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Inhibition of membrane-bound acetylcholinesterase by d-tubocurarine and its reversal by bivalent cations
Life Sciences Vol . 9, Part I, pp . 259-287, 1970 . Printed in Great Britain
Pergamon Press
INHIBITION OF MEMHRANE-BOUND ACETYLCHOLINBSTERAS$ - BY d-TUHOCURARIME AND ITS REYßRSAL BY HIYALENT CATIONS
P . Wina, ß . Schoffenlela and J .-M . Foidart Department of Biochemistry, University of Liège, Helgiuo
(In final form SO November 1969)
THS affecta of biaquaternary nitrogendarivativea on the activity of acetylcholineateraae (AChase) * ere of special interest since their inhibitory action cannot be fully interpreted on the basis of simple competitive kinetics . It has already been pointed out by Wilson (1) that certain effects of compounds of the type (CH3) 3N+(CH2 )nN* (CH3 ) 3 suggest the presence of negative charges distinct from the anionic catalytic Bite . Such an interpretation was strongly supported by the observation (2) that d-tubocurarine and flaxedil antagonize the inhibition of decamethonium . Further studies by Changeux and associates (3) led to the conception that AChase is an ellosteric enzyme which, in addition to the active site, possesses n number of noncatalytic anionic centers . The latter also appear to bind ACh and its analogues . Binding at these sites has a regulatory effect on the enzyme activity . The results presented in this paper show that, especially in the presence of curare, bivalent cationa exert a marked activating effect on the activity of the membrane-bound AChase, obtained from electric organs of Torpedo . Our data also indicate the presence of noncatalytic anionic sites, distinct from the active site of the enzyme . : Acetylcholine hydrolaxe, E .C . 3 .1 .1 .7 .
259
880
INSIHITION OF AChase
Yol. 9, No . 5
Methods Frozen electric organs Eros Torpedo marmosiata L . and Torpedo nobiliaaa Honaparte were used as eazyne source . A piece of organ is minced xith aclasora during thaxing . To the resulting paste xere added 4 voluaes of 0 .3 M sucrose, pH ~ .0 . The suspension xaa hasogenized is a Potter-Slvehjen homogenizer, xith a loose-fitting pestle, sad centrifuged . The particulate fraction aedisentiag betxeea 1 .500 sad 12 .000 g xaa resuspeaded in 0 .3 M sucrose and used as eazyse preparation" AChase is also present in the other f~actioas . Routine enzyme assays xere performed by estimating ACh (4) after 15 sin . incubation at 37' is a medium containing ACh 6 mM ; isidazole-maleate buffer, pH 7 .0, 25 sM ; sad enough eazyse to split about one third of the ACh during the incubation . As a control, xe also used the pH-stat assay sethodY , essentially as described by Earlin (5), ezcept that the medium did not contain NaCl at the beginning oP the reaction . Re salt a Fig . 1 ahoxs the effects of d-tubocurariae chloride on the esterase activity is the absence and in the presence of MgC1 2 10 s1t .Srrea in the absence of curare, Mg ++ activates the enzyme by about 60-80 per cent . This fact has been observed for sany years, at least xhsa nonpurified AChase preparations xere used (6) . The present results ahox that, in the presence of Mg ++ , the inhibitory -3 M effect of curare is greatly reduced . Ia the presence of 10 curare, the inhibition is nearly cosplete in the abaeace of Mg ++ but is less than 30 per coat in the presence of this ion . = Radioseter TTT 1 pH-stet device
Vol. 9, No . 5
IIdHIHITION OF ACi~ase
281
~o e++
a ~ rlY+i,O ++ O dp
~o
~~x+~b lox o a tip, ôo rl ~mo N
N
Ir
M~~iôti h O +~ F
NÔ~~ " O v o O ,é, bOp
ô
ôg " rl
~x,q" a++ N
`~ a~o~oto +q+^" +~+~q~
WflIwIY EIJ~jDQI1~U1 {p
1
~411
J>I~ ~DJ
pA
°
b~ .17~~~ w 0 +~1 ~ Ô ,q Ov ~~~ b O O No o~+bx ~ âeci ~x ~~
r o ~
~ôo
ôm
rl i~
ô °
ô v
N
I O~ b b m w o 0
Oe q 10 0 4 p
~s a 0 w ob
m~
282
INHIBITION OF AChase
Vol . 9, No . 5
The effects of Ca++ , Sr ++ and Min++ ions xere found to be Mg++ . emery siailar to those of In the ezperi4eat reported in Fig . S, xe have a continuous recording of the easymatic hydrolysis of ~Ch using the pH-stat technique . It can be seen that the reaction is blocked by the addition of curare
~ .$ z 10- ~ M (the ACh con-
centration being 2 z 10 - ~) but a reactivation of the eaara is observed upon addition of CaCl a 20 s~M, even though the inhibition induced by curare was cosplete . Obviously, it is difficult, if not impossible, to interpret the present findings only on the basis of the model outlined by Nachmanaoha and Wileon (7) . Indeed, is this model, there is only one type of anionic center on the enzyme molecule and, if Mg ++ or ~++ xere bound to these aiteae they should competitively inhibit the enzyme rather than activate it . At calcium concentrations which were stimulatory, we found no evidence for competition with ACh .
It is thus most likely that bivalent cationa are bound to
noacatalytic anionic centers . It is of interest to know xhather d-tubocurarine binds to the same sites as the bivalent cationa . The data of Fig . 1 seem to support this interpretation since the apparent inhibition constant . for curare (al ) considerably increases in the presence of Ca ++ Now, if we plot the enzyme activity as a function of Ca+ concen-
tration (Fig . 3), we nee that the amount of Ca ++ needed for halfsazinal activation (apparent iCM) increases in the presence of curare, but only by a small factor . If Ca ++ and curare bind to the sane sites, this would suggest that the affinity of these sites for Ca ++ is considerably higher than for curare . On the other hand, the shape of the activation curves is not hyperbolic, suggesting that Ca ++ actually binds to several categories of sites .
Vol. 9, No . 5
283
INÜIHITION OF AC~ase
e w
~ro
e e e e O ++ aAa e o. N
b:
,C b rl
in
wv O O e Ô~ w O +~ql~ u ee w ô~ ws.o 0o w a
P~I~~PR4 4~ Y ~ »d
'o u E eo ~ w ~+ a ~ x a w ~
a e~ Fi tr.
,C ..i ~+
0~ Ô 1 ~ e N
a a V m U i GI O e O w~ w m ~ .+ r, .+ u p, i v v W e ~. L W !el ++ 0
284
Vol. 9, No . 5
INHIBITION OF AChaee
40
to-
o
tar'
:xtQ`
M decamsttwnium
FzsvxE 5
Effects of decaetethonius on the AChaae activity lathe presence (o--~) and abaeace (e---+) of CaCla 20 sM . 1~loreover, ire have seen that Ca++ activates even is the abaeace of curare ; it is thus difficult to interpret the effects of calcius only is tsrtaa of sidpla competitive kinetics . Finally, the effects of curare have been compared with those
of tKO other agents, p-chlorostercuribenzoate (FCMH) and decataethoniuta . As shown in Fig . 4, PCMB at high concentrations tends to block the esterase activity and this inhibition is relieved by ~++ ions . 111e action of pCMB is thus similar to that of curare, but now it can be seen that the apparent affinity for Ca++ ie increased rather than decreased is the presence of the inhibitor .
Vol . 9, No. 5
INHIBITION OF AChaee
285
Fig . $ shown that inhibitory effect of decavetha~nium, a bisquaternary competitive inhibitor, ie not reversed in the presence of of Ca ++ 1ona .The inhibitory effect is even store marked in the pre pence of calcium . Thin is is contrast with the effects observed with curare and PCMB . Dincueaion The significance of the effects of bivalent cationa on AChaee is difficult to appreciate as long as the physiological meaning of the alloateric properties of the enzyme remains uncertain . As pointed out by Changeuz et al .(3), there are striking similarities between the regulatory sites of AChase - or, at least,
some of
them - and the ACh receptor aite(a) involved in electrical activity of the conducting membrane . The possibility was thus considered, that the ACh receptor would actually be a noncatalytic subuait of the AChase (3) . Our results may support this hypothesis since they also show an antagonism between the effects of receptor inhibitors and recepfor activatôra
oa the esterase activity : indeed, as shown above,
bivalent cationa antagonize the effects of curare and PCMB, which are receptor inhibitors (8), but they do not antagonize the inhibit~.on
due to decamethonium, which is a receptor activator . More-
over, if the curare-binding regulatory sites of the AChaae are really involved in receptor activation sad inhibition, we can expect, on the basis of our results that Mg++ and Ca ++ should antagonize the blocking effect of curare on electrical activity . Thin has indeed been demonstrated by Jenkinaon (9) is motor end-platen and confirmed by Dettbarn (10) in lobster axons .
288
INFIIHITION OF AChase
Vol. 9, No . 5
There is n fact, however, that might shake the above hypothesis - the concentration of curare and PCMB used in our experimanta are considerably higher than those required to block the electrical activity of intact poateynaptic membranes . It should be e~phaaized, however, that our experiments are performed in the presence of very high aoouata of ACh . Anyway ; even though no definite conclusion can be drawn at present, a more detailed study of the effects of cetiona on AChase activity will most probably be a useful approach to elucidate the biological function of the regulatory sites of the enzyme . Acknowledpementa P .W . is Chargé de Recherches du Fonda National de la Recherche Scientifique . Thin work wan aided by a grant from the Fonde de la Recherche Scientifique Fondamentale Collective to E .S . Our thanks are due to Dr . W . Van den Hergh, Director of the "Société Royale de Zoologie d'Anvers", for providing the Torpedo . Referencéa 1 . I .B . WILSON in "The Ehzymea", editAd by P .D . H07ER, H . LARDY and ä . MYRBACa, Vol . 4, p .501-519, Acadeoic Press, New York (1960) . 2 . J .-P . CHANGEUä, Mol . Pharatacol . ~, 369 (1966) . 3 . J .-P . CHANGEUR, T . PODLESaI sad J .-C . MEUNIER, J .Gea . Physiol . ~4, 225 S (1969) " 4 .s . HESTRIN, J . Biol .Chem .~8~, 249 (1949) " 5- A . (CARLIN, Biochim . Biophya .Acta ~~~, 359 (1967) " 6 . D . NACNMANSOf1N and I .B . WILSON, Methods ~aymol .,
,642 (1955) "
7 . D . NACIiMANSOEIN and I .B . WILSON, Advances in Bn~ymol ., ~~, 259 (1951) .
INHIBITION OF AChase
Vol . 9, No . 5
8 . A . ICARLIN
and S .
HARTSLS,
Biochic .
Hiophya .Acta
287
~26, $25
(1966) . 9.
D .H .
JSNgIN30N,
10 . fi .D . DSTTHARN,
J.
Phyeiol .(Loadoa )1~2, 309 (19~) " s
Life Sci ., ~, 910 (1963) .
Pergamon Press
INHIBITION OF MEMHRANE-BOUND ACETYLCHOLINBSTERAS$ - BY d-TUHOCURARIME AND ITS REYßRSAL BY HIYALENT CATIONS
P . Wina, ß . Schoffenlela and J .-M . Foidart Department of Biochemistry, University of Liège, Helgiuo
(In final form SO November 1969)
THS affecta of biaquaternary nitrogendarivativea on the activity of acetylcholineateraae (AChase) * ere of special interest since their inhibitory action cannot be fully interpreted on the basis of simple competitive kinetics . It has already been pointed out by Wilson (1) that certain effects of compounds of the type (CH3) 3N+(CH2 )nN* (CH3 ) 3 suggest the presence of negative charges distinct from the anionic catalytic Bite . Such an interpretation was strongly supported by the observation (2) that d-tubocurarine and flaxedil antagonize the inhibition of decamethonium . Further studies by Changeux and associates (3) led to the conception that AChase is an ellosteric enzyme which, in addition to the active site, possesses n number of noncatalytic anionic centers . The latter also appear to bind ACh and its analogues . Binding at these sites has a regulatory effect on the enzyme activity . The results presented in this paper show that, especially in the presence of curare, bivalent cationa exert a marked activating effect on the activity of the membrane-bound AChase, obtained from electric organs of Torpedo . Our data also indicate the presence of noncatalytic anionic sites, distinct from the active site of the enzyme . : Acetylcholine hydrolaxe, E .C . 3 .1 .1 .7 .
259
880
INSIHITION OF AChase
Yol. 9, No . 5
Methods Frozen electric organs Eros Torpedo marmosiata L . and Torpedo nobiliaaa Honaparte were used as eazyne source . A piece of organ is minced xith aclasora during thaxing . To the resulting paste xere added 4 voluaes of 0 .3 M sucrose, pH ~ .0 . The suspension xaa hasogenized is a Potter-Slvehjen homogenizer, xith a loose-fitting pestle, sad centrifuged . The particulate fraction aedisentiag betxeea 1 .500 sad 12 .000 g xaa resuspeaded in 0 .3 M sucrose and used as eazyse preparation" AChase is also present in the other f~actioas . Routine enzyme assays xere performed by estimating ACh (4) after 15 sin . incubation at 37' is a medium containing ACh 6 mM ; isidazole-maleate buffer, pH 7 .0, 25 sM ; sad enough eazyse to split about one third of the ACh during the incubation . As a control, xe also used the pH-stat assay sethodY , essentially as described by Earlin (5), ezcept that the medium did not contain NaCl at the beginning oP the reaction . Re salt a Fig . 1 ahoxs the effects of d-tubocurariae chloride on the esterase activity is the absence and in the presence of MgC1 2 10 s1t .Srrea in the absence of curare, Mg ++ activates the enzyme by about 60-80 per cent . This fact has been observed for sany years, at least xhsa nonpurified AChase preparations xere used (6) . The present results ahox that, in the presence of Mg ++ , the inhibitory -3 M effect of curare is greatly reduced . Ia the presence of 10 curare, the inhibition is nearly cosplete in the abaeace of Mg ++ but is less than 30 per coat in the presence of this ion . = Radioseter TTT 1 pH-stet device
Vol. 9, No . 5
IIdHIHITION OF ACi~ase
281
~o e++
a ~ rlY+i,O ++ O dp
~o
~~x+~b lox o a tip, ôo rl ~mo N
N
Ir
M~~iôti h O +~ F
NÔ~~ " O v o O ,é, bOp
ô
ôg " rl
~x,q" a++ N
`~ a~o~oto +q+^" +~+~q~
WflIwIY EIJ~jDQI1~U1 {p
1
~411
J>I~ ~DJ
pA
°
b~ .17~~~ w 0 +~1 ~ Ô ,q Ov ~~~ b O O No o~+bx ~ âeci ~x ~~
r o ~
~ôo
ôm
rl i~
ô °
ô v
N
I O~ b b m w o 0
Oe q 10 0 4 p
~s a 0 w ob
m~
282
INHIBITION OF AChase
Vol . 9, No . 5
The effects of Ca++ , Sr ++ and Min++ ions xere found to be Mg++ . emery siailar to those of In the ezperi4eat reported in Fig . S, xe have a continuous recording of the easymatic hydrolysis of ~Ch using the pH-stat technique . It can be seen that the reaction is blocked by the addition of curare
~ .$ z 10- ~ M (the ACh con-
centration being 2 z 10 - ~) but a reactivation of the eaara is observed upon addition of CaCl a 20 s~M, even though the inhibition induced by curare was cosplete . Obviously, it is difficult, if not impossible, to interpret the present findings only on the basis of the model outlined by Nachmanaoha and Wileon (7) . Indeed, is this model, there is only one type of anionic center on the enzyme molecule and, if Mg ++ or ~++ xere bound to these aiteae they should competitively inhibit the enzyme rather than activate it . At calcium concentrations which were stimulatory, we found no evidence for competition with ACh .
It is thus most likely that bivalent cationa are bound to
noacatalytic anionic centers . It is of interest to know xhather d-tubocurarine binds to the same sites as the bivalent cationa . The data of Fig . 1 seem to support this interpretation since the apparent inhibition constant . for curare (al ) considerably increases in the presence of Ca ++ Now, if we plot the enzyme activity as a function of Ca+ concen-
tration (Fig . 3), we nee that the amount of Ca ++ needed for halfsazinal activation (apparent iCM) increases in the presence of curare, but only by a small factor . If Ca ++ and curare bind to the sane sites, this would suggest that the affinity of these sites for Ca ++ is considerably higher than for curare . On the other hand, the shape of the activation curves is not hyperbolic, suggesting that Ca ++ actually binds to several categories of sites .
Vol. 9, No . 5
283
INÜIHITION OF AC~ase
e w
~ro
e e e e O ++ aAa e o. N
b:
,C b rl
in
wv O O e Ô~ w O +~ql~ u ee w ô~ ws.o 0o w a
P~I~~PR4 4~ Y ~ »d
'o u E eo ~ w ~+ a ~ x a w ~
a e~ Fi tr.
,C ..i ~+
0~ Ô 1 ~ e N
a a V m U i GI O e O w~ w m ~ .+ r, .+ u p, i v v W e ~. L W !el ++ 0
284
Vol. 9, No . 5
INHIBITION OF AChaee
40
to-
o
tar'
:xtQ`
M decamsttwnium
FzsvxE 5
Effects of decaetethonius on the AChaae activity lathe presence (o--~) and abaeace (e---+) of CaCla 20 sM . 1~loreover, ire have seen that Ca++ activates even is the abaeace of curare ; it is thus difficult to interpret the effects of calcius only is tsrtaa of sidpla competitive kinetics . Finally, the effects of curare have been compared with those
of tKO other agents, p-chlorostercuribenzoate (FCMH) and decataethoniuta . As shown in Fig . 4, PCMB at high concentrations tends to block the esterase activity and this inhibition is relieved by ~++ ions . 111e action of pCMB is thus similar to that of curare, but now it can be seen that the apparent affinity for Ca++ ie increased rather than decreased is the presence of the inhibitor .
Vol . 9, No. 5
INHIBITION OF AChaee
285
Fig . $ shown that inhibitory effect of decavetha~nium, a bisquaternary competitive inhibitor, ie not reversed in the presence of of Ca ++ 1ona .The inhibitory effect is even store marked in the pre pence of calcium . Thin is is contrast with the effects observed with curare and PCMB . Dincueaion The significance of the effects of bivalent cationa on AChaee is difficult to appreciate as long as the physiological meaning of the alloateric properties of the enzyme remains uncertain . As pointed out by Changeuz et al .(3), there are striking similarities between the regulatory sites of AChase - or, at least,
some of
them - and the ACh receptor aite(a) involved in electrical activity of the conducting membrane . The possibility was thus considered, that the ACh receptor would actually be a noncatalytic subuait of the AChase (3) . Our results may support this hypothesis since they also show an antagonism between the effects of receptor inhibitors and recepfor activatôra
oa the esterase activity : indeed, as shown above,
bivalent cationa antagonize the effects of curare and PCMB, which are receptor inhibitors (8), but they do not antagonize the inhibit~.on
due to decamethonium, which is a receptor activator . More-
over, if the curare-binding regulatory sites of the AChaae are really involved in receptor activation sad inhibition, we can expect, on the basis of our results that Mg++ and Ca ++ should antagonize the blocking effect of curare on electrical activity . Thin has indeed been demonstrated by Jenkinaon (9) is motor end-platen and confirmed by Dettbarn (10) in lobster axons .
288
INFIIHITION OF AChase
Vol. 9, No . 5
There is n fact, however, that might shake the above hypothesis - the concentration of curare and PCMB used in our experimanta are considerably higher than those required to block the electrical activity of intact poateynaptic membranes . It should be e~phaaized, however, that our experiments are performed in the presence of very high aoouata of ACh . Anyway ; even though no definite conclusion can be drawn at present, a more detailed study of the effects of cetiona on AChase activity will most probably be a useful approach to elucidate the biological function of the regulatory sites of the enzyme . Acknowledpementa P .W . is Chargé de Recherches du Fonda National de la Recherche Scientifique . Thin work wan aided by a grant from the Fonde de la Recherche Scientifique Fondamentale Collective to E .S . Our thanks are due to Dr . W . Van den Hergh, Director of the "Société Royale de Zoologie d'Anvers", for providing the Torpedo . Referencéa 1 . I .B . WILSON in "The Ehzymea", editAd by P .D . H07ER, H . LARDY and ä . MYRBACa, Vol . 4, p .501-519, Acadeoic Press, New York (1960) . 2 . J .-P . CHANGEUä, Mol . Pharatacol . ~, 369 (1966) . 3 . J .-P . CHANGEUR, T . PODLESaI sad J .-C . MEUNIER, J .Gea . Physiol . ~4, 225 S (1969) " 4 .s . HESTRIN, J . Biol .Chem .~8~, 249 (1949) " 5- A . (CARLIN, Biochim . Biophya .Acta ~~~, 359 (1967) " 6 . D . NACNMANSOf1N and I .B . WILSON, Methods ~aymol .,
,642 (1955) "
7 . D . NACIiMANSOEIN and I .B . WILSON, Advances in Bn~ymol ., ~~, 259 (1951) .
INHIBITION OF AChase
Vol . 9, No . 5
8 . A . ICARLIN
and S .
HARTSLS,
Biochic .
Hiophya .Acta
287
~26, $25
(1966) . 9.
D .H .
JSNgIN30N,
10 . fi .D . DSTTHARN,
J.
Phyeiol .(Loadoa )1~2, 309 (19~) " s
Life Sci ., ~, 910 (1963) .