Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.1.7 (acetylcholinesterase)
28,390 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Comparison of published LD50 or LC50 levels for a variety of insecticides in several vertebrate species indicate that a wide range of toxicity levels exist, and these cannot be easily predicted within either a chemical group or within a species. There is a relatively limited data base documenting interactions between insecticides and other chemicals, either agricultural or non-agricultural; however, the fact that all major insecticide groups perturb nervous system function as their primary mechanism of acute toxicity suggests the potential for interactions. Studies in our laboratories on a select group of phosphorothionate insecticides in rats indicated that brain acetylcholinesterase sensitivity to inhibition by the oxons, the active metabolites of the phosphorothionates, does not correlate with acute toxicity levels. The activities and properties of hepatic cytochrome P450-mediated activation (desulfuration) and detoxication (dearylation) of the phosphorothionates as well as of A-esterase-mediated hydrolysis of oxons contribute substantially to understanding the acute toxicity levels in rats, as does the sensitivity of the protective aliesterases to phosphorylation. However, in the channel catfish, the acetylcholinesterase sensitivity to oxon inhibition reflects the acute toxicity level of these same insecticides, and may be largely responsible for determining the acute toxicity level in this species. Thus, metabolism of insecticides appears to be far more influential in some species than others in determining the toxicity elicited.
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PMID:Biochemical mechanisms contributing to species differences in insecticidal toxicity. 857 66

Phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of paraoxon and related acetylcholinesterase inhibitors with rate enhancements that approach 10(12). The enzyme requires a binuclear metal center for activity and as isolated contains 2 equiv of zinc per subunit. Here we describe the three-dimensional structure of the Zn2+/Zn2+-substituted enzyme complexed with the substrate analog diethyl 4-methylbenzylphosphonate. Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.6 A, b = 91.4 A, c = 69.4 A, beta = 91.9 degrees, and two subunits in the asymmetric unit. The model was refined by least-squares analysis to a nominal resolution of 2.1 A and a crystallographic R-factor of 15.4% for all measured X-ray data. As in the previously reported structure of the cadmium-containing enzyme, the bridging ligands are a carbamylated lysine residue (Lys 169) and a hydroxide. The zinc ions are separated by 3.3 A. The more buried zinc ion is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging hydroxide in a trigonal bipyramidal arrangement as described for the cadmium-substituted enzyme. Unlike the octahedral coordination observed for the more solvent-exposed cadmium ion, however, the second zinc is tetrahedrally ligated to Lys 169, His 201, His 230, and the bridging hydroxide. The diethyl 4-methylbenzylphosphonate occupies a site near the binuclear metal center with the phosphoryl oxygen of the substrate analog situated at 3.5 A from the more solvent-exposed zinc ion. The aromatic portion of the inhibitor binds in a fairly hydrophobic pocket. A striking feature of the active site pocket is the lack of direct electrostatic interactions between the inhibitor and the protein. This most likely explains the broad substrate specificity exhibited by phosphotriesterase. The position of the inhibitor within the active site suggests that the nucleophile for the hydrolysis reaction is the metal-bound hydroxide.
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PMID:Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate. 863 43

Annealed murine erythrocytes were employed as a carrier model to antagonize the toxic effects of organophosphorus agents. These resealed cells containing a recombinant phosphotriesterase provided striking protection against the lethal effect of paraoxon, an active metabolite of an agricultural pesticide, parathion. Phosphotriesterase hydrolyzes paraoxon to the less-toxic 4-nitrophenol and diethylphosphate. This enzyme was encapsulated into carrier erythrocytes by hypotonic dialysis with subsequent resealing and annealing. These carrier cells were administered to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recipient animals were subsequently challenged with paraoxon and a marked protection was noted. Protection of free enzyme and encapsulated enzyme was compared and the encapsulated enzyme was found to persist longer and possess much greater efficacy. Less serum cholinesterase inhibition also was observed with this enhanced protection. These results indicate that the erythrocyte carrier alone is quite effective in the antagonism of organophosphorus intoxication. Moreover, when these carrier cells were administered in combination with 2-PAM and/or atropine, a marked synergism was observed.
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PMID:Antagonism of the lethal effects of paraoxon by carrier erythrocytes containing phosphotriesterase. 883 30

A novel therapy against organophosphate exposure, the combination of a carbamate eptastigmine and an organophosphate hydrolase (phosphotriesterase) was studied in mice against diisopropylfluorophosphate (DFP) (1.75 mg/kg) exposure. Mice received eptastigmine (0.9 mg/kg; iv) 10 min prior to the ip injection of DFP. Phosphotriesterase (83 U/g body weight) was injected iv 10 min after DFP. Eptastigmine (1.5 mg/kg; iv) inhibited the acetylcholinesterase activities in brain and erythrocytes for a longer time than physostigmine. Eptastigmine caused only minor changes in the behavior and activity of the animals, whereas physostigmine clearly reduced their activity for about 30 min. The eptastigmine pretreatment clearly supplemented the protective effect of phosphotriesterase against DFP: the plasma butyrylcholinesterase activity was doubled and the activity recovered faster than in animals treated with phosphotriesterase alone. In lung, butyrylcholinesterase activity was initially lower after eptastigmine-phosphotriesterase than phosphotriesterase treatment alone. However, the activity returned 24 hr later to normal in eptastigmine-phosphotriesterase-treated groups. With phosphotriesterase only, it recovered only to 75% of the control level. Presumably eptastigmine, by preventing the binding of DFP to cholinesterases, caused an elevation of free DFP levels in body fluids and promoted phosphotriesterase hydrolysis of DFP.
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PMID:Eptastigmine-phosphotriesterase combination in DFP intoxication. 888 53

Many organophosphorus compounds (OPs) are potent cholinesterase inhibitors, accounting for their use as insecticides and, unfortunately, also as nerve agents. Each year there are approximately 3 million pesticide poisonings world-wide resulting in 220,00 deaths. In 1990, there were 1.36 million kg of chlorpyrifos, 4.67 million kg of diazinon and 1.23 million kg of ethyl parathion manufactured in the USA (data supplied by the USEPA). In addition to exposure risks during pesticide manufacturing, distribution and use, there are risks associated with the major international effort aimed at destroying the arsenals of nerve agents, including soman and sarin. The United States has pledged to destroy approximately 25,000 tons of chemical agents by the end of the decade. The high density lipoprotein (HDL)-associated enzyme paraoxonase (PON1) contributes significantly to the detoxication of several OPs (Fig. 1). The insecticides parathion, chlorpyrifos and diazinon are bioactivated to potent cholinesterase inhibitors by cytochrome P-450 systems. The resulting toxic oxon forms can be hydrolysed by PON1, which also hydrolyses the nerve agents soman and sarin (Fig. 1). PON1 is polymorphic in human populations and different individuals also express widely different levels of this enzyme. The Arg192 (R192) PON1 isoform hydrolyses paraoxon rapidly, while the Gln192 (Q191) isoform hydrolyses paraoxon slowly. Both isoforms hydrolyse chlorpyrifos-oxon and phenylacetate at approximately the same rate. The role of PON1 in OP detoxication is physiologically significant. Injected PON1 protects against OP poisoning in rodent model systems and interspecies differences in PON1 activity correlate well with observed median lethal dose (LD50) values. We report here a simple enzyme analysis that provides a clear resolution of PON1 genotypes and phenotypes allowing for a reasonable assessment of an individual's probable susceptibility or resistance to a given OP, extending earlier studies on this system. We also show that the effect of the PON1 polymorphism is reversed for the hydrolysis of diazoxon, soman and especially sarin, thus changing the view of which PON1 isoform is considered to be protective.
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PMID:The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin. 889 66

The protective action of i.v. administered eptastigmine, an organophosphate hydrolase (phosphotriesterase), or pralidoxime-2-chloride (2-PAM) and their combination in acute diisopropylfluorophosphate (DFP) intoxication were evaluated in mice. The mice received the physostigmine derivative, eptastigmine (0.9 mg/kg body wt, i.v.), 10 min prior to the i.p. injection of DFP (1.8 mg/kg body wt). Phosphotriesterase (66 micromol/min x ml/g and 6 microg/g body wt) or 2-PAM (30 mg/kg body wt) were given i.v. 30 min after DFP. The animals also received atropine sc (37.5 mg/kg body wt) immediately after DFP. The cholinesterase (ChE) activities were not protected or reactivated by 2-PAM alone. The ChE activities in brain and plasma were protected by phosphotriesterase. Eptastigmine alone assisted the recovery of the brain ChE activities. Also the combination of eptastigmine-phosphotriesterase protected the brain enzymes. It did not, however, provide any additional protection compared with phosphotriesterase-treatment on its own. In brain, the combination of eptastigmine with 2-PAM resulted in partly restored enzyme activities 24 hr after DFP exposure. In plasma, eptastigmine did not prevent the inhibition of ChE by DFP. However, when it was combined with phosphotriesterase, it significantly promoted the recovery of plasma ChE activity. In lung and in erythrocytes, the various combinations of antidotes caused only minor changes in the ChE activities.
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PMID:Phosphotriesterase, pralidoxime-2-chloride (2-PAM) and eptastigmine treatments and their combinations in DFP intoxication. 897 81

The extensive international use of organophosphorus compounds (OP) results in numerous acute intoxications each year. OPs inhibit acetylcholinesterase, the enzyme responsible for breaking down the neurotransmitter acetylcholine. The World Health Organization recognizes cholinesterase (ChE) biomonitoring as a preventive measure against OP overexposure. The aim of this study was to determine if dermal OP contamination could interfere with current field ChE biomonitoring assays, which use a fingerstick blood sample. In this study we also sought to determine if high levels of a plasma enzyme, A-esterase, could protect ChE from inhibition by hydrolyzing environmentally generated oxons potentially present in a fingerstick sample. A heparinized venous blood sample was collected from a volunteer. Erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (PChE) activities were measured using a field-based colorimetric cholinesterase kit. ChE dose-response curves were constructed by allowing 10-microliters blood samples to contact environmentally realistic levels of OP thioate and oxon for 10 s. An inhibition threshold could not be established for PChE when exposed to oxon within the time necessary to perform a fingerstick analysis. AChE was also inhibited by trace amounts of oxon consistent with previously reported environmental levels. These findings suggest that the reliability of field-based biomonitoring results is limited if OP residues remain on a skin surface at the time of sample collection. A-esterase's role in protecting ChE activity was investigated using capillary and venous blood from 30 unexposed individuals. Baseline ChE activities were measured, as were individual A-esterase activities using paraoxon, diazoxon, and phenylacetate as substrates. Results were then compared to ChE activities measured after 10 s of contact with an environmentally realistic amount of OP, containing 1% oxon. Both ChE activities were significantly inhibited, with capillary values being significantly more inhibited than their venous counterparts. However, no protective effect could be associated between the degree of A-esterase activity and the subsequent level of ChE inhibition observed in an individual's blood. These results suggest that (1) if there is any uncertainty about OP skin contamination, venous blood would be a more appropriate specimen to employ when using field ChE biomonitoring kits--it is collected in larger volumes and has essentially no direct contact to dermal surfaces; and (2) A-esterase activity demonstrates no protective effect against ChE inhibition upon a blood droplet's brief contact with an OP residue containing traces of oxon.
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PMID:Simulated dermal contamination with capillary samples and field cholinesterase biomonitoring. 916 60

Chlorpyrifos (CPF), a commonly used cholinesterase-inhibiting insecticide, is lethal at much lower doses to young animals than adults. To explain this higher sensitivity in younger animals, we hypothesized that young rats have less chlorpyrifos-oxonase (CPFOase) activity than adults. To test this hypothesis, CPFOase activity was measured in the brain, plasma, and liver of male, postnatal day 4 (PND4) and adult (PND90) Long-Evans rats. CPFOase is biochemically defined as a Ca(2+)-dependent A-esterase that hydrolyzes chlorpyrifos-oxon (CPFO), the active metabolite of CPE. No brain CPFOase activity was detected at either age. Plasma and liver CPFOase activities were markedly lower at PND4 compared to adult: PND4 plasma and liver CPFOase activities were 1/11 and 1/2 the adult plasma and liver activities, respectively. Because the Km of CPFOase activity was high (i.e., 210-380 microM), it was important to determine if this CPFOase activity could hydrolyze physiologically relevant concentrations (i.e., nM to low microM) of CPFO. This was accomplished by comparing the shifts in the tissue acetylcholinesterase (AChE) IC50 for CPFO in the presence or absence of CPFOase activity. One would expect an increase in the "apparent" IC50 if CPFOase hydrolyzes substantial amounts of CPFO during the 30 minutes the tissue is preincubated with the CPFO. In the adult, both plasma and liver AChE apparent IC50 values were higher in the presence of CPFOase activity, suggesting that the CPFOase in those tissues was capable of hydrolyzing physiologically relevant concentrations of CPFO within 30 minutes. In young animals, however, there was less of a shift in the IC50 curves compared to the adult, confirming that the young animal has less capacity than the adult to detoxify physiologically relevant concentrations of CPFO via CPFOase.
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PMID:Maturational differences in chlorpyrifos-oxonase activity may contribute to age-related sensitivity to chlorpyrifos. 926 78

Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had < 10(-5) of wild-type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon.
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PMID:A combinatorial library for the binuclear metal center of bacterial phosphotriesterase. 940 51

1. The effects of two model inducers of the cytochrome P450 system, phenobarbital (PB) and beta-naphthoflavone (NF), on the toxicity of paraoxon were studied in rats. 2. Paraoxon toxicity was measured by inhibition of brain acetylcholinesterase (AChE) activity. 3. PB treatment did not affect the toxicity of paraoxon, whereas NF increased the inhibition of brain AChE. PB administration slightly increased the activities of some peripheral cholinesterases and carboxylesterases, as well as liver microsomal paraoxonase (Pxase). 4. NF administration, in contrast, decreased the activities of peripheral esterases. Serum Pxase activity was reduced by both inducers. 5. Hepatic CYP2B and CYP1A were markedly induced by PB and NF, respectively. 6. Cytochrome P450 isoenzymes induced by PB or NF seemed not to be critical in the detoxification of paraoxon in vivo. NF caused a general reduction of peripheral esterases, which led to an increase in paraoxon toxicity. 7. The results indicated the great importance of peripheral cholinesterases and carboxylesterases as a detoxifying mechanism of paraoxon. The role of serum paraoxonase was not critical.
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PMID:Effect of phenobarbital and beta-naphthoflavone on activities of different rat esterases after paraoxon exposure. 968 78


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