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Properties of partly preinhibited hen brain neuropathy target esterase
Chem.-Biol. Interactions, 87 (1993) 417- 423
417
Elsevier Scientific Publishers Ireland Ltd.
PROPERTIES OF PARTLY PREINHIBITED HEN BRAIN NEUROPATHY TARGET ESTERASE
J.L. VICEDO, V. CARRERA, J. BARRIL and E. VILANOVA
Department of Neurochemistry, Alicante University, P.O. Box 374, Alicante (Spain)
SUMMARY
NTE inhibitors cause different toxicological consequences (protection, induction or potentiation/promotion of neuropathy) depending on the order of dosing. These effects might be explained in terms of several phosphorylable sites with 'allosteric irreversible' behaviour. Brain neuropathy target esterase (NTE) has been preinhibited with phenylmethylsulphonyl fluoride (PMSF) (0, 5, 10, 15, 30 and 60 ~M) or with diisopropylphoshoro fluoridate (DFP) (0, 0.2, 0.5, and 1 #M) at 37°C for 30 min. After washing by centrifugation, tissues were then reinhibited with a range of PMSF (0 to 80 #M) or DFP (0 to 1 ~M) concentrations. The slopes of the inhibition curves (log % activity vs. concentration) of pretreated tissues were identical to those of the non-pretreated tissues, with non-distinguishable I50 values. It is concluded that allosteric effects are not likely to be involved in membrane-bound NTE of hen brain.
Key words: Organophosphorus -- Neuropathy Target Esterase -- Diisopropylphosphorofluoridate
INTRODUCTION
Neuropathy target esterase (NTE) is the suggested target protein involved in the mechanism of initiation of organophosphorus (OP) induced delayed polyneuropathy (OPIDP). NTE inhibition and phosphorylation is not enough in itself to induce OPIDP. Specific modifications affecting more than 70-80% of NTE sites are needed [1,2]. Considerable evidence suggests that most organophosphates satisfy this specific requirement by dealkylating the phosphorylated NTE, a process usually called 'aging' [2,3] but some phosphoramidate analogues can induce OPIDP without requiring the 'aging' reaction [4,5]. Correspondence to: J.L. Vicedo, Department of Neurochemistry, Alicante University, P.O. Box 374, Alicante, Spain. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
418
Some sulphonyl fluorides, such as phenylmethylsulphonyl fluoride (PMSF), carbamates and phosphinates can also covalently and irreversibly inhibit NTE without inducing neuropathy. When they are given to hens at doses blocking (inhibiting) more than 30 - 40% of NTE, they protect against the effect of a subsequent high neuropathic dose of a neuropathic OP [6]. It has recently been reported that when 'protective' NTE inhibitors such as PMSF are dosed after a low non-neuropathic dose of a neuropathic OP (causing > 40 - 50% NTE inhibition), its neurotoxicity is 'potentiated' [7] or 'promoted' [8,9], causing severe neuropathy. The aim of this work was to determine the sensitivity to DFP and PMSF of samples of NTE partly preinhibited either with the same compound or with the other. We tested the hypothesis that NTE may have more than one site for binding inhibitors which could exhibit 'allosteric' interactions related to protection, induction and/or promotion effects. MATERIAL AND METHODS
Tissue preparation and inhibitor preincubation Brains obtained from adult hens (Gallus domesticus, 1.5 - 2 kg, 20 - 22 months old) were homogenized in 50 mM Tris/HC1 buffer (pH 8.0) at a concentration of 100 mg/ml using a Polytron homogenizer with a PTA 10S head. Aliquots of 3 ml homogenate were put in 30-ml centrifuge tubes, adding 45 ~1 of PMSF or DFP to reach the desired final concentration. The mixtures were incubated at 37°C for 3 min. The inhibition reaction was then stopped by dilution up to 30 ml with ice cold buffer. To remove the remaining inhibitor the diluted solution was centrifuged at 30 000 x g for 30 min, in a JA 21 Beckman centrifuge. The pellets were resuspended in 30 ml and centrifuged again. The final pellets were resuspended in a volume of 37.5 ml of Tris/HC1, i mM EDTA (pH 8.0) buffer and stored in an ice-water bath at 0 - 5°C until use the next day for the second inhibition and NTE assay as described below. Second inhibition and N T E assay A solution of 0.925 ml containing the appropriate DFP or PMSF concentration in 10 mM Tris/citrate/1 mM EDTA (pH 6.0) buffer was added to test tubes. A volume of 0.025 ml of 3.2 mM paraoxon (final concentration 40 ~M) in acetone was added to all tubes. In paired tubes we then added either 0.050 ml of Tris/citrate (pH 6) buffer or 0.050 ml of 10 mM mipafox (final 250 ~M) in the same buffer. In all tubes we added 1 ml of the diluted tissue resulting from pretreatment as described above. After 30 min preincubation at 37°C, 2 ml of substrate were added (1 ml of stock 30 mg/ml phenyl valerate in dimethylformamide diluted with 30 ml distilled water). The reaction was stopped by adding 2 ml of a solution containing 2% SDS and 0.25 ml aminoantipirine in 50 mM Tris/HC1 pH 8 buffer. The mixture was shaken and then 1 ml of 0.4% potassium ferricyanide was added in water. Absorbance was read at 510 nm and we estimated the final phenol concentration by comparing with the calibration curve. Activity was calculated and expressed as nmol phenol liberated/min/g
419
original fresh tissue. Percentage activity was calculated over the control without DFP or PMSF in the pretreatment and in the second inhibition. Log (% activity) was plotted versus inhibitor concentration and fitted to straight lines using the Sigma Plot programme (V.4.0). RESULTS
Hen brain homogenates were pretreated with 0, 0.2, 0.5 and 1.0 ~M DFP or with 0, 10, 30 and 60 ~M PMSF and samples washed by two centrifugations to
Pretreotmenf: 2.0 ,,@,=. , I
,,@,=.
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/~M
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._J
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v
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,
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Fig. 1. Sensitivity to DFP of brain NTE partly preinhibited with DFP or PMSF. Each line represents data obtained from a sample of brain particles that have been pretreated with the concentration indicated for each set of point symbols. The concentrations of DFP for the second inhibition are indicated on the abscissa. Activity is expressed for all the points as the percentage over the control with 0 inhibitor concentration in pretreatment and at the second inhibition (first point in black-filled circles).
420
remove the remaining inhibitor as indicated in Methods. The remaining uninhibited NTE activity was then tested for sensitivity to inhibition by itself and by the other inhibitor. Figure 1 shows the results of the study of sensitivity to the inhibition by DFP of samples pretreated either with DFP or PMSF, plotting log (% activity) vs. concentration. The origin indicates the log of the remaining pretreatment activity. The value of the origin decreased with the pretreatment concentration. Each set of pretreated samples exhibited straight lines parallel to the controls, suggesting that the remaining activity shows sensitivity similar to that of the nonpretreated samples. Table I shows the parameters obtained from fitting data to the straight lines. No significant differences were obtained comparing the values of I50 deduced from the slopes. Sensitivity to PMSF was also unmodified by pretreatment with either DFP or PMSF (Fig. 2, Table II). DISCUSSION
We have observed that samples of particulate brain NTE partly preinhibited by pretreatment with DFP or PMSF show the same sensitivity to posterior inhibition by either the same compound or the other. This suggests that NTE molecules either have only one site for inhibition or else show insufficient allosteric interaction to modify sensitivity to a further inhibition assay. As described in the Introduction, NTE inhibitors cause different effects: induction, protection and promotion. The chronological sequence in which events are experimentally induced critically affects the final results. The involvement of NTE in the protection and induction mechanism is generally accepted on the basis of multiple evidences [6]. However, the molecular site and mechanism involved in promotion has not been clarified. One possible hypothesis is that the TABLE I S E N S I T I V I T Y TO D F P Pretreatment
Origin L o g ( E ' 0 × 100)
Slope (~M -1
DFP 0.0 0.2 0.5 1.0
1.959 1.765 1.615 1.197
± ± ± ±
0.028 0.024 0.024 0.029
0.511 0.513 0.554 0.460
± 0.055 ± 0.058 =e 0.046 ± 0.056
0.59 0.59 0.54 0.65
± ± ± ±
0.06 0.07 0.04 0.08
1.966 1.790 1.560 1.305
± ± ± +
0.017 0.017 0.011 0.030
0.577 0.613 0.598 0.575
+ ± ± ±
0.52 0.49 0.50 0.52
+ ± + ±
0.03 0.03 0.02 0.05
PMSF
~M #M ~M ~M
0 10 30 60
~M ~M ~M ~M
I50 (~M)
0.034 0.034 0.020 0.058
P a r a m e t e r s obtained from fitting data of Fig. 1 to straight lines using the Sigma Plot programme. I5o values obtained from the slope as follows: I50 = log(100/50)/slope = 0.301030/slope.
421
2.0
,4-,,
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~
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o
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-41
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20
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40
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,
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80
PMSr Fig. 2. Sensitivity to PMSF of brain NTE partly preinhibited with DFP or PMSF. Each line represents data obtained from a sample of brain particles that have been pretreated with the concentration indicated for each set of point symbols. The concentrations of PMSF for the second inhibition are indicated on the abscissa. Activity is expressed for all the points as the percentage over the control with 0 inhibitor concentration in pretreatment and at the second inhibition (first point in blackfilled circles).
same protein could be modified irreversibly to two different states (conformations?) by the covalently binding of protectors or inducers to 'allosteric' sites. The final state obtained would mainly depend on the kind of initial inhibitor employed. The first inhibitor could direct modification to either the 'neuropathic' or 'non-neuropathic' state. With such a mechanism it could be expected 0out not
422 TABLE II SENSITIVITY TO PMSF Pretreatment
Origin Log(E'o x 100)
Slope (~M -1)
I50 (#M)
DFP 0.0 0.2 0.5 1.0
#M t~M t~M ~M
1.931 1.844 1.744 1.495
± ± ± ±
0.043 0.033 0.031 0.050
0.0124 0.0141 0.0140 0.0129
± ± ± ±
0.0011 0.0009 0.0008 0.0013
24.3 21.4 21.5 23.3
± ± ± ±
2.0 1.4 1.2 2.4
PMSF0 10 30 60
~M tLM ~M t~M
1.927 2.001 1.767 1.683
± ± + ±
0.026 0.019 0.025 0.062
0.0142 0.0159 0.0163 0.0164
± + ± ±
0.00007 0.00005 0.00007 0.00016
21.2 18.9 18.4 18.4
+ + ± ±
1.1 0.6 0.8 1.8
Parameters obtained from fitting data of Fig. 1 to straight lines using the Sigma Plot programme. I50 values obtained from the slope as follows: I50 = log(100/50)/slope = 0.301030/slope.
necessarily so) that NTE partly modified by an inhibitor would undergo modification of affinity/sensitivity for the binding of the remaining sites on the same proteins. To test this hypothesis and determine whether NTE may have more than one inhibitory site, we considered it an interesting task to test whether the remaining activity of in vitro partly-inhibited NTE (by either PMSF or DFP) shows identical or different properties to native NTE regarding inhibition either by itself or the other compound (DFP or PMSF). Our results do not confirm such a hypothesis. However, it cannot be ruled out, due to the following reasons: (a) an allosteric interaction could affect affinity but not the phosphorylation rate, and this could be the limiting step of inhibition rate. (b) Binding of the first inhibitor (PMSF or DFP) to a site could perhaps irreversibly stabilize a protein state (conformation?) without causing detectable modification in sensitivity to the binding to the other sites. (c) In this study only particulate brain NTE was considered. However, a soluble form (S-NTE) has been described in peripheral nerve [10]. Further studies will be needed to confirm or reject the discussed hypothesis. ACKNOWLEDGEMENTS
Work partly supported by grant FISS 89/0874 and FISS 92/0811. REFERENCES 1 2
M.K. Johnson, Structure-activity relationships for substrates and inhibitors of hen brain neurotoxic esterase, Biochem. Pharmacol., 24 (1975) 797-805. M.K. Johnson, The target for initiation of delayed neurotoxicity by organophosphorus esters: biochemical and toxicological applications, Rev. Biochem. Toxicol., 4 (1982) 141-212.
423 3 4
5
6 7 8
9
10
B. Clothier and M.K. Johnson, Reactivation and aging of neurotoxic esterase inhibited by a variety of organophosphorus esters, Biochem. J., 185 (1980) 739- 747. M.K. Johnson, E. Vilanova and D.J. Read, Anomalous biochemical responses in tests of the delayed neuropathic potential of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved isomers and some higher O-alkyl homologues, Arch. Toxicol., 65 (1991) 618- 624. E. Vilanova, M.K. Johnson and J.L. Vicedo, Interaction of some unsubstituted phosphoramidate analogues of methamidophos (O,S-dimethyl phosphorothioamidate) with acetylcholinesterase and neuropathy target esterase of hen brain, Pestic. Biochem. Physiol., 28 (1987) 224 - 238. M.K. Johnson, Receptor or enzyme: the puzzle of NTE and organophosphate-induced delayed polyneuropathy, Trends Pharmacol. Sci., 8 (1987) 174-179. C.N. Pope and S. Padilla, Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride, J. Toxicol. Environ. Health, 31 (1990) 261-273. M. Lotti, S. Caroldi, E. Capodicasa and A. Moretto, Promotion of organophosphate-induced delayed polyneuropathy by phenylmethanesulfonyl fluoride, Toxicol. Appl. Pharmacol., 108 (1991) 234-241. M. Peraica, M. Capodicasa, M.L. Scapellato, M. Bertolazzi, A. Moretto and M. Lotti, Organophosphate induced delayed polyneuropathy (OPIDP) in chicks: induction, promotion and recovery, Toxicologist, 11 (1991) 306. E. Vilanova, J. Barril, V. Carrera and M.C. Pellin, Soluble and Particulate Forms of the Organophosphorus neuropathy target esterase in hen sciatic nerve. J. Neurochem., 55 (October 1990) 1258-1265.
417
Elsevier Scientific Publishers Ireland Ltd.
PROPERTIES OF PARTLY PREINHIBITED HEN BRAIN NEUROPATHY TARGET ESTERASE
J.L. VICEDO, V. CARRERA, J. BARRIL and E. VILANOVA
Department of Neurochemistry, Alicante University, P.O. Box 374, Alicante (Spain)
SUMMARY
NTE inhibitors cause different toxicological consequences (protection, induction or potentiation/promotion of neuropathy) depending on the order of dosing. These effects might be explained in terms of several phosphorylable sites with 'allosteric irreversible' behaviour. Brain neuropathy target esterase (NTE) has been preinhibited with phenylmethylsulphonyl fluoride (PMSF) (0, 5, 10, 15, 30 and 60 ~M) or with diisopropylphoshoro fluoridate (DFP) (0, 0.2, 0.5, and 1 #M) at 37°C for 30 min. After washing by centrifugation, tissues were then reinhibited with a range of PMSF (0 to 80 #M) or DFP (0 to 1 ~M) concentrations. The slopes of the inhibition curves (log % activity vs. concentration) of pretreated tissues were identical to those of the non-pretreated tissues, with non-distinguishable I50 values. It is concluded that allosteric effects are not likely to be involved in membrane-bound NTE of hen brain.
Key words: Organophosphorus -- Neuropathy Target Esterase -- Diisopropylphosphorofluoridate
INTRODUCTION
Neuropathy target esterase (NTE) is the suggested target protein involved in the mechanism of initiation of organophosphorus (OP) induced delayed polyneuropathy (OPIDP). NTE inhibition and phosphorylation is not enough in itself to induce OPIDP. Specific modifications affecting more than 70-80% of NTE sites are needed [1,2]. Considerable evidence suggests that most organophosphates satisfy this specific requirement by dealkylating the phosphorylated NTE, a process usually called 'aging' [2,3] but some phosphoramidate analogues can induce OPIDP without requiring the 'aging' reaction [4,5]. Correspondence to: J.L. Vicedo, Department of Neurochemistry, Alicante University, P.O. Box 374, Alicante, Spain. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
418
Some sulphonyl fluorides, such as phenylmethylsulphonyl fluoride (PMSF), carbamates and phosphinates can also covalently and irreversibly inhibit NTE without inducing neuropathy. When they are given to hens at doses blocking (inhibiting) more than 30 - 40% of NTE, they protect against the effect of a subsequent high neuropathic dose of a neuropathic OP [6]. It has recently been reported that when 'protective' NTE inhibitors such as PMSF are dosed after a low non-neuropathic dose of a neuropathic OP (causing > 40 - 50% NTE inhibition), its neurotoxicity is 'potentiated' [7] or 'promoted' [8,9], causing severe neuropathy. The aim of this work was to determine the sensitivity to DFP and PMSF of samples of NTE partly preinhibited either with the same compound or with the other. We tested the hypothesis that NTE may have more than one site for binding inhibitors which could exhibit 'allosteric' interactions related to protection, induction and/or promotion effects. MATERIAL AND METHODS
Tissue preparation and inhibitor preincubation Brains obtained from adult hens (Gallus domesticus, 1.5 - 2 kg, 20 - 22 months old) were homogenized in 50 mM Tris/HC1 buffer (pH 8.0) at a concentration of 100 mg/ml using a Polytron homogenizer with a PTA 10S head. Aliquots of 3 ml homogenate were put in 30-ml centrifuge tubes, adding 45 ~1 of PMSF or DFP to reach the desired final concentration. The mixtures were incubated at 37°C for 3 min. The inhibition reaction was then stopped by dilution up to 30 ml with ice cold buffer. To remove the remaining inhibitor the diluted solution was centrifuged at 30 000 x g for 30 min, in a JA 21 Beckman centrifuge. The pellets were resuspended in 30 ml and centrifuged again. The final pellets were resuspended in a volume of 37.5 ml of Tris/HC1, i mM EDTA (pH 8.0) buffer and stored in an ice-water bath at 0 - 5°C until use the next day for the second inhibition and NTE assay as described below. Second inhibition and N T E assay A solution of 0.925 ml containing the appropriate DFP or PMSF concentration in 10 mM Tris/citrate/1 mM EDTA (pH 6.0) buffer was added to test tubes. A volume of 0.025 ml of 3.2 mM paraoxon (final concentration 40 ~M) in acetone was added to all tubes. In paired tubes we then added either 0.050 ml of Tris/citrate (pH 6) buffer or 0.050 ml of 10 mM mipafox (final 250 ~M) in the same buffer. In all tubes we added 1 ml of the diluted tissue resulting from pretreatment as described above. After 30 min preincubation at 37°C, 2 ml of substrate were added (1 ml of stock 30 mg/ml phenyl valerate in dimethylformamide diluted with 30 ml distilled water). The reaction was stopped by adding 2 ml of a solution containing 2% SDS and 0.25 ml aminoantipirine in 50 mM Tris/HC1 pH 8 buffer. The mixture was shaken and then 1 ml of 0.4% potassium ferricyanide was added in water. Absorbance was read at 510 nm and we estimated the final phenol concentration by comparing with the calibration curve. Activity was calculated and expressed as nmol phenol liberated/min/g
419
original fresh tissue. Percentage activity was calculated over the control without DFP or PMSF in the pretreatment and in the second inhibition. Log (% activity) was plotted versus inhibitor concentration and fitted to straight lines using the Sigma Plot programme (V.4.0). RESULTS
Hen brain homogenates were pretreated with 0, 0.2, 0.5 and 1.0 ~M DFP or with 0, 10, 30 and 60 ~M PMSF and samples washed by two centrifugations to
Pretreotmenf: 2.0 ,,@,=. , I
,,@,=.
I.i
t4
DFP
* 0.0 ffM
•
-
~
v
0.2
/~M
"
0.5 #M
1.6 1.2 r]" " " - ' - -
"''"--N..
-..
0
._J
0.8 I
,
I
,
I
,
Pretreotmenh
2.0
I
,
I
PMSF
-. ....
[]
i
I
•
0
v
,.......
50 t~M
.,iI o ~ , m ...@.
I,J
.,d:
t~M
10 /zM
60 tzM
1.6 1.2
I::l'l 0
[3"'''''-...
_..I
~ -V
0.8 I
0.0
,
I
0.2
,
I
0.4
DFP
,
I
0.6
(p,U)
~
I
0.8
"''--.
[]
,
I
,
1.0
Fig. 1. Sensitivity to DFP of brain NTE partly preinhibited with DFP or PMSF. Each line represents data obtained from a sample of brain particles that have been pretreated with the concentration indicated for each set of point symbols. The concentrations of DFP for the second inhibition are indicated on the abscissa. Activity is expressed for all the points as the percentage over the control with 0 inhibitor concentration in pretreatment and at the second inhibition (first point in black-filled circles).
420
remove the remaining inhibitor as indicated in Methods. The remaining uninhibited NTE activity was then tested for sensitivity to inhibition by itself and by the other inhibitor. Figure 1 shows the results of the study of sensitivity to the inhibition by DFP of samples pretreated either with DFP or PMSF, plotting log (% activity) vs. concentration. The origin indicates the log of the remaining pretreatment activity. The value of the origin decreased with the pretreatment concentration. Each set of pretreated samples exhibited straight lines parallel to the controls, suggesting that the remaining activity shows sensitivity similar to that of the nonpretreated samples. Table I shows the parameters obtained from fitting data to the straight lines. No significant differences were obtained comparing the values of I50 deduced from the slopes. Sensitivity to PMSF was also unmodified by pretreatment with either DFP or PMSF (Fig. 2, Table II). DISCUSSION
We have observed that samples of particulate brain NTE partly preinhibited by pretreatment with DFP or PMSF show the same sensitivity to posterior inhibition by either the same compound or the other. This suggests that NTE molecules either have only one site for inhibition or else show insufficient allosteric interaction to modify sensitivity to a further inhibition assay. As described in the Introduction, NTE inhibitors cause different effects: induction, protection and promotion. The chronological sequence in which events are experimentally induced critically affects the final results. The involvement of NTE in the protection and induction mechanism is generally accepted on the basis of multiple evidences [6]. However, the molecular site and mechanism involved in promotion has not been clarified. One possible hypothesis is that the TABLE I S E N S I T I V I T Y TO D F P Pretreatment
Origin L o g ( E ' 0 × 100)
Slope (~M -1
DFP 0.0 0.2 0.5 1.0
1.959 1.765 1.615 1.197
± ± ± ±
0.028 0.024 0.024 0.029
0.511 0.513 0.554 0.460
± 0.055 ± 0.058 =e 0.046 ± 0.056
0.59 0.59 0.54 0.65
± ± ± ±
0.06 0.07 0.04 0.08
1.966 1.790 1.560 1.305
± ± ± +
0.017 0.017 0.011 0.030
0.577 0.613 0.598 0.575
+ ± ± ±
0.52 0.49 0.50 0.52
+ ± + ±
0.03 0.03 0.02 0.05
PMSF
~M #M ~M ~M
0 10 30 60
~M ~M ~M ~M
I50 (~M)
0.034 0.034 0.020 0.058
P a r a m e t e r s obtained from fitting data of Fig. 1 to straight lines using the Sigma Plot programme. I5o values obtained from the slope as follows: I50 = log(100/50)/slope = 0.301030/slope.
421
2.0
,4-,,
•
Prefreafmenf:
DFPe
0.0 /~M
~
0.2 /~M
1.6
o
1.2 V
O~ 0
0.8
°
._1
0.4 I
2.0
.,-
,
I
=
I
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Prefreafmenf:
-41
I
,
PMSF
•
I
0 pM
1.6
o
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~0
0.8
_1
0.4 I
0
,
I
,
20
I
40
,
I
60
,
I
80
PMSr Fig. 2. Sensitivity to PMSF of brain NTE partly preinhibited with DFP or PMSF. Each line represents data obtained from a sample of brain particles that have been pretreated with the concentration indicated for each set of point symbols. The concentrations of PMSF for the second inhibition are indicated on the abscissa. Activity is expressed for all the points as the percentage over the control with 0 inhibitor concentration in pretreatment and at the second inhibition (first point in blackfilled circles).
same protein could be modified irreversibly to two different states (conformations?) by the covalently binding of protectors or inducers to 'allosteric' sites. The final state obtained would mainly depend on the kind of initial inhibitor employed. The first inhibitor could direct modification to either the 'neuropathic' or 'non-neuropathic' state. With such a mechanism it could be expected 0out not
422 TABLE II SENSITIVITY TO PMSF Pretreatment
Origin Log(E'o x 100)
Slope (~M -1)
I50 (#M)
DFP 0.0 0.2 0.5 1.0
#M t~M t~M ~M
1.931 1.844 1.744 1.495
± ± ± ±
0.043 0.033 0.031 0.050
0.0124 0.0141 0.0140 0.0129
± ± ± ±
0.0011 0.0009 0.0008 0.0013
24.3 21.4 21.5 23.3
± ± ± ±
2.0 1.4 1.2 2.4
PMSF0 10 30 60
~M tLM ~M t~M
1.927 2.001 1.767 1.683
± ± + ±
0.026 0.019 0.025 0.062
0.0142 0.0159 0.0163 0.0164
± + ± ±
0.00007 0.00005 0.00007 0.00016
21.2 18.9 18.4 18.4
+ + ± ±
1.1 0.6 0.8 1.8
Parameters obtained from fitting data of Fig. 1 to straight lines using the Sigma Plot programme. I50 values obtained from the slope as follows: I50 = log(100/50)/slope = 0.301030/slope.
necessarily so) that NTE partly modified by an inhibitor would undergo modification of affinity/sensitivity for the binding of the remaining sites on the same proteins. To test this hypothesis and determine whether NTE may have more than one inhibitory site, we considered it an interesting task to test whether the remaining activity of in vitro partly-inhibited NTE (by either PMSF or DFP) shows identical or different properties to native NTE regarding inhibition either by itself or the other compound (DFP or PMSF). Our results do not confirm such a hypothesis. However, it cannot be ruled out, due to the following reasons: (a) an allosteric interaction could affect affinity but not the phosphorylation rate, and this could be the limiting step of inhibition rate. (b) Binding of the first inhibitor (PMSF or DFP) to a site could perhaps irreversibly stabilize a protein state (conformation?) without causing detectable modification in sensitivity to the binding to the other sites. (c) In this study only particulate brain NTE was considered. However, a soluble form (S-NTE) has been described in peripheral nerve [10]. Further studies will be needed to confirm or reject the discussed hypothesis. ACKNOWLEDGEMENTS
Work partly supported by grant FISS 89/0874 and FISS 92/0811. REFERENCES 1 2
M.K. Johnson, Structure-activity relationships for substrates and inhibitors of hen brain neurotoxic esterase, Biochem. Pharmacol., 24 (1975) 797-805. M.K. Johnson, The target for initiation of delayed neurotoxicity by organophosphorus esters: biochemical and toxicological applications, Rev. Biochem. Toxicol., 4 (1982) 141-212.
423 3 4
5
6 7 8
9
10
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