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Sheep brain pseudocholinesterase: Inhibition kinetics of the partially purified enzyme by some substrate analogues
Chem.-Biol. Interactions, 87 (1993) 259-264 Elsevier Scientific Publishers Ireland Ltd.
259
SHEEP BRAIN PSEUDOCHOLINESTERASE: INHIBITION KINETICS OF THE PARTIALLY PURIFIED ENZYME BY SOME SUBSTRATE ANALOGUES
A. NE~E CeOKU(]RA~and E. FERHAN TEZCAN Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara (Turkey)
SUMMARY
Pseudocholinesterase (ChE) (acylcholineacylhydrolase, EC 3.1.1.8) has been partially purified (about 270-fold) from sheep brain. The procedure included ammonium sulfate fractionation (20- 80%), DEAE-Trisacryl M chromatography and procainamide-Sepharose 4B affinity chromatography. The molecular weight of purified ChE was found to be 290 000 by gel filtration. Kinetic properties of the enzyme have been studied using the substrate analogues choline, succinylcholine and benzoylcholine. It was shown that the inhibition was partially competitive.
Key words: Sheep brain -- Pseudocholinesterase inhibition -- Choline Succinylcholine -- Benzoylcholine
INTRODUCTION
Pseudocholinesterase (ChE) (acylcholineacylhydrolase, EC 3.1.1.8) has been purified from human and horse serum. Molecular weights given for the human serum enzyme are between 300 000 - 340 000. The enzyme consists of four identical subunits containing an active center on each subunit [1- 3]. Recently, the enzyme has been cloned and the structure of the gene examined [4]. ChE, generally called butyrylcholinesterase, catalyzes the hydrolysis of both hydrophilic and hydrophobic choline esters [5,6]. The aim of this study was to investigate the effect of choline (Ch), succinylcholine (SuCh) and benzoylcholine (BeCh) on the activity of the partially purified enzyme from the soluble fraction of sheep brain. However, water soluble forms of acetylcholinesterase (ACHE) in mammalian brain have also been reported [7]. Correspondence to: A. Ne}e ~okug~ra~, Department of Biochemistry, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
260
ChE activity could be separated from AChE by using procainamide-Sepharose 4B gel [3]. MATERIALS AND METHODS
Materials Fresh sheep brains were obtained from the Ankara slaughter house. Reagents used were analytical grade.
Enzyme assay Enzyme activities were measured spectrophotometrically with Beckman Spectrophotometer Model 25 at 37°C, according to the procedure of Ellman et al. [8] with butyrylthiocholine (BuTCh) as substrate. Initial velocities were measured using 0.25 mM 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) in 5 mM 3-(Nmorpholino) propane sulfonic acid (MOPS) buffer (pH 7.5) and different concentrations of BuTCh from 0.3 mM to 4 mM. The enzyme used was 40 ~g per ml of assay mixture. In the inhibition studies, appropriate amounts of Ch, SuCh or BeCh have been added into the mixture. The hydrolysis was measured up to 90 s. The reaction was linear during this time period. All assays were carried out in duplicate.
Protein assay Protein concentrations were determined by the method of Bradford [9].
Purification of ChE Sheep brains were homogenized in 10 mM potassium phosphate buffer (pH 6.9) containing 0.5 mM EDTA and centrifuged at 100 000 × g for one hour. The ammonium sulfate fraction (20-80%) of the supernatant was dissolved in and dialyzed against the same buffer. The dialysate was applied onto a column of DEAE-Trisacryl M (3x37 cm) equilibrated with the buffer mentioned above. The enzyme was eluted by a linear gradient of NaC1 (0-0.6 M). The ChE activity peaked at 0.15 M NaC1. In the final step, the eluate was applied to a column of procainamide-Sepharose 4B affinity gel (1.6 × 3 cm), prepared according to the method described by Lockridge and La Du [10]. The enzyme was eluted as a single peak with 0.35 M NaC1 prepared in the buffer mentioned above and concentrated using XM 50 paper in Amicon Ultrafiltration Cell Mode 12. RESULTS
Sheep brain ChE was purified according to the procedure given in Materials and Methods and the purification procedure is summarized in Table I. Molecular weight of the enzyme was estimated [11] as 290 000 by Sephadex G-200 gel filtration (Fig. 1). The enzyme sample was used to examine the inhibition effects of Ch, SuCh and BeCh on BuTCh hydrolysis. In Fig. 2, the control line and the lines obtained in
2655.0 2226.0 1840.0
20 600 x g supernatant 100 000 x g supernatant Ammonium sulfate fractionation (20- 80%) DEAE-Triacryl M chromatography Procainamide-Sepharose 4B affinity chromatography 3.838 2.559
23.162 23.162 20.000
Total activity (units)
12.26 2364.9
8.7 10.4 10.9
Specific activity (IU/mg x 10 -~)
1.2 1.25 1.41 271.8
--
Purification (fold)
16.6 11.0
100 100 86.3
Yield (%)
Purifications and yields have been calculated according to the results obtained for the 20 600 x g supernatant. The procedure has been repeated three times, each time with similar results.
313.2 3.97
Total protein (mg)
Steps
SUMMARY OF A TYPICAL PURIFICATION PROCEDURE
TABLE I
262
serum
50
0
I 20
kinase, dog muscle
"~uvote
C'h'E,brain
~ e
albumin,trimer ~0
3
Lactate dehydrogenose ~ pig muscle ~ovine albumin~ dimer ~l~.!utothione. reductose~ broln inose ~ ye0st
I0
0 0
I
I
1
I
I
0.1
0.2
0.:5
0.4
0.5
K av
Fig. 1. Calibration curve of molecular weight determination by gel filtration on Sephadex G-200. Column size, 1.6 x 75 cm; buffer, 50 mM phosphate buffer (pH 7.5) containing 0.1 M NaC1; flow rate, 8.8 ml/hr. Reference proteins (1 mg in 1 ml of the buffer mentioned above) were applied to the column separately.
the presence of different concentrations of SuCh intersect on 1/v-axis suggesting that inhibition is competitive. Similar results have been obtained for Ch and BeCh. But, when the concentrations of these compounds were increased at a fixed concentration of BuTCh, velocities decreased and gave limiting finite values at high concentrations. This result indicates that inhibitions are partially competitive. Otherwise, in pure competitive inhibition, curves would approach zero at high concentrations of the compounds [12]. Binding of these compounds to enzyme decreases the affinity of BuTCh molecules to enzyme by a factor of a, and Ks (dissociation constant of E S complex) becomes aKs. Likewise, when BuTCh is bound to enzyme first, Ki (dissociation constant for EI) also changes into od~i. The secondary plot, slope versus concentrations of inhibitor, is a hyperbola; and Ki values were determined from the plot of 1/A slope versus 1/[I](inset of Fig. 2). A Slope values were calculated by subtracting the slope of the control line from the slopes of the lines obtained at different concentrations of inhibitor, thereby transforming the hyperbola into a straight line. From the control line, Ks has been determined as 0.15 mM and Vm, 1.42 #mol per minute per mg protein. Ki and c~ values obtained for all inhibitors are shown in Table II.
263
2.0
I
/ ,/
/.
K~(
I)
//"
~-~ ~-',
I
~ ~ ~ I/[SuCh],
I
///// "6
5'
mM -I
I
//////
-4
.=_ E
"
;
/J//~
/
/~/
-2
2 I/
[BuTCh]
, mM -I
Fig. 2. Reciprocal plot in the absence of SuCh (1~ - - 1%);in the presence of 0.2 mM SuCh (A A); 0.4 mM SuCh (O - - O) and 0.6 mM SuCh (O - - 0). The linear regions of the reciprocal plots have been given. The inset shows the plot of 1/A slope versus l/[SuCh]. & slopes were calculated by subtracting the slope of control line from the slopes of the lines with SuCh.
DISCUSSION
Because the enzyme exhibits negative cooperativity with respect to BuTCh ( n H = 1.2), at high BuTCh concentrations double reciprocal plots deviate from linearity [13]. Therefore, the control lines and the other lines with Ch, SuCh and BeCh have been obtained by linear regression analysis of the linear regions of the double reciprocal plots.
binding
TABLE II KINETIC P A R A M E T E R S OF S H E E P BRAIN ChE Inhibitors
Ki (mM)
c~
Ch SuCh BeCh
0.35 0.02 0.02
3 13 48
264
It was shown that inhibition type of these compounds is partially competitive. Therefore, they must be bound to a secondary site and induce a conformational change especially at the substrate binding site. Due to the order of binding, substrate or inhibitor affects the binding affinity of the other, and Ks is converted to Cd~s, K i to aKi. From the K i or cd~i values, it can be said that the affinity of SuCh or BeCh to enzyme is greater than that of Ch. If the cd/s values are compared, it can also be said that the inhibition effect of BeCh is greater than that of the others. REFERENCES 1 2 3 4
5 6 7 8 9 10
11 12 13
D. Boutin and J. Brodeur, Human serum pseudocholinesterase: molecular weight estimation of a subunit structure, Can. J. Physiol. Pharmacol., 49 (1971) 777-779. O. Lockridge, H.W. Eckerson and B.N. La Du, Interchain disulfide bonds and subunit organization in human serum cholinesterase, J. Biol. Chem., 254(17) (1979) 8324-8330. J.S. Ralston, A.R. Main, B.F. Kilpatrick and A.L. Chasson, Use of procainamide gels in the purification of human and horse serum cholinesterase, Biochem. J., 211 (1983) 243-250. M. Arpagaus, M. Kott, K.P. Vatsis, C.F. Barrels, B.N. La Du and O. Lockridge, Structure of the gene for human butyrylcholinesterase. Evidence for a single copy, Biochemistry 29 (1990) 124-131. S.S. Brown, W. Kalow, W. Pilz, M. Whittaker and C.L. Woronick, The plasma cholinesterases: a new perspective, Adv. Clin. Chem., 22 (1981) 1 - 123. J.R. Wetherell and M.C. French, The hydrolysis of succinylthiocholine and related thiocholine esters by human plasma and purified enzyme, Biochem. Pharmacol., 35(6) (1986) 939-945. A. Chatonnet and O. Lockridge, Review article: comparison of butyrylcholinesterase and acetylcholinesterase, Biochem. J., 260 (1989) 625-634. G.C. Ellman, K.P. Courtney, A. Andres Jr and R.M. Featherstone, A new rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248- 254. O. Lockridge and B.N. La Du, Comparison of atypical and usual human serum cholinesterase. Purification, number of active sites, substrate affinity and turnover number, J. Biol. Chem., 253 (1978) 361 - 366. P. Andrews, The gel filtration behaviour of proteins related to their molecular weights over a wide range, Biochem. J., 96 (1965) 595-605. I.H. Segel, Enzyme Kinetics, John Wiley and Sons Inc., Toronto, 1975, pp. 161-226. A.N. Qoku~ra~ and E.F. Tezcan, Aluminum inhibition of brain pseudocholinesterase, in: G.T. Yfire~ir, O. Donma and L. Kayrin (Eds.), Trace Elements in Health and Disease, (~ukurova University Publishing Company, Adana, 1991, pp. 415-420.
259
SHEEP BRAIN PSEUDOCHOLINESTERASE: INHIBITION KINETICS OF THE PARTIALLY PURIFIED ENZYME BY SOME SUBSTRATE ANALOGUES
A. NE~E CeOKU(]RA~and E. FERHAN TEZCAN Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara (Turkey)
SUMMARY
Pseudocholinesterase (ChE) (acylcholineacylhydrolase, EC 3.1.1.8) has been partially purified (about 270-fold) from sheep brain. The procedure included ammonium sulfate fractionation (20- 80%), DEAE-Trisacryl M chromatography and procainamide-Sepharose 4B affinity chromatography. The molecular weight of purified ChE was found to be 290 000 by gel filtration. Kinetic properties of the enzyme have been studied using the substrate analogues choline, succinylcholine and benzoylcholine. It was shown that the inhibition was partially competitive.
Key words: Sheep brain -- Pseudocholinesterase inhibition -- Choline Succinylcholine -- Benzoylcholine
INTRODUCTION
Pseudocholinesterase (ChE) (acylcholineacylhydrolase, EC 3.1.1.8) has been purified from human and horse serum. Molecular weights given for the human serum enzyme are between 300 000 - 340 000. The enzyme consists of four identical subunits containing an active center on each subunit [1- 3]. Recently, the enzyme has been cloned and the structure of the gene examined [4]. ChE, generally called butyrylcholinesterase, catalyzes the hydrolysis of both hydrophilic and hydrophobic choline esters [5,6]. The aim of this study was to investigate the effect of choline (Ch), succinylcholine (SuCh) and benzoylcholine (BeCh) on the activity of the partially purified enzyme from the soluble fraction of sheep brain. However, water soluble forms of acetylcholinesterase (ACHE) in mammalian brain have also been reported [7]. Correspondence to: A. Ne}e ~okug~ra~, Department of Biochemistry, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
260
ChE activity could be separated from AChE by using procainamide-Sepharose 4B gel [3]. MATERIALS AND METHODS
Materials Fresh sheep brains were obtained from the Ankara slaughter house. Reagents used were analytical grade.
Enzyme assay Enzyme activities were measured spectrophotometrically with Beckman Spectrophotometer Model 25 at 37°C, according to the procedure of Ellman et al. [8] with butyrylthiocholine (BuTCh) as substrate. Initial velocities were measured using 0.25 mM 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) in 5 mM 3-(Nmorpholino) propane sulfonic acid (MOPS) buffer (pH 7.5) and different concentrations of BuTCh from 0.3 mM to 4 mM. The enzyme used was 40 ~g per ml of assay mixture. In the inhibition studies, appropriate amounts of Ch, SuCh or BeCh have been added into the mixture. The hydrolysis was measured up to 90 s. The reaction was linear during this time period. All assays were carried out in duplicate.
Protein assay Protein concentrations were determined by the method of Bradford [9].
Purification of ChE Sheep brains were homogenized in 10 mM potassium phosphate buffer (pH 6.9) containing 0.5 mM EDTA and centrifuged at 100 000 × g for one hour. The ammonium sulfate fraction (20-80%) of the supernatant was dissolved in and dialyzed against the same buffer. The dialysate was applied onto a column of DEAE-Trisacryl M (3x37 cm) equilibrated with the buffer mentioned above. The enzyme was eluted by a linear gradient of NaC1 (0-0.6 M). The ChE activity peaked at 0.15 M NaC1. In the final step, the eluate was applied to a column of procainamide-Sepharose 4B affinity gel (1.6 × 3 cm), prepared according to the method described by Lockridge and La Du [10]. The enzyme was eluted as a single peak with 0.35 M NaC1 prepared in the buffer mentioned above and concentrated using XM 50 paper in Amicon Ultrafiltration Cell Mode 12. RESULTS
Sheep brain ChE was purified according to the procedure given in Materials and Methods and the purification procedure is summarized in Table I. Molecular weight of the enzyme was estimated [11] as 290 000 by Sephadex G-200 gel filtration (Fig. 1). The enzyme sample was used to examine the inhibition effects of Ch, SuCh and BeCh on BuTCh hydrolysis. In Fig. 2, the control line and the lines obtained in
2655.0 2226.0 1840.0
20 600 x g supernatant 100 000 x g supernatant Ammonium sulfate fractionation (20- 80%) DEAE-Triacryl M chromatography Procainamide-Sepharose 4B affinity chromatography 3.838 2.559
23.162 23.162 20.000
Total activity (units)
12.26 2364.9
8.7 10.4 10.9
Specific activity (IU/mg x 10 -~)
1.2 1.25 1.41 271.8
--
Purification (fold)
16.6 11.0
100 100 86.3
Yield (%)
Purifications and yields have been calculated according to the results obtained for the 20 600 x g supernatant. The procedure has been repeated three times, each time with similar results.
313.2 3.97
Total protein (mg)
Steps
SUMMARY OF A TYPICAL PURIFICATION PROCEDURE
TABLE I
262
serum
50
0
I 20
kinase, dog muscle
"~uvote
C'h'E,brain
~ e
albumin,trimer ~0
3
Lactate dehydrogenose ~ pig muscle ~ovine albumin~ dimer ~l~.!utothione. reductose~ broln inose ~ ye0st
I0
0 0
I
I
1
I
I
0.1
0.2
0.:5
0.4
0.5
K av
Fig. 1. Calibration curve of molecular weight determination by gel filtration on Sephadex G-200. Column size, 1.6 x 75 cm; buffer, 50 mM phosphate buffer (pH 7.5) containing 0.1 M NaC1; flow rate, 8.8 ml/hr. Reference proteins (1 mg in 1 ml of the buffer mentioned above) were applied to the column separately.
the presence of different concentrations of SuCh intersect on 1/v-axis suggesting that inhibition is competitive. Similar results have been obtained for Ch and BeCh. But, when the concentrations of these compounds were increased at a fixed concentration of BuTCh, velocities decreased and gave limiting finite values at high concentrations. This result indicates that inhibitions are partially competitive. Otherwise, in pure competitive inhibition, curves would approach zero at high concentrations of the compounds [12]. Binding of these compounds to enzyme decreases the affinity of BuTCh molecules to enzyme by a factor of a, and Ks (dissociation constant of E S complex) becomes aKs. Likewise, when BuTCh is bound to enzyme first, Ki (dissociation constant for EI) also changes into od~i. The secondary plot, slope versus concentrations of inhibitor, is a hyperbola; and Ki values were determined from the plot of 1/A slope versus 1/[I](inset of Fig. 2). A Slope values were calculated by subtracting the slope of the control line from the slopes of the lines obtained at different concentrations of inhibitor, thereby transforming the hyperbola into a straight line. From the control line, Ks has been determined as 0.15 mM and Vm, 1.42 #mol per minute per mg protein. Ki and c~ values obtained for all inhibitors are shown in Table II.
263
2.0
I
/ ,/
/.
K~(
I)
//"
~-~ ~-',
I
~ ~ ~ I/[SuCh],
I
///// "6
5'
mM -I
I
//////
-4
.=_ E
"
;
/J//~
/
/~/
-2
2 I/
[BuTCh]
, mM -I
Fig. 2. Reciprocal plot in the absence of SuCh (1~ - - 1%);in the presence of 0.2 mM SuCh (A A); 0.4 mM SuCh (O - - O) and 0.6 mM SuCh (O - - 0). The linear regions of the reciprocal plots have been given. The inset shows the plot of 1/A slope versus l/[SuCh]. & slopes were calculated by subtracting the slope of control line from the slopes of the lines with SuCh.
DISCUSSION
Because the enzyme exhibits negative cooperativity with respect to BuTCh ( n H = 1.2), at high BuTCh concentrations double reciprocal plots deviate from linearity [13]. Therefore, the control lines and the other lines with Ch, SuCh and BeCh have been obtained by linear regression analysis of the linear regions of the double reciprocal plots.
binding
TABLE II KINETIC P A R A M E T E R S OF S H E E P BRAIN ChE Inhibitors
Ki (mM)
c~
Ch SuCh BeCh
0.35 0.02 0.02
3 13 48
264
It was shown that inhibition type of these compounds is partially competitive. Therefore, they must be bound to a secondary site and induce a conformational change especially at the substrate binding site. Due to the order of binding, substrate or inhibitor affects the binding affinity of the other, and Ks is converted to Cd~s, K i to aKi. From the K i or cd~i values, it can be said that the affinity of SuCh or BeCh to enzyme is greater than that of Ch. If the cd/s values are compared, it can also be said that the inhibition effect of BeCh is greater than that of the others. REFERENCES 1 2 3 4
5 6 7 8 9 10
11 12 13
D. Boutin and J. Brodeur, Human serum pseudocholinesterase: molecular weight estimation of a subunit structure, Can. J. Physiol. Pharmacol., 49 (1971) 777-779. O. Lockridge, H.W. Eckerson and B.N. La Du, Interchain disulfide bonds and subunit organization in human serum cholinesterase, J. Biol. Chem., 254(17) (1979) 8324-8330. J.S. Ralston, A.R. Main, B.F. Kilpatrick and A.L. Chasson, Use of procainamide gels in the purification of human and horse serum cholinesterase, Biochem. J., 211 (1983) 243-250. M. Arpagaus, M. Kott, K.P. Vatsis, C.F. Barrels, B.N. La Du and O. Lockridge, Structure of the gene for human butyrylcholinesterase. Evidence for a single copy, Biochemistry 29 (1990) 124-131. S.S. Brown, W. Kalow, W. Pilz, M. Whittaker and C.L. Woronick, The plasma cholinesterases: a new perspective, Adv. Clin. Chem., 22 (1981) 1 - 123. J.R. Wetherell and M.C. French, The hydrolysis of succinylthiocholine and related thiocholine esters by human plasma and purified enzyme, Biochem. Pharmacol., 35(6) (1986) 939-945. A. Chatonnet and O. Lockridge, Review article: comparison of butyrylcholinesterase and acetylcholinesterase, Biochem. J., 260 (1989) 625-634. G.C. Ellman, K.P. Courtney, A. Andres Jr and R.M. Featherstone, A new rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248- 254. O. Lockridge and B.N. La Du, Comparison of atypical and usual human serum cholinesterase. Purification, number of active sites, substrate affinity and turnover number, J. Biol. Chem., 253 (1978) 361 - 366. P. Andrews, The gel filtration behaviour of proteins related to their molecular weights over a wide range, Biochem. J., 96 (1965) 595-605. I.H. Segel, Enzyme Kinetics, John Wiley and Sons Inc., Toronto, 1975, pp. 161-226. A.N. Qoku~ra~ and E.F. Tezcan, Aluminum inhibition of brain pseudocholinesterase, in: G.T. Yfire~ir, O. Donma and L. Kayrin (Eds.), Trace Elements in Health and Disease, (~ukurova University Publishing Company, Adana, 1991, pp. 415-420.