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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
In vitro effect of dimethoate on the activity of tryptophan pyrrolase in rat liver a
Ashraf A. M. Hassan , Shehata M. El‐Sewedy a
b
, Yohsuke Minatogawat & Ryo Kido
b
a
Applied Medical Chemistry Department, Medical Research Institute , Alexandria university , Egypt b
Biochemistry Department , Wakayama Medical College , Japan Published online: 21 Nov 2008.
To cite this article: Ashraf A. M. Hassan , Shehata M. El‐Sewedy , Yohsuke Minatogawat & Ryo Kido (1991) In vitro effect of dimethoate on the activity of tryptophan pyrrolase in rat liver, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 26:3, 333-338 To link to this article: http://dx.doi.org/10.1080/03601239109372739
PLEASE SCROLL DOWN FOR ARTICLE
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Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
J. ENVIRON. SCI. HEALTH, B26(3), 333-338 (1991)
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
IN VITRO EFFECT OF DIMETHOATE ON THE ACTIVITY OF TRYPTOPHAN PYRROLASE IN RAT LIVER
Key words: Dimethoate, Tryptopohan pyrrolase, Total-form , Holo-form Ashraf A. M. Hassan¶*, Shehata M. El-Sewedy¶, Yohsuke Minatogawat† and Ryo Kido†* ¶ Applied Medical Chemistry Department, Medical Research Institute, Alexandria university, Egypt, † Biochemistry Department, Wakayama Medical College, Japan.
ABSTRACT Total and holo-enzyme activities of tryptophan 2,3dioxygenase were measured in vitro in the presence and absence of the organophosphorous insecticide, dimethoate. Addition of dimethoate to the reaction mixture decreased the activities of both total and holo-forms. Total and holoenzyme activities were decreased by 34% and 26%, respectively, by 1 mM dimethoate. On the other hand, 5 mM dimethoate resulted in 56% and 34% inhibition to total and holo-enzyme activities, respectively. Lineweaver-Burk plot of the total-enzyme activity at different tryptophan concentration in the presence of 2 mM dimethoate gave uncompetitive type of inhibition.
* To whom correspondence should be adressed 333 Copyright© 1991 by Marcel Dekker, Inc.
334
HASSAN ET AL.
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
INTRODUCTION The hepatic enzyme tryptophan pyrrolase (L-tryptophan 2,3-dioxygenase, EC 1.13.11.11) which catalyzes the oxidative cleavage of L-tryptophan to N-formylkynurenine is the first and probably the rate limiting enzyme in the catabolism of L-tryptophan (Knox, 1951). In rat liver, tryptophan pyrrolase exists in two forms, the active heme-containing holo-enzyme and the inactive heme-free apo-enzyme. It has been postulated that the relative changes in the holo- and apoenzyme activities reflect the availability of "free" heme of the intracellular "regulatory" heme pool (Badawy, 1978, 1979; Kikuchi and Yoshida, 1983; Litman and Correia, 1985) . It has been reported that tryptophan pyrrolase (TPO) activity is regulated by pancreatic hormones as well as glucocorticoids; giucagon or dibutyryi cAMP greatly enhances the induction by dexamethasone, whereas insulin supresses it (Nakamura et al., 1980). Epinephrine supresses the enzyme induction by giucagon and dexamethasone (Noda et al., 1983). Our previous study showed that TPO activity significantly decreased in vivo after oral administration of the carbamate insecticide "carbaryl" to rats. The in vitro addition of carbaryl to the reaction mixture exerted noncompetitive inhibition to the enzyme activity (Hassan et al., 1990).
EXPERIMENTAL PROCEDURF M a t e r i a l s : Dimethoate was provided from Sumitomo Chemical Co. Ltd., Osaka, Japan. Hematin was purchased from Sigma chemicals Co., St. Louis, USA. L-Kynurenine was from Nacalai tesque, Inc., Kyoto, Japan. All other chemicals used were commercial products of the highest grade available.
335
EFFECT OF DIMETHOATE ON TRYPTOPHAN PYRROLASE
100Holoenzyme 90-
ö
80-
'S
70·
£
60·
Downloaded by [University of Toronto Libraries] at 06:41 20 December[ivilj 2014
Έ ou
Totale niyme
60·
υ
30
2.5
1.0
Figure Tryptophan
pyrrolase
holo-
and
5.0
[Dlmelhoale] (mM)
1 total
activity
at
different
concentrations Of dimethoate. Dlmethoale concentrallon varied Irom 0.5 · 6.0 mM In the reacllon mixture which contained 0.5 mM L-tryptophan. The assay was performed as described under Materials and Methods. Each value Is the average ol duplicate experiments.
Enzyme assay: The enzyme activity was measured according to the principle of Feigelson and Greengard, (1961). The reaction mixture (1 ml) contains 5 mM ascorbate, 2 μ Μ hematin, 0.1 M potassium phosphate buffer, pH 7.0, enzyme preparation and different concentrations of tryptophan (as cited in the figures). After incubation at 37°C for 60 minutes, the reaction was stopped by adding 200 μΙ of 30% trichloroacetic acid, then the reaction tube was placed in boiling water bath for 5 minutes to allow the hydrolysis of Nformylkynurenine to L-kynurenine. The latter was measured spectrophotometrically at 480 nm after the addition of 0.7 ml p-dimethylaminobenzaldhyde to an equal volume of the supernatent (Takikawa et al., 1988).
336
HASSAN ET AL.
je
Without dimelhonte «2 mM Dlmethoate
α>
S
o. σι
2·
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
ι 2 o e 0.2
0.0
0.4
o.e
0.6
Figure
1.0
[Tryptophan](mM
2
Tryptophan pyrrolase total-activity at different concentrations of L-tryptophan in the presence and absence of dimethoate. L-Tryptophan concentrallon In the reaction mixture varied from 0.2 · 1.0 mM In the presence and absence of 2.0 mM dimethoate. The assay was performed as described under Materials and Methods. Each value is the average of duplicate experiments. U.3-
0.4-
O
Without dimethoate
•
+2 mM Dimethoate
0.3-
0.2-
0.1 -
1/[Tryptopha 0.0•β
•5
-3
-2
Figure Uncompetitive
3
inhibition of rat liver tryptophan pyrrolase total-
activity. Double-reciprocal plots of the rates of the reaction versus variée concentrations of L-tryplophan In the presence of 2 mM of dimethoate. The assay was perlormed as described under Figure (2). Each value Is the average of duplicate experiments.
EFFECT OF DIMETHOATE ON TRYPTOPHAN PYRROLASE
337
RESULTS AND DISCUSSION
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
Total and holo-enzyme activities were inhibited by 34% and 26%, respectively, by 1 mM dimethoate. The inhibition of totai-enzyme activity reached 56% by 5 mM dimethoate, whereas only 34% inhibition was observed in the holo-enzyme (Figure 1). This result shows that the apo-enzyme is more sensitive than the holo-enzyme to dimethoate. The conversion of apo-enzyme to holo-enzyme may be affected by the insecticide. Figure (2) shows the hyperbolic curve of tryptophan concentration versus total-enzyme activity. The activity of the enzyme in the presence of dimethoate did not reach the maximum velocity. The inhibition of the total-enzyme activity was found to be uncompetitive as shown in Figure (3). Dimethoate decreased the Km value from 0.29 mM to 0.24 mM, but the product release from substrate-product complex might be decreased. In the control reaction, high concentration of tryptophan (0.8-1.0 mM) showed a substrate inhibition, but dimethoate set free from this inhibition (Figure 3). In our previous data (Hassan et al., 1990), carbaryl in a final concentration of 500 μΜ showed noncompetitive type of inhibition to tryptophan pyrrolase activity. The same concentration of dimethoate in this study did not inhibit the enzyme activity (data was not shown), but 1 mM dimethoate resulted in an inhibition of the total-enzyme activity more than the holo-enzyme activity. In conclusion, dimethoate may interact with the holo-form of tryptophran pyrrolase and increase the affinity of the enzyme to L-tryptophan, but" decrease the turnover rate. The interaction of the insecticide with the enzyme apo-form may decrease its conversion to the holo-form.
338
HASSAN ET AL.
REFERENCES Badawy Α Α Β. Biochem J, 1 7 2 : 481. (1978). Badawy A A B. Biochem S o c Trans, 7: 575. (1979).
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
Feigelson P, Greengard O. J Biol Chem. 236 (1): 153-157. (1961). Hassan A M, Minatogawa Y, El-Sewedy S M, Kido R. J Environ B25 (3): 333-346. (1990).
Sci
Health
Kikuchi G, Yoshida Y. Molec Cell Biochem, 5 3 : 163. (1983). Knox W E. Br J Exp Path, 32: 462. (1951). Litman D A, Correia M A. J Pharmac Exp Ther, 232: 337. (1985). Nakamura T, Shinno H, Ichihara A. J Biol Chem, 2 5 5 : 7533-7535. (1980). Noda C, Nakamura T, Ichihara A. J Biol Chem, 258: 1520-1525.(1983). Takikawa O, Kuroiwa T, Yamazaki F, Kido R. J Biol Chem, 263 (4): 20412048. (1988).
Received: December 5, 199
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
In vitro effect of dimethoate on the activity of tryptophan pyrrolase in rat liver a
Ashraf A. M. Hassan , Shehata M. El‐Sewedy a
b
, Yohsuke Minatogawat & Ryo Kido
b
a
Applied Medical Chemistry Department, Medical Research Institute , Alexandria university , Egypt b
Biochemistry Department , Wakayama Medical College , Japan Published online: 21 Nov 2008.
To cite this article: Ashraf A. M. Hassan , Shehata M. El‐Sewedy , Yohsuke Minatogawat & Ryo Kido (1991) In vitro effect of dimethoate on the activity of tryptophan pyrrolase in rat liver, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 26:3, 333-338 To link to this article: http://dx.doi.org/10.1080/03601239109372739
PLEASE SCROLL DOWN FOR ARTICLE
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
J. ENVIRON. SCI. HEALTH, B26(3), 333-338 (1991)
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
IN VITRO EFFECT OF DIMETHOATE ON THE ACTIVITY OF TRYPTOPHAN PYRROLASE IN RAT LIVER
Key words: Dimethoate, Tryptopohan pyrrolase, Total-form , Holo-form Ashraf A. M. Hassan¶*, Shehata M. El-Sewedy¶, Yohsuke Minatogawat† and Ryo Kido†* ¶ Applied Medical Chemistry Department, Medical Research Institute, Alexandria university, Egypt, † Biochemistry Department, Wakayama Medical College, Japan.
ABSTRACT Total and holo-enzyme activities of tryptophan 2,3dioxygenase were measured in vitro in the presence and absence of the organophosphorous insecticide, dimethoate. Addition of dimethoate to the reaction mixture decreased the activities of both total and holo-forms. Total and holoenzyme activities were decreased by 34% and 26%, respectively, by 1 mM dimethoate. On the other hand, 5 mM dimethoate resulted in 56% and 34% inhibition to total and holo-enzyme activities, respectively. Lineweaver-Burk plot of the total-enzyme activity at different tryptophan concentration in the presence of 2 mM dimethoate gave uncompetitive type of inhibition.
* To whom correspondence should be adressed 333 Copyright© 1991 by Marcel Dekker, Inc.
334
HASSAN ET AL.
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
INTRODUCTION The hepatic enzyme tryptophan pyrrolase (L-tryptophan 2,3-dioxygenase, EC 1.13.11.11) which catalyzes the oxidative cleavage of L-tryptophan to N-formylkynurenine is the first and probably the rate limiting enzyme in the catabolism of L-tryptophan (Knox, 1951). In rat liver, tryptophan pyrrolase exists in two forms, the active heme-containing holo-enzyme and the inactive heme-free apo-enzyme. It has been postulated that the relative changes in the holo- and apoenzyme activities reflect the availability of "free" heme of the intracellular "regulatory" heme pool (Badawy, 1978, 1979; Kikuchi and Yoshida, 1983; Litman and Correia, 1985) . It has been reported that tryptophan pyrrolase (TPO) activity is regulated by pancreatic hormones as well as glucocorticoids; giucagon or dibutyryi cAMP greatly enhances the induction by dexamethasone, whereas insulin supresses it (Nakamura et al., 1980). Epinephrine supresses the enzyme induction by giucagon and dexamethasone (Noda et al., 1983). Our previous study showed that TPO activity significantly decreased in vivo after oral administration of the carbamate insecticide "carbaryl" to rats. The in vitro addition of carbaryl to the reaction mixture exerted noncompetitive inhibition to the enzyme activity (Hassan et al., 1990).
EXPERIMENTAL PROCEDURF M a t e r i a l s : Dimethoate was provided from Sumitomo Chemical Co. Ltd., Osaka, Japan. Hematin was purchased from Sigma chemicals Co., St. Louis, USA. L-Kynurenine was from Nacalai tesque, Inc., Kyoto, Japan. All other chemicals used were commercial products of the highest grade available.
335
EFFECT OF DIMETHOATE ON TRYPTOPHAN PYRROLASE
100Holoenzyme 90-
ö
80-
'S
70·
£
60·
Downloaded by [University of Toronto Libraries] at 06:41 20 December[ivilj 2014
Έ ou
Totale niyme
60·
υ
30
2.5
1.0
Figure Tryptophan
pyrrolase
holo-
and
5.0
[Dlmelhoale] (mM)
1 total
activity
at
different
concentrations Of dimethoate. Dlmethoale concentrallon varied Irom 0.5 · 6.0 mM In the reacllon mixture which contained 0.5 mM L-tryptophan. The assay was performed as described under Materials and Methods. Each value Is the average ol duplicate experiments.
Enzyme assay: The enzyme activity was measured according to the principle of Feigelson and Greengard, (1961). The reaction mixture (1 ml) contains 5 mM ascorbate, 2 μ Μ hematin, 0.1 M potassium phosphate buffer, pH 7.0, enzyme preparation and different concentrations of tryptophan (as cited in the figures). After incubation at 37°C for 60 minutes, the reaction was stopped by adding 200 μΙ of 30% trichloroacetic acid, then the reaction tube was placed in boiling water bath for 5 minutes to allow the hydrolysis of Nformylkynurenine to L-kynurenine. The latter was measured spectrophotometrically at 480 nm after the addition of 0.7 ml p-dimethylaminobenzaldhyde to an equal volume of the supernatent (Takikawa et al., 1988).
336
HASSAN ET AL.
je
Without dimelhonte «2 mM Dlmethoate
α>
S
o. σι
2·
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
ι 2 o e 0.2
0.0
0.4
o.e
0.6
Figure
1.0
[Tryptophan](mM
2
Tryptophan pyrrolase total-activity at different concentrations of L-tryptophan in the presence and absence of dimethoate. L-Tryptophan concentrallon In the reaction mixture varied from 0.2 · 1.0 mM In the presence and absence of 2.0 mM dimethoate. The assay was performed as described under Materials and Methods. Each value is the average of duplicate experiments. U.3-
0.4-
O
Without dimethoate
•
+2 mM Dimethoate
0.3-
0.2-
0.1 -
1/[Tryptopha 0.0•β
•5
-3
-2
Figure Uncompetitive
3
inhibition of rat liver tryptophan pyrrolase total-
activity. Double-reciprocal plots of the rates of the reaction versus variée concentrations of L-tryplophan In the presence of 2 mM of dimethoate. The assay was perlormed as described under Figure (2). Each value Is the average of duplicate experiments.
EFFECT OF DIMETHOATE ON TRYPTOPHAN PYRROLASE
337
RESULTS AND DISCUSSION
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
Total and holo-enzyme activities were inhibited by 34% and 26%, respectively, by 1 mM dimethoate. The inhibition of totai-enzyme activity reached 56% by 5 mM dimethoate, whereas only 34% inhibition was observed in the holo-enzyme (Figure 1). This result shows that the apo-enzyme is more sensitive than the holo-enzyme to dimethoate. The conversion of apo-enzyme to holo-enzyme may be affected by the insecticide. Figure (2) shows the hyperbolic curve of tryptophan concentration versus total-enzyme activity. The activity of the enzyme in the presence of dimethoate did not reach the maximum velocity. The inhibition of the total-enzyme activity was found to be uncompetitive as shown in Figure (3). Dimethoate decreased the Km value from 0.29 mM to 0.24 mM, but the product release from substrate-product complex might be decreased. In the control reaction, high concentration of tryptophan (0.8-1.0 mM) showed a substrate inhibition, but dimethoate set free from this inhibition (Figure 3). In our previous data (Hassan et al., 1990), carbaryl in a final concentration of 500 μΜ showed noncompetitive type of inhibition to tryptophan pyrrolase activity. The same concentration of dimethoate in this study did not inhibit the enzyme activity (data was not shown), but 1 mM dimethoate resulted in an inhibition of the total-enzyme activity more than the holo-enzyme activity. In conclusion, dimethoate may interact with the holo-form of tryptophran pyrrolase and increase the affinity of the enzyme to L-tryptophan, but" decrease the turnover rate. The interaction of the insecticide with the enzyme apo-form may decrease its conversion to the holo-form.
338
HASSAN ET AL.
REFERENCES Badawy Α Α Β. Biochem J, 1 7 2 : 481. (1978). Badawy A A B. Biochem S o c Trans, 7: 575. (1979).
Downloaded by [University of Toronto Libraries] at 06:41 20 December 2014
Feigelson P, Greengard O. J Biol Chem. 236 (1): 153-157. (1961). Hassan A M, Minatogawa Y, El-Sewedy S M, Kido R. J Environ B25 (3): 333-346. (1990).
Sci
Health
Kikuchi G, Yoshida Y. Molec Cell Biochem, 5 3 : 163. (1983). Knox W E. Br J Exp Path, 32: 462. (1951). Litman D A, Correia M A. J Pharmac Exp Ther, 232: 337. (1985). Nakamura T, Shinno H, Ichihara A. J Biol Chem, 2 5 5 : 7533-7535. (1980). Noda C, Nakamura T, Ichihara A. J Biol Chem, 258: 1520-1525.(1983). Takikawa O, Kuroiwa T, Yamazaki F, Kido R. J Biol Chem, 263 (4): 20412048. (1988).
Received: December 5, 199
