0020-71 IX/91 53.00 + 0.00 Copyright 0 1991 Pergamon Press plc
1~. J. Biochem. Vol. 23, No. 2, pp. 169-174, 1991 Printed in Great Britain. All rights reserved
BIOCHEMICAL STUDIES OF THE ACTIONS OF ETHANOL ON ACETYLCHOLINESTERASE ACTIVITY: ETHANOL-ENZYME-SOLVENT INTERACTION SOOK SHIN,’ PORTIA Wu’ and CHANG-HWEI CHEN’~ ‘Wadsworth Center for Laboratories and Research, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509, U.S.A. *Department of Biomedical Sciences, State University of New York at Albany, Albany, New York, U.S.A. (Received 2 April 1990) Abstract-l. Biochemical studies of the actions of ethanol on the activity of acetylcholinesterase (AChE), isolated from electric eel (Electrophorus electricus) and purified by affinity chromatography, were. performed to elucidate ethanol+nzyme+solvent interactions. 2. Ethanol at a low concentration ([EtOH] = 2.7-200 mM) was found to enhance AChE activity slightly and systematically. 3. This observation was consistent with the result from enzyme-kinetic studies that ethanol might noncompetitively activate AChE activity at this lower concentration range. 4. If ethanol alters the hydrophobic site interaction on the enzyme and subsequently induces a favorable conformation for the active center of the enzyme, then a slight increase in the AChE activity in the presence of a low concentration of ethanol will be observed. 5. This speculation was supported by the finding of ethanol’s ability to perturb the inhibition of AChE activity by tetrabutylammonium bromide and to affect hydrophobic interaction between this salt and AChE, as investigated by enzyme activity and microcalorimetric measurements. 6. The ethanol effect on the activity of this soluble AChE was found to be distinguishable from that on a membrane-bound AChE. 7. Furthermore, to elucidate the effect of ethanol-solvent interaction on AChE activity, enzyme activity in the presence of much higher concentrations of ethanol was also examined. 8. At [EtOH] > 800 mM, ethanol can perturb the structure of water around hydrophobic areas of AChE, causing an instability in the enzyme conformation and subsequently decreasing AChE activity.
INTRODUCI’ION
In the synaptic transmission, the arrival of an action potential at the terminus of the presynaptic cell stimulates Ca*+ uptake, triggering the release of acetylcholine (Ach) from vesicles into the synaptic cleft (Rubin, 1974). The binding of Ach molecules with acetylcholine receptor (AchR) in the postsynaptic membrane triggers depolarization, thus propagating an action potential in the postsynaptic cell (Conti-Tronconi and Raftery, 1982). Rapid hydrolysis of Ach bound to the postsynaptic membrane is catalyzed by acetylcholinesterase (AChE). This reduces the number of Ach-AchR complexes and repolarizes the membrane. The level of Ach in the synaptic cleft is dependent on the ability of AChE to catalyze Ach hydrolysis. To examine the action of ethanol on synaptic events, it is important to investigate the effect of ethanol on the activity of AChE and to elucidate the ethanol-AChE interaction, Although the effects of ethanol on the activity of AChE isolated from several sources [such as erythrocyte membrane (Haboubi and Thumham, 1986), honeybee head (Lewis, 1967), cockroach (Colhoun, 1961) and calf brain (Kalant, 1967)] have been investigated, no attempt was made to elucidate the ethanol-AChE interaction. Electric eel (Elecrrophorous electricus) is rich in homogeneous nicotinic
choline@ synapses. AChE isolated from this source may serve as a model system to study the mechanism of the Ach hydrolysis reaction in the mammalian nervous system. However, there is a lack of systematic study of the ethanol effect on AChE isolated from electric eel, and consequently no elucidation of the ethanol-AChE interaction has been made in this system either. Ethanol is believed to act on the hydrophobic portion of an enzyme (Hunt, 1985; Tabakoff et al., 1987). Investigations of the effect of ethanol on inhibitor-AChE reactions involving hydrophobic interaction would be helpful in elucidating the nature of ethanol-AChE interaction. Enzyme-kinetics studies of AChE activity in the presence of inhibitors such as quaternary ammonium salts have been employed to evaluate the nature of AChEcatalyzed Ach hydrolysis reaction (Taylor and Lapp, 1975; Krupka and Hellenbrand, 1974). Hydrophobic interaction between bulky alkyl groups of these salts [such as butyl groups in tetrabutylammonium bromide (Bu,NBr)] and hydrophobic areas of AChE has been found to play an important role in determining their inhibition ability. Such an interaction can lead to a stronger binding of Bu,NBr to AChE and a subsequently larger inhibition of its activity. Investigation of the effect of ethanol on the Bu,NBr-AChE interaction should provide important information to 169
SOK SHINet al.
170
support the proposal that ethanol primary alters the hydrophobic site interaction of an enzyme. In a continuation of our efforts to understand the action of ethanol on proteins, membranes and enzymes (Chen, 1980; Chen er al., 1988; Baker and Chen; 1988, Chen, 1989), we have purified highactivity AChE (from electric eel) by affinity chromatography. Using the purified AChE, we have performed enzyme activity and enzyme kinetics measurements to examine the actions of ethanol on AChE activity and the ethanol-AChE-solvent interaction. Our motivation in the present investigation is threefold: (1) to systematically examine how this AChE is sensitive to a wide concentration range of ethanol; (2) to elucidate the nature of the ethanol-AChE interaction; and (3) to examine the effect of ethanol-solvent interaction on the AChE activity. Such biochemical studies may serve as a model system to investigate alcohol-related alterations in the activities of isolated enzymes in the nervous system. MATERIALS AND METHODS
Crude electric eel acetylcholinesterase (type VI-S), bovine erythrocyte acetylcholinesterase. (type XII-S), acetylthiocholine iodide, 5,-5’dithiobis(2-nitrobenzoic acid), e-aminon-caproic acid and lethyl-3-(3dimethylaminopropyl) earbodiimide were obtained from Sigma Chemical Co. Sepharose 4B was a product of Pharmacia Fine Chemicals. Cyanogen bromide and decamethonium bromide were obtained from Aldrich Chemical Co. 9-(B-aminopropylamino) acridine was synthesized in our laboratory. Reagent-grade Bu,NBr and tetraethylammonium bromide (Et,NBr) were purchased from Eastman Kodak Co. Ethanol was obtained from U.S. Industrial Chemicals Co., BCA protein assay reagent was from Pierce Chemical Co. and collodion membrane (type UH 100/75) from Schleicher and Schuell, Inc. Double distilled water was used in all experiments. Other chemicals were of reagent grade and obtained from commercial sources. Purl@cation of AChE by ajinity chromatography Preparation of ajinity gel. The preparation of affinity gel followed by the procedure of Taylor et al. (1983) with minor modification. A 100 ml sample of washed sepharose 4B was collected on a sintered glass tilter and resuspended in lOOm1 water at 4°C. The resin was activated by adding 10 g of CNBr dissolved in 10 ml of N-methylpyrolidinone and quickly adjusted to ca pH 11 using 1 M NaOH. The gel suspension was immediately filtered and washed with 1 1. of cold water and then with the same volume of cold 0.1 M NaHCO,, pH 9.0. The activated sepharose 4B was suspended in 100 ml water containing 10 g of c-amino-n-caproic acid. The pH of the suspension was adjusted to 9.0 with 1 M NaOH. The coupling reaction was gently shaken for 3 hr and then allowed to stand at 4°C overnight. The reaction product was washed with 2 1. of cold water and 1 1. of 10% acetic acid (v/v), and resuspended in lOOm1 water. To this gel suspension (ca pH 5.0), lOOm1 of water containing 38.9 mg of our synthesized 9-(/I-aminopropylamino) acridine_HCl 800 mM, an inhibition of AChE activity occurs as ethanol gradually becomes an inhibitor. This is shown in Figs 1 and 3. As [EtOH] increases to high concentration such as 1.6 or 2.0 M (Fig. 3), Lineweaver-Burke plots no longer share a common intercept on either l/V or l/[ATC] axis, suggesting that ethanol becomes a mixed-type inhibitor in this high [EtOH]. Hydrophobic interaction of Bu,NBr with AChE Ethanol is believed to act on the hydrophobic portion of a protein. It primarily alters hydrophobic site interactions of an enzyme (Tabakoff et al., 1987; Hunt, 1985). Examinations of the important role of hydrophobic interaction in the activity of AChE and the effect of ethanol on such an interaction are essential to the elucidation of ethanol-AChE interaction. For this reason, we have also investigated the inhibition of AChE activity by Bu,NBr and the effect
1
0
J 200
400
800
[EIOH]
(mM)
1200
1600
Fig. 1. The effect of ethanol on AChE activity in 0.1 M potassium phosphate buffer pH 8.0, where [ATC] = 75 mM and AChE activity is 1560 U/mg protein. The activity in the absence of ethanol is taken as the control 100%.
M l/[ATC].
IO’M-’
Fig. 3.‘The effect of higher concentrations of ethanol on the enzyme kinetics of AChEcatalyzed ATC hydrolysis reaction. See Fig. 2 for experimental conditions.
SooK SmN et al.
I72
Table I. E&t of ethanol on K, of ACbEcatalyzed acetyltbiocholine hydrolysis reaction inhibited by Bu,NBr’
(Bu,NBr] (p=f 0 :: 20
0.2
0.0
CONCENTRATION
Fig. 4. Comparison
0.4 OF INHIBITOR
of the inhibition
(mhi)
m 1~~ Ob 0 2.7 5.4
x; 10-3M 1.82 f 0.20 0.67 f 0.10 0.40 f 0.06
‘Enzyme reaction was carried out as described in the Materials and Methods section. SE of the slopes of the straight-line plots in Figs 2, 5 and 7 are 0.5%. bK,=(2.07+0.03)x 10m4M and V,,=(l.l9 + 0.03) x lo-’ M/mm.
of AChE activity
by Bu,NBr with Et,NBr. See Figs 1 and 2 for other experimental conditions. of ethanol on such an inhibition reaction. Figure 4 demonstrate that Bu.,NBr inhibits AChE activity and the degree of inhibition increases, as its concentration increases from 20 to 4OOpM. Comparative studies of the inhibition of AChE activity by Bu,NBr and tetraethylammonium bromide (Et,NBr) as shown in Fig. 4 reveal that BgNBr is a stronger inhibitor than Et.,NBr at a finite salt concentration, due to a larger size and a greater hydrophobicity of Bu (butyl) than Et (ethyl) group. The inhibitory effect of Bu,NBr on AChE activity is further supported by eke-kinetic measurements as presented in Fig. 5. The l/V data in the presence of Bu,NBr are above those in the absence of Bu,NBr (as the control), suggesting the inhibition of AChE activity by Bu,NBr. The figure shows a common intercept in the l/ATC axis in Lineweaver-Burke plots obtained from a computer least square analysis, indicating that Bu,NBr is a noncompetititve inhibitor (Krupka and Hellenbrand, 1974; Segel, 1975). The value of K, is determined as (1.82 f 0.20) x 10-r M (Table 1). The presence of ethanol significantly decreases the value of K,. Hydrophobic interaction between ByNBr and AChE is further demonstrated in microcalorimetric measurements on the heat of Ach hydrolysis reaction in the presence of quaternary ammonium salts o() containing various alkyl chain length. Table 2 shows
Fig. 5. The effect of Bu,NBr on the enzyme kinetics of AChE-catalyzed ATC hydrolysis reaction. LineweaverBurke plot of l/Y vs l/[ATC’j was performed in the absence and presence of Bu, NBr. Each data point is a mean value of 34 measurements with an experimental error of & 3%.
Table 2. Effects of quatemary ammonium salt (X) on the heat of Ach hydrolysis reaction’ Salt (Qb None Et,NBr
Pr,NBr Bu,NBr
Heat of reaction @.I) 12.65 f 0.63 20.36 f 1.02
3.53t0.18 -3.29 + 0.16
“Microcalorimetric mixing scheme is [Ach (1OOmM) + X(lf#lmM)] in 0.4ml bulk + AChE (1 mg) in 0.1 ml buffer + [Ach (SOmM) t X (80 mM) t AChE (I mg)] in 0.5 ml buffer, where the commerciaI AChE was used. bEt = ethyl, Pr = propyl and Bu = butyl. The ability for hydrophobic interaction is in the order of Bu,NBrr Pr,NBr > EtdNBr.
that the hydrolysis reaction is accompanied by an endothermal heat. In the presence of X, the heat of hydrolysis reaction is affected by: an inhibition of Ach hydrolysis reaction and the binding of X to AChE. As the hydrophobicity of X is enhanced from Et,NBr to BbNBr, the degree of Ach hydrolysis reaction is decreased, while hydrophobic interaction of X with AChE is increased. The observed heat change from a positive sign (endothe~~) to a negative sign (exothermal) confirms a strong hydrophobic interaction between Bu,NBr and AChE. The effect of ethanol on the inhibition of AChE activity by &NBr As indicated above, the study of the effect of ethanol on Bu,NBr inhibition of AChE activity is useful in the elucidation of the nature of ethanolAChE interaction. In the presence of a low concentration of ethanol (5.4 mM or less), Fig. 6 reveals that ethanol enhances the inhibition of AChE activity by Bu,NBr. In other words, this low [EtOH] makes Bu,NBr a more effective inhibitor. This finding is consistent with the results of enzyme kinetics studies as shown in Fig. 7, since the figure reveals that the data of l/V in Lineweaver-Burke plots in the presence of ethanol are systematically above those in the absence of ethanol (as the control), Nevertheless, the basic enzyme kinetics of Bu,NBr as a noncompetitive inhibitor is not affected by ethanol. From the common intercept in these plots as obtained from a least square computer analysis, the value of Ki in the presence of ethanol was found to be significantly lower than that in the absence of ethanol, decreasing from 1.82 to 0.40 x 10e3 M (Table 1). These results
173
Actions of ethanol on AChE
0
100
200
300
400
[Bu.NBrl (VW
Fig. 6. The effect of ethanol of the inhibition of AChE activity by Bu,NBr. See Figs 1 and 2 for experimental conditions.
confirm that the presence of a low concentration of ethanol enhances the binding of Bu,NBr to AChE, making Bu,NBr a more effective inhibitor. [Note: at a higher concentration of ethanol such as 200mM, more ethanol molecules are available to compete with Bu.,NBr for the hydrophobic site of AChE. Consequently, the interaction of Bu,NBr with AChE is weakened, causing a reduction in the inhibitory ability of Bu,NBr (S. Shin, L. Roth and C.-H. Chen; unpublished data).] DISCUSSION
The nature of the ethanoLAChE interaction For the assay of the ethanol effect on AChE activity, a dilute solution of the purified AChE was divided into two equal volumes, adding a volume of ethanol to one and an equal volume of buffer to the other. Thus, the ionic strength was kept constant and the dilution effect was considered over various concentrations of ethanol. The presence of ethanol increases AChE activity by IO%, which is small but systematic. It may be significant in consideration of the experimental error of f 3%. A similar observation
of a small increase in AChE activity in the presence of ethanol was also reported for AChE isolated from honeybee head and cockroach (Lewis, 1967; Colhoun, 1961). Our observation is not caused by a change in the pH of the medium, since the pH of the buffer solution is essentially unchanged in the presence of [EtOH] Q 200 mM. A low concentration of ethanol can affect enzyme activity by interacting with hydrophobic areas of an enzyme (Tabakoff et al., 1987). If the perturbation by ethanol of these areas causes an alteration in the hydrophobic site interaction on AChE and subsequently induces a favorable conformation for the active center of the enzyme (Taylor and Lappi, 1975; Berman et al., 1980; Wilson and Silman, 1977), then an increase in the activity of AChE will be observed. This speculation is supported by our observations: (1) ethanol’s noncompetitive modulation of the enzyme kinetics of AChE-catalyzed reaction; and (2) ethanol’s ability to perturb the inhibition of AChE activity by Bu,NBr. Besides ethanol-+nzyme interaction, another important consideration is the ethanol-solvent interaction. It has been reported that, at a low ethanol concentration, ethanol molecules are able to tie up many water molecules around them through hydrogen bonding (Franks and Ives, 1966; Franks and Smith, 1969). As a result of this, less water molecules will be available for an enzyme. This could provide AChE with a more hydrophobic environment for maintaining its conformation. On the other hand, at a high ethanol concentration, ethanol molecules can perturb the structure of water around hydrophobic areas of AChE (the dielectric constant of ethanol is 24 vs that of water, 78). Such a perturbation can weaken the hydrogen bonding between water molecules and therefore the structure of water (Franks, 1974). As a results of this, the presence of ethanol can induce an instability in the hydrophobic conformation of the enzyme, therefore causing a decrease in AChE activity at [EtOH] > 800 mM (see Figs 1 and 3). Distinction of the ethanoLAChE interaction from ethanol-lipid interaction AChE used in our studies is a soluble enzyme. To demonstrate that the above ethanol-AChE I
l/[ATC], 10’M“
Fig. 7. The effect of ethanol on the enzyme kinetics of AChE-catalyzed ATC hydrolysis inhibited by Bu,NBr. Lineweaver-Burke plot of l/V vs l/[ATC] was carried out in the absence and presence of ethanol and Bu,NBr. Each point is a mean value of 34 experiments with an experimental error of +3%. See Fig. 1 for other experimental conditions.
0
I
200 lEoHI
400 FM)
Fig. 8. The effect of ethanol on the activity of membranebound AChE isolated from bovine erythrocytes in 0.1 M potassium phosphate buffer pH 8.0, where [ATC] = 75 mM. AChE activity in the absence of ethanol is taken as the control, 100%.
1~. J. Biochem. Vol. 23, No. 2, pp. 169-174, 1991 Printed in Great Britain. All rights reserved
BIOCHEMICAL STUDIES OF THE ACTIONS OF ETHANOL ON ACETYLCHOLINESTERASE ACTIVITY: ETHANOL-ENZYME-SOLVENT INTERACTION SOOK SHIN,’ PORTIA Wu’ and CHANG-HWEI CHEN’~ ‘Wadsworth Center for Laboratories and Research, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509, U.S.A. *Department of Biomedical Sciences, State University of New York at Albany, Albany, New York, U.S.A. (Received 2 April 1990) Abstract-l. Biochemical studies of the actions of ethanol on the activity of acetylcholinesterase (AChE), isolated from electric eel (Electrophorus electricus) and purified by affinity chromatography, were. performed to elucidate ethanol+nzyme+solvent interactions. 2. Ethanol at a low concentration ([EtOH] = 2.7-200 mM) was found to enhance AChE activity slightly and systematically. 3. This observation was consistent with the result from enzyme-kinetic studies that ethanol might noncompetitively activate AChE activity at this lower concentration range. 4. If ethanol alters the hydrophobic site interaction on the enzyme and subsequently induces a favorable conformation for the active center of the enzyme, then a slight increase in the AChE activity in the presence of a low concentration of ethanol will be observed. 5. This speculation was supported by the finding of ethanol’s ability to perturb the inhibition of AChE activity by tetrabutylammonium bromide and to affect hydrophobic interaction between this salt and AChE, as investigated by enzyme activity and microcalorimetric measurements. 6. The ethanol effect on the activity of this soluble AChE was found to be distinguishable from that on a membrane-bound AChE. 7. Furthermore, to elucidate the effect of ethanol-solvent interaction on AChE activity, enzyme activity in the presence of much higher concentrations of ethanol was also examined. 8. At [EtOH] > 800 mM, ethanol can perturb the structure of water around hydrophobic areas of AChE, causing an instability in the enzyme conformation and subsequently decreasing AChE activity.
INTRODUCI’ION
In the synaptic transmission, the arrival of an action potential at the terminus of the presynaptic cell stimulates Ca*+ uptake, triggering the release of acetylcholine (Ach) from vesicles into the synaptic cleft (Rubin, 1974). The binding of Ach molecules with acetylcholine receptor (AchR) in the postsynaptic membrane triggers depolarization, thus propagating an action potential in the postsynaptic cell (Conti-Tronconi and Raftery, 1982). Rapid hydrolysis of Ach bound to the postsynaptic membrane is catalyzed by acetylcholinesterase (AChE). This reduces the number of Ach-AchR complexes and repolarizes the membrane. The level of Ach in the synaptic cleft is dependent on the ability of AChE to catalyze Ach hydrolysis. To examine the action of ethanol on synaptic events, it is important to investigate the effect of ethanol on the activity of AChE and to elucidate the ethanol-AChE interaction, Although the effects of ethanol on the activity of AChE isolated from several sources [such as erythrocyte membrane (Haboubi and Thumham, 1986), honeybee head (Lewis, 1967), cockroach (Colhoun, 1961) and calf brain (Kalant, 1967)] have been investigated, no attempt was made to elucidate the ethanol-AChE interaction. Electric eel (Elecrrophorous electricus) is rich in homogeneous nicotinic
choline@ synapses. AChE isolated from this source may serve as a model system to study the mechanism of the Ach hydrolysis reaction in the mammalian nervous system. However, there is a lack of systematic study of the ethanol effect on AChE isolated from electric eel, and consequently no elucidation of the ethanol-AChE interaction has been made in this system either. Ethanol is believed to act on the hydrophobic portion of an enzyme (Hunt, 1985; Tabakoff et al., 1987). Investigations of the effect of ethanol on inhibitor-AChE reactions involving hydrophobic interaction would be helpful in elucidating the nature of ethanol-AChE interaction. Enzyme-kinetics studies of AChE activity in the presence of inhibitors such as quaternary ammonium salts have been employed to evaluate the nature of AChEcatalyzed Ach hydrolysis reaction (Taylor and Lapp, 1975; Krupka and Hellenbrand, 1974). Hydrophobic interaction between bulky alkyl groups of these salts [such as butyl groups in tetrabutylammonium bromide (Bu,NBr)] and hydrophobic areas of AChE has been found to play an important role in determining their inhibition ability. Such an interaction can lead to a stronger binding of Bu,NBr to AChE and a subsequently larger inhibition of its activity. Investigation of the effect of ethanol on the Bu,NBr-AChE interaction should provide important information to 169
SOK SHINet al.
170
support the proposal that ethanol primary alters the hydrophobic site interaction of an enzyme. In a continuation of our efforts to understand the action of ethanol on proteins, membranes and enzymes (Chen, 1980; Chen er al., 1988; Baker and Chen; 1988, Chen, 1989), we have purified highactivity AChE (from electric eel) by affinity chromatography. Using the purified AChE, we have performed enzyme activity and enzyme kinetics measurements to examine the actions of ethanol on AChE activity and the ethanol-AChE-solvent interaction. Our motivation in the present investigation is threefold: (1) to systematically examine how this AChE is sensitive to a wide concentration range of ethanol; (2) to elucidate the nature of the ethanol-AChE interaction; and (3) to examine the effect of ethanol-solvent interaction on the AChE activity. Such biochemical studies may serve as a model system to investigate alcohol-related alterations in the activities of isolated enzymes in the nervous system. MATERIALS AND METHODS
Crude electric eel acetylcholinesterase (type VI-S), bovine erythrocyte acetylcholinesterase. (type XII-S), acetylthiocholine iodide, 5,-5’dithiobis(2-nitrobenzoic acid), e-aminon-caproic acid and lethyl-3-(3dimethylaminopropyl) earbodiimide were obtained from Sigma Chemical Co. Sepharose 4B was a product of Pharmacia Fine Chemicals. Cyanogen bromide and decamethonium bromide were obtained from Aldrich Chemical Co. 9-(B-aminopropylamino) acridine was synthesized in our laboratory. Reagent-grade Bu,NBr and tetraethylammonium bromide (Et,NBr) were purchased from Eastman Kodak Co. Ethanol was obtained from U.S. Industrial Chemicals Co., BCA protein assay reagent was from Pierce Chemical Co. and collodion membrane (type UH 100/75) from Schleicher and Schuell, Inc. Double distilled water was used in all experiments. Other chemicals were of reagent grade and obtained from commercial sources. Purl@cation of AChE by ajinity chromatography Preparation of ajinity gel. The preparation of affinity gel followed by the procedure of Taylor et al. (1983) with minor modification. A 100 ml sample of washed sepharose 4B was collected on a sintered glass tilter and resuspended in lOOm1 water at 4°C. The resin was activated by adding 10 g of CNBr dissolved in 10 ml of N-methylpyrolidinone and quickly adjusted to ca pH 11 using 1 M NaOH. The gel suspension was immediately filtered and washed with 1 1. of cold water and then with the same volume of cold 0.1 M NaHCO,, pH 9.0. The activated sepharose 4B was suspended in 100 ml water containing 10 g of c-amino-n-caproic acid. The pH of the suspension was adjusted to 9.0 with 1 M NaOH. The coupling reaction was gently shaken for 3 hr and then allowed to stand at 4°C overnight. The reaction product was washed with 2 1. of cold water and 1 1. of 10% acetic acid (v/v), and resuspended in lOOm1 water. To this gel suspension (ca pH 5.0), lOOm1 of water containing 38.9 mg of our synthesized 9-(/I-aminopropylamino) acridine_HCl 800 mM, an inhibition of AChE activity occurs as ethanol gradually becomes an inhibitor. This is shown in Figs 1 and 3. As [EtOH] increases to high concentration such as 1.6 or 2.0 M (Fig. 3), Lineweaver-Burke plots no longer share a common intercept on either l/V or l/[ATC] axis, suggesting that ethanol becomes a mixed-type inhibitor in this high [EtOH]. Hydrophobic interaction of Bu,NBr with AChE Ethanol is believed to act on the hydrophobic portion of a protein. It primarily alters hydrophobic site interactions of an enzyme (Tabakoff et al., 1987; Hunt, 1985). Examinations of the important role of hydrophobic interaction in the activity of AChE and the effect of ethanol on such an interaction are essential to the elucidation of ethanol-AChE interaction. For this reason, we have also investigated the inhibition of AChE activity by Bu,NBr and the effect
1
0
J 200
400
800
[EIOH]
(mM)
1200
1600
Fig. 1. The effect of ethanol on AChE activity in 0.1 M potassium phosphate buffer pH 8.0, where [ATC] = 75 mM and AChE activity is 1560 U/mg protein. The activity in the absence of ethanol is taken as the control 100%.
M l/[ATC].
IO’M-’
Fig. 3.‘The effect of higher concentrations of ethanol on the enzyme kinetics of AChEcatalyzed ATC hydrolysis reaction. See Fig. 2 for experimental conditions.
SooK SmN et al.
I72
Table I. E&t of ethanol on K, of ACbEcatalyzed acetyltbiocholine hydrolysis reaction inhibited by Bu,NBr’
(Bu,NBr] (p=f 0 :: 20
0.2
0.0
CONCENTRATION
Fig. 4. Comparison
0.4 OF INHIBITOR
of the inhibition
(mhi)
m 1~~ Ob 0 2.7 5.4
x; 10-3M 1.82 f 0.20 0.67 f 0.10 0.40 f 0.06
‘Enzyme reaction was carried out as described in the Materials and Methods section. SE of the slopes of the straight-line plots in Figs 2, 5 and 7 are 0.5%. bK,=(2.07+0.03)x 10m4M and V,,=(l.l9 + 0.03) x lo-’ M/mm.
of AChE activity
by Bu,NBr with Et,NBr. See Figs 1 and 2 for other experimental conditions. of ethanol on such an inhibition reaction. Figure 4 demonstrate that Bu.,NBr inhibits AChE activity and the degree of inhibition increases, as its concentration increases from 20 to 4OOpM. Comparative studies of the inhibition of AChE activity by Bu,NBr and tetraethylammonium bromide (Et,NBr) as shown in Fig. 4 reveal that BgNBr is a stronger inhibitor than Et.,NBr at a finite salt concentration, due to a larger size and a greater hydrophobicity of Bu (butyl) than Et (ethyl) group. The inhibitory effect of Bu,NBr on AChE activity is further supported by eke-kinetic measurements as presented in Fig. 5. The l/V data in the presence of Bu,NBr are above those in the absence of Bu,NBr (as the control), suggesting the inhibition of AChE activity by Bu,NBr. The figure shows a common intercept in the l/ATC axis in Lineweaver-Burke plots obtained from a computer least square analysis, indicating that Bu,NBr is a noncompetititve inhibitor (Krupka and Hellenbrand, 1974; Segel, 1975). The value of K, is determined as (1.82 f 0.20) x 10-r M (Table 1). The presence of ethanol significantly decreases the value of K,. Hydrophobic interaction between ByNBr and AChE is further demonstrated in microcalorimetric measurements on the heat of Ach hydrolysis reaction in the presence of quaternary ammonium salts o() containing various alkyl chain length. Table 2 shows
Fig. 5. The effect of Bu,NBr on the enzyme kinetics of AChE-catalyzed ATC hydrolysis reaction. LineweaverBurke plot of l/Y vs l/[ATC’j was performed in the absence and presence of Bu, NBr. Each data point is a mean value of 34 measurements with an experimental error of & 3%.
Table 2. Effects of quatemary ammonium salt (X) on the heat of Ach hydrolysis reaction’ Salt (Qb None Et,NBr
Pr,NBr Bu,NBr
Heat of reaction @.I) 12.65 f 0.63 20.36 f 1.02
3.53t0.18 -3.29 + 0.16
“Microcalorimetric mixing scheme is [Ach (1OOmM) + X(lf#lmM)] in 0.4ml bulk + AChE (1 mg) in 0.1 ml buffer + [Ach (SOmM) t X (80 mM) t AChE (I mg)] in 0.5 ml buffer, where the commerciaI AChE was used. bEt = ethyl, Pr = propyl and Bu = butyl. The ability for hydrophobic interaction is in the order of Bu,NBrr Pr,NBr > EtdNBr.
that the hydrolysis reaction is accompanied by an endothermal heat. In the presence of X, the heat of hydrolysis reaction is affected by: an inhibition of Ach hydrolysis reaction and the binding of X to AChE. As the hydrophobicity of X is enhanced from Et,NBr to BbNBr, the degree of Ach hydrolysis reaction is decreased, while hydrophobic interaction of X with AChE is increased. The observed heat change from a positive sign (endothe~~) to a negative sign (exothermal) confirms a strong hydrophobic interaction between Bu,NBr and AChE. The effect of ethanol on the inhibition of AChE activity by &NBr As indicated above, the study of the effect of ethanol on Bu,NBr inhibition of AChE activity is useful in the elucidation of the nature of ethanolAChE interaction. In the presence of a low concentration of ethanol (5.4 mM or less), Fig. 6 reveals that ethanol enhances the inhibition of AChE activity by Bu,NBr. In other words, this low [EtOH] makes Bu,NBr a more effective inhibitor. This finding is consistent with the results of enzyme kinetics studies as shown in Fig. 7, since the figure reveals that the data of l/V in Lineweaver-Burke plots in the presence of ethanol are systematically above those in the absence of ethanol (as the control), Nevertheless, the basic enzyme kinetics of Bu,NBr as a noncompetitive inhibitor is not affected by ethanol. From the common intercept in these plots as obtained from a least square computer analysis, the value of Ki in the presence of ethanol was found to be significantly lower than that in the absence of ethanol, decreasing from 1.82 to 0.40 x 10e3 M (Table 1). These results
173
Actions of ethanol on AChE
0
100
200
300
400
[Bu.NBrl (VW
Fig. 6. The effect of ethanol of the inhibition of AChE activity by Bu,NBr. See Figs 1 and 2 for experimental conditions.
confirm that the presence of a low concentration of ethanol enhances the binding of Bu,NBr to AChE, making Bu,NBr a more effective inhibitor. [Note: at a higher concentration of ethanol such as 200mM, more ethanol molecules are available to compete with Bu.,NBr for the hydrophobic site of AChE. Consequently, the interaction of Bu,NBr with AChE is weakened, causing a reduction in the inhibitory ability of Bu,NBr (S. Shin, L. Roth and C.-H. Chen; unpublished data).] DISCUSSION
The nature of the ethanoLAChE interaction For the assay of the ethanol effect on AChE activity, a dilute solution of the purified AChE was divided into two equal volumes, adding a volume of ethanol to one and an equal volume of buffer to the other. Thus, the ionic strength was kept constant and the dilution effect was considered over various concentrations of ethanol. The presence of ethanol increases AChE activity by IO%, which is small but systematic. It may be significant in consideration of the experimental error of f 3%. A similar observation
of a small increase in AChE activity in the presence of ethanol was also reported for AChE isolated from honeybee head and cockroach (Lewis, 1967; Colhoun, 1961). Our observation is not caused by a change in the pH of the medium, since the pH of the buffer solution is essentially unchanged in the presence of [EtOH] Q 200 mM. A low concentration of ethanol can affect enzyme activity by interacting with hydrophobic areas of an enzyme (Tabakoff et al., 1987). If the perturbation by ethanol of these areas causes an alteration in the hydrophobic site interaction on AChE and subsequently induces a favorable conformation for the active center of the enzyme (Taylor and Lappi, 1975; Berman et al., 1980; Wilson and Silman, 1977), then an increase in the activity of AChE will be observed. This speculation is supported by our observations: (1) ethanol’s noncompetitive modulation of the enzyme kinetics of AChE-catalyzed reaction; and (2) ethanol’s ability to perturb the inhibition of AChE activity by Bu,NBr. Besides ethanol-+nzyme interaction, another important consideration is the ethanol-solvent interaction. It has been reported that, at a low ethanol concentration, ethanol molecules are able to tie up many water molecules around them through hydrogen bonding (Franks and Ives, 1966; Franks and Smith, 1969). As a result of this, less water molecules will be available for an enzyme. This could provide AChE with a more hydrophobic environment for maintaining its conformation. On the other hand, at a high ethanol concentration, ethanol molecules can perturb the structure of water around hydrophobic areas of AChE (the dielectric constant of ethanol is 24 vs that of water, 78). Such a perturbation can weaken the hydrogen bonding between water molecules and therefore the structure of water (Franks, 1974). As a results of this, the presence of ethanol can induce an instability in the hydrophobic conformation of the enzyme, therefore causing a decrease in AChE activity at [EtOH] > 800 mM (see Figs 1 and 3). Distinction of the ethanoLAChE interaction from ethanol-lipid interaction AChE used in our studies is a soluble enzyme. To demonstrate that the above ethanol-AChE I
l/[ATC], 10’M“
Fig. 7. The effect of ethanol on the enzyme kinetics of AChE-catalyzed ATC hydrolysis inhibited by Bu,NBr. Lineweaver-Burke plot of l/V vs l/[ATC] was carried out in the absence and presence of ethanol and Bu,NBr. Each point is a mean value of 34 experiments with an experimental error of +3%. See Fig. 1 for other experimental conditions.
0
I
200 lEoHI
400 FM)
Fig. 8. The effect of ethanol on the activity of membranebound AChE isolated from bovine erythrocytes in 0.1 M potassium phosphate buffer pH 8.0, where [ATC] = 75 mM. AChE activity in the absence of ethanol is taken as the control, 100%.
