Department of Biochemistry, College of Science, King Saud University, P.O. Box: 2455, Riyadh 11451, Saudi Arabia

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1 Vol. 43, No. I, September 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages EFFECT OF MALATHION ON KINETIC PARAMETERS OF ACETYLCHOLINESTERASE (EC )IN VITRO MOHAMMAD AMJAD KAMAL Department of Biochemistry, College of Science, King Saud University, P.O. Box: 2455, Riyadh 11451, Saudi Arabia Received April l, 1997 Received alter revision May 28, 1997 SUMMARY Kinetic analysis of the interaction of malathion with camel erythrocyte acetylcholinesterase was investigated in the present study. The Michaelis-Menten constant (Kin) for the hydrolysis of acetylthiocholine iodide (ASCh) was found to be ~tm and the Vm, was gmol/min/mg protein. The Kmapp and V... pp were both decreased by increased malathion concentration. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the pure uncompetitive type with K~ value estimated as ppm. The Kiapp decreased while gmaxiap p increased by an increased concentration in ASCh. Key words: Acetylcholinesterase; malathion; inhibition; erythrocyte; kinetics INTRODUCTION Generally, acetylcholinesterase (ACHE; EC ) is an externally oriented, membranebound and highly efficient hydrolase enzyme which terminates the action of neurotransmitter acetylcholine (ACh) after its release at cholinergic synapses in the central and peripheral nervous systems, in this way allowing precise temporal control of muscle contraction [1]. It is a target enzyme for a variety of pharmacological agents, insecticides in widespread use, and nerve gases. The AChE occurs in erythrocytes as well as in neurons and is reported to be similar structurally and enzymically to the brain enzyme [2]. The erythrocyte AChE provides a readily available source of the enzyme to act as a model for studying the biochemical mechanisms by which inhibitors of this enzyme act [3-6] /97/ /0 Copyright by Academic Press Australia. All rights af reproduction in any farm reserved.

2 Vol. 43, No. 1, ] 997 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL The AChE is a very stable enzyme due to conformational plasticity of its active site [7]. In vivo, it is polymorphic in nature, consists of globular catalytic subunits [8]. in case of the dimer, it consists of two subunits, catalytic and regulatory subunits that have masses of 75 kd [7]. The sizes of the two subtmits of the AChE axe identical and these are linked with each other covalently through disulphide bridges. There are five domains in the AChE molecule: (1) an anionic locus, which is used for binding the substrate due to possessing a choline-binding pocket; (2) an esteratic locus, comprised of the active site serine and histidine that are involved in catalysis; (3) a hydrophobic region that is contiguous with or near the esteratic and anionic loci and that is important in binding aryl substrates and (hydrophobic) active site ligands (aromatic cations); (4) an allosteric site, located in the regulatory subunit of the ACHE, that is capable of binding cationic ligands such as gallamine, d- tubocurarine, decamethonium, noncompetitive, uncompetitive and mixed type inhibitors. When ligands bind with this site, t]mquently conformation of the active site is altered. Due to this allosteric site, complex reaction dynamics and active site conformation dynamics take place which are hallmarks of this enzyme. It involve in the regulation of the AChE activity by ligand binding with it. The one characteristic property of AChE is that inhibition by high concentration of substrate is well established and it is suggested that the peripheral anionic site is involved in this type of the inhibition [7-11]; (5) an acidic site of the active site of AChE [12]. These five important points are illustrated in figure 1. Inhibition studies can be used to determine the nature of the binding site of enzymes and are helpful in understanding the enzyme's catalytic characteristics. Moreover, in some cases the inhibition study is beneficial in therapy of some disorders, e.g., slow reversible inhibitors of AChE are used as a pretreatment drug for nerve-agent poisoning [ 13] and for the treatment of Alzheimer's disease [14], glaucoma and myasthenia gravis [15]. Malathion has been used as a pesticide since 1980, due to its low toxicity to humans [7]. The aim of the present investigation is to study the interaction of malathion with a mammalian source of AChE which is very common in Gulf countries, i.e. camel erythrocyte membrane bound AChE to provide some insight in its mechanism of action as well as its binding with the domains of ACHE. MATERIALS AND METHODS Materials: All reagents were of analytical grade. Acetylthiocholine (used as substrate, ASCh) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bovine serum albumin and Triton X-100 were obtained from Fluka Chemika-Bio Chemika. 90

3 BIOCHEMISTRYend MOLECULAR BIOLOGY INTERNATIONAL Ac~ po ACh E ( a dimer ) Fig. 1 Proposed model for the representation of the binding sites of AChE for substrate and some inhibitors. Imidazole, hydroxyl of the histidine (His) and serine (Ser) are shown within the esteratic site. While within the anionic and allosteric subsite, (COO')n, represents negative charges due to carboxylic amino acids. The A. S, E. S, A1. S, M. t, Un. c, N. c, ACh, A. C. B. S represents anionic subsite, esteratic subsite, allosteric subsite, mixed type, uncompetitive, non-competitive inhibitor, acetylcholine and aromatic cation binding subsite respectively. Enzyme Preparation." Camel blood was collected in the presence of citrate phosphate-dextroseadenine solution as an anticoagulant from healthy young males (Camelus dromedarius) during slaughtering at a local slaughter house. Erythrocytes were separated from plasma by centrifugation at 1000 x g for 30 min at 4 ~ C, the plasma and the buffy coat removed by aspiration. The cells were washed twice with 10 vol. of 175 mm, Tris/HC1 buffer, ph 7.6. The washed cells were re-suspended in the same 175 mm, isotonic Tris buffer to a hematocrit of 50% and mixed well by inversion for 1 min. The 1 vol. of 50% hematocrit was added dropwise with constant stirring in 7.5 vol. of lysis buffer (10 mm Tris/HC1 buffer, ph 7.8). After lysis the RBC, "ghosts" were harvested by centrifugation at 100,000 x g, 60 min at 4 ~ C and the supematant removed by suction. The ghosts was homogenized using a tissue homogenizer by applying 4/5 strokes (8000/min) in the presence ofprecooled 0.05 M sodium phosphate buffer, ph 7.2, to produce the homogenate. The homogenate was centrifuged at 100,000 x g for 60 min at 4 ~ C. The supernatant was discarded and the pellet suspended in Triton X-100 (1.0 %). The mixture was stirred, re-homogenized and clarified by recentrifugation. The supernatant was collected and the AChE activity in the supernatant was used as "membrane bound ACHE". 91

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Enzyme assays. AChE-catalyzed hydrolysis of the ASCh was followed by the spectrophotometric assay of Ellman et al. [16]. The 5 min. incubation time was selected for the enzyme assay at 22 ~ C. The concentration of DTNB in the assay was same as reported earlier [ 17-19]. Michaelis-Menten constants were estimated by means of Lineweaver-Burk plot [20] using initial velocities obtained over a substrate concentration range from 0.02 to 0.2 mm. The assay conditions for determining the residual activities in the presence of malathion were identical to the above assay procedure, except that a fixed concentration (50.0 to ppm) of malathion was used in the assay medium. Estimation of protein. The protein content of the enzyme preparation was estimated according to the method of Lowry et al. [21], using bovine serum albumin as standard. The detergent, Triton X- 100 interfered with this estimation, but this problem was overcome as mentioned previously [22-24]. The graphs and correlation coefficients were obtained by using GraFit program [25]. RESULTS The value of Km for AChE was determined to be gm in the control system; a value decreased by % in the presence of the malathion ( ppm) (Fig. 2). In association with this change in Kin, there was a decrease in V... of % (Table 1). The (i ~ 024 5" E ~ r 0 0s o ~6 [>,, ~ 1 / 1 L... I i~o,.n,,~ LA --1 [Malathion (ppm) < I -D I ~G k > 16 _ - j I i I I I I J I ~ f' I I / [ASCh] (mm) 1 Fig. 2 Lineweaver-Burk plot of reciprocal of the initial AChE velocity versus reciprocal of the ASCh concentration in the absence and presence of malathion. The correlation coefficient in case of o, e, rt, m, ~x and 9 was , , , , and respectively. Each point represents the mean of four determinations. A inset Hyperbolic plot of v versus [ASCh] to represent the pattern of inhibition through original data i.e. without any transformation. B inset Secondary replot of the Lineweaver-Burk plot, where correlation coefficient was found as and for l/vmaxapp and 1/Kmapp respectively. 92

5 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL TABLE 1 Effect of malathion on kinetic parameters of camel erythrocyte acetylcholinesterase [Malathion] Kmapp % decreased V... uu % decreased (ppm) (gm) (gm/min/mg) The Kmapp and V... pp were determined by their respective regression equations as well as by the Lineweaver-Burk plot (Fig. 2). The Km.pp is equal to reciprocal of the abscissa intercept while V... pv is the reciprocal of the ordinate intercept for each malathion concentration within the Lineweaver-Burk plot. Vmax was also determined by the secondary reptot (1/V... pp versus malathion concentration) of the Lineweaver-Burk plot (Fig. 2 B inset) and the secondary replot i.e. 1/Vm~x~apu versus 1/[ASCh] of the Dixon plot in Fig. 3, and are represented in the Fig. 3 inset. The Kmapp and V... up were determined by their respective regression equations. The Kmapp is the reciprocal of the abscissa intercept while V... pp the reciprocal of the ordinate intercept for each malathion concentration within the Lineweaver-Burk plot. The nature of the inhibition was of the uncompetitive type in this case. The inhibition constant Ki was estimated by applying the method of Dixon [26] in which 1/v was plotted versus the malathion concentration for each ASCh concentration (Fig. 3), from the secondary replots (1/Km~pp and 1/V... ppversus malathion concentration) of the Lineweaver Burk plot (Fig. 2 B inset) and secondary replot (K~,ppVersus 1/[ASCh]) of the Dixon plot (Fig. 3 inset) [27]. The values of the apparent inhibition constants, Kiapp and Vmaxiaup which were determined by their respective regression equation are presented in Table 2. The Kiapp is equal to the abscissa intercept while Vm,x~,Up is equal to the reciprocal of the ordinate axis for each ASCh concentration in the Dixon plot (Fig. 3). As indicated in tabte 2, the K~,pp decreased from % while Vm,~pp increased between and 172 % over the range of ASCh concentration used while the slope was unchanged. The value of the three important parameters such as K~, Vm,~ and K~ were determined by different methods and presented in table 3. 93

6 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL DISCUSSION The decrease in K m and V... values would suggest that malathion increases substrate affinity for the anionic substrate binding site with decreasing catalytic activity (V~ due to conforrnational changes in the ACHE. However, the decrease in Kmapp value with malathion occurred because the reaction (AChE-ASCh + Malathion - AChE - ASCh - Malathion) removes some of the AChE - ASCh complex thereby causing the reaction (AChE + ASCh -, AChE - ASCh) to proceed to the right. Therefore the Km value was decreased in the presence of malathion as in the case of AChE in older bovine erythrocytes [28]. The nature of the inhibition of AChE by malathion is pureuncompetitive since the 1/V... pp versus [malathion] is linear (Fig. 2 B inset) [29]. The malathion decreases the V... pp and Kmapp in a similar fashion as expected in the case of pure uncompetitive inhibition, as clearly observed from the style of the primary plots in figures 2 A inset and 3. According to the substrate hydrolysis scheme for AChE [30], malathion can interact with the AChE-ASCh complex stage or the acetylated-ache stage. The former possibility is more probable, because if malathion was bound to acetylated-ache, thiocholine (first product of hydrolysis; PI) X w 2 -~'rg [ASCh] (mm) E "e ;> tz II G ~ I f r I I r i I I I [Malathion] (ppm) Fig. 3. Dixon plot for camel erythrocyte membrane-bound AChE at six concentrations of ASCh as shown in legend box. The correlation coefficient in case ofo, e, m, L zx and 9 was , , , , and respectively. Each point represents the mean of four experiments. inset Secondary replot of the Dixon plot, where correlation coefficient was and for 1/Vmaxiap p and Kiapp respectively. 94

7 BIOCHEMISTRY(rod MOLECULAR BIOLOGY INTERNATIONAL TABLE 2 Effect of ASCh on kinetic parameters of camel erythrocyte acetylcholinesterase [ASCh] Kiapp % decreased Vrnaxiapp % increased (mm) (ppm) (gm/min/mg) , , , The K~,pp and Vma were determined by their respective regression equations and Dixon plot (Fig. 3). The Kiapp is the abscissa intercept while Vmaxi,pp is the reciprocal of the ordinate intercept for each ASCh concentration within the Dixon plot. TABLE 3 Kinetic constants estimated by various methods Method Km V~ax K~ (~tm) (gm/min/mg) (ppm) Primary plot Secondary replot ~LBP Secondary replot 2LBP Secondary replot mr Secondary replot 2DP Mean value ,1 S. E.M , The details of primary plot and secondary replots have been given in the text while LBP means Lineweaver-Burk plot and DP means Dixon plot. 1L~p and 2LBP represents replot of 1/V... pp and 1/Km,pp (from Lineweaver-Burk plot) versus malathion concentration respectively (Fig. 2 B inset). ~DP,,d 2DP represents replot of Kiapp and 1/Vmaxiap p (from Dixon plot)versus 1/[ASCh] respectively (Fig. 3 inset). 95

8 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL would not be affected, while the results show that PI is affected (because optical density was decreased at 412 nan due to a decrease in the color product) by the increase of the concentration of malathion. This means that malathion did not bind with acetylated-ache and surely bound with AChE-ASCh complex. Now, considering the case of AChE-ASCh complex, the anionic site is occupied by the trimethylammonium head of choline of the ASCh and thus is not available to a second ligand. Therefore malathion must bind to another site known as the allosteric peripheral anionic site. The binding of malathion to the peripheral allosteric site within the AChE-ASCh complex stage, is also supported by one latest report, in which it is stated that the nature of inhibition of human erythrocyte AChE by organophosphorus insecticides phosphamidon and malathion was uncompetitive [31]. Moreover, there is one general statement that organophosphorus inhibitors of AChE may also react at the peripheral site [32-34]. The reports about this type of inhibition system (i.e. pure uncompetitive) are rare in the literature, e.g. only HSR-803, two parallel plane acridine araphanes, propidium, cisplatin and malathion are reported as uncompetitive inhibitors of AChE [11, 27, 31, 35-37]. These results reflect that malathion produces a conformational change in the camel erythrocyte AChE by binding with the AChE-ASCh complex, yielding a non-productive ACHE- ASCh-Malathion complex. In this way, malathion decreases the activity of the enzyme since it prevents the proper positioning of the catalytic center for acetylation with substrate to form acetylated ACHE. REFERENCES 1. Eichler, J., Anselmeet, A., Sussman, J.L., Massoulie, J. and Silman, I. (1994) Mol. Pharmacol., 45, Kamal, M.A. and A1-Jafari, A.A. (1996) Cell. Pharm., 3, A1-Jafari, A. A., Duhaiman, A. S. and Kamal, M. A. (1995) Toxicology, 96, A1-Jafari, A.A., Kamal, M.A. and Duhaiman, A.S. (1995) J. Enz. Inhib. 8, A1-Khweetyer, F., Kamal, M.A. and A1-Jafari, A.A. (1996) Toxicol. Lett. 87/2, 3, Kamal, M.A and A1-Jafari, A.A. (1996) Prep. Biochem. Biotech. 26, Quinn, D.M. (1987) Chem. Rev. 87, Shafai, J. and Cortner, J.A. (1971) Biochem. Biophys. Acta, 236, Ott, P., Jeny, B. and Brodbeck, U. (1975) Eur. J. Biochem. 57, Sussman J.L., Harel M., Frolow F., Oefner C., Goldman A., Toker L. and Silman I. (1991) Science, 253, Taylor, J.L., Mayer, R.T. and ttimel, C.M. (1994) Mol. Pharmacol. 45, Krupka, R.M. and Laidler, K.J. (1961) J. Med. Chem. 83, Green, A.L. (1983) Biochem. Pharmacol., 32,

9 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Giacobini, E., Becker, R., Elbe, R., Mattio, T., Mcllhany, M. and Scaxsella, G. (1987) Neurobiology of acetylcholine (Dun, N., ed.), pp , Plenmn Press, New York. Gray, P. (1991) Biochem. J., 274, Ellman, G. L., Courtney, D., Andres, V. and Featherston, R.M. (1961) Biochem. Pharmacol., 7, A1-Jafari, A.A and Kamal, M.A. (1996)Biochem. Mol. Biol. Int. 38, A1-Jafari, A.A, Kamal, M.A., Duhaiman, A. S. and Al-homaida, A.S. (1996) J. Enz. Inhibit. 1996, 11, A1-Jafari, A.A, Kamal, M.A., Duhaiman, A. S. and Al-homaida, A.S. (1995) Mol. Cell. Biochem., 151, Lineweaver, H. and Burk, D. (1934) J. Amer. Chem. Soc. 56, Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1953) J. Biol. Chem.193, Kamal, M.A., Nasim, F.H. and A1-Jafari, A.A. (1996) Mol. Cell. Biochem., 159, Kamal, M.A., Nasim, F.H. and A1-Jafari, A.A. (1996) Biochem. Mole. Biol. Int., 39 (2), A1-Jafari, A.A, A1-Khweetyer, F., Kamal, M.A. and Al-homaida, A.S. (1996) Japn. J. Pharmacol., 72 (1), A1-Jafari, A.A, Kamal, M.A. and Al-homaida, A.S. (1997) J. Enz. Inhibit.,11, Dixon, M. (1953) Biochem. J., 55, Kamal, M.A. (1996) Anticancer Res., 16, Grzelinska, E., Bartosz, G. and Bartkowiak, A. (1983) Enzyme 30, Segel, I.H. (1975) Behaviour and analysis of rapid equilibrium and steadystate enzyme systems: Enzyme kinetics, pp New York: John Wiley and Sons. Cohen, S. G., Chishti, S. B., Bell, D. A., Howard, S. I., Salih, E. and Cohen, J.B. (1991) Biochem. Biophys. Acta. 1076, Datta, C., Gupta, G. and Sengupta, D. (1994) Ind. J. Med. Res., 100, Friboulet, A., Goudou, D. and Rieger, F. (1986)Neurochem. Int., 9, Friboulet, A., Rieger, F., Goudou, D., Amitai, G. and Taylor, P. (1990) Biochem, 29, Michaelson, S. and Gray, P. (1991) Biochem. Pharmacol., 42, Kishibayashi, N., Ishii, A. and Karasawa, A. (1994) Jpn. J. Pharmacol., 66, Taylor, P. and Lappi, S. (1975) Biochem., 14, Shin, S., Roth, L.G. and Chen, C.H. (1991) Int. J. Biochem., 23,