- Email: [email protected]
Amitriptyline: a potent inhibitor of butyrylcholinesterase from human serum
ISSN 0306-3623/97 $17.00 + .00 PII S0306-3623 (97)00004-9 All rights reserved
Gen. Pharmac. Vol. 29, No. 5, pp. 835-838, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
Amitriptyline: A Potent Inhibitor of Butyrylcholinesterase from Human Serum A. Ne~e (~oku~ca4and E. Ferhan Tezcan* HACETTEPE UNIVERSITY, FACULTY OF MEDICINE, DEPARTMENT OF BIOCHEMISTRY, ANKARA06100, TURKEY [TEL:+90 312 324 58 85; FAX:+90 312 310 05 80]
ABSTRACT. 1. The effect of amitriptyline on human serum butyrylcholinesterase (acylcholine acylhydrolase E.C.3.1.1.8) has been investigated. From the Lineweaver-Burk plot and the plot of v versus amitriptyline concentration, it was concluded that amitriptyline inhibition is partially competitive, and the kinetic parameters have been calculated as Ks=O.11 mM, a = 1425 and K/=O.O1 mM. 2. Because amitriptyline is a partial competitive inhibitor of butyrylcholinesterase, acquired deficiency may be seen in patients treated with amitriptyline and may cause complications in operations. OEN PHARMAC29;5:835--838, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Amitriptyline, butyrylcholinesterase INTRODUCTION Amitriptyline (AMI) is widely used for the treatment of major depressive disorders and chronic pain and for the prophylactic therapy of migraine (Schmider et 02., 1995). The drug has the anticholinergic side effects manifested in symptoms such as dry mouth, blurred vision, constipation and sinus tachycardia (Aaltonen et al., 1985). It can be suggested that these side effects are due to the antagonizing effect of tricyclic antidepressants on the cholinergic receptors in the brain and periphery (Schein and Smith, 1978) or to the inhibition of cholinesterases (Perkinson et 02., 1969) or to both. Vertebrates have two different cholinesterases: acetylcholinesterase (ACHE, E.C.3.1.1.7) and butyrylcholinesterase (BChE, EC.3.1.1.8). There are similarities in the protein sequence and in the molecular forms of AChE and BChE but differences in their kinetic properties (Chatonnet and Lockridge, 1989). They are exactly distinct enzymes encoded by two different but related genes (Arpagaus et 02., 1990). BChE, also called cholinesterase, nonspecific cholinesterase or pseudocholinesterase, is present in all tissues and body fluids (Prody et 02., 1987; Ryh~inen, 1983). It catalyzes the hydrolysis of both hydrophilic and hydrophobic choline esters (Hijikata-Okunomiya et al., 1988; Whetherell and French, 1986). BChE is the most abundant cholinesterase in human serum synthesized by the liver (Prody eta/., 1987). Although the exact physiological function of BChE is still unclear, the assay of serum BChE activity is especially used in the diagnosis of pesticide poisoning and in the assessment of patients with prolonged apnea after administration of succinytcholine during anesthesia (Nelson and Burritt, 1986). In the present study, a detailed kinetic analysis of human serum BChE inhibition by AMI was carried out, and the inhibition mechanism was described. MATERIALS A N D METHODS
Mater/a/s Human serum BChE was purchased from Sigma Chemical Co. (St Louis, MO, U.S.A). It was further purified by using Sepharose CI-6B *To whom correspondence should be addressed. Received 16 July 1996; revised 19 December 1996; accepted 9 January 1997.
column and kept frozen in 10 mM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer (pH 7.5). AMI also was obtained from Sigma. All other reagents used were analytical grade.
E n z y m e assay Enzyme activities were measured spectrophotometrically with a Beckman spectrophotometer Model 25 according to the procedure of Ellman et o2. (1961). Initial velocities were measured at 37°C by using 0.25 mM 5,5'-dithio-bis (2-nitro benzoic acid) in 5 mM MOPS buffer (pH 7.5) and in increasing concentrations of butyrylthiocholine (BuTCh) iodide from 0.3 to 4.0 mM in the absence and in the presence of 0.2, 0.4, 0.6, 0.8 mM AMI. The amount of enzyme used was 32 I~g/ml of assay mixture. Assays were carried out in duplicate and the activities were measured for up to 90 sec. The reaction was linear during this period. In the measurements, a blank tube containing all compounds except enzyme was used. So the spontaneous hydrolysis of BuTCh has been eliminated.
Protein assay Protein concentration was calculated from absorbance at 280 nm, using an extinction coefficient of 1.8 cm 1 for a 1 mg/ml solution (Lockridge and LaDu, 1978). RESULTS The effect of AMI on serum BChE has been investigated in detail. The 1/v versus I/[BuTCh] plot, in the absence (control) and in the presence of different fixed concentrations of AMI intersected on the 1/v axis (Fig. 1). This behavior indicates that inhibition of the enzyme by AMI is purely or partially competitive. Partial competitive inhibition could be distinguished from pure competitive inhibition by plotting v versus [AMI] at a fixed [BuTCh] (Fig. 2). It is seen that the curve reaches a plateau, suggesting that inhibition was partially competitive. Otherwise, if the inhibition were purely competitive, the curve would approach zero (Segel, 1975).
836
A . N . (7oku~ra~ and E. F. Tezcan
1.1
0.9
0.7 FIGURE 1. Reciprocal plot in the absence of AMI control (solid circles) and in the presence of 0.2 mM (triangles), 0.4 mM (asterisks) 0.6 mM (squares) 0.8 mM (open circles) AMI concentrations.
E d 0.5
E --,...A
E
z~k
o.3
0.1
-2
-1
0
1
2
3
l l l B u T C h ] , mM
In this situation, the secondary plot, slope versus [AMI], must be a hyperbola. For this reason, c~ and Ki values were determined from the plot of 1/A slope versus 1/[AMI] (Fig. 3). So, the hyperbola transforms into a straight line. The A slope values were calculated by subtracting the slope of the control line from the slopes of the lines obtained at different concentrations of AMI in Fig. 1. From the intercepts of the straight line, Ki (the dissociation constant of the enzyme-AMI complex) was found to be 0.01 mM and a (the factor by which Ks and Ki change when AMI occupies the enzyme) was calculated as 1425. Ks (the dissociation constant of enzyme-BuTCh complex) was determined as 0.11 mM from the control line shown in Fig. 1. Linear regression analysis was used in the calculation of the kinetic parameters. DISCUSSION The inhibitions of cholinesterases from dog plasma and from rat brain by tricyclic antidepressants have been reported (Perkinson et al., 1969). But there have not been any detailed studies of the effect of AMI on serum BChE. In this study, we have shown that AMI partially inhibits the enzyme competitively. BChE is a tetramer and the subunits are arranged as a dimer of dimers. Each dimer is composed of two identical subunits joined by interchain disulfide bonds (Lockridge et al., 1979). It has also been shown that the enzyme exhibits negative cooperativity with respect
to BuTCh (q2oku~ra§ and Tezcan, 1993a). Because the enzyme is an allosteric enzyme and the inhibition of AMl is partially competitive, it can be said that AMI is bound to a secondary site and not to the active sites of the subunits of BChE. The active site of BChE consists of two parts: the anionic site, which binds the choline moiety of the substrates, and the esteratic site, which hydrolyzes the ester bond (LaDu, 1971). Additionally, near the active site, there is a hydrophobic pocket that interacts with huge hydrophobic parts of the substrates (Kabachnik et al., 1970) (Fig. 4). AMI has a side chain with a dimethylamino group that resembles choline (Fig. 5). It may be that this side chain is bound to a choline binding site described previously for sheep brain BChE (~oku~ra§ and Tezcan, 1993b) or that the tricyclic part of AMI interacts with the hydrophobic pocket of the enzyme or both. So these interactions disturb the steric properties of the enzyme, and AMI acts as a partial competitive inhibitor. A large number of allelic variants of the human BChE gene have been described. The best-known allelic variants of the enzyme are the usual, atypical, fluoride-resistant and the silent phenotypes. Whereas the atypical and fluoride-resistant variants have lower BChE activity than does the usual variant, the silent phenotype has no activity (Massouli~ et al., 1993). Individuals with inactive or less-active BChE enjoy normal health unless they undergo surgery. Succinyldicholine is a neuromuscular blocking agent commonly used during anesthesia in operations, and its effect is terminated by hydrolysis by plasma BChE (Hobbiger and Peck, 1969). Inherited or
Amitriptyline Inhibition of Butylcholinesterase
837
12 100 10
80 e~
8
o
rJ 3
<1 - 6 60 4
.g < 40
Vm Ks (et- I)
/T
I
I
I
I
i
1
2
3
4
5
1 c~Ki
20
I/[AMI], mM"~ I
I
I
I
0.5
1.0
1.5
2.0
F I G U R E 3. The plot of 1/A slope versus 1/[AMI]. The A slopes were calculated by subtracing the slope of the control line from the slopes of the lines with A M I in the reciprocal plot.
(AMI), mM F I G U R E 2. The effect of AMI on BChE activity at 4.0 mM BuTCh.
acquired deficiency of BChE causes prolonged apnea and even death after a patient receives succinylcholine (Nelson and Burritt, 1986). Because AMI is a partial competitive inhibitor of BChE, acquired deficiency may be seen in patients treated for a long term with AMI. If these patients have to be operated on because of an emergency, the possibility of succinylcholine apnea must be considered. References Aaltonen L., Syv~ilahtiE., Iisalo E. and Peltom~ikiT. (1984) Anticholinergic activity in the serum of patients receiving maintenance amitriptyline or doxepin therapy. Acre Pharmac. Toxicol. 56, 75-80. Arpagaus M., Knott M., Vatsis K. P., Bartels C. F., LaDu B. N. and Lockridge O. (1990) Structure of the gene for human butyrylcholinesterase: evidence for a single copy. Biochemistry 29, 124-131. Chatonnet A. and Lockridge O. (1989) Review: comparison of butyrylcholinesterase and acetylcholinesterase. Biochem. J. 260, 625-634. (~oku~ra~ A. N. and Tezcan E. F. (1993a) Inhibition kinetics of brain butyrylcholinesterase by Cd > and gn 2+, Ca 2+ or Mg> reactivates the inhibited enzyme. Int. J. Biochem. 25, 1115-1120. ~oku~ra~ A. N. and Tezcan E. F. (1993b) Sheep brain pseudocholinesterase: inhibition kinetics of the partially purified enzyme by some substrate analogues. Chem.-Biol.fnteract. 87, 259-264. Ellman G. C., Courmey K. P., Andres V. Jr. and Featherstone R. M. (1961) A new rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88-95. Hijikata-Okunomiya A., Okamoto S., Tamao Y. and Kikumoto R. (1988) N-Dansyl-r-arginine 4-phenylpiperidine amide: a potent and selective inhibitor of horse serum cholinesterase. J. Biol. Chem. 23, 11,26911,273.
Anionic Site
Esteratic Site
~Hydrophobi~ }tff,lll, l,lf,lP°cket
/I/11/1-~
.I V _
H3C
}
C// ~CH2_CH2_
O/
N~H 2 -CH 3
F I G U R E 4. Active center of BChE monomer [modified from LaDu (1971)].
H2-~ CH-CH2-CH2-CHa N II
/CH3
F I G U R E 5. Chemical structure of AMI.
838 Hobbiger F. and Peck A. W. (1969) Hydrolysis of suxamethonium by different types of plasma. Br. J. Pharmac. 37, 258-271. Kabachnik M. I., Brestkin A. P., Godovikov N. N., Michelsen M. N., Rozengart E. V. and Rozengart V. I. (1970) Hydrophobic areas on the active surface of chotinesterases. Pharmac. Rev. 22, 355-388. LaDu B. N. ( 1971) Plasma esterase activity and the metabolism of drags with ester groups. Ann. N. Y. Acad. Sci. 179, 684-694. Lockridge O., Eckerson H. W. and LaDu B.N. (1979) lnterchain disulfide bonds and subunit organization in human serum cholinesterase. J. Biol. Chem. 254, 8324-8330. Lockridge O. and LaDu B. N. (1978) Comparison of atypical and usual human serum cholinesterase: purification, number of active sites, substrate affinity and turnover number. J. Biol. Chem. 253, 361-366. Massouli~ J., Pezzementi L., Bon S., Krejci E. and Vallette F. M. (1993) Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 31-91. Nelson T. C. and Burritt M. F. (1986) Pesticide poisoning, succinylcholineinduced apnea, and pseudocholinesterase. Mayo Clin. Proc. 61,750--755. Perkinson E., Ruckart R. and Davanzo J. P. (1969) Pharmacological and bio-
A . N . ~oku~ra~ and E. F. Tezcan chemical comparison of lithium and reference antidepressants. Proc. Exp. Biol. bled. 131,685-689. Prody C. A., Zevin-Sonkin D., Gnatt A., Golberg O. and Soreq H. (1987) Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues. Proc. Natl. Acad. Sci. U.S.A. 84, 3555-3559. Ryh~inen R. J. J. (1983) Pseudocholinesterase activity in some human body fluids. Gen. Pharmac. 14, 459-460. Schein K. and Smith S.E. (1978) Structure-activity relationship for the anticholinoceptor action of tricyclic antidepressants. Br. J. Pharmac. 62, 567-571. Schmider J., Greenblatt D. J., Von Moltke L. L., Harmatz J. S. and Shader R.I. (1995) N-Demethylation of amitriptyline in vitro: role of cytochrome P-4503A (CYP 3A) isoforms and effect of metabolic inhibitor. J. Pharmac. Exp. Ther. 275, 592-597. Segel I. H. (1975) Enzyme Kinetics. pp. 161-166. Wiley, Toronto. Whetherell J. R. and French M. C. (1986) The hydrolysis of succinyldithiocholine and related thiocholine esters by human plasma and purified cholinesterase. Biochem. Pharmac. 35, 939-945.
Gen. Pharmac. Vol. 29, No. 5, pp. 835-838, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
Amitriptyline: A Potent Inhibitor of Butyrylcholinesterase from Human Serum A. Ne~e (~oku~ca4and E. Ferhan Tezcan* HACETTEPE UNIVERSITY, FACULTY OF MEDICINE, DEPARTMENT OF BIOCHEMISTRY, ANKARA06100, TURKEY [TEL:+90 312 324 58 85; FAX:+90 312 310 05 80]
ABSTRACT. 1. The effect of amitriptyline on human serum butyrylcholinesterase (acylcholine acylhydrolase E.C.3.1.1.8) has been investigated. From the Lineweaver-Burk plot and the plot of v versus amitriptyline concentration, it was concluded that amitriptyline inhibition is partially competitive, and the kinetic parameters have been calculated as Ks=O.11 mM, a = 1425 and K/=O.O1 mM. 2. Because amitriptyline is a partial competitive inhibitor of butyrylcholinesterase, acquired deficiency may be seen in patients treated with amitriptyline and may cause complications in operations. OEN PHARMAC29;5:835--838, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Amitriptyline, butyrylcholinesterase INTRODUCTION Amitriptyline (AMI) is widely used for the treatment of major depressive disorders and chronic pain and for the prophylactic therapy of migraine (Schmider et 02., 1995). The drug has the anticholinergic side effects manifested in symptoms such as dry mouth, blurred vision, constipation and sinus tachycardia (Aaltonen et al., 1985). It can be suggested that these side effects are due to the antagonizing effect of tricyclic antidepressants on the cholinergic receptors in the brain and periphery (Schein and Smith, 1978) or to the inhibition of cholinesterases (Perkinson et 02., 1969) or to both. Vertebrates have two different cholinesterases: acetylcholinesterase (ACHE, E.C.3.1.1.7) and butyrylcholinesterase (BChE, EC.3.1.1.8). There are similarities in the protein sequence and in the molecular forms of AChE and BChE but differences in their kinetic properties (Chatonnet and Lockridge, 1989). They are exactly distinct enzymes encoded by two different but related genes (Arpagaus et 02., 1990). BChE, also called cholinesterase, nonspecific cholinesterase or pseudocholinesterase, is present in all tissues and body fluids (Prody et 02., 1987; Ryh~inen, 1983). It catalyzes the hydrolysis of both hydrophilic and hydrophobic choline esters (Hijikata-Okunomiya et al., 1988; Whetherell and French, 1986). BChE is the most abundant cholinesterase in human serum synthesized by the liver (Prody eta/., 1987). Although the exact physiological function of BChE is still unclear, the assay of serum BChE activity is especially used in the diagnosis of pesticide poisoning and in the assessment of patients with prolonged apnea after administration of succinytcholine during anesthesia (Nelson and Burritt, 1986). In the present study, a detailed kinetic analysis of human serum BChE inhibition by AMI was carried out, and the inhibition mechanism was described. MATERIALS A N D METHODS
Mater/a/s Human serum BChE was purchased from Sigma Chemical Co. (St Louis, MO, U.S.A). It was further purified by using Sepharose CI-6B *To whom correspondence should be addressed. Received 16 July 1996; revised 19 December 1996; accepted 9 January 1997.
column and kept frozen in 10 mM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer (pH 7.5). AMI also was obtained from Sigma. All other reagents used were analytical grade.
E n z y m e assay Enzyme activities were measured spectrophotometrically with a Beckman spectrophotometer Model 25 according to the procedure of Ellman et o2. (1961). Initial velocities were measured at 37°C by using 0.25 mM 5,5'-dithio-bis (2-nitro benzoic acid) in 5 mM MOPS buffer (pH 7.5) and in increasing concentrations of butyrylthiocholine (BuTCh) iodide from 0.3 to 4.0 mM in the absence and in the presence of 0.2, 0.4, 0.6, 0.8 mM AMI. The amount of enzyme used was 32 I~g/ml of assay mixture. Assays were carried out in duplicate and the activities were measured for up to 90 sec. The reaction was linear during this period. In the measurements, a blank tube containing all compounds except enzyme was used. So the spontaneous hydrolysis of BuTCh has been eliminated.
Protein assay Protein concentration was calculated from absorbance at 280 nm, using an extinction coefficient of 1.8 cm 1 for a 1 mg/ml solution (Lockridge and LaDu, 1978). RESULTS The effect of AMI on serum BChE has been investigated in detail. The 1/v versus I/[BuTCh] plot, in the absence (control) and in the presence of different fixed concentrations of AMI intersected on the 1/v axis (Fig. 1). This behavior indicates that inhibition of the enzyme by AMI is purely or partially competitive. Partial competitive inhibition could be distinguished from pure competitive inhibition by plotting v versus [AMI] at a fixed [BuTCh] (Fig. 2). It is seen that the curve reaches a plateau, suggesting that inhibition was partially competitive. Otherwise, if the inhibition were purely competitive, the curve would approach zero (Segel, 1975).
836
A . N . (7oku~ra~ and E. F. Tezcan
1.1
0.9
0.7 FIGURE 1. Reciprocal plot in the absence of AMI control (solid circles) and in the presence of 0.2 mM (triangles), 0.4 mM (asterisks) 0.6 mM (squares) 0.8 mM (open circles) AMI concentrations.
E d 0.5
E --,...A
E
z~k
o.3
0.1
-2
-1
0
1
2
3
l l l B u T C h ] , mM
In this situation, the secondary plot, slope versus [AMI], must be a hyperbola. For this reason, c~ and Ki values were determined from the plot of 1/A slope versus 1/[AMI] (Fig. 3). So, the hyperbola transforms into a straight line. The A slope values were calculated by subtracting the slope of the control line from the slopes of the lines obtained at different concentrations of AMI in Fig. 1. From the intercepts of the straight line, Ki (the dissociation constant of the enzyme-AMI complex) was found to be 0.01 mM and a (the factor by which Ks and Ki change when AMI occupies the enzyme) was calculated as 1425. Ks (the dissociation constant of enzyme-BuTCh complex) was determined as 0.11 mM from the control line shown in Fig. 1. Linear regression analysis was used in the calculation of the kinetic parameters. DISCUSSION The inhibitions of cholinesterases from dog plasma and from rat brain by tricyclic antidepressants have been reported (Perkinson et al., 1969). But there have not been any detailed studies of the effect of AMI on serum BChE. In this study, we have shown that AMI partially inhibits the enzyme competitively. BChE is a tetramer and the subunits are arranged as a dimer of dimers. Each dimer is composed of two identical subunits joined by interchain disulfide bonds (Lockridge et al., 1979). It has also been shown that the enzyme exhibits negative cooperativity with respect
to BuTCh (q2oku~ra§ and Tezcan, 1993a). Because the enzyme is an allosteric enzyme and the inhibition of AMl is partially competitive, it can be said that AMI is bound to a secondary site and not to the active sites of the subunits of BChE. The active site of BChE consists of two parts: the anionic site, which binds the choline moiety of the substrates, and the esteratic site, which hydrolyzes the ester bond (LaDu, 1971). Additionally, near the active site, there is a hydrophobic pocket that interacts with huge hydrophobic parts of the substrates (Kabachnik et al., 1970) (Fig. 4). AMI has a side chain with a dimethylamino group that resembles choline (Fig. 5). It may be that this side chain is bound to a choline binding site described previously for sheep brain BChE (~oku~ra§ and Tezcan, 1993b) or that the tricyclic part of AMI interacts with the hydrophobic pocket of the enzyme or both. So these interactions disturb the steric properties of the enzyme, and AMI acts as a partial competitive inhibitor. A large number of allelic variants of the human BChE gene have been described. The best-known allelic variants of the enzyme are the usual, atypical, fluoride-resistant and the silent phenotypes. Whereas the atypical and fluoride-resistant variants have lower BChE activity than does the usual variant, the silent phenotype has no activity (Massouli~ et al., 1993). Individuals with inactive or less-active BChE enjoy normal health unless they undergo surgery. Succinyldicholine is a neuromuscular blocking agent commonly used during anesthesia in operations, and its effect is terminated by hydrolysis by plasma BChE (Hobbiger and Peck, 1969). Inherited or
Amitriptyline Inhibition of Butylcholinesterase
837
12 100 10
80 e~
8
o
rJ 3
<1 - 6 60 4
.g < 40
Vm Ks (et- I)
/T
I
I
I
I
i
1
2
3
4
5
1 c~Ki
20
I/[AMI], mM"~ I
I
I
I
0.5
1.0
1.5
2.0
F I G U R E 3. The plot of 1/A slope versus 1/[AMI]. The A slopes were calculated by subtracing the slope of the control line from the slopes of the lines with A M I in the reciprocal plot.
(AMI), mM F I G U R E 2. The effect of AMI on BChE activity at 4.0 mM BuTCh.
acquired deficiency of BChE causes prolonged apnea and even death after a patient receives succinylcholine (Nelson and Burritt, 1986). Because AMI is a partial competitive inhibitor of BChE, acquired deficiency may be seen in patients treated for a long term with AMI. If these patients have to be operated on because of an emergency, the possibility of succinylcholine apnea must be considered. References Aaltonen L., Syv~ilahtiE., Iisalo E. and Peltom~ikiT. (1984) Anticholinergic activity in the serum of patients receiving maintenance amitriptyline or doxepin therapy. Acre Pharmac. Toxicol. 56, 75-80. Arpagaus M., Knott M., Vatsis K. P., Bartels C. F., LaDu B. N. and Lockridge O. (1990) Structure of the gene for human butyrylcholinesterase: evidence for a single copy. Biochemistry 29, 124-131. Chatonnet A. and Lockridge O. (1989) Review: comparison of butyrylcholinesterase and acetylcholinesterase. Biochem. J. 260, 625-634. (~oku~ra~ A. N. and Tezcan E. F. (1993a) Inhibition kinetics of brain butyrylcholinesterase by Cd > and gn 2+, Ca 2+ or Mg> reactivates the inhibited enzyme. Int. J. Biochem. 25, 1115-1120. ~oku~ra~ A. N. and Tezcan E. F. (1993b) Sheep brain pseudocholinesterase: inhibition kinetics of the partially purified enzyme by some substrate analogues. Chem.-Biol.fnteract. 87, 259-264. Ellman G. C., Courmey K. P., Andres V. Jr. and Featherstone R. M. (1961) A new rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88-95. Hijikata-Okunomiya A., Okamoto S., Tamao Y. and Kikumoto R. (1988) N-Dansyl-r-arginine 4-phenylpiperidine amide: a potent and selective inhibitor of horse serum cholinesterase. J. Biol. Chem. 23, 11,26911,273.
Anionic Site
Esteratic Site
~Hydrophobi~ }tff,lll, l,lf,lP°cket
/I/11/1-~
.I V _
H3C
}
C// ~CH2_CH2_
O/
N~H 2 -CH 3
F I G U R E 4. Active center of BChE monomer [modified from LaDu (1971)].
H2-~ CH-CH2-CH2-CHa N II
/CH3
F I G U R E 5. Chemical structure of AMI.
838 Hobbiger F. and Peck A. W. (1969) Hydrolysis of suxamethonium by different types of plasma. Br. J. Pharmac. 37, 258-271. Kabachnik M. I., Brestkin A. P., Godovikov N. N., Michelsen M. N., Rozengart E. V. and Rozengart V. I. (1970) Hydrophobic areas on the active surface of chotinesterases. Pharmac. Rev. 22, 355-388. LaDu B. N. ( 1971) Plasma esterase activity and the metabolism of drags with ester groups. Ann. N. Y. Acad. Sci. 179, 684-694. Lockridge O., Eckerson H. W. and LaDu B.N. (1979) lnterchain disulfide bonds and subunit organization in human serum cholinesterase. J. Biol. Chem. 254, 8324-8330. Lockridge O. and LaDu B. N. (1978) Comparison of atypical and usual human serum cholinesterase: purification, number of active sites, substrate affinity and turnover number. J. Biol. Chem. 253, 361-366. Massouli~ J., Pezzementi L., Bon S., Krejci E. and Vallette F. M. (1993) Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 31-91. Nelson T. C. and Burritt M. F. (1986) Pesticide poisoning, succinylcholineinduced apnea, and pseudocholinesterase. Mayo Clin. Proc. 61,750--755. Perkinson E., Ruckart R. and Davanzo J. P. (1969) Pharmacological and bio-
A . N . ~oku~ra~ and E. F. Tezcan chemical comparison of lithium and reference antidepressants. Proc. Exp. Biol. bled. 131,685-689. Prody C. A., Zevin-Sonkin D., Gnatt A., Golberg O. and Soreq H. (1987) Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues. Proc. Natl. Acad. Sci. U.S.A. 84, 3555-3559. Ryh~inen R. J. J. (1983) Pseudocholinesterase activity in some human body fluids. Gen. Pharmac. 14, 459-460. Schein K. and Smith S.E. (1978) Structure-activity relationship for the anticholinoceptor action of tricyclic antidepressants. Br. J. Pharmac. 62, 567-571. Schmider J., Greenblatt D. J., Von Moltke L. L., Harmatz J. S. and Shader R.I. (1995) N-Demethylation of amitriptyline in vitro: role of cytochrome P-4503A (CYP 3A) isoforms and effect of metabolic inhibitor. J. Pharmac. Exp. Ther. 275, 592-597. Segel I. H. (1975) Enzyme Kinetics. pp. 161-166. Wiley, Toronto. Whetherell J. R. and French M. C. (1986) The hydrolysis of succinyldithiocholine and related thiocholine esters by human plasma and purified cholinesterase. Biochem. Pharmac. 35, 939-945.