inhibition of acetylcholinesterase activity by aromatic chelating agents

Eur. J. Biochem. 35,499-506 (1973)

Inhibition of Acetylcholinesterase Activity by Aromatic Chelating Agents

Bendicht WERMUTH and Urs BRODBECE Medizinisch-Chemisches Institut der Universitlit, Bern

(Received November 23, 1972/March 12, 1973)

1. Form B of acetylcholinesterase from toluene-treated electric organs of the electric eel was inhibited in a non-competitive manner by chelating agents such as 1,lO-phenanthroline, 8-hydroxyquinoline, 2,2’-dipyridyl and salicylic aldehyde. The apparent inhibition constants ranged between 1.3 mM and 4.8 mM. Secondary plots revealed hyperbolic inhibition for 1,lO-phenanthroline, slope-parabolic-intercept-linear inhibition for 8-hydroxyquinoline, slope-linear-intercept-hyperbolic inhibition for 2,2’-dipyridyl and a linear inhibition pattern for salicylic aldehyde.

2. The direct participation of metal cations in the catalytic process could be ruled out on the following basis: the inhibition of the enzyme by chelating agents was rapid and reversible; it did not show any time-dependent irreversible inactivation. Excess metal did not reverse the inhibition: metal * chelate complexes inhibited the enzyme similarly or even stronger as did the complexing agents by themselves. No inhibition, even at concentrations up to 0.1 M was observed with EDTA.

3. With 8-hydroxyquinoline a t pH 6.0 non-linear double-reciprocal plots were observed. The Hill coefficients were 1.01, 1.20, 1.30 and 1.32 for 0, 0.75, 1.50 and 3.00 mM 8-hydroxyquinoline.

4. The inhibitory effects of these compounds were explained in terms of hydrophobic adsorption of aromatic nuclei at the active center and in terms of structural similarities to the substrate. The type of inhibition observed by these compounds is indicative of co-operative binding of these ligands to acetylcholinesterase.

The activation of a large number of enzymes by cations is of interest from structural, mechanistic and regulatory points of view. It is well established that the activity of acetylcholinesterase strongly depends on the presence of monovalent or divalent cations in the reaction medium [1,2]. I n general a t low concentrations an increase in salt concentration activates the enzyme whereas media of high ionic strength cause an increase in the apparent Michaelis constant and a decrease in maximal velocities [3,4]. The inhibition obtained a t high ionic strength was attributed to competing interactions of cations with the substrate a t the anionic center located in the active site of acetylcholinesterase [5,6]. It is unclear however, if the observed activation by cations is due to a specific effect on the structure around the active center of this enzyme or a t a site distinctly Merent from the catalytic center. Such effects on a regulatory site of acetylcholinesterase were postu- lated by Changeux [7] and by Wombacher and Wolf PI .

This paper is dedicated to Professor Hugo Aebi in honor

Enzymes. Acetylcholinesterase or acetylcholine hydrolase of his receiving the Otto N6geli Preis 1972.

(EC 3.1.1.7). 33+

As shown by Leuzinger et al. [9] acetylcholinesterase from the electric organ of Electrophorus electricus has a tetrameric structure with a subunit composition of More recently Wermuth et al. [lo] showed that the enzyme can exist in three and possibly more oligomeric forms with the a,!?-dimer as the protomeric structure. I n view of the growing evidence for the presence of a regulatory site in acetylcholinesterase and the activation of the enzyme by cations the effects of chelating agents on the catalytic activity of this enzyme were determined, This paper presents evidence that acetylcholinesterase from the electric organ of Electrophorus electricus is inhibited by chelating agents in a non-competitive manner and that under certain conditions co-operative effects of acetylcholine on the catalytic activity can be observed. A preliminary account of these hd ings was presented earlier [ll].

MATERIALS AND METHODS

Enzyme Acetylcholinesterase (form B) was prepared by

affinity chromatography followed by gel filtration from toluene-treated electric tissue of Electrophorus

500 Inhibition of Acetylcholinesterase Activity by Chelating Agents Eur. J. Biochem.

electricus [lo]. The enzyme was electrophoretically pure and had a specific activity of 12000-15000 IU/ mg protein.

Other Reagents Bovine serum albumin was obtained from Poviet

Producten (Amsterdam). 5,5’-Dithiobis(2-nitrobenzoic acid), acetylcholine chloride, acetylthiocholine iodide, salicylic aldehyde and 2,2’-dipyridyl came from Fluka AG (Buchs). 1,lO-Phenanthroline chloride 8-hydroxyquinoline and all metal salts were supplied by Merck AG (Darmstadt).

All other chemicals employed were standard commercial products. De-ionized water was used for the purification steps and double-distilled water for analytical procedures.

Enzyme Assays Acetylcholinesterase activity was determined a t

30 “C either by following the production of thiocholine (assay 1) according to the method of Ellman et al. [12] or by measuring the amount of acetic acid produced (assay 2). Enzyme activity was expressed in international units (IU = pmol product produced per min).

Assay 1. In a total volume of 3.0 ml the standard assay mixture contained 25 mM sodiumphosphate or 0.1 M Tris-C1 pH 7.4, 0.125 mM 5,5‘-dithiobis(2- nitrobenzoic acid), 0.01 O l i o bovine serum albumin, varying concentrations of inhibitors and metal ions and 0.15- 1.5 I U acetylcholinesterase. The reaction was started by addition of varying amounts of acetylthiocholine iodide and followed spectrophotometrically by measuring the increase in absorbance at 412 nm on a Beckman DB-G spectrophotometer equipped with a W + W recorder 3002.

Assay 2. In a total volume of 20 ml the standard mixture contained 0.1 M NaCl, 0.01 bovine serum albumin, varying concentrations of inhibitors and metal ions and 1 - 10 IU acetylcholinesterase. The reaction was started by adding varying amounts of acetylcholine chloride. The pH was kept constant at 7.4 by addition of 0.01 M NaOH using a Metrohm pH-meter E 300 B, equipped with Impulsomat E 473 and Dosimat E 425. The reaction mixture was kept under C0,-free nitrogen.

Inhibition Experiments Results of inhibition experiments are presented

as Lineweaver-Burk plots [13] with the secondary plots as insets [14]. Lines were fitted to the points by the method of the least squares using a Hewelett Packard 9100 A computer. Apparent Ki values were determined according to Cleland [ 141 and Dixon [ i5]. Following the rules of Cleland the term competitive inhibition will be used to describe increases in slope caused by the inhibitor and the term uncompetitive

inhibition will be used in describing inhibitor- dependent increases in intercept. If both slopes and intercepts are increased by the inhibitor, the term non-competitive inhibition will be used. Non-linear inhibition patterns will be called hyperbolic or parabolic for convex or concave upward curvature of the secondary plots of slopes or intercepts versus inhibitor concentration.

RESULTS When the effect of EDTA on acetylcholinesterase

activity was investigated, no inhibition even a t concentrations up to 0.1 M could be detected. However, aromatic chelating agents such as 8-hydroxyquinoline, 1,lO-phenanthroline, 2,2’-dipyridyl and salicylic aldehyde inhibited the enzymatic hydrolysis of acetylcholine at concentrations ranging from the micromolar to the millimolar level. The rates of acetylthiocholine hydrolysis a t substrate concentrations between 0.1 and 0.01 mM in the presence of varying concentrations of inhibitor were determined (Fig. 1-4). Under the conditions employed all double-reciprocal plots were linear and showed non- competitive inhibition, When the slopes or the intercepts with the ordinate in the Lineweaver-Burk plots were drawn as a function of inhibitor concentrations the following types of inhibition were observed. With 1,lO-phenanthroline both slopes and intercepts were hyperbolic functions of the inhibitor concentration (insets of Fig. 1). On the other hand slope-parabolic- intercept-linear-inhibition patterns were found with 8-hydroxyquinoline (insets of Fig. 2). 2,2’-Dipyridyl gave a slope-linear-intercept-hyperbolic inhibition (insets of Fig. 3) and for salicylic aldehyde both slope and intercept were linear functions of the inhibitor concentration (insets of Fig.4). A summary of the results of these inhibition experiments is shown in Table 1. These data might suggest that metal cations play an essential role in the hydrolysis of choline esters. However, as cautioned by Vallee [16], the identification of a metalloenzyme should not be based solely on the inhibition by metal-complexing agents. If an agent reacts specifically with a metal ion of an enzyme, inhibition should be prevented by occupying the chelation site of the agent with a metal ion prior to exposure of the enzyme [16]. Further when a mixed metalloenzyme * inhibitor complex has form- ed, a critical excess of metal ions should reverse the inhibition by competing with the metalloenzyme for the inhibitor bound to its metal. Removal of the inhibitor from the metalloenzyme by mass action should restore activity. As shown in Table 2, except for 1,lO-phenanthroline, no significant difference in the inhibition of acetylcholinesterase was observed when the decrease in enzymatic activities due to addition of chelating agents alone was compared to the one obtained when mixtures of metal ions and

Vo1.35, No.3, 1973 E. WERMUTH and U. ERODBECK 501

Fig. 1. Lineweaver-Burk plot of acetylcholinesterase inhibited by 1 ,lo-phenanthroline. Acetylcholinesterase activity was determined spectrophotometrically a t 412 nm by following the production of thiocholine according to the method of Ellman et al. In a total volume of 3.0 ml the assay mixture contained 0.1 M Tris-C1 pH 7.4, 0.125 mM 5,5’-dithiobis-

(2-nitrobenzoic acid), 0.01 O/,, bovine serum albumin and 1.2 IU acetylcholinesterase. Inhibitor concentrations were (0) zero, (A) 0.05 mM, (W) 0.1 mM, ( 0 ) 0.5mM and ( A ) 1.0 mM 1,lO-phenanthroline. The reactions were initiated by addition of varying amounts of acetylthiocholine. The insets show secondary plots of slopes and intercepts versus inhibitor

concentration

‘ 4

0 2 4 [Inhibitorj( mM1

i

Fig. 2. Lineweaver-Burk plot of acetylcholinesterase inhibited by 8-hydroxyquinoline. The conditions are the same as described in legend to Fig. 1 except that 0.025 M sodium phosphate buffer pH 7.4 was used. Inhibitor concentrations were (0) zero, (A) 2.0mM, (m) 4.0mM and (0 ) 5.0mM 8-hydroxyquino-line. The insets show secondary plots of intercepts and slopes versus

inhibitor concentration

chelating agents were added to the assay medium. I n the case of the 1,lO-phenanthroline - Fez+ complex (3 : l ) the inhibition was even stronger than the one observed with 1,lO-phenanthroline alone. Thus, the effects of increasing amounts of Fez+ on the enzymatic activity in presence and absence of 1,lO-phen-

anthroline were investigated, As shown in Fig.5, optimal inhibition was observed when the ratio of the 1,10-phenanthroline to the Fez+ concentration was 3 : 1.

Additional experiments showed that the inhibition of acetylcholinesterase by chelating agents was

502 Inhibition of Acetylcholinesterase Activity by Chelating Agents

I 1 I I I

0 10 25 50 75 100

Bur. J. Biwhem.

I/[Sl (rnM-’)

Fig. 3. Lineweaver-Burk plot of acetylcholine-sterme inhibited by 2,2’-dipyridyl. The conditions are the same as described in legend to Fig.1. Inhibitor concentrations were (0) zero, (A) 1 mM, (m) 3 mM and (0 ) lOmM 2,Y-dipyridyl. The insets

show secondary plots of slopes and intercepts versus inhibitor concentration

100 - - E c

2 Y

5 . .- 50

0 0 2 10 20 25 33

l /[S] (rnM-’)

Fig.4. Lineweaver-Burk plot of acetylcholinesterase inhibited by salicylic aldehyde. The conditions are the same as described in legend to Fig. 1, except that 0.025 M sodiumphosphate buffer pH 7.4 was used. Inhibitor concentrations were (0) zero, (A) 1 mM, (m) 2 mM and (0 ) 4 m M salicylic aldehyde. The insets show secondary plots of slopes and intercepts versw-s

inhibitor concentration

Table 1. Summary of results of inhibition of acetylcholinesterme by chelating agents L : linear, H: hyperbolic, P: parabolic, NC : non-competitive. The inhibition constant was determined from secondary plots of

slopes and intercepte v e r m [I] according to the method of Cleland [14]. a d . = not determinable

Type of inhibition Inhibition constant from Inhibitor

l / V w.4 l/[S] Intercept we. [I] Slope w.9. [I1 Intercept w.4. [I] Slope w.4. [I]

1,lO-Phenanthroline 8-Hydroxyquinoline 2,2’-Dipyridyl Salicvlic aldehvde

NC H H NC L P

mM mM

n.d. n.d. 1.5 n.d.

NC H L n.d. 1.3 NC L L 4.8 2.0

Vo1.35, No.3, 1973 B. WERMUTH and U. BRODBECK 503

Table 2. Effect of metal cations on the inhibition of acetylc7toEilzesterase by chelating agents Acetylcholinesterase activity was determined using assay 2. The assay mixture contained in a total volume of 20 ml, 0.1 M NaCI, 0.01 o/o bovine serum albumin and 6.0 IU acetylcholinesterase. To this mixture 1: 1 complexes of metal cations with chelating agents were added and the reaction was started by the addition of 50 pmol acetylcholine. The concentrations of the chelating agents were adjusted to give 50% enzyme activity in absence of inhibitor. In the absence of chelating agents, the salt concentrations were 3 mM. The concentrations of the inhibitors, 1,lO-phenanthroline, 2,2'-dipyridyl and 8-hydroxyquinoline

were 1.0, 3.1 and 3.8 mM, respectively

Metalious No inhibitor 1,lO-Phenanthroline 2,2 -Dipyridyl 8-Hydroxyquinoline

IU/rnl Qlo IUIrnl "lo IU/ml O l O IUlrnl ' I . Control 0.30 100 0.15 100 0.15 100 0.15 100

0.35 117 0.16 107 0.17 113 0.16 107 0.32 106 0.16 107 0.15 100 0.15 100

MgC4 CaCI, FeCI, 0.29 97 0.04 27 0.12 80 0.15 100 FeC1, 0.28 93 0.13 87 0.11 73 0.14 93

1 .o

0 3. . 3

0.5

0 J I I L 0 0.6 1 .2 1.8

[FeC~zl (mM)

Fig. 5. Inhibition of acetylcholinesterase by 1,lO-phenanthroline * Fez+ wmplexes. Acetylcholinesterase activity was determined titrimetrically a t pH 7.4 by measuring the amount of acetic acid produced. In a total volume of 20 ml the assay mixture contained 0.1 M NaCl, 0.010/, bovine serum albumin 4.5 IU acetylcholinesterase and varying amounts of FeCI, without (0 ) and in the presence (A) of 0.6 mM 1,lO-phenanthroline. The reactions were initiated by addition of 20 ymol acetylcholine. v,, represents initial velocity in the absence of FeCI, and 1,lO-phenanthroline, v, in the presence of FeCI, and FeCl, plus 1,lO-phenanthroline

respectively

an instantaneous and reversible reaction (as measured under steady-state conditions) and not a time- dependent irreversible inactivation, a phenomenon often observed with metalloenzymes [17,18]. Thus, these results can be taken as presumptive evidence that the inhibition of acetylcholinesterase by chelating agents is not due to interactions with cations a t the active site. Structural similarities to the parent substrate might thus account for the inhibitory effect of these substances. In order to test this hypothesis the effects of pH on the inhibition by 8-hydroxyquinoline were determined (Fig. 6). Chan- ges in pH effect the state of protonation of 8-hydroxyquinoline (pK, = 5.13) thus rendering a compound less similar to acetylcholine at high pH and one more

0.4 -

0.3 -

P . 0.2 -

0.1 -

O 5 1 PH 7 9

Fig. 6. pH dependence of acetylcholinesterase inhibition by 8-hydroxypuinoline. Acetylcholinesterase activity was determined spectrophotometrically as described in legend to Fig. 1. The buffer contained 25 mM acetic acid, 25 mM NaH,PO, and 25mM Tris. The pH was adjusted with NaOH. The inhibitor concentration was 4mM and the reactions were started by the addition of (0 ) 0.3 pmol or (A) 3.0 ymol acetylthiocholine. v, represents initial velocities in the absence

and v in the presence of inhibitor

closely related to the substrate a t decreased pH. As shown in Fig.6, 8-hydroxyquinoline becomes an increasingly more potent inhibitor with decreasing pH. Below pH 6 this effect levels off, probably due to an increased protonation of the anionic site at the active site of acetylcholinesterase. The Ki values obtained were 0.8 mM at pH 7.4 and decreased to

Inhibition of Acetylcholinesterase Activity by Chelating Agents Eur. J. Biochem.

7. C 1 I L 0 10 25 50 75 10

r,

l / [ S ] ( m M ' ' )

Fig. 7. Lineweaver-Burk plot of ucetylcholinesteruse inhibited by 8-hydroxyquinoline at p H 6.0. The conditions are the same as described for Fig.1 except that 0.1 M sodium citrate p H 6.0 was used. Inhibitor concentrations were (0) zero, (A) 0.75 mM, (m) 1.5 mM and(.) 3.0mM 8-hydroxyquinoline. The inset shows a hill plot of the corresponding values from

the Lineweaver-Burk plot

0.2 mM a t pH 5.5. With salicylic aldehyde, a compound that does not contain a nitrogen atom which can be protonized, no such pH dependence was found.

When assayed a t pH 6.0 the double-reciprocal plots of enzymatic activity vs substrate concentration became non-linear in the presence of 8-hydroxyquinoline, thus deviating from normal steady-state kinetics (Fig.7). An increase in the Hill coefficients was obtained with increasing amounts of 8-hydroxyquinoline. Thus a t concentrations of 8-hydroxyquinoline of 0.0, 0.75, 1.50 and 3.00 mM, the corresponding Hill coefficients were 1.01, 1.20, 1.30 and 1.32.

DISCUSSION Nature of Inhibition

Although it was shown that aromatic chelating agents inhibited acetylcholinesterase from the electric eel, no evidence was found that the obtained inhibitory effects were due to chelation of a metal cation essential for catalysis: the inhibition was instantaneous and reversible upon dilution and no

time-dependent irreversible inactivation could be observed. Furthermore no reversal of the inhibition could be seen when excess metal ions were added t o the enzyme * inhibitor complex. Protonation of a tertiary nitrogen atom, rendering a positively charged ammonium group, able to interact with an anionic site of the enzyme, might thus be responsible for the observed inhibition. This hypothesis could be sup- ported by the fact that the inhibition by 8-hydroxyquinoline was pH dependent. Although the pK, for 8-hydroxyquinoline is 5.13 the compound exists in a partially protonated form above p H 5 due to its tautomeric structure. By the binding to hydrophobic regions a t the active site [22], substrates and inhibitors are transferred from water to a less polar medium. This in turn brings about a decrease in activity of the proton and a concomitant increase in pK, of 8-hydroxyquinoline. On the other hand a decrease in pH could also result in the protonation of a histidine residue being possibly responsible for the binding of the inhibitor to the enzyme. The inhibition of acetylcholinesterase by salicylic aldehyde, however, is not pH dependent. This compound contains no nitrogen atom and has no positive charge around neutral pH, yet it inhibits the enzyme with an apparent inhibition constant comparable to the one for choline and other quaternary nitrogen containing compounds. Thus it is more likely that the pH-dependent increase in inhibition by 8-hydroxyquinoline is due to an increased protonation of this inhibitor and that hydrophobic adsorption of salicylic aldehyde alone accounts for the inhibitory effect of the latter compound.

The interaction of the cationic head of acetylcholine with the anionic site a t the active center of the enzyme involves not only coulombic forces. The three methyl groups attached to the nitrogen atom are also important factors facilitating binding of substrates or inhibitors to the enzyme [19,20]. Moreover, 3,3-dimethylbutylacetate, whose tertiary butyl group sterically imitates the trimethylammo- nium group of acetylcholine is hydrolyzed only slightly less rapidly than acetylcholine [Zl]. Thus hydrophobic adsorption of methyl groups of substrates and inhibitors gives additional strength in the binding of these compounds to the active site of the enzyme. This concept of hydrophobic adsorption has been reviewed by Kabachnik et al. [22]. More recently Mayer and Himel [23] extended it to the binding of active-site-directed fluorescent probes.

Analysis of Inhibition Patterns As shown in Fig.1-4, the type of inhibition varies

with the nature of the inhibitor employed. Following the nomenclature of Cleland [I41 the course of hydrolysis of acetylcholine is a uni-bi reaction (cf. scheme 1 upper path).

Vo1.35, N0.3, 1973 B. WERMUTR and U. BRODBECK 505

A P I I

Q I

J \

Scheme 1

The substrate is represented by A, P and Q repre- sent the products released. E is the free enzyme and EQ the acylated enzyme intermediate. 1,lO-Phen- anthroline, 8-hydroxyquinoline, 2,2’-dipyridyl and salicylic aldehyde are neither substrates nor products of the above reaction. Thus these compounds can act as dead-end inhibitors or ligands that introduce alternative reaction sequences. For dead-end inhibitors, non-competitive inhibition is observed in the above reaction scheme if the compound acts in a competitive as well as uncompetitive manner during the reaction sequence.

Formation of a dead-end EI complex yields the competitive component in which the slopes in the Lineweaver-Burk plot are a function of inhibitor concentration. Formation of a dead-end EQI complex gives the competitive part in which the intercepts with the ordinate are a function of inhibitor concentration. Such an uncompetitive inhibition of acetylcholinesterase has been observed by Rosen- berry and Bernhard [24] in the case of the inhibition of 1-naphthyl acetate hydrolysis by 2-pyridinecarb- aldoxime methiodide. The sum of the competitive and uncompetitive results results in non-competitive inhibition [24]. The effects of salicylic aldehyde that shows both slope and intercept linear non-competitive inhibition can possibly be explained in these terms. Thus a molecule of salicylic aldehyde binds either to the free enzyme or to the acylated form of acetylcholinesterase. It is of interest to note that this neutral, nitrogen-less compound gives linear inhibition whereas the nitrogen-containing compounds 1,iO-phenanthroline, 8-hydroxyquinoline and 2,2’-dipyridyl show more complex inhibition patterns. 8-Hydroxyquinoline gives rise to slope-parabolic- intercept-linear non-competitive inhibition. Accord- ing to the rules of Cleland [14] parabolic effects on the secondary plots are only seen if two or more molecules of an inhibitor combine in dead-end fashion with the same enzyme form. Thus with 8-hydroxyquinoline the EI complex probably can combine with a second molecule of the inhibitor to form a dead-end EI, complex.

A third situation arises with 2,2’-dipyridyl which gives a slope linear intercept hyperbolic inhibition. This pattern is observed if the inhibitor combines with free enzyme in dead-end fashion and forms a non-abortive EQI complex.

Finally, with 1,lO-phenanthroline both slopes and intercepts appear as hyperbolic functions of the inhibitor concentration. Thus slower alternative reaction sequences are introduced by both the EI and the EQI complexes as shown in Scheme 1.

As shown by Krupka and coworkers [25] inhibitors of acetylcholinesterase containing quaternary nitrogen atoms strongly bind to the acylated form of the enzyme. Nicotine, tensilone and related compounds are inhibitors of this type. These ligands act in a non-competitive fashion thus binding to the free as well as to the acylated form of acetyleholinester- ase. They show linear behavior in the secondary plots and thus form abortive complexes only. It is however conceivable that certain compounds can form non-abortive complexes with the acylated enzyme. As shown by Krupka and Laidler [26] substrate inhibition can be explained in these terms.

Although it is conceivable that the slope and intercept hyperbolic inhibition by 1 ,lo-phenanthroline can be described in terms of alternate reaction sequences, a more likely explanation is that there is more than one site to which the inhibitor can bind. The one a t the active site leads to formation of dead- end complexes in the course of reaction. If the inhibitor is also bound to a regulatory site, non-linear inhibition can result from such a situation. Hyper- bolic inhibition of membrane-bound acetylcholinesterase from erythrocyte ghosts by gallamine and d-tubocurarine were reported by Wombacher and Wolf [8]. This situation however constitutes only presumptive evidence of the existence of an allosteric site of erythrocyte acetylcholinesterase. On the other hand the double inhibitor studies of Changeux [7] clearly showed allosteric properties when a modified Dixon plot (v/vo w.s [I]) was used as diagnostic test for co-operativity [27]. More recently Kato et al. [as] found similar co-operative effects when the inhibition of acetylcholinesterase from head ganglia of squid by atropine a t high substrate levels was determined.

These and other results [29-321 are complement- ed by the finding that at pH 6.0 in the presence of 8-hydroxyquinoline a deviation from normal Micha- elis-Menten kinetics is observed with Hill coefficients depending on the effector concentration. The studies of Krupka and Laidler [26] showed that for substrates or inhibitors of acetylcholinesterase the distance between a cationic nitrogen atom and an electron donating group such as a carbonyl group is about 0.5 nm. The theoretical considerations of Beers and Reich [33] disclosed that the distance between the center of a positively charged atom and the van der Waals surface of an atom proposed to be

506 B. WEBMUTH and U. BRODBEUK: Inhibition of Acetylcholinesterase Activity by Chelating Agents Eur. J. Biochem.

the electron donor is 0.59 nm for nicotinic and 0.43-0.44 nm for muscarinic agents. For 8-hydroxy- guinoline the distance between the hydroxyl group and the nitrogen atom is about 0.3nm. The same distance separates the two nitrogen atoms in 1 , l O - phenanthroline. On the other hand the potent acetylcholinesterase inhibitor 3-hydroxuyphenyltri- methylammonium chloride has a distance of about 0.5 nm between its hydroxyl and ammonium group. On the basis of these considerations the acridine derivatives proflavine and acrifiavine should also act as cholinesterase inhibitors. Studies with these two compounds are detailed in a following paper (in preparation).

The authors are indebted to Dr M. Rottenberg for helpful discussions and valuable suggestions regarding this work, to Professor H. Aebi for providing financial assistance and for his continuous interest in this work and to Mrs Michel for skilful technical assistance. This work was made possible by Dr W. Leuzinger whom the authors thank for the very generous gift of toluene-treated electroplax organs.

REFERENCES 1. Augustinson, K. B. (1948) Acta Physiol. Scand. 15, Suppl.

2. Cohen, J. A. & Oosterbaan, R. A. (1963) in Handbuch der Experimentellen Pharmakologie, Erganzungswerk X V (Eichler, 0. & Farah, F., eds) pp. 315-316, Springer-Verlag, Berlin-Heidelberg-New York.

3. Brestkin, A. P. & Ivanova, L. A. (1970) Biokhimiya, 35,

4. Lundin, S. J., Gentinetta, R. & Brodbeck, U., Acta

5. Myers, D. K. (1952) Arch. Biochem. Biophys. 37, 469-

6. Hofstee, B. H. J. (1960) J . Pharmacol. Exp. Ther. 128,

7. Changeux, J . P. (1966) Mol. Pharmacol. 2, 369-392. 8. Wombacher, H. & Wolf, H. U. (1971) Mol. Phizrmmol. 7,

9. Leuzinger, W., Goldberg, M. & Cauvin, E. (1969) J. Mol.

52, 27-28.

652 - 656.

Chem. Scand. in press.

487.

299-304.

554-566.

Biol. 40, 217-225.

10. Wermuth, B. & Brodbeck, U. (1972) Experientia (Baael)

11.

12.

13.

28, 740-471. Wermuth, B., Gentinetta, R. & Brodbeck, U. (1971)

Experientia (Basel) 27, 731. Ellman, G. L., Courtney, D. K., Andres, V. & Feather-

stone, R. M. (1961) Biochem. Pharmawl. 7 , 88-95. Lineweaver. H. & Burk. D. (19341 J. Am. Chem. Soc. 56,

I , - , 658-666:

14. Cleland. W. W. (1970) in The Enzumes 3rd edn, (Boger,

15.

16.

17.

18.

19.

20. 21.

22.

23.

24.

25. 26.

27.

28.

29.

30. 31.

32.

33.

P. D.; ed.) vol. 2, 'pp. 1-65, Academic Press, s e w York.

Dixon,M. & Webb,E.C. (1964) Enzymes, 2nd edn, pp. 327-331, Longmans Green and Co., London.

Vallee, B. L. (1960) in The Enzymes 2nd edn, (Boyer, P. D., Lardy, H. & Myrback, K., eds) vol. 3, pp. 225- 276, Academic Press, New York.

Hoch, F. L., Williams, R. J. P. & Vallee, B. L. (1958) J. Biol. Ghem. 232, 453-464.

Williams, R. J. P., Hoch, F. L. & Vallee, B. L. (1958) J. Biol. Chem. 232, 465-474.

Bergmann, F. & Segal, R. (1954) Biochem. J. 58, 692- 698.

Wilson, I. B. (1952) J . Biol. Chem. 197, 215-225. Adama, D. N. & Whittaker, V. P. (1949) Biochim. Bio-

phys. Acta, 3, 358-366. Kabachnik, M. I., Brestkin, A. P., Godvikov, N. N.,

Michelson, M. J., Rozengart, E. V. & Rozengart, V. I. (1970) Pharmol. Rev. 22, 355-388.

Mayer, R. T. & Himel, C. M. (1972) Biochemistry, 11,

Rosenberry, T. L. & Bernhard, S. A. (1972) Biochemistry,

Krupka, R. M. (1964) Biochemistry, 3, 1749-1754. Krupka, R. M. & Laidler, K. J. (1961) J . Am. Chem.

Koshland, D.E., Jr. (1970) in The Enzymes, 3rd edn (Boyer, P. D., ed.) vol. 1, pp. 341-396, Academic Press, New York.

Kato, G., Tm, E. & Yung, J. (1972) J . Bwb. Chm. 247,

Kitz, R. J., Braswell, L. M. & Ginsburg, S. (1970) Mol.

!hpan;iE, A. 0. (1970) FEBS Lett. 11, 277-280. Rosenberry, T. L. & Bernhard, S. A. (1971) Biochemistry,

Mooser, G. & Sigman, D. S. (1972) Biochem. Bwphys. Res.

Beers, W. H. & Reich, E. (1970) Nature (Lond.) 228,

2082 -2090.

11, 4308-4321.

SOC. 83, 1445-1460.

3 186 -3189.

Phamnac~l. 6 , 108-121.

10, 4114-4120.

Commun. 48, 559-564.

917 -922.

B. Wermuth and U. Brodbeok, Medizinisch-Chemisches Institut der Universittt, Biihlstrasse 28, CH-3012 Bern, Switzerland

inhibition of acetylcholinesterase activity by aromatic chelating agents

Documents