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Interaction of imidazolium and pyridinium dioximes with human erythrocyte acetylcholinesterase
Chem.-Biol. Interactions, 87 (1993) 323-328
323
Elsevier Scientific Publishers Ireland Ltd.
INTERACTION OF IMIDAZOLIUM AND PYRIDINIUM DIOXIMES WITH HUMAN ERYTHROCYTE ACETYLCHOLINESTERASE
LENKA FRANCISKOVI(~ a, MIRA SKRINJARI(~ SPOLJAR b and ELSA REINER b
aFaculty of Agriculture, University of Osijek, Osijek and blnstitute for Medical Research and Occupational Health, University of Zagreb, Zagreb (Croatia)
SUMMARY
Two pyridinium and two imidazolium dioximes were tested as reversible inhibitors of human erythrocyte acetylcholinesterase (ACHE), as protectors of the enzyme against phosphylation and as reactivators of the phosphylated ACHE. All four dioximes reversibly inhibited ACHE, protected the enzyme against phosphylation by soman and tabun and reactivated AChE after phosphylation by sarin, VX and tabun. From the experimental results the enzyme/dioxime dissociation constants were evaluated for the catalytically active enzyme and for phosphylated enzyme. The evaluation constants have shown that all four dioximes have about the same affinity for the catallytically active as for the phosphylated ACHE. Obtained results also indicate that imidazolium dioximes probably bind only to the allosteric, while pyridinium dioximes bind to both, the catalytic and the allosteric site of the enzyme.
Key words: Acetylcholinesterase -- Reversible inhibition -
Binding-site specificity -- Protection against organophosphates -- Reactivation -- Oximes -Organophosphorus compounds -- Sarin - Soman -- Tabun -- VX
Four dioximes (Table I) were tested as reversible inhibitors of acetylcholinesterase (ACHE; EC 3.1.1.7), as protectors of AChE against phosphylation by organophosphorus compounds (OP) and as reactivators of the phosphylated ACHE. MATERIALS AND METHODS
The enzyme source were native human erythrocytes. The oximes were kindly prepared by Drs. Vjera Deljac and Zlatko Binenfeld (Laboratory of Organic ChemCorrespondence to: M. Skrinjari6 Spoljar, Institute for Medical Research and Occupational Health, University of Zagreb, P.O. Box 291, 41001 Zagreb, Croatia. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
324 TABLE I ABBREVIATIONS A N D STRUCTURAL F O R M U L A E OF OXIMES. BDB-106 AND BDB-108a WERE CHLORIDES; BDB-110 A N D BDB-108b WERE BROMIDES Imidazolium dioximes: BDB-106 (trans) Y: C H ~ C H
I
I
H3C-N+
i
CH 2 I
CH=CH-
N
BDB-108a (cis) BDB-108b (trans)
CH=NOH CH 2 I
lq
0
Pyridinium dioximes:
N-CH 3 I
CH=NOH
BDB-110 Y: C H 2 - - C H 2
]
N-CH2-Y-CH2... N+
G
I CH=NOH
I CH=NOH
istry, Faculty of Science and Mathematics, University of Zagreb) according to published procedures [1-3]. The enzyme activities were measured in 0.1 M phosphate buffer pH 7.6 at 37°C with acetylthiocholine as substrate, using the spectrophotometric method of Ellman et al. [4]. Reversible inhibition was measured in a reaction medium (3.0 ml) which contained the erythrocytes suspended in buffer, the thiol reagent DTNB (final concentration 0.33 mM), the oxime and the substrate. The increase in absorbance (at 412 nm) was followed up to 2 rain. Control samples contained no oxime. The effect of oximes upon phosphylation of AChE was measured in a reaction medium (3.0 ml) which contained erythrocytes in buffer, DTNB, oxime and OP solution. After a given time of inhibition (up to 3 rain) the substrate was added, and the activity measured as above. Control samples contained no oxime. Reactivation of the phosphylated AChE was measured in the following way: undiluted erythrocytes were incubated for 10 min with the OP compound. The OP concentration was chosen to inhibit about 80% of the enzyme activity. The erythrocytes were then diluted 400-fold with buffer containing the oxime. At suitable time intervals aliquots (3.0 ml) were withdrawn, DTNB and substrate added, and the enzyme activity was measured as above. RESULTS AND DISCUSSION
All four dioximes were reversible inhibitors of AChE. The apparent enzyme/oxime dissociation c o n s t a n t s Kapp were calculated from: Kapp = vi •
i/(Vo
-
vi) = K i
• [1 +
s/K(S)]
(1)
325
Vo and vi are the enzyme activities in absence and presence of the oxime, i and s are the oxime and substrate concentrations respectively. Ki is the enzyme/ oxime dissociation constant in absence of the substrate. At each substrate concentration, 3 - 1 0 different oxime concentrations were tested giving between 20% and 80% of inhibition. The substrate concentrations were 0.1 mM and 1.0 mM. Higher substrate concentrations could not be used because all four oximes reacted with acetylthiocholine [5] and this reaction interfered with the enzyme assay. Equation 1 applies to reversible inhibitors which bind only to one site in AChE [6- 8]. If the inhibitor binds to the catalytic site only, K(S) should correspond to the Km of the substrate. If the inhibitor binds to the allosteric site only, K(S) should correspond to the substrate inhibition constant Kss. Under the given experimental conditions, Km and Kss for acetylthiocholine are 0.054 mM and 14 mM respectively [9]. This means that for an increase in substrate concentration from 0.1 mM to 1.0 mM, the Kapp should increase 6.8-fold (if the inhibitor binds only to the catalytic site) or 1.1-fold (if the inhibitor binds only to the allosteric site). For the inhibition with BDB-106 and BDB-110 the Kapp w a s the same at 0.1 mM and 1.0 mM substrate. The obtained Kap p w a s therefore taken to represent the enzyme/oxime dissociation constant for the allosteric site of the enzyme (Table II). For inhibition with BDB-108a and BDB-108b the Kapp increased twofold (when s was increased from 0.1 mM to 1.0 raM) which indicated that these oximes probably bind to both sites on the enzyme [6], as it was shown earlier for some other bispyridinium compounds [10-13]. An extrapolation of the Kap p to zero substrate concentration was taken to represent the inhibitory potency of BDB-108a and BDB-108b for AChE (Table II).
TABLE II ENZYME/OXIME DISSOCIATION CONSTANTS (Ki) OBTAINED FROM REVERSIBLE INHIBITION AND FROM THE EFFECT OF DIOXIMES UPON PHOSPHYLATION OF AChE BY THE INDICATED ORGANOPHOSPHORUS COMPOUNDS (PROTECTION) Oxime
KilgM Reversible inhibition
(n)
Protection
BDB-106
24
(16)
BDB-110
11
(17)
BDB-108a
52
(9)
BDB-108b
45
(7)
Soman Tabun Soman Tabun Soman Tabun Soman Tabun
(n) Is the number of experiments.
(n)
23 21 15 9.3 45 50 42 44
(3) (3) {4) (4) (3) (3) (3) (3)
326
The effect of the oximes upon phosphylation of AChE by soman and tabun was tested using oxime concentrations which corresponded to their Ki constants. The following equation was used to interpret the results: kJk'~ = 1 + i/Ki
(2)
ka and k'a are the second-order rate constants of phosphylation in absence and presence of the oxime. The calculated enzyme/oxime dissociation constants Ki obtained from these experiments (Table II) were the same as the Ki values obtained from reversible inhibition. This indicates that both reactions follow the same mechanism, and it also indicates that protection of the enzyme by BDB-106 and BDB-110 is achieved through the allosteric binding only. All four oximes reactivated AChE phosphylated by sarin, VX or tabun. The rate constants were calculated from: in (epo/ept)
= k2
• i
• t/(Kr
+ i)
(3)
or
In (epo/ept)= kr • i • t
(4)
epo and ept stand for the phosphylated enzyme at time zero and time t respectively, and i is the oxime concentration, k2 and kr are the first-order and the
TABLE III REACTIVATION OF ACHE PHOSPHYLATED BY THE INDICATED ORGANOPHOSPHORUS (OP) C O M P O U N D S . K r I S T H E P H O S P H Y L A T E D - A C h E / O X I M E DISSOCIATION CONSTANT
Oxime
OP
Kr tLM
k2 min - 1
BDB-106
VX Sarin Tabun VX Sarin Tabun VX Sarin Tabun VX Sarin Tabun
-56 -7.3 21 3.8 --11 13 13 --
-0.460 -0.060 0.106 0.016 --0.049 0.593 0.593 --
BDB-110
BDB-108a
BDB-108b
k × 10 .3 tool - 11 rain - 1
5.6 8.3 3.6 8.2 5.0 4.1 21 21 4.4 45 35 7.7
(n)
(6) (5) (3) (4) (4) (3) (5) (4) (3) (4) (4) (3)
k 2 a n d k r a r e t h e f i r s t - o r d e r a n d t h e s e c o n d - o r d e r r a t e c o n s t a n t s of r e a c t i v i t y , (n) is the n u m b e r of
experiments
327
second-order rate constants of reactivation. Kr is the phosphylated-AChE/oxime dissociation constant. In equation 4 the kr constant corresponds to the ratio kz / Kr. For all studied reactions the time course of reactivation deviated from the above equations after a certain reactivation time (depending on the oxime and OP compound). The rate constants were calculated from the initial slopes of the reactivation curves. Comparing the k r constants (Table III) it follows that the pyridinium dioximes are better reactivators than the imidazolium dioximes. Further, the VX- and sarin-phosphylated enzyme can be more easily reactivated than the tabun-phosphylated enzyme. The best reactivator was BDB-108b as it was also shown by Kova~evi~ et al. [14]. For some of the studied reactions it was possible to evaluate separately the K r and k 2 constants (equation 3, Table III). The Kr constant represents the affinity of a compound for the phosphylated enzyme, while the Ki constant represents the affinity of a compound for the free, non-phosphylated, enzyme. A comparison of these two constants (Tables II and III) reveals that for a given dioxime these constants are either about the same or differ up to five-fold. It seems therefore that the four dioximes have affinities of the same order of magnitude for the phosphylated AChE as for the catalytically active enzyme. The same holds for several other types of reversible inhibitors of AChE such as propidium and coumarin [15,16]. REFERENCES 1 J. Pato~ka, J. Bielavsky and F. Ornst, Reactivating effect of c~,~-bis-(4-pyridinealdoxime)-2-transbutene dibromide on isopropyl-methylphosphonylated acetylcholinesterase, FEBS Letts., 10 (1970) 182-184. 2 V. Deljae, V. Kovarevir, M. Maksimovi~ and Z. Binenfeld, Alkene bis-pyridinium and bisimidazolium oximes as reactivators of AChE inhibited with Tabun, Sarin and VX, International Meeting on Esterases Hydrolyzing Organophosphorus Compounds, Dubrovnik, Croatia. Abstracts p20, 1988. 3 C.D. Bedford, R.N. Harris, R.A. Howd, D.A. Goff, G.A. Koolpe, M. Petesch, A. Miller, H.W. Nolen, H.A. Musallam, R.O. Pick, D.E. Jones and J. Koplovitz, Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 2. Preparation and in vitro and in vivo evaluation of 1-(alkoxymethyl)2-[(hydroxyimino)methyl]-3-methylimidazolium halides for reactivation of organophosphorusinhibited acetylcholinesterases, J. Med. Chem., 32 (1989) 493- 503. 4 G.L. Ellman, K.D. Courtney, V. Andres, Jr. and V.M. Featherstone~ A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 5 L. Francis'kovi6, Interaction of Pyridinium and Imidazolium dioximes with Native and Phosphylated Acetylcholinesteras¢, M. Sc. Thesis (in Croatian), Faculty of Science and Mathematics, University of Zagreb, Croatia, 1990. 6 W.N. Aldridge and E. Reiner, Enzyme Inhibitors as Substrates. Interaction of Esterases with Esters of Organophosphorus and Carbamic Acids, XV+328 pp., North Holland Publishing Co., Amsterdam, 1972. 7 E. Reiner and V. Simeon, Kinetic study of the effect of substrates on reversible inhibition of cholinesterase and acetylcholinesterase by two coumarin derivatives, Croat. Chem. Acta. 47 (1975) 321-331. 8 Z. Radi~, E. Reiner and V. Simeon, Binding sites on acetylcholinesterase for reversible ligands and phosphorylating agents, Biochem. Pharmacol., 33 (1984)671-667.
328 9 10 11 12
13
14
15 16
Z. Radi6 and E. Reiner, Effect of atropine on esterases from human blood and pig liver, Acta Pharm, Jugosl., 36 (1986) 1-7. V. Simeon, Z. Radi6 and E. Reiner, Inhibition of cholinesterases by the oximes P2AM and Toxogonin, Croat. Chem. Acta, 54 (1981) 473-480. E. Reiner, Inhibition of acetylcholinesterase by 4,4'-bipyridiae and its effect upon phosphylation of the enzyme, Croat. Chem. Acta, 59 (1986) 925-931. M. Skrinjaric Spoljar, V. Simeon, E. Reiner and B. Krauthacker, Bispyridinium compounds: inhibition of human erythrocyte acetylcholinesterase and protection of the enzyme against phosphylation, Acta Pharm. Jugosl., 38 (1988) 101-109. M. Skrinjari6, Spoljar, E. Reiner, V. Simeon and B. Krauthacker, 1,3-(Hydroxyiminopyridiniumcyclohexyl-carbonylpyridinium)-bismethylene oxide position isomers: inhibition of bovine erythrocyte acetylcholinesterase and reactivation of the phosphylated enzyme, Acta Pharm. Jugosl., 38 (1988) 111-117. V. Kova~evi6, M. Maksimovi6, V. Deljac and Z. Binenfeld, The efficacy of 1,1 '-(l,4-buten)-bis- (4hydroxyiminomethylpyridinium) dibromide in the treatment of organophosphate poisoning, Acta Pharm. Jugosl., 39 (1989) 167-170. P. Taylor and S. Lappi, Interaction of fluorescent probes with acetylcholinesterase: the site and specificity of propidium binding, Biochemistry, 14 (1975) 1989-1997. Z. Radi6, E. Reiner and P. Taylor, Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives, Mol. Pharmacol., 39 (1991) 98-104.
323
Elsevier Scientific Publishers Ireland Ltd.
INTERACTION OF IMIDAZOLIUM AND PYRIDINIUM DIOXIMES WITH HUMAN ERYTHROCYTE ACETYLCHOLINESTERASE
LENKA FRANCISKOVI(~ a, MIRA SKRINJARI(~ SPOLJAR b and ELSA REINER b
aFaculty of Agriculture, University of Osijek, Osijek and blnstitute for Medical Research and Occupational Health, University of Zagreb, Zagreb (Croatia)
SUMMARY
Two pyridinium and two imidazolium dioximes were tested as reversible inhibitors of human erythrocyte acetylcholinesterase (ACHE), as protectors of the enzyme against phosphylation and as reactivators of the phosphylated ACHE. All four dioximes reversibly inhibited ACHE, protected the enzyme against phosphylation by soman and tabun and reactivated AChE after phosphylation by sarin, VX and tabun. From the experimental results the enzyme/dioxime dissociation constants were evaluated for the catalytically active enzyme and for phosphylated enzyme. The evaluation constants have shown that all four dioximes have about the same affinity for the catallytically active as for the phosphylated ACHE. Obtained results also indicate that imidazolium dioximes probably bind only to the allosteric, while pyridinium dioximes bind to both, the catalytic and the allosteric site of the enzyme.
Key words: Acetylcholinesterase -- Reversible inhibition -
Binding-site specificity -- Protection against organophosphates -- Reactivation -- Oximes -Organophosphorus compounds -- Sarin - Soman -- Tabun -- VX
Four dioximes (Table I) were tested as reversible inhibitors of acetylcholinesterase (ACHE; EC 3.1.1.7), as protectors of AChE against phosphylation by organophosphorus compounds (OP) and as reactivators of the phosphylated ACHE. MATERIALS AND METHODS
The enzyme source were native human erythrocytes. The oximes were kindly prepared by Drs. Vjera Deljac and Zlatko Binenfeld (Laboratory of Organic ChemCorrespondence to: M. Skrinjari6 Spoljar, Institute for Medical Research and Occupational Health, University of Zagreb, P.O. Box 291, 41001 Zagreb, Croatia. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
324 TABLE I ABBREVIATIONS A N D STRUCTURAL F O R M U L A E OF OXIMES. BDB-106 AND BDB-108a WERE CHLORIDES; BDB-110 A N D BDB-108b WERE BROMIDES Imidazolium dioximes: BDB-106 (trans) Y: C H ~ C H
I
I
H3C-N+
i
CH 2 I
CH=CH-
N
BDB-108a (cis) BDB-108b (trans)
CH=NOH CH 2 I
lq
0
Pyridinium dioximes:
N-CH 3 I
CH=NOH
BDB-110 Y: C H 2 - - C H 2
]
N-CH2-Y-CH2... N+
G
I CH=NOH
I CH=NOH
istry, Faculty of Science and Mathematics, University of Zagreb) according to published procedures [1-3]. The enzyme activities were measured in 0.1 M phosphate buffer pH 7.6 at 37°C with acetylthiocholine as substrate, using the spectrophotometric method of Ellman et al. [4]. Reversible inhibition was measured in a reaction medium (3.0 ml) which contained the erythrocytes suspended in buffer, the thiol reagent DTNB (final concentration 0.33 mM), the oxime and the substrate. The increase in absorbance (at 412 nm) was followed up to 2 rain. Control samples contained no oxime. The effect of oximes upon phosphylation of AChE was measured in a reaction medium (3.0 ml) which contained erythrocytes in buffer, DTNB, oxime and OP solution. After a given time of inhibition (up to 3 rain) the substrate was added, and the activity measured as above. Control samples contained no oxime. Reactivation of the phosphylated AChE was measured in the following way: undiluted erythrocytes were incubated for 10 min with the OP compound. The OP concentration was chosen to inhibit about 80% of the enzyme activity. The erythrocytes were then diluted 400-fold with buffer containing the oxime. At suitable time intervals aliquots (3.0 ml) were withdrawn, DTNB and substrate added, and the enzyme activity was measured as above. RESULTS AND DISCUSSION
All four dioximes were reversible inhibitors of AChE. The apparent enzyme/oxime dissociation c o n s t a n t s Kapp were calculated from: Kapp = vi •
i/(Vo
-
vi) = K i
• [1 +
s/K(S)]
(1)
325
Vo and vi are the enzyme activities in absence and presence of the oxime, i and s are the oxime and substrate concentrations respectively. Ki is the enzyme/ oxime dissociation constant in absence of the substrate. At each substrate concentration, 3 - 1 0 different oxime concentrations were tested giving between 20% and 80% of inhibition. The substrate concentrations were 0.1 mM and 1.0 mM. Higher substrate concentrations could not be used because all four oximes reacted with acetylthiocholine [5] and this reaction interfered with the enzyme assay. Equation 1 applies to reversible inhibitors which bind only to one site in AChE [6- 8]. If the inhibitor binds to the catalytic site only, K(S) should correspond to the Km of the substrate. If the inhibitor binds to the allosteric site only, K(S) should correspond to the substrate inhibition constant Kss. Under the given experimental conditions, Km and Kss for acetylthiocholine are 0.054 mM and 14 mM respectively [9]. This means that for an increase in substrate concentration from 0.1 mM to 1.0 mM, the Kapp should increase 6.8-fold (if the inhibitor binds only to the catalytic site) or 1.1-fold (if the inhibitor binds only to the allosteric site). For the inhibition with BDB-106 and BDB-110 the Kapp w a s the same at 0.1 mM and 1.0 mM substrate. The obtained Kap p w a s therefore taken to represent the enzyme/oxime dissociation constant for the allosteric site of the enzyme (Table II). For inhibition with BDB-108a and BDB-108b the Kapp increased twofold (when s was increased from 0.1 mM to 1.0 raM) which indicated that these oximes probably bind to both sites on the enzyme [6], as it was shown earlier for some other bispyridinium compounds [10-13]. An extrapolation of the Kap p to zero substrate concentration was taken to represent the inhibitory potency of BDB-108a and BDB-108b for AChE (Table II).
TABLE II ENZYME/OXIME DISSOCIATION CONSTANTS (Ki) OBTAINED FROM REVERSIBLE INHIBITION AND FROM THE EFFECT OF DIOXIMES UPON PHOSPHYLATION OF AChE BY THE INDICATED ORGANOPHOSPHORUS COMPOUNDS (PROTECTION) Oxime
KilgM Reversible inhibition
(n)
Protection
BDB-106
24
(16)
BDB-110
11
(17)
BDB-108a
52
(9)
BDB-108b
45
(7)
Soman Tabun Soman Tabun Soman Tabun Soman Tabun
(n) Is the number of experiments.
(n)
23 21 15 9.3 45 50 42 44
(3) (3) {4) (4) (3) (3) (3) (3)
326
The effect of the oximes upon phosphylation of AChE by soman and tabun was tested using oxime concentrations which corresponded to their Ki constants. The following equation was used to interpret the results: kJk'~ = 1 + i/Ki
(2)
ka and k'a are the second-order rate constants of phosphylation in absence and presence of the oxime. The calculated enzyme/oxime dissociation constants Ki obtained from these experiments (Table II) were the same as the Ki values obtained from reversible inhibition. This indicates that both reactions follow the same mechanism, and it also indicates that protection of the enzyme by BDB-106 and BDB-110 is achieved through the allosteric binding only. All four oximes reactivated AChE phosphylated by sarin, VX or tabun. The rate constants were calculated from: in (epo/ept)
= k2
• i
• t/(Kr
+ i)
(3)
or
In (epo/ept)= kr • i • t
(4)
epo and ept stand for the phosphylated enzyme at time zero and time t respectively, and i is the oxime concentration, k2 and kr are the first-order and the
TABLE III REACTIVATION OF ACHE PHOSPHYLATED BY THE INDICATED ORGANOPHOSPHORUS (OP) C O M P O U N D S . K r I S T H E P H O S P H Y L A T E D - A C h E / O X I M E DISSOCIATION CONSTANT
Oxime
OP
Kr tLM
k2 min - 1
BDB-106
VX Sarin Tabun VX Sarin Tabun VX Sarin Tabun VX Sarin Tabun
-56 -7.3 21 3.8 --11 13 13 --
-0.460 -0.060 0.106 0.016 --0.049 0.593 0.593 --
BDB-110
BDB-108a
BDB-108b
k × 10 .3 tool - 11 rain - 1
5.6 8.3 3.6 8.2 5.0 4.1 21 21 4.4 45 35 7.7
(n)
(6) (5) (3) (4) (4) (3) (5) (4) (3) (4) (4) (3)
k 2 a n d k r a r e t h e f i r s t - o r d e r a n d t h e s e c o n d - o r d e r r a t e c o n s t a n t s of r e a c t i v i t y , (n) is the n u m b e r of
experiments
327
second-order rate constants of reactivation. Kr is the phosphylated-AChE/oxime dissociation constant. In equation 4 the kr constant corresponds to the ratio kz / Kr. For all studied reactions the time course of reactivation deviated from the above equations after a certain reactivation time (depending on the oxime and OP compound). The rate constants were calculated from the initial slopes of the reactivation curves. Comparing the k r constants (Table III) it follows that the pyridinium dioximes are better reactivators than the imidazolium dioximes. Further, the VX- and sarin-phosphylated enzyme can be more easily reactivated than the tabun-phosphylated enzyme. The best reactivator was BDB-108b as it was also shown by Kova~evi~ et al. [14]. For some of the studied reactions it was possible to evaluate separately the K r and k 2 constants (equation 3, Table III). The Kr constant represents the affinity of a compound for the phosphylated enzyme, while the Ki constant represents the affinity of a compound for the free, non-phosphylated, enzyme. A comparison of these two constants (Tables II and III) reveals that for a given dioxime these constants are either about the same or differ up to five-fold. It seems therefore that the four dioximes have affinities of the same order of magnitude for the phosphylated AChE as for the catalytically active enzyme. The same holds for several other types of reversible inhibitors of AChE such as propidium and coumarin [15,16]. REFERENCES 1 J. Pato~ka, J. Bielavsky and F. Ornst, Reactivating effect of c~,~-bis-(4-pyridinealdoxime)-2-transbutene dibromide on isopropyl-methylphosphonylated acetylcholinesterase, FEBS Letts., 10 (1970) 182-184. 2 V. Deljae, V. Kovarevir, M. Maksimovi~ and Z. Binenfeld, Alkene bis-pyridinium and bisimidazolium oximes as reactivators of AChE inhibited with Tabun, Sarin and VX, International Meeting on Esterases Hydrolyzing Organophosphorus Compounds, Dubrovnik, Croatia. Abstracts p20, 1988. 3 C.D. Bedford, R.N. Harris, R.A. Howd, D.A. Goff, G.A. Koolpe, M. Petesch, A. Miller, H.W. Nolen, H.A. Musallam, R.O. Pick, D.E. Jones and J. Koplovitz, Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 2. Preparation and in vitro and in vivo evaluation of 1-(alkoxymethyl)2-[(hydroxyimino)methyl]-3-methylimidazolium halides for reactivation of organophosphorusinhibited acetylcholinesterases, J. Med. Chem., 32 (1989) 493- 503. 4 G.L. Ellman, K.D. Courtney, V. Andres, Jr. and V.M. Featherstone~ A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 5 L. Francis'kovi6, Interaction of Pyridinium and Imidazolium dioximes with Native and Phosphylated Acetylcholinesteras¢, M. Sc. Thesis (in Croatian), Faculty of Science and Mathematics, University of Zagreb, Croatia, 1990. 6 W.N. Aldridge and E. Reiner, Enzyme Inhibitors as Substrates. Interaction of Esterases with Esters of Organophosphorus and Carbamic Acids, XV+328 pp., North Holland Publishing Co., Amsterdam, 1972. 7 E. Reiner and V. Simeon, Kinetic study of the effect of substrates on reversible inhibition of cholinesterase and acetylcholinesterase by two coumarin derivatives, Croat. Chem. Acta. 47 (1975) 321-331. 8 Z. Radi~, E. Reiner and V. Simeon, Binding sites on acetylcholinesterase for reversible ligands and phosphorylating agents, Biochem. Pharmacol., 33 (1984)671-667.
328 9 10 11 12
13
14
15 16
Z. Radi6 and E. Reiner, Effect of atropine on esterases from human blood and pig liver, Acta Pharm, Jugosl., 36 (1986) 1-7. V. Simeon, Z. Radi6 and E. Reiner, Inhibition of cholinesterases by the oximes P2AM and Toxogonin, Croat. Chem. Acta, 54 (1981) 473-480. E. Reiner, Inhibition of acetylcholinesterase by 4,4'-bipyridiae and its effect upon phosphylation of the enzyme, Croat. Chem. Acta, 59 (1986) 925-931. M. Skrinjaric Spoljar, V. Simeon, E. Reiner and B. Krauthacker, Bispyridinium compounds: inhibition of human erythrocyte acetylcholinesterase and protection of the enzyme against phosphylation, Acta Pharm. Jugosl., 38 (1988) 101-109. M. Skrinjari6, Spoljar, E. Reiner, V. Simeon and B. Krauthacker, 1,3-(Hydroxyiminopyridiniumcyclohexyl-carbonylpyridinium)-bismethylene oxide position isomers: inhibition of bovine erythrocyte acetylcholinesterase and reactivation of the phosphylated enzyme, Acta Pharm. Jugosl., 38 (1988) 111-117. V. Kova~evi6, M. Maksimovi6, V. Deljac and Z. Binenfeld, The efficacy of 1,1 '-(l,4-buten)-bis- (4hydroxyiminomethylpyridinium) dibromide in the treatment of organophosphate poisoning, Acta Pharm. Jugosl., 39 (1989) 167-170. P. Taylor and S. Lappi, Interaction of fluorescent probes with acetylcholinesterase: the site and specificity of propidium binding, Biochemistry, 14 (1975) 1989-1997. Z. Radi6, E. Reiner and P. Taylor, Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives, Mol. Pharmacol., 39 (1991) 98-104.