Chicken Serum Albumin Hydrolyzes Dichlorophenyl

Chem. Res. Toxicol. 1998, 11, 1441-1446

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Chicken Serum Albumin Hydrolyzes Dichlorophenyl Phosphoramidates by a Mechanism Based on Transient Phosphorylation Miguel A. Sogorb, Antonio Monroy, and Eugenio Vilanova* Unidad de Toxicologı´a y Seguridad Quı´mica, Instituto de Bioingenierı´a, Universidad Miguel Herna´ ndez, Alicante, Spain Received January 22, 1998

The hydrolyzing activities of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) and p-nitrophenyl butyrate (p-NPB) in chicken serum had been found to copurify in the same protein, identified as albumin. The hydrolyzing activities of both chicken serum and commercial serum albumins from different species were inhibited in a dose-dependent manner by short chain fatty acids. On simultaneous incubation of chicken serum with HDCP and p-NPB, a competitive interaction was detected between the two substrates. This behavior suggests that both are hydrolyzed in the same albumin active site. When chicken serum was preincubated with one of the substrates, and the latter were withdrawn by large dilution, the hydrolyzing activities with both substrates were found to be reduced. This reduction was in turn dependent upon the time of preincubation with the first substrate. These results suggest that HDCP and p-NPB are hydrolyzed by the same albumin active site, via a mechanism based on transient phosphorylation/acylation of the active site. The proposed hydrolysis mechanism would account for the hydrolytic kinetics of both substrates.

Introduction Organophosphorus compounds (OPs)1 are widely used as pesticides. As such, they constitute the principal chemical agents implicated in the intoxication of domestic and wild animals (1). The main enzymes involved in OP detoxification are some esterases of unknown physiological function (2, 3) called phosphotriesterases (PTEs) (EC 3.1.8) (2). The highest levels of PTEs have been reported in serum and liver of mammals (2, 4). In general, PTEs are almost undetectable in avian and insect species (2, 5). This is in turn reflected by the marked susceptibility of birds and insects to the toxic effects of OPs, and the greater resistance of mammalian (2). The best characterized PTE is the enzyme called paraoxonase, which hydrolyzes the insecticide paraoxon (O,O′-diethyl pnitrophenyl phosphate). This PTE has been mainly studied in mammalian tissues (6, 7) and in the bacterium Pseudomonas diminuta (8, 9). Aldridge (10) classified the carboxyl esterases (CbEs) as a function of their interaction with OPs. In this sense, A-esterases hydrolyze carboxyl esters and OPs without being inhibited by the latter. The B-esterases likewise hydrolyze carboxyl esters and OPs but are inhibited by the latter. In the case of the type B CbEs, the OPs act * To whom correspondence should be addressed: Divisio´n de Toxicologı´a, UPD de Gene´tica, Nutriciı´on y Toxicologı´a, Facultad de Medicina, Universidad Miguel Herna´ndez, E-03550, San Juan de Alicante, Alicante, Spain. Phone: +34-96-5919477. Fax: +34-965919484. E-mail: [email protected]. 1 Abbreviations: CbE, carboxyl esterase; CSA, chicken serum albumin; DCP, 2,5-dichlorophenol; HDCP, O-hexyl O-2,5-dichlorophenyl phosphoramidate; HDCPase, O-hexyl O-2,5-dichlorophenyl phosphoramidate hydrolyzing activity; HSA, human serum albumin; OP, organophosphorus compound; p-NP, p-nitrophenol; p-NPA, p-nitrophenyl acetate; p-NPB, p-nitrophenyl butyrate; p-NPBase, p-nitrophenyl butyrate hydrolyzing activity; PTE, phosphotriesterase activity.

as suicide substrates, and the interaction between CbE and OP produces the irreversible inhibition by phosphorylation of a serine group within the active site of the enzyme (11). The B-esterases may also be regarded as proteins involved in OP detoxification because each molecule of B-esterase is able to react with an OP molecule, scavenging the latter and thus preventing it from reaching its targets within the central nervous system. In some cases, no clear differentiation exists between A-esterases and PTEs. Thus, paraoxonase activities that hydrolyze carboxyl esters (A-esterases) have been described (6), while in other cases, the protein only hydrolyzes the OP (12). Our laboratory has described the hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) in plasma, liver, and brain of chickens and rats (13). The kinetics of the HDCP hydrolyzing activity (HDCPase) in plasma exhibited an initial fast exponential component (k1 ) 1.603 × 10-3 min-1/µL of plasma) and a slower exponential component (k2 ) 0.144 × 10-3 min-1/µL of plasma) (14). The reaction rate of HDCP hydrolysis in the initial fast phase increased with the concentrations of substrate and enzyme, though the reaction rate did not seem to be dependent upon substrate concentration in the slow phase (14). When chicken serum HDCPase activity was purified to apparent homogeneity, HDCP was found to be hydrolyzed in this tissue by a single protein subsequently identified as albumin (15). The hydrolyzing activities of HDCP and p-nitrophenyl butyrate (p-NPB) were copurified in chicken serum albumin (CSA) (15). In addition, other phosphoramidates were also hydrolyzed by CSA (15). This study investigates the interactions between CSA, HDCP, and p-NPB. The results obtained allow us to propose a mechanism for the hydrolysis of HDCP based

10.1021/tx980015z CCC: $15.00 © 1998 American Chemical Society Published on Web 11/12/1998

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Sogorb et al.

Table 1. Effect of Fatty Acids on HDCPase and p-NPBase Activities in Chicken Serum and Albumins from Different Speciesa percentage activity tissue

substrate controlb

caprylic acid

palmitic acid

chicken serum chicken serum albumin bovine serum albumin rabbit serum albumin human serum albumin

HDCP HDCP HDCP HDCP HDCP

15 ( 1 28 ( 9 46 ( 9 38 ( 5 25 ( 5

46 ( 15 100 ( 19 18 ( 16 103 ( 20 33 ( 13 92 ( 18 22 ( 8 33 ( 15 105 ( 21

chicken serum chicken serum albumin bovine serum albumin rabbit serum albumin human serum albumin

p-NPB p-NPB p-NPB p-NPB p-NPB

101 ( 13 156 ( 20 169 ( 54 130 ( 54 113 ( 16

78 ( 18 111 ( 23 64 ( 45 103 ( 20 61 ( 20 103 ( 20 60 ( 19 66 ( 25 81 ( 16

a The tissue samples were preincubated at 37 °C for 60 min alone (controls) or with 4 mM caprylic acid or 0.08 mM palmitic acid. Afterward, HDCP or p-NPB was added to concentrations of 400 and 500 µM, respectively, and the HDCPase and p-NPBase activities were assayed. The recorded results were expressed as a percentage of activity with respect to the respective control incubated without fatty acid. The results correspond to the mean ( SD of three independent experiments, each one performed in triplicate. b Nanomoles of product per 30 min per milligram of protein.

on transient phosphorylation of CSA. This mechanism would account for the observed hydrolytic kinetics of HDCP.

Experimental Procedures Biological Material and Reagents. A racemic mixture (96% pure) of HDCP was synthesized by J. Pardo and C. Na´jera of the Departamento de Quı´mica Orga´nica (Universidad de Alicante, Alicante, Spain). Caution: HDCP is a hazardous chemical and should be handled carefully. All reagents and biological tissues were obtained from Sigma Chemicals (Madrid, Spain) and were of analytical grade. The biological preparations were chicken serum (Sigma product S6773), chicken serum albumin (Sigma product A-3686), human serum albumin (Sigma product A-3782), bovine serum albumin (Sigma product A-0281), and rabbit serum albumin (Sigma product A-0764). According to the supplier catalog, the purity of all albumin samples was approximately 99% (based on agarose electrophoresis criterion). Measurement of Enzymatic Activities. Hydrolyzing activities of HDCP (HDCPase) and p-nitrophenyl butyrate (pNPBase) were analyzed according to previously described methods based on the detection of the released 2,5-dichlorophenol (DCP) and p-nitrophenol (p-NP) (14-16). Measurement of Protein. Bradford’s method was employed (17) using bovine serum albumin as the standard.

Results Effect of Fatty Acids on HDCPase and p-NPB Hydrolyzing Activities. It was reported that short chain fatty acids inhibit the hydrolysis of p-nitrophenyl acetate (p-NPA) in human serum albumin (HSA) (18, 19). Since the HDCPase and hydrolyzing activities of p-NPB (p-NPBase) were found to copurify in chicken serum albumin (CSA) (15), we compared the effect of fatty acids on both activities. For palmitic acid, and at the maximum concentration that can be achieved in the medium (0.08 mM), no significant inhibition was observed in any of the samples assayed with either activity (Table 1). Caprylic acid inhibited both activities of all the samples assayed, in a concentration-dependent manner. Table 1 shows the

results obtained corresponding to the caprylic acid concentration that produced maximum inhibition (4 mM). This inhibition was not dependent upon preincubation time for either activity (data not shown). Both activities (i.e., HDCPase and p-NPBase) were inhibited to a similar degree in all albumins. The percentage of inhibition in each tissue was higher for HDCPase than for p-NPBase (Table 1). These observations could be considered to indicate that both activities of albumin, CbE and PTE, are affected by short chain fatty acids. Concurrent Competitive Interaction between HDCP and p-NPB. HDCPase and p-NPBase of chicken serum were copurified in the same protein (albumin) (15). Moreover, Ca2+ and EDTA affected neither HDCPase (16, 20) nor p-NPBase (15), and both activities were inhibited by caprylic acid (Table 1). In addition, no significant differences between the kinetics of HDCP hydrolysis by whole serum and purified albumin have been detected (unpublished results). All these observations suggest that HDCP and p-NPB might be hydrolyzed by the same active site. To test this hypothesis, we studied the potential inhibitory interaction between both substrates. We had previously studied the kinetics of chicken serum HDCPase. The use of increasing substrate concentrations produced an increase in the first phase of the reaction rate (14). Due to the limited solubility of HDCP, the maximum possible concentration does not cause sufficient saturation to estimate the Michaelis kinetic parameters. It is therefore not possible to determine the inhibition with reasonable accuracy in terms of modifications in the Michaelis curve kinetic parameters. Consequently, we adopted a different approach to test if both substrates are able to interact competitively. The HDCPase activity of chicken serum was recorded employing as the substrate 100 µM HDCP in the presence of p-NPB at several concentrations. The carboxyl ester inhibited HDCPase in a concentration-dependent manner. The maximum level of inhibition reached 50% for a concentration of 100 µM p-NPB (Figure 1A). Similarly, HDCP inhibited p-NPBase, the maximum level of inhibition being slightly more than 50% (Figure 1B). Therefore, the substrate of the CbE inhibited HDCPase, and the substrate of HDCPase inhibited p-NPBase. A characteristic of the competitive interaction is the reversion of inhibition by an increase in substrate concentration. When HDCPase was assayed in samples of serum with several HDCP concentrations both with and without 200 µM p-NPB, we found that the inhibition of p-NPB could be forced to revert by increasing the HDCP concentration (Figure 1C). In a similar experiment, HDCP also inhibited p-NPBase in a manner that could be reversed by increasing the p-NPB concentration (Figure 1D). This competitive interaction confirms that both substrates are most probably hydrolyzed by the same active site in the albumin molecule. Sequential Interaction between HDCP and pNPB. The kinetics of HDCP hydrolysis by chicken serum (14) and p-NPA by HSA (18) and by bovine mercaptalbumin (21) were similar. In the latter two cases, the fast hydrolytic phase was ascribed to the existence in the albumin of a fast acylation site. The acylation of this site would release p-NP. Since HDCPase and p-NPBase possess similar properties with regard to kinetic behavior (14, 18, 21) and fatty acid (Table 1) and Ca2+/EDTA effects (15, 16, 20), experiments were conducted to determine whether the

Hydrolysis of Phosphoramidates by Albumin

Figure 1. Concurrent competitive interaction between HDCP and p-NPB. The substrate of the assayed activity was added at the same time as the tested inhibitor. Control values (activities without inhibitor) were expressed as the mean of activity ( SD (n ) 4) [*statistically different from control (p < 0.005) and **statistically different from control (p < 0.001)]. (A) Effect of p-NPB on HDCPase. Control activity ) 5.5 ( 0.5 nmol of DCP per 30 min per milligram of protein. (B) Effect of HDCP on p-NPBase. Control activity ) 14.5 ( 0.8 nmol of p-NP per 30 min per milligram of protein. (C) Reversion of the inhibition of HDCPase induced by p-NPB. Control activities ) 3.6 ( 0.6, 7.7 ( 0.3, 10.9 ( 0.6, and 19.1 ( 2.3 nmol of DCP per 30 min per milligram of protein for 50, 100, 200, and 400 µM HDCP, respectively. (D) Reversion of the inhibition of p-NPBase induced by HDCP. Control activities ) 15.4 ( 0.6, 24.6 ( 2.3, 37.4 ( 5.1, and 60.0 ( 7.7 nmol of p-NP per 30 min per milligram of protein for 25, 100, 200, and 400 µM p-NPB, respectively.

catalytic mechanism of HDCP hydrolysis involves a covalent phosphorylated intermediate and to obtain further confirmation that both substrates are hydrolyzed in the same active site. Simultaneously, we attempted to obtain a mechanistic interpretation of the HDCPase reaction time course. The working hypothesis we sought to confirm was that the initial fast phase could be attributed to a quick phosphorylating reaction and that the second phase reflects the dephosphorylating reaction. To “dissect” both phases and to test the existence of the potential covalent intermediate stage, we adopted the following approach. Samples of undiluted serum were preincubated with one substrate (either p-NPB or HDCP) for a short time, to allow most of the albumin to be acylated or phosphorylated. Samples were then diluted, followed by incubation with the substrate to assay either HDCPase or p-NPBase. If albumin were in the covalent phosphorylated or acylated stage, the rate of hydrolysis of the assayed substrate would be expected to be lower than in the control sample (i.e., not preincubated with the first substrate). Specifically, samples of 360 µL of undiluted chicken serum were mixed with 4.8 µL of 100 mM p-NPB (yielding 1.3 mM p-NPB in the mixture) and the mixtures preincubated at 37 °C for 15 or 60 min. Samples were afterward diluted by adding 5.09 mL of 10 mM Tris (pH 7.0). The theoretical maximum possible p-NPB concentration was 88 µM (estimated assuming that it had not been degraded during the previous incubation period). This hypothetical maximum possible p-NPB remaining in the solution should not competitively interact with HDCPase, according to Figure 1C. Moreover, after preincubation for 60 min, we found that practically all the p-NPB had been hydrolyzed. Immediately after

Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1443

Figure 2. Sequential interaction between HDCP and p-NPB. (A) Effect of preincubation with p-NPB on HDCPase. Chicken serum was incubated at 37 °C with 1.3 mM p-NPB and diluted. Subsequently, HDCP was added and HDCPase assayed. The control was preincubated with solvent. No significant differences were observed between controls preincubated for 15 and 60 min. (B) Effect of preincubation with HDCP on p-NPBase. Chicken serum was incubated at 37 °C with 1.3 mM p-NPB and diluted. Afterward, p-NPB was added and p-NPBase assayed. No significant differences were observed between controls preincubated for 15 and 60 min.

preincubation and dilution of p-NPB, HDCP was added to the medium to obtain a concentration of 400 µM, and the HDCPase time course was monitored. Results show that the extent of HDCP hydrolysis was lower than the control (not preincubated with p-NPB) when samples had been preincubated for 15 min with p-NPB (Figure 2A). This is consistent with the hypothesis that all the p-NPB has been hydrolyzed and that the catalytic site of HDCP hydrolysis remains butyrylated some time after interacting with p-NPB. However, this apparent inhibition was forced to revert in part when initial preincubation was prolonged for 60 min (Figure 2A). This could be interpreted by considering that by this time (60 min) most of the catalytic sites had been debutyrylated. When in a similar experiment p-NPB was used as the second substrate, similar results were observed (data not shown), thus providing further confirmation that the acylated (butyrylated) albumin is temporally blocked at the catalytic site of hydrolysis, this site being common to both substrates. Similar experiments were performed by preincubating serum with 1.3 mM HDCP, diluting, and assaying for p-NPBase or HDCPase. After preincubation for 15 min at 37 °C, p-NPBase showed less activity than the nonpretreated controls (Figure 2B). However, in this case, the apparent inhibition did not disappear when preincubation was prolonged to 60 min (Figure 2B). Similar results were observed when samples were preincubated for 180 min and HDCP was added as the second substrate (data not shown). However, when the concentration of HDCP in the preincubation medium was less than 1.3 mM (0.5 mM), the apparent inhibition disappeared after preincubation for 120 min (Figure 3). This indicates that HDCP can phosphorylate the catalytic site of p-NPB and cause HDCP hydrolysis but that the dephosphorylating reaction was slower than the deacylating reaction, and only if lower HDCP concentrations are used is it

1444 Chem. Res. Toxicol., Vol. 11, No. 12, 1998

Figure 3. Effect of preincubation of chicken serum with 0.5 mM HDCP on HDCPase activity. Chicken serum was incubated at 37 °C for 120 min with 0.5 mM HDCP and diluted. Afterward, HDCP was added and HDCPase assayed. The control was preincubated with solvent.

possible to detect the recovery of the active nonphosphorylated enzyme.

Discussion The hydrolysis of esters by serum albumins has been previously reported. Examples include the hydrolysis of p-NPA by HSA (18, 19), bovine serum albumin (22), and mercaptalbumin of the same species (21), the hydrolysis of naphthol acetate by human and bovine serum albumins (23), and the hydrolysis of p-nitrophenyl 4-guanidinobenzoate by HSA (24). This study shows that p-NPB is hydrolyzed by chicken, rabbit, human, and bovine serum albumins (Table 1). The study also shows that the organophosphorus compound HDCP is hydrolyzed at the same active site as p-NPB by a mechanism based on transient phosphorylation. The hydrolysis of p-NPB by HSA was inhibited by fatty acids (Table 1), the inhibiting effect of the latter decreasing as the the hydrocarbon chain was lengthened (18, 19). These results are similar to those described for the HDCPase and p-NPBase activities in chicken serum, though in the case of p-NPBase the inhibition percentages were somewhat lower than for HDCPase (Table 1). When the amino acid sequence of albumin was known, the Tyr residue (position 410 in bovine serum albumin and 411 in HSA) of the sequence Arg-Tyr-Thr-Arg was identified as the residue which is acylated in the reaction between p-NPA and albumin (25). The sequence ArgTyr-Thr-Lys exists in the CSA amino acid sequence (boldface and double-underlined in Figure 4). In this sequence, Tyr is also placed in position 411. As may be observed, the CSA sequence differs from bovine or human serum albumin sequences in the last amino acid (Lys instead of Arg). Both amino acids have basic character, and therefore, both might play a similar role in a hypothetical catalysis of p-NPB hydrolysis. Thus, the sequence Arg-Tyr-Thr-Lys appears to be the main candidate for being the target of acylation by p-NPB and phosphorylation by HDCP. We have previously described that HDCPase in chicken plasma exhibits an initial fast hydrolytic phase and a posterior slow hydrolytic phase (14). This behavior is similar to that described for p-NPA hydrolysis in HSA (18) and in bovine mercaptalbumin (21). The authors interpreted that the fast initial reaction corresponded to the formation of an acylated covalent intermediate followed by a slower phase corresponding to the hydrolysis

Sogorb et al.

of this intermediate with subsequent new acylation. To clarify the possible involvement of an acylating/phosphorylating mechanism in p-NPB and HDCP hydrolysis, we used an experimental approach to detect the covalent intermediate by sequential interaction with substrates. Samples were incubated with a first substrate (to allow formation of the hypothesized covalent intermediate) and diluted immediately before assaying activities. This approach gave further confirmation that both substrates act upon the same active site. The experiments generated data that are consistent with a mechanism based on acylating/phosphorylating binding, which also would explain the initial fast release of the leaving group (pNP or DCP), followed by a slower deacylating/dephosphorylating reaction that would account for the slow hydrolytic phase. When samples where preincubated with the first substrate (either p-NPBase or HDCP) for a short time and then diluted, the activity with the second substrate (either p-NPBase or HDCP) was lower than the nonpretreated controls. This confirms the hypothesis that the active site remains covalently acylated or phosphorylated. Therefore, the reaction rate after adding the second substrate could be interpreted as being dependent on the deacylating/dephosphorylating reaction of the first substrate which generates free active sites that are ready for new catalytic cycles. This apparent inhibition is forced to revert if preincubation is prolonged long enough to hydrolyze all of the first substrate and allow the active sites to be deacylated or dephosphorylated. When the first substrate in the preincubation stage was HDCP, longer times and lower concentrations were required to recover the activity than when the first substrate was p-NPB, showing that the dephosphorylation rate is slower than the deacylation rate. The bond between the phosphorus moiety of HDCP and albumin is cleaved very slowly. This could suggest that albumin is phosphorylated by HDCP in an irreversible manner. However, our previous experiments demonstrate that chicken serum is able to hydrolyze 100% of the substrate when the substrate concentration is 3-fold higher than the albumin concentration (14). Hence, as albumin is the only protein with HDCPase activity in chicken serum (15), it had to be phosphorylated and dephosphorylated several times in the course of the reaction, in what can be considered a catalytic cycle. The above results all suggest that HDCP hydrolysis might be explained by the mechanism shown in Figure 5. In this sense, HDCP would be hydrolyzed by the same site acylated during the hydrolysis of p-NPB and p-NPA (probably tyrosine in position 411). When the results shown in Figure 1 are taken into account, the target affinity of p-NPB would be greater than the affinity of HDCP. However, the bond of CSA with the phosphoryl moiety would be more stable than that with the butyryl moiety. The initial phosphorylation would account for the fast initial HDCP hydrolysis phase previously described (14). After this fast phosphorylation, the active site would be slowly dephosphorylated (at a slower rate than would be expected with deacylated after p-NPB acylation), thus generating new free active sites that are ready for rephosphorylation, thereby beginning a new catalytic cycle. The reported slow HDCP hydrolysis phase (14) would thus depend on the rate of dephosphorylation.

Hydrolysis of Phosphoramidates by Albumin

Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1445

Figure 4. Amino acid sequence of chicken serum albumin. The amino acids are abbreviated according to one-letter international nomenclature. Boldface type and double underlining mark the candidate sequence suggested to be responsible for the hydrolysis of p-NPB and HDCP (taken from ref 26).

Figure 5. Proposed mechanism for the hydrolysis of HDCP and p-NPB by chicken serum albumin.

Tildon and Ogilvie (21) suggested that slow phase p-NPB hydrolysis by mercaptalbumin takes place at an active site different from that responsible for fast phase hydrolysis. However, in the case of HDCP hydrolysis by CSA, the fact that phosphorylation of the latter is not irreversible, and the observation that the apparent initial rate of the HDCP slow hydrolytic phase is not dependent upon substrate concentration (14), suggest that no such hypothetical second active site exists in the case of HDCPase, or at least that it need not be considered to account for the reported kinetics. It has recently been shown that the substitution of glycine in position 117 with histidine in human butyrylcholinesterase provides the latter (a B-esterase) with PTE activity. The authors propose that following phosphorylation of the serine group in the active site, the introduced amino acid (histidine) activates a water molecule that in turn attacks the phosphoric group, releasing it from the active site (27). This mechanism should be similar to that proposed in this study for the CSA hydrolysis of HDCP, although in our case tyrosine would be phosphorylated in place of serine. To our knowledge, the mechanism of hydrolysis of paraoxon by mammalian paraoxonase is unknown. The mechanism of hydrolysis of the paraoxonase found in Pseudomonas diminuta has been recently investigated, and it seems that this enzyme does not act through a phosphorylation (8). On the other hand, it is believed that the mechanism of hydrolysis of acetylcholine (a B-esterase) by acetylcholinesterase is based on the acylation of a serine residue of the protein, following deacylation through attack of an activated water molecule (11). The inhibition of acetylcholinesterase by OPs is based in the phosphorylation of the same serine residue acylated during acetylcholine hydrolysis. However, after this phosphorylation, the protein is unable to be dephosphorylated by the attack of a water molecule (2, 11). Due to all these considerations, it might be concluded that serum albumin hydrolyzes HDCP by a mechanism different from the mechanism employed for other known PTEs but

similar to the mechanism of hydrolysis of cholinesterases. It also might be concluded that this mechanism is also similar to the mechanism of inhibition of B-esterases by OPs. This conclusion is also supported by the fact that when a histidine (an amino acid with an ability to activate water molecules) is introduced in the appropriate place in a B-esterase (butyrylcholinesterase), this protein receives the ability to hydrolyze OPs (27). Insects have traditionally been thought to lack PTE (2). It has been demonstrated that the resistance showed by certain insect strains such as the cockroach Blattella germanica (28, 29) and several mosquito species (3032) toward the toxic effects of OPs is due to increments in the synthesis of CbEs (B-esterases) capable of scavenging an OP molecule through the irreversible phosphorylation of a serine group within the active site (11). The efficacy of this detoxification system is a result of the high CbE concentration found in the tissues of the insect, rather than the result of the catalytic efficacy of the system (i.e., a single OP molecule hydrolyzed by one CbE molecule). In the same way, the efficacy of albumin in the phosphoramidate detoxification can be attributed to its high concentration in the plasma of all vertebrates (between 50 and 66%) (33). The above-mentioned OP toxicity resistance mechanism in insects is based on the irreversible phosphorylation of CbEs. However, to our knowledge, there have been no studies to date of the possible reversibility of these phosphorylations. The results obtained in this study suggest that research is required to evaluate this possible reversibility. In effect, the existence of reversibility, even if very slow, would imply an important enhancement of the efficiency of the detoxification process.

Acknowledgment. This study was financed by DGICT SAF Grant SAF96/0168. M.A.S. was a holder of a grant from the Spanish Ministry of Education and Science and A.M. from AECI and CONACYT.

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