Neurotoxicoh~gyand Teratology, Vol. 12, pp. 611~614. ,7 PergamonPress pie, 1990. Printed in the U.S.A.
0892-0362i90 $3.00 ~ .[X)
Distribution and Some Biochemical Properties of Rat Paraoxonase Activity M. C. P E L L I N , * A. MORETI"O,'I" M. LOTTI+ A N D E. V I L A N O V A *
*Departamento de Neuroquhnica, Universidad de Alicante, Apartado 99, Alicante, Spain 4Istituto di Medicina di Lavoro, Universit& di Padova, Via Facciolati, 71, Padova, Itah"
PELLIN, M. C.. A. MORETTO, M. LOTTI AND E. VILANOVA. Distribution and some biochemicalproperties of rat paruoxonase activity. NEUROTOXICOL TERATOL 12(61 611~614. 1990.--The calcium-dependent enzyme activity which hydrolyzes the p-nitrophenyI-O-P bond of paraoxon (paraoxonase) has been studied in several rat and human tissues. Rat plasma and liver showed the highest activities (1.31 '-0.19, 0.82 _+0.09 nmol/min mg protein -+-SEM, respectively), while other tissues showed less than 2~ plasma activity. The Arrhenius plot showed monophasic patterns in both tissues with activation energy values of Ea = 57 - 3 and 69 -+4 kcal/mol °K for rat liver and plasma, respectively. Rat plasma and liver paraoxonase lost about 80% activity after 24-hr storage at 27-30~C and was not restored by calcium addition. There was no loss of activity in human serum after 3 days and only 33% after 5 days. The pH optimum Ik)r paraoxonase activities was about 7.4 tbr both rat tissues. It is concluded that plasma paraoxonase is similar to the liver enzyme and is a good mirror for total body detoxifying activity. Organophosphorus
A-esterase
Paraoxonase
Rat
Paraoxon
Detoxitication
slightly higher (10eh-) than for pH 7. In the heat inactivation study 0.8 ml tissue solution (100 mg/ml) was preincubated at the temperature of assay (37, 4 5 . 4 7 , 50, 55 and 60°C). After the preincubation time (range of 2-9[) rain), 0.1 ml of cold 10 mM EDTA or water was added and the tubes put into an iced water bath during 1 rain betore adding substrate (0.1 ml 10 mM paraoxon) and continuing the paraoxonase assay in the usual manner at 37"C. In plasma and liver of rat the tormation of p-nitrophenol was linear with tissue concentrations from 16 to 80 mg/ml in the assay mixture, up to 120 rain.
ALDRIDGE and Reiner (2) defined "'A-esterases" the enzymes that hydrolyse organophosphorus compounds much faster than B-esterases. An alternative name such as "phosphoric triester hydrolases'" was also suggested (16). We report the distribution and some biochemical properties of paraoxonase in human and rat tissues. The general aim of this work is to understand the role of paraoxonase in paraoxon detoxification, and to ascertain whether the level of accessible plasma paraoxonase is representative of the total body activity. METHOD
RESULTS Paraoxon (o,o-diethyl p-nitrophenyl phosphate) was purchased from Sigma Chemical Co. and purified as already described (8). Wistar rats were anaesthetized with chloral hydrate IP (0.3 g/kg) and heparinized by cardiac puncture. Blood was withdrawn and tissues perfused with 0.9ok NaCI. The dissected tissues were homogenized in 100 mM Tris/HC1 buffer using a Polymm homogenizer. Paraoxonase was assayed as described by Traverso et al. (15) and Playfer et al. (13), at pH 7.4 and 37°C, except when specifically indicated. For the study of the pH effect the reaction was followed in buffer at the desired pH. For this, tissue was homogenated at 3(1% in KCI 1.15~ without buffer and diluted with buffer of the desired pH. The final pH was checked in parallel control experiments and the deviations from the nominal pH were less than 0.1 unit in the extreme pHs with lowest buffer capacity. The pH during the colorimetric measurement was always the same and determined by the perchloric/acetate and 0.5 M Tris pH 10 buffer added alter the enzymatic reaction. Blanks were used to correct for the underground colour (substrate and tissue) and for spontaneous hydrolysis. The spontaneous hydrolysis for the highest pH used was only
Rat plasma and liver showed the highest paraoxonase activities (I.31 and 0.82 nmol/min mg protein), while other tissues showed less than 2% of Table I. No correlation was lbund when individual plasma activities were plotted versus the corresponding liver activities, either expressed as g tissue or mg protein. Human liver assayed 24 hr alter death showed 0.301 tunol/min mg protein of paraoxonase activity. Hmnan plasma, when sampled 24 hr alter death, had activity similar to that of fresh plasma which showed 0.357 _+0.042 nmol/min nag protein ( n = 19). Other human tissues had no detectable activity. The time-dependent heat inactivation of rat plasma and liver paraoxonase was studied, in the range of 2-90 rain at 37, 45, 47. 50, 55 and 60~C. The Arrhenius plot showed monophasic patterns (Figs. I and 2). with Ea values of 57 = 3 and 69 -+ 4 kcal/mol °K for rat liver and plasma respectively. The high Ea values suggest that the long decay of rat paraoxonase during storage is not entirely due to thermal inactivation but other cofactors like calcium might be involved. Rat plasma and liver paraoxonase lost 80c/~ activity alter 24 hr
61]
612
PELLIN E l A L .
TABLE 1 I)ISTRIBUTION OF PARAOXONASE ACTIVITY IN RAT TISSUES
nmol/min g Tissue* -'- SEM
Tissue Plasma laver Kidney Heart Diaphragm Intercostal muscle Lung Brain Spleen Testes (.)vau
83.3 82.4 1.7 1.1 1.0 0.7 0.3 0.2 0.1 0.6 2.4
: = _" = :_-'_+ ± _-_ _ -'-
nmol/min mg Protein ± SEM
13.6 10.4 0.5 0.3 0.3 0.4 0.5 0.2 0.6 0.3
1.31 0.82 0.02 0.02 0.02 0.01 0.01 0.01 (I.00 (I.01 0.07
1.2
_" 0.19 -* 0.09 _+ 0.0 ± 0.0 _+ 0,0 ± 0.0 _+ 0.0 -'- 0.o = 0.o _" o.0 = 0.04
*Data are means ± SEM from 8 different animals except for testes and o v a l ' (n =4).
at 30°C and the activity was not restored by calcium (Table 2). T h e s e results contrast with those o f h u m a n p l a s m a obtained over 5 days at 27°C (Table 3), and s h o w i n g a relative stability for 3 days. The pH profile for rat liver and p l a s m a p a r a o x o n a s e is s h o w n in Fig. 3. Both liver and p l a s m a p a r a o x o n a s e s h o w e d similar o p t i m u m pH o f about 7.4. Michaelis constant was d e t e r m i n e d for liver and p l a s m a paraoxonase u s i n g 16 different paraoxon concentrations in the range of 0.03 to 4 m M and u s i n g standard conditions for p a r a o x o n a s e assay. Higher concentration w a s not possible to a s s a y due to the solubility o f paraoxon. Data were fitted to the Michaelis equation by a nonlinear direct c o m p u t e r i z e d fitting m e t h o d based on the least square principle. For p l a s m a , in two separate experim e n t s , K . , values o f 3.7 and 4 . 0 m M were obtained, while a value o f 2.7 m M w a s obtained w h e n a s s a y e d at pH 10. For liver 9.9, 5.3 and 7.1 m M were obtained from 3 separate e x p e r i m e n t s . In one e x p e r i m e n t u s i n g 1 m M c a l c i u m a K,, o f 5.0 m M was obtained. T h e standard deviation o f the residuals o f the fitting was less than 1.5c~ o f the calculated V m (except one liver e x p e r i m e n t that was 4 ~ ).
n. ~3T'0
0 E~I
,, ..I ." ~
t/T
FIG. 2. Arrhenius plot of the heat inactivation of rat plasma paraoxonase. K = first order rate constant of inactivation. T = Absolute temperature f ~'KI.
DISCUSSION
P a r a o x o n a s e activity in rats has the greatest activity in plasma and liver. Significant activities in other tissues such as spleen and kidney have been reported (1,6), but the a n i m a l s in these experim e n t s were not peffused, s u g g e s t i n g a possible c o n t a m i n a t i o n by plasma. O u r results c o n c e r n i n g rat p l a s m a p a r a o x o n a s e (83-+ 1.4 n m o l / m i n ml plasma) are similar to those already reported [57 -'- 4 n m o l / m i n ml s e r u m (4), 6 1 - ' - 4 n m o l / m i n / m l serum (10) and 89 n m o l / m i n ml p l a s m a (12)]. Data for K,,, determination fitted the Michaelis equation. H o w e v e r . the reproducibility of the calculated K m was limited by the lack o f saturation of the reaction because o f the solubility of paraoxon (about 4 m M ) . O u r values o f K,,, calculated for rat plasma and liver were m u c h h i g h e r than those reported for s e r u m of scveral species (0.6 m M for rat) (18). and for liver (9000 × g supernatant o f rat and mice livers, 0.18 and 0.13 m M , respectively) (17). Several reasons might account for these differences. In one case. clear description o f the paraoxon concentrations is described (17), and in another only 5 different concentrations up to 5 m M at 25°C were used (18). Paraoxon concentrations higher than its solubility (about 4 m M at 37°C) could explain the apparent
TABLE 2 STABILITY O k RAT PLASMA ,AND LIVER PARAOXONASE AT 3(YC D
|)lasma l,i~er Initial Activity Initial Activit,.. Activity After 24 hr Activit,, After 24 hr
D
"2 :) 01_, ~ 0
0.'30.= I
O., Hj ~,2
tit
FIG. I. Arrheniu,, plot of the heat inactivation of rat liver paraoxonase. K = first order rate constant of inactivation. T = Absolute temperature (°K).
Tissue Tissue .- EDTA 1 mM
2.52 0.00
0.57 0.0(I
7btal .ctivitv - EDTA activity
2.52
0.57
0..t3
0.07
Tissue + EDTA 1 mM + Ca I mM Tissue + EDTA 1 mM + Ca 5 mM Tissue - Ca I mM Tissue - Ca 5 mM
2.65
0.55
(.I.76
0.05
3.34
0.74
1.52
0.16
3.30 3.40
0.75 0.g7
1.38 1.51
0.O8 (].16
Activity is expressed in nmol/min mg protein.
0.33 (t.IX)
(I.07 0.00
RAT P A R A O X O N A S E ACTIVITY
613
TABLE 3 STABILITY OF HUMAN SERUM PARAOXONASE Experimental Conditions
100 Day 1
Day 2
Day 3
Day 4
Day 5
Serum Serum ..e EDTA 1 mM
(I.93 0.21
0.92 0.13
0.81 0.09
0.74 0.08
0.53 0.115
Total activity EDTA activin"
0.72
0.79
0.72
0.67
0.48
Serum + EDTA 1 mM + Ca 1 mM Serum + EDTA 1 mM + Ca 5 mM Serum - Ca 1 mM Serum - Ca 5 mM
0.95
0.92
0.83
0.79
0.55
1.23
1.25
1.18
1.17
0.83
1.24 1.25
1.22 1.29
1.14 1.21
1.13 1.20
0.79 0.87
60
20
Activity is expressed in nmol/min mg protein. (Enzyme activity was assayed in I(XI mM glycine pH 10.) Serum was kept undiluted at 27"C and tested over a period of five days.
saturation o f the reaction resulting in an apparent lower K,,. The differences in Ca concentration can be ruled out because the K ~ values were in the same order o f magnitude with or without Ca addition. The pH profile showed pH optimum o f about 7.4 in rat plasma and liver paraoxonase. This is similar to that reported for rabbit plasma paraoxonase (1,3), but is different than that o f human serum paraoxonase which does not show a pH optimum (11,15). However, in a Danish population, it was described that the low activity group did not have a pH optimum, while the high activity group had a pH optimum at about 8.5 (7). In another study (I 1), however, such differences were not found. The apparent activation energy (Ea) o f the heat inactivation process, calculated by the Arrhenius plot shows that the sensitivity o f liver paraoxonase is somewhat higher than that o f plasma. The first order rate constant observed for inactivation at 50°C for plasma paraoxonase ( K / m i n - ~ = 0 . 0 3 8 ) was similar to that described by Traverso et al. (15) for human plasma paraoxonase (K/rain '=-0.024) and that described by Reiner et al. (141. At
!
!
~
r
I
7
8
9
10
11
FIG. 3. Effect of pH in the activity of plasma and liver rat paraoxonasc. Maximal activities were 2.08 and 1.52 nmol/min mg protein respectively.
55°C the rate o f inactivation highly increases (0.169 min -~) similarly to what was shown by Traverso et al. (15). Suggestions have been made about the possibility to predict whether subjects with low serum activity are more susceptible to the toxic effects o f a given OP (9) and also to use this enzyme therapeutically in OP intoxications (5). This paper shows that rat plasma paraoxonase is similar to the liver enzyme and represents about one half o f "total b o d y " paraoxonase. Therefore, it seems a good mirror for such detoxifying activity. ACKNOWLEDGEMENTS This work was supported by FISSS research grants (FISSS No. 89/0874). M. C. Pellin is supported by a postdoctoral fellowship from "'Ministerio de Educaci6n y Ciencia."
REFERENCF~ 1. Aldridge, W. N. Serum esterases. 2 An enzyme hydrolysing diethyl p-nitrophenyl phosphate (E600) and its identity with the A-esterase of mammalian sera. Biochem. J. 53:117-124: 1953. 2. Aldridge, W. N.; Reiner, E. Enzyme inhibitors as substrates. Interactions of esterases with esters of organophosphorus and carbamic acids. Amsterdam: North-Holland Publishing Company; 1972, 3. Becker, E. L.; Barbaro, . I . F . The enzymatic hydrolysis of pnitrophenyl ethyl phosphonates by mammalian plasmas. Biochem. Pharmacol. 13:1219-1227: 1964. 4. Brea/ey, C. J.; Walker, C. H.; Baldwin. B. C. A-esterase activities in relation to the differential toxicity of pirimiphos-methyl to birds and mammals. Pest. Sci. 11:546-554; 1980. 5. Chemnitius, J. M.; Haselmeyer, K. H.; Schilling, P.; Zech, R. Organophosphate detoxification by covalently i.lmobilized phosphorylphosphatases. Dubrovnik: International meeting on esterases hydrolysing organophosphorus compounds (Abstract p. 101; 1988. 6. Chemnitius, J. M.; Losch, H.; Losch, K.: Zech, R. Organophosphate detnxicating hydrolases in different vertebrate species. Comp. Biochem. Physiol. 76C:85-93; 1983. 7. Eiberg, H.; Mohr, J. Genetics of paraoxonase. Ann. Hum. Genet. 45:323-330; 1981. 8. Johnson, M. K. Improved assay of neurotoxic estcrase tor screening organophosphates for delayed neurotoxicity potential. Arch. Toxicol.
37:113-115; 1977. 9. Omenn, G. S. The role of genetic differences in human susceptibility to pesticides. In: Costa, L. G.; Galli. C. L.; Murphy. S. D., eds. Toxicology of pesticides: Experimental, clinical and regulatory perspectives. Nato Asi. Series vol. H 13. Berlin: Springer; 1987:93-107. 10. Mackness. M. I.; Thompson, H. M.: Hardy, A. R.: Walker, C. H. Distinction between A-esterases and arylesterases. Implications for esterase classification. Biochem. J. 245:293-296; 1987. 11. Mueller, R. F.; Hornung, S.; Furlong, C. E.: Anderson. J.; Giblen, E. R.: Motulsky. A. O. Plasma paraoxonase polymorphism; a new enzyme assay population, family, biochemicaL, and linkage studies. Am. J. Hum. Genet. 35:393-408; 1983. 12. Pla, A.; Johnson, M. K. Degradation by rat tissues in vitro of organophosphorus esters which inhibit cholinesterase. Biochem. Pharmacol. 38:1535-1542; 1989. 13. Playfer, J. R,: Eze. L. C.; Bullen, M. F.; Evans, D. A. P. Genetic polymorphism and interethnic variability of plasma paraoxonase activity. J. Med. Genet. 13:337-342; 1976. 14. Reiner, E.; Simeon, V.; Skrinjaric-Spoljar, M. Hydrolysis of o,odimethyl-2,2-dichlorovinyl phosphate (DDVP) by estcrases in parasitic helminths, and in vertebrate plasma and erythrocytes. Comp. Biochem. Physiol. 66C:149-152; 1980. 15. Traverso. R.; Moreno, A.; Loni, M. Human serum "A"-esterases
614
hydrolysis of o,o-dimethyl-2,2-dichloroviny] phosphate. Bi~xzhem. Pharmacol. 38:671-676: 1989. 16. Walker. C. H. The development t~t"an improved system for nomenclature and classification of esterases. Dubrovnik: International meeting on esterases hydrolysing organophosphorus compounds (Abstract L, 25); 1988.
PELLIN ET AL.
17. Wallace, K. B., Dargan, J. E. Intrinsic metabolic clearance of parathion and paraoxon by livers from fish and rodents. Toxicol. Appl. Pharmacol. 90:235-242: 198"/. 18 Zcch. R.: Zurcher. K. Organophosphate splitting serum enzymes m diflerent mammals. Comp. Biochem. Physiol. 48t3:427--1.33: 1974.
0892-0362i90 $3.00 ~ .[X)
Distribution and Some Biochemical Properties of Rat Paraoxonase Activity M. C. P E L L I N , * A. MORETI"O,'I" M. LOTTI+ A N D E. V I L A N O V A *
*Departamento de Neuroquhnica, Universidad de Alicante, Apartado 99, Alicante, Spain 4Istituto di Medicina di Lavoro, Universit& di Padova, Via Facciolati, 71, Padova, Itah"
PELLIN, M. C.. A. MORETTO, M. LOTTI AND E. VILANOVA. Distribution and some biochemicalproperties of rat paruoxonase activity. NEUROTOXICOL TERATOL 12(61 611~614. 1990.--The calcium-dependent enzyme activity which hydrolyzes the p-nitrophenyI-O-P bond of paraoxon (paraoxonase) has been studied in several rat and human tissues. Rat plasma and liver showed the highest activities (1.31 '-0.19, 0.82 _+0.09 nmol/min mg protein -+-SEM, respectively), while other tissues showed less than 2~ plasma activity. The Arrhenius plot showed monophasic patterns in both tissues with activation energy values of Ea = 57 - 3 and 69 -+4 kcal/mol °K for rat liver and plasma, respectively. Rat plasma and liver paraoxonase lost about 80% activity after 24-hr storage at 27-30~C and was not restored by calcium addition. There was no loss of activity in human serum after 3 days and only 33% after 5 days. The pH optimum Ik)r paraoxonase activities was about 7.4 tbr both rat tissues. It is concluded that plasma paraoxonase is similar to the liver enzyme and is a good mirror for total body detoxifying activity. Organophosphorus
A-esterase
Paraoxonase
Rat
Paraoxon
Detoxitication
slightly higher (10eh-) than for pH 7. In the heat inactivation study 0.8 ml tissue solution (100 mg/ml) was preincubated at the temperature of assay (37, 4 5 . 4 7 , 50, 55 and 60°C). After the preincubation time (range of 2-9[) rain), 0.1 ml of cold 10 mM EDTA or water was added and the tubes put into an iced water bath during 1 rain betore adding substrate (0.1 ml 10 mM paraoxon) and continuing the paraoxonase assay in the usual manner at 37"C. In plasma and liver of rat the tormation of p-nitrophenol was linear with tissue concentrations from 16 to 80 mg/ml in the assay mixture, up to 120 rain.
ALDRIDGE and Reiner (2) defined "'A-esterases" the enzymes that hydrolyse organophosphorus compounds much faster than B-esterases. An alternative name such as "phosphoric triester hydrolases'" was also suggested (16). We report the distribution and some biochemical properties of paraoxonase in human and rat tissues. The general aim of this work is to understand the role of paraoxonase in paraoxon detoxification, and to ascertain whether the level of accessible plasma paraoxonase is representative of the total body activity. METHOD
RESULTS Paraoxon (o,o-diethyl p-nitrophenyl phosphate) was purchased from Sigma Chemical Co. and purified as already described (8). Wistar rats were anaesthetized with chloral hydrate IP (0.3 g/kg) and heparinized by cardiac puncture. Blood was withdrawn and tissues perfused with 0.9ok NaCI. The dissected tissues were homogenized in 100 mM Tris/HC1 buffer using a Polymm homogenizer. Paraoxonase was assayed as described by Traverso et al. (15) and Playfer et al. (13), at pH 7.4 and 37°C, except when specifically indicated. For the study of the pH effect the reaction was followed in buffer at the desired pH. For this, tissue was homogenated at 3(1% in KCI 1.15~ without buffer and diluted with buffer of the desired pH. The final pH was checked in parallel control experiments and the deviations from the nominal pH were less than 0.1 unit in the extreme pHs with lowest buffer capacity. The pH during the colorimetric measurement was always the same and determined by the perchloric/acetate and 0.5 M Tris pH 10 buffer added alter the enzymatic reaction. Blanks were used to correct for the underground colour (substrate and tissue) and for spontaneous hydrolysis. The spontaneous hydrolysis for the highest pH used was only
Rat plasma and liver showed the highest paraoxonase activities (I.31 and 0.82 nmol/min mg protein), while other tissues showed less than 2% of Table I. No correlation was lbund when individual plasma activities were plotted versus the corresponding liver activities, either expressed as g tissue or mg protein. Human liver assayed 24 hr alter death showed 0.301 tunol/min mg protein of paraoxonase activity. Hmnan plasma, when sampled 24 hr alter death, had activity similar to that of fresh plasma which showed 0.357 _+0.042 nmol/min nag protein ( n = 19). Other human tissues had no detectable activity. The time-dependent heat inactivation of rat plasma and liver paraoxonase was studied, in the range of 2-90 rain at 37, 45, 47. 50, 55 and 60~C. The Arrhenius plot showed monophasic patterns (Figs. I and 2). with Ea values of 57 = 3 and 69 -+ 4 kcal/mol °K for rat liver and plasma respectively. The high Ea values suggest that the long decay of rat paraoxonase during storage is not entirely due to thermal inactivation but other cofactors like calcium might be involved. Rat plasma and liver paraoxonase lost 80c/~ activity alter 24 hr
61]
612
PELLIN E l A L .
TABLE 1 I)ISTRIBUTION OF PARAOXONASE ACTIVITY IN RAT TISSUES
nmol/min g Tissue* -'- SEM
Tissue Plasma laver Kidney Heart Diaphragm Intercostal muscle Lung Brain Spleen Testes (.)vau
83.3 82.4 1.7 1.1 1.0 0.7 0.3 0.2 0.1 0.6 2.4
: = _" = :_-'_+ ± _-_ _ -'-
nmol/min mg Protein ± SEM
13.6 10.4 0.5 0.3 0.3 0.4 0.5 0.2 0.6 0.3
1.31 0.82 0.02 0.02 0.02 0.01 0.01 0.01 (I.00 (I.01 0.07
1.2
_" 0.19 -* 0.09 _+ 0.0 ± 0.0 _+ 0,0 ± 0.0 _+ 0.0 -'- 0.o = 0.o _" o.0 = 0.04
*Data are means ± SEM from 8 different animals except for testes and o v a l ' (n =4).
at 30°C and the activity was not restored by calcium (Table 2). T h e s e results contrast with those o f h u m a n p l a s m a obtained over 5 days at 27°C (Table 3), and s h o w i n g a relative stability for 3 days. The pH profile for rat liver and p l a s m a p a r a o x o n a s e is s h o w n in Fig. 3. Both liver and p l a s m a p a r a o x o n a s e s h o w e d similar o p t i m u m pH o f about 7.4. Michaelis constant was d e t e r m i n e d for liver and p l a s m a paraoxonase u s i n g 16 different paraoxon concentrations in the range of 0.03 to 4 m M and u s i n g standard conditions for p a r a o x o n a s e assay. Higher concentration w a s not possible to a s s a y due to the solubility o f paraoxon. Data were fitted to the Michaelis equation by a nonlinear direct c o m p u t e r i z e d fitting m e t h o d based on the least square principle. For p l a s m a , in two separate experim e n t s , K . , values o f 3.7 and 4 . 0 m M were obtained, while a value o f 2.7 m M w a s obtained w h e n a s s a y e d at pH 10. For liver 9.9, 5.3 and 7.1 m M were obtained from 3 separate e x p e r i m e n t s . In one e x p e r i m e n t u s i n g 1 m M c a l c i u m a K,, o f 5.0 m M was obtained. T h e standard deviation o f the residuals o f the fitting was less than 1.5c~ o f the calculated V m (except one liver e x p e r i m e n t that was 4 ~ ).
n. ~3T'0
0 E~I
,, ..I ." ~
t/T
FIG. 2. Arrhenius plot of the heat inactivation of rat plasma paraoxonase. K = first order rate constant of inactivation. T = Absolute temperature f ~'KI.
DISCUSSION
P a r a o x o n a s e activity in rats has the greatest activity in plasma and liver. Significant activities in other tissues such as spleen and kidney have been reported (1,6), but the a n i m a l s in these experim e n t s were not peffused, s u g g e s t i n g a possible c o n t a m i n a t i o n by plasma. O u r results c o n c e r n i n g rat p l a s m a p a r a o x o n a s e (83-+ 1.4 n m o l / m i n ml plasma) are similar to those already reported [57 -'- 4 n m o l / m i n ml s e r u m (4), 6 1 - ' - 4 n m o l / m i n / m l serum (10) and 89 n m o l / m i n ml p l a s m a (12)]. Data for K,,, determination fitted the Michaelis equation. H o w e v e r . the reproducibility of the calculated K m was limited by the lack o f saturation of the reaction because o f the solubility of paraoxon (about 4 m M ) . O u r values o f K,,, calculated for rat plasma and liver were m u c h h i g h e r than those reported for s e r u m of scveral species (0.6 m M for rat) (18). and for liver (9000 × g supernatant o f rat and mice livers, 0.18 and 0.13 m M , respectively) (17). Several reasons might account for these differences. In one case. clear description o f the paraoxon concentrations is described (17), and in another only 5 different concentrations up to 5 m M at 25°C were used (18). Paraoxon concentrations higher than its solubility (about 4 m M at 37°C) could explain the apparent
TABLE 2 STABILITY O k RAT PLASMA ,AND LIVER PARAOXONASE AT 3(YC D
|)lasma l,i~er Initial Activity Initial Activit,.. Activity After 24 hr Activit,, After 24 hr
D
"2 :) 01_, ~ 0
0.'30.= I
O., Hj ~,2
tit
FIG. I. Arrheniu,, plot of the heat inactivation of rat liver paraoxonase. K = first order rate constant of inactivation. T = Absolute temperature (°K).
Tissue Tissue .- EDTA 1 mM
2.52 0.00
0.57 0.0(I
7btal .ctivitv - EDTA activity
2.52
0.57
0..t3
0.07
Tissue + EDTA 1 mM + Ca I mM Tissue + EDTA 1 mM + Ca 5 mM Tissue - Ca I mM Tissue - Ca 5 mM
2.65
0.55
(.I.76
0.05
3.34
0.74
1.52
0.16
3.30 3.40
0.75 0.g7
1.38 1.51
0.O8 (].16
Activity is expressed in nmol/min mg protein.
0.33 (t.IX)
(I.07 0.00
RAT P A R A O X O N A S E ACTIVITY
613
TABLE 3 STABILITY OF HUMAN SERUM PARAOXONASE Experimental Conditions
100 Day 1
Day 2
Day 3
Day 4
Day 5
Serum Serum ..e EDTA 1 mM
(I.93 0.21
0.92 0.13
0.81 0.09
0.74 0.08
0.53 0.115
Total activity EDTA activin"
0.72
0.79
0.72
0.67
0.48
Serum + EDTA 1 mM + Ca 1 mM Serum + EDTA 1 mM + Ca 5 mM Serum - Ca 1 mM Serum - Ca 5 mM
0.95
0.92
0.83
0.79
0.55
1.23
1.25
1.18
1.17
0.83
1.24 1.25
1.22 1.29
1.14 1.21
1.13 1.20
0.79 0.87
60
20
Activity is expressed in nmol/min mg protein. (Enzyme activity was assayed in I(XI mM glycine pH 10.) Serum was kept undiluted at 27"C and tested over a period of five days.
saturation o f the reaction resulting in an apparent lower K,,. The differences in Ca concentration can be ruled out because the K ~ values were in the same order o f magnitude with or without Ca addition. The pH profile showed pH optimum o f about 7.4 in rat plasma and liver paraoxonase. This is similar to that reported for rabbit plasma paraoxonase (1,3), but is different than that o f human serum paraoxonase which does not show a pH optimum (11,15). However, in a Danish population, it was described that the low activity group did not have a pH optimum, while the high activity group had a pH optimum at about 8.5 (7). In another study (I 1), however, such differences were not found. The apparent activation energy (Ea) o f the heat inactivation process, calculated by the Arrhenius plot shows that the sensitivity o f liver paraoxonase is somewhat higher than that o f plasma. The first order rate constant observed for inactivation at 50°C for plasma paraoxonase ( K / m i n - ~ = 0 . 0 3 8 ) was similar to that described by Traverso et al. (15) for human plasma paraoxonase (K/rain '=-0.024) and that described by Reiner et al. (141. At
!
!
~
r
I
7
8
9
10
11
FIG. 3. Effect of pH in the activity of plasma and liver rat paraoxonasc. Maximal activities were 2.08 and 1.52 nmol/min mg protein respectively.
55°C the rate o f inactivation highly increases (0.169 min -~) similarly to what was shown by Traverso et al. (15). Suggestions have been made about the possibility to predict whether subjects with low serum activity are more susceptible to the toxic effects o f a given OP (9) and also to use this enzyme therapeutically in OP intoxications (5). This paper shows that rat plasma paraoxonase is similar to the liver enzyme and represents about one half o f "total b o d y " paraoxonase. Therefore, it seems a good mirror for such detoxifying activity. ACKNOWLEDGEMENTS This work was supported by FISSS research grants (FISSS No. 89/0874). M. C. Pellin is supported by a postdoctoral fellowship from "'Ministerio de Educaci6n y Ciencia."
REFERENCF~ 1. Aldridge, W. N. Serum esterases. 2 An enzyme hydrolysing diethyl p-nitrophenyl phosphate (E600) and its identity with the A-esterase of mammalian sera. Biochem. J. 53:117-124: 1953. 2. Aldridge, W. N.; Reiner, E. Enzyme inhibitors as substrates. Interactions of esterases with esters of organophosphorus and carbamic acids. Amsterdam: North-Holland Publishing Company; 1972, 3. Becker, E. L.; Barbaro, . I . F . The enzymatic hydrolysis of pnitrophenyl ethyl phosphonates by mammalian plasmas. Biochem. Pharmacol. 13:1219-1227: 1964. 4. Brea/ey, C. J.; Walker, C. H.; Baldwin. B. C. A-esterase activities in relation to the differential toxicity of pirimiphos-methyl to birds and mammals. Pest. Sci. 11:546-554; 1980. 5. Chemnitius, J. M.; Haselmeyer, K. H.; Schilling, P.; Zech, R. Organophosphate detoxification by covalently i.lmobilized phosphorylphosphatases. Dubrovnik: International meeting on esterases hydrolysing organophosphorus compounds (Abstract p. 101; 1988. 6. Chemnitius, J. M.; Losch, H.; Losch, K.: Zech, R. Organophosphate detnxicating hydrolases in different vertebrate species. Comp. Biochem. Physiol. 76C:85-93; 1983. 7. Eiberg, H.; Mohr, J. Genetics of paraoxonase. Ann. Hum. Genet. 45:323-330; 1981. 8. Johnson, M. K. Improved assay of neurotoxic estcrase tor screening organophosphates for delayed neurotoxicity potential. Arch. Toxicol.
37:113-115; 1977. 9. Omenn, G. S. The role of genetic differences in human susceptibility to pesticides. In: Costa, L. G.; Galli. C. L.; Murphy. S. D., eds. Toxicology of pesticides: Experimental, clinical and regulatory perspectives. Nato Asi. Series vol. H 13. Berlin: Springer; 1987:93-107. 10. Mackness. M. I.; Thompson, H. M.: Hardy, A. R.: Walker, C. H. Distinction between A-esterases and arylesterases. Implications for esterase classification. Biochem. J. 245:293-296; 1987. 11. Mueller, R. F.; Hornung, S.; Furlong, C. E.: Anderson. J.; Giblen, E. R.: Motulsky. A. O. Plasma paraoxonase polymorphism; a new enzyme assay population, family, biochemicaL, and linkage studies. Am. J. Hum. Genet. 35:393-408; 1983. 12. Pla, A.; Johnson, M. K. Degradation by rat tissues in vitro of organophosphorus esters which inhibit cholinesterase. Biochem. Pharmacol. 38:1535-1542; 1989. 13. Playfer, J. R,: Eze. L. C.; Bullen, M. F.; Evans, D. A. P. Genetic polymorphism and interethnic variability of plasma paraoxonase activity. J. Med. Genet. 13:337-342; 1976. 14. Reiner, E.; Simeon, V.; Skrinjaric-Spoljar, M. Hydrolysis of o,odimethyl-2,2-dichlorovinyl phosphate (DDVP) by estcrases in parasitic helminths, and in vertebrate plasma and erythrocytes. Comp. Biochem. Physiol. 66C:149-152; 1980. 15. Traverso. R.; Moreno, A.; Loni, M. Human serum "A"-esterases
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hydrolysis of o,o-dimethyl-2,2-dichloroviny] phosphate. Bi~xzhem. Pharmacol. 38:671-676: 1989. 16. Walker. C. H. The development t~t"an improved system for nomenclature and classification of esterases. Dubrovnik: International meeting on esterases hydrolysing organophosphorus compounds (Abstract L, 25); 1988.
PELLIN ET AL.
17. Wallace, K. B., Dargan, J. E. Intrinsic metabolic clearance of parathion and paraoxon by livers from fish and rodents. Toxicol. Appl. Pharmacol. 90:235-242: 198"/. 18 Zcch. R.: Zurcher. K. Organophosphate splitting serum enzymes m diflerent mammals. Comp. Biochem. Physiol. 48t3:427--1.33: 1974.
