unable to discover what slight procedural changes may have produced this result. In runs performed with fresh particulate preparations without Triton X-100 (as in Methods but without solubilizing with Triton X-100), excellent linearity, implying one high-affinity component was always seen; the average in four runs was Kd = 13.9 f 7.7 nM. In particulates from material stored 5 months in liquid nitrogen, twocomponent systems were again seen; in three runs the Kd values averaged 1.77 nM and 0.198 pM, and the high affinity represented 46%. In purified preparations of fresh T. californica receptor, prepared ac-
460
O’BRIEN
AND GIBSON
a
LL,,
1
,
2 3
,
.L .5
1
J
6 .7 6 .9 10
b
6 Lt
FIG. 1. Acetylcholine binding to unpurified extracts of fresh electroplax membranes. B = binding (nmol/g original plax). L = acetylcholine concentration (FM); lowest L = 0.0005. (a) Commonest form: one component, no positive cooperativity. (b) Example in which positive cooperativity is seen even with lowest L = 0.5 nM. (c) Frequent form: two components, no positive cooperativity (high-affinity = 33%).
cording to the procedure developed for T. marmorata (9) and with only traces of residual detergent, low-affinity binding was always the predominant form. In three runs, the Kd values averaged 17.2 nM and 0.48 PM, with the high affinity representing 32% (the Scatchard plots have been published elsewhere (13) but without numerical analysis). When crude preparations from T. californica electroplax were heated at 40°C for 40 min, or treated with 10m4M PCMB or DTNB, they were converted to a material containing a mixture of high- and lowaffinity binding components (Fig. 2a, b, cl, with the latter much in excess. In seven
cases of heat treatment the average value for the high-affinity dissociation constant, KH, was 9.34 f 7.65 nM, and for the low-affinity constant, KL, it was 0.848 f 0.44 FM; the high-affinity binding contributed 4% of the total binding. For PCMBtreated electroplax (four experiments) the binding constants were KH = 4.96 * 2.47 nM and K, = 1.026 & 0.64 PM and the high-affinity form contributed 6%. The values of K, and K, (from two separate experiments) for DTNB-treated receptor were 6.81 nM and 0.561 PM, and the highaffinity form represented 8% of the observed binding. The values of KH and KL obtained for the three different treatments
CONVERSION
OF ACETYLCHOLINE
02
OL
06
06
10
12
461
RECEPTOR
1L
16
16
b
OL I
,,
OL
1,.
08
12
16
20
2L
.2t3
32
36
08
12
16
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2L
B
FIG. 2. Effect of treatments upon single-component preparations of Triton extracts of fresh electroplax membranes. B, L as in Fig. 1. (a) Heating, 40°C for 40 min. (b) 1 mM PCMB. (c) 1 mM DTNB. The B of high-affinity in these examples was 3, 13, and 5%.
did not differ significantly from each other or the estimates of K, and K, obtained from two-component control curves. These constants are also in good agreement with KH and K, determined for aged T. marmorata. With heat treatment, there was, in addition to conversion of high- and low-affinity forms, a net loss of activity averaging 71%, a finding which is capatible with our previous report (14) for T. marmorata that the half-life at 40°C in Triton X-100 is about 45 min for low and 4 min for high-affinity binding. We would have wished to show that highly purified receptor was also convertible from all-high-affinity to mostly-lowaffinity by heat. Unfortunately, as indicated above, all purified receptor contains much low-affinity binding. Furthermore, Triton itself reduces binding strongly so that heating under conditions strictly comparable to work with crude preparations (i.e., in 1% Triton) yielded very low activities (Fig. 3b). When pure receptor was heated without detergent, the preparation was far less heat-sensitive than in the
presence of detergent (Fig. 3a), an observation which we shall elaborate upon in a future paper. Particulate preparations of T. californica were also relatively insensitive to heat, a phenomenon which might reflect innate stability, or perhaps only the lack of detergent. Other variables, covering procedures used in other laboratories or publications, were explored: one-component binding was found with (a) Triton-soluble and particulate preparations from freshly killed T. californica prepared (5) in the way used for the T. marmorata studies referred to in the first paragraph of the present paper, i.e., homogenization of the plax in water instead of 0.4 M NaCl, and centrifugation for 90 min instead of 15 min (b) treatment of fresh T. californica before and during dialysis with lo-” M diisopropyl phosphorofluoridate instead of lo-“ M Tetram as an anticholinesterase. In view of the fact that a mercurial SH-agent (PCMB) and an SH-oxidizing agent (DTNB) could mimic the effect of heating, it seemed plausible to suppose that SH-oxidation was responsible for the
462
O’BRIEN
AND GIBSON b
FIG. 3. Effect of heat, 40°C for 40 min, on highly purified receptor. B = binding (in nmol/mg (PM). (a) In the absence of added detergent. Note protein). L = acetylcholine concentration relative insensitivity. (b) In the presence of 1% Triton X-100. That detergent greatly reduces control binding, and increases heat-sensitivity. Note different ordinate scales for (a) and (b).
heating effect. However, not all SH-agents Form B has a binding constant of 0.85, have an effect upon receptor. Sodium arse- 1.03, or 0.56 PM for these three treatments, nite, N-ethyl maleimide and iodoacetavery similar to (a) that with Kd = 5 or 2.3 mide at 10m3 M had little or no effect. PM reported (by different procedures) for We explored the possibility that appro- purified T. californica (7, 8); (b) the lowpriate reducing conditions during heating affinity component of T. marmorata recepmight lessen the heat denaturation. We tor, reported as 1.97 FM for highly purified hoped that such experiments would also material without excess detergent or as 0.6 enable us to find conditions for preparation PM for unpurified Triton extracts of T. of controls which always had the highmarmorata (4); (c) the low-affinity compoaffinity binding only. But concentrations of nent of cholate extracts of T. marmorata l-100 mM of mercaptoethanol, ascorbate, reported (9) as 3 PM. dithiothreitol, sodium bisulfite, sodium It is a plausible hypothesis that the sulfite, or reduced glutathione did not native form of receptor is Form A, found in lessen the production of low-affinity bindfresh particulates or fresh detergent exing, as judged by the relative amounts of tracts of T. californica. This form is oxidizbinding assayed at lOmEand 1Om6M acetylable under fairly mild conditions, and choline. presumably the purified preparations described above are partially or fully oxidized to the B form, as is the long-stored T. DISCUSSION marmorata described in the opening paraTriton extracts of fresh T. californica graph. SH groups are probably involved in plax usually display only one form (Form this oxidation. It has been shown (15) that A) of high-affinity binding for acetylchoour aged T. marmorata has half of the free line. Its dissociation constant (17 nM) sug- SH groups per mole of acetylcholine-bindgests that it is the same as (a) that with Kd ing sites as does fresh T. californica pre= 8 nM observed (6) in T. californica plax pared under identical conditions, perhaps particulate using either Naja toxin dis- accounting for the differences in their conplacement or acetylcholine dialysis; (b) the tent of A and B forms. Furthermore T. high-affinity component of aged T. marmorata receptor purified under deoxmarmorata (4), with Kd = 11.3 nM in 1% ygenating conditions had 6.4 SH groups Triton X-100 or of highly purified Tritonper mole, as compared with 2.4 for that free T. marmorata (11) with Kd = 20 nM. purified under normal conditions. It should be stressed that cu-bunHeating, or treatment with PCMB or DTNB, converts most of the receptor (i.e., garotoxin binds strongly to both A and B 8596%) to a form (Form B) with a drastiforms so that the toxin cannot detect their cally reduced affinity for acetylcholine. interconversion; acetylcholine is the rea-
CONVERSION
OF ACETYLCHOLINE
gent of choice to distinguish the forms, because it is the ligand for which the difference in affinity is maximal (4). Our proposal that Form A is the native form of the receptor, and is typically present alone in fresh particulate and detergent extracts, runs counter to the proposal of Cohen, Weber and Changeux (9). They conclude that (a) the occurrence of Form A is related to the removal of the receptor from the membrane environment by detergent. But we find that such removal usually gives preparation containing only A; and that the fresh membrane preparations without detergent invariably contain only A; (b) the physiologically significant form is B, because of certain time constraints associated with binding in the nM range; but our findings suggest that B is simply a denatured form of A; (c) that the A form loses affinity on solubilization; they had earlier found (6) Kd = 8 nM in particulate preparations, and later (9) found Kd = 40-70 nM for cholate-solubilized receptor. But it should be stressed that the variance is high in all these binding studies, and also different techniques give somewhat different values, e.g., for the B form, Kd = 0.8 FM and 3 PM in two different procedures (9). We find no significant changes in solubilization, e.g., from Kd = 14 nM in an all-A particulate to 17 nM in an all-A soluble. The B form had Kd = 0.2 PM in particulates and 0.5 PM in soluble preparations. Detergents are well known to modify protein properties, and Triton X-100 inhibits acetylcholine-binding to purified receptor (Fig. 3 and Ref. 16). Yet paradoxically the crude fresh soluble receptor in 1% Triton X-100 is a better model of fresh membrane-bound receptor (i.e., both giving only-A preparations) than the highly purified receptor, which is mostly-B in spite of its small detergent content.
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ACKNOWLEDGMENTS We are grateful to the National Institutes of Health Grant NS 09144, which funded this work in large part, to National Institutes of Health Post-doctoral Award GM 53317 to R. E. G., and to Nancy Baston and Carol Timpone for excellent technical assistance. Drs. M. E. and A. T. Eldefrawi kindly provided the purified receptor. REFERENCES 1. ELDEFRAWI, M. E., BRITTEN, A: G., AND ELDEFRAWI, A. T. (1971) Science 173, 338-340. 2. ELDEFRAWI, M. E., ELDEFRAWI, A. T., SEIFERT, S., AND O’BRIEN, R. D. (1972) Arch. Biochem. Biophys. 150, 210-218. 3. ELDEFRAWI, M. E., AND ELDEFRAWI, A. T. (1973) Biochem. Pharmacol. 22, 31453150. 4. O’BRIEN, R. D., AND GIBSON, R. E. (1974) Arch. Biochem. Biophys. 165, 681-690. 5. O’BRIEN, R. D., GILMOUR, L. P., AND ELDEFRAWI, M. E. (1970) Proc. Nat. Acad. Sci. USA 65, 438-445. 6. WEBER, M., AND CHANGEUX, J.-P. (1974) Mol. Pharmacol. 10, 15-24. 7. MARTINEZ-CARRION, M., AND RAFTERY, M. A. (1973) Biochem. Biophys. Res. Commun. 55, 1156-1164. 8. MOODY, T., SCHMIDT, J., AND RAFTERY, M. A. (1973) Biochem. Biophys. Res. Commun. 53, 761-772. 9. COHEN, J. B., WEBER, M., AND CHANGEUX, J.-P. (1974) Mol. Pharmacol. 10, 904-932. 10. MILEDI, R., MOLISOFF, P., AND POTTER, L. T. (1971) Nature (London) 229, 554-557. 11. ELDEFRAWI, M. E., AND ELDEFRAWI, A. T. (1973) Arch. Biochem. Biophys. 159, 362-373. 12. GIBSON, R. E., in preparation. 13. ELDEFRAU’I, M. E., ELDEFRAWI, A. T., PENFIELD, L. A., O’BRIEN, R. D., AND VAN CAMPEN, D. (1975) Life Sci. 16, 925-936. 14. O’BRIEN, R. D., THOMPSON, W. R., ANDGIBSON, R. E. (1974) in Neurochemistry of Cholinergic Receptors (De Robertis, E.. and Schacht, J., eds.), Raven Press, New York. 15. ELDEFRAWI, M. E., ELDEFRA~I, A. T., AND WILSON, D. (1975) Biochemistry (submitted). 16. EDELSTEIN. S. J., BEYER, W. B., ELDEFRAWI, A. T., AND ELDEFRAWI, M. E. (1975) J. Biol. Chem., in press.