The effect of acetylcholinesterase on outgrowth of dopaminergic neurons in organotypic slice culture of rat mid-brain

This study has investigated the possibility that acetylcholinesterase could play a non-classical role as an adhesion factor or growth factor in the development of dopaminergic neurons in organotypic slice culture of postnatal day 1 rats. When the culture medium was supplemented with acetylcholinesterase (3 U/ml), outgrowth of tyrosine hydroxylase-immunoreactive neurites was significantly enhanced. Addition of a specific inhibitor of acetylcholinesterase, BW284c51, caused a decrease in the number of tyrosine hydroxylase neurons and a reduction in the cell body size and extent of neurite outgrowth of remaining neurons. However, echothiophate which also inhibits AChE activity, did not produce these effects. Therefore acetylcholinesterase could act as a growth enhancing factor for dopaminergic neurons, and disruption of an as yet unidentified site on the acetylcholinesterase molecule by BW284c51 could decrease the survival and outgrowth of these neurons.     

Chicken retinospheroids as developmental and pharmacological in vitro models: acetylcholinesterase is regulated by its own and by butyrylcholinesterase activity

Summary The phylo- and ontogenetically related enzymes butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are expressed consecutively at the onset of avian neuronal differentiation. In order to investigate their possible co-regulation, we have studied the effect of highly selective inhibitors on each of the cholinesterases with respect to their expression in rotary cultures of the retina (retinospheroids) and stationary cultures of the embryonic chick tectum. Adding the irreversible BChE inhibitor iso-OMPA to reaggregating retinal cells has only slight morphological effects and fully inhibits BChE expression. Unexpectedly, iso-OMPA also suppresses the expression of AChE to 35%–60% of its control activity. Histochemically, this inhibition is most pronounced in fibrous regions. The release of AChE into the media of both types of cultures is inhibited by iso-OMPA by more than 85%. Control experiments show that AChE suppression by the BChE inhibitor is only partially explainable by direct cross-inhibition of iso-OMPA on AChE. In contrast, the treatment of retinospheroids with the reversible AChE inhibitor BW284C51 first accelerates the expression of AChE and then leads to a rapid decay of the spheroids. After injection of BW284C51 into living embryos, we find that AChE is expressed prematurely in cells that normally express BChE. We conclude that the cellular expression of AChE is regulated by the amount of both active BChE and active AChE within neuronal tissues. Thus, direct interaction with classical cholinergic systems is indicated for the seemingly redundant BChE.     

Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism

Cholinesterases present homologies with some cell adhesion molecules; however, it is unclear whether and how they perform adhesive functions. Here, we provide the first direct evidence showing that neurite growth in vitro from various neuronal tissues of the chick embryo can be modified by some, but not all, anticholinesterase agents. By quantifying the neuritic G4 antigen in tectal cell cultures, the effect of anticholinesterases on neurite growth is directly compared with their cholinesterase inhibitory action. BW 284C51 and ethopropazine, inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively, strongly decrease neurite growth in a dose-dependent manner. However, echothiophate which inhibits both cholinesterases, does not change neuritic growth. These quantitative data are supplemented by morphological observations in retinal explant cultures grown on striped laminin carpets, viz., defasciculation of neurite bundles by BW 284C51 and Bambuterol occurs, indicating that these drugs disturb adhesive mechanisms. These data strongly suggest that a) cholinesterases can participate in regulating axonal growth, b) both AChE and BChE can perform such a nonsynaptic function, and c) this function is not the result of the enzyme activity per se, since at least one drug was found that inhibits all cholinesterase activities but not neurite growth. Thus, a secondary site on cholinesterase molecules must be responsible for adhesive functions.     

Cholinergic development in chick brain reaggregated cell cultures

Reaggregated cell cultures from dissociated 7-day-old chick embryo whole brains were prepared, and the developmental profiles of acetylcholinesterase and choline acetyltransferase, in the aggregates, determined over a 30-day period. Enzyme activities in vitro, at different times of culture, typically lie between 30 and 60% of the values obtained for embryos or chicks of the same developmental age, up to day-10 posthatching. The increase in acetylcholinesterase activity over a 24-day period of culture/incubation is fourfold in the aggregates vs. sixfold for embryos, while the choline acetyltransferase values increase, during the same period of time, 32-fold in the aggregates vs. 17-fold in vivo. Choline acetyltransferase activity seems to be more dependent on good cell-to-cell contact than acetylcholinesterase activity. On the other hand, morphological studies on the aggregates with light and electron microscopy reveal a number of structural features characteristic of well-developed nervous tissue. It is suggested that aggregate cultures of chick brain cells are an adequate model system that is especially useful in analyzing developmental phenomena requiring free tridimensional interaction.Abbreviations AChE acetylcholinesterase - ChAT choline acetyltransferase - BW284 C51 dibromide 1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide - ACh acetylcholine     

Biphasic Effect of Eserine and Other Acetylcholinesterase Inhibitors on the Nicotinic Response to Acetylcholine in Cultured Adrenal Chromaffin Cells

Abstract: A pharmacological study was made of the effects of various anticholinesterases (anti-ChEs) on the release of [³H]noradrenaline ([³H]NA) evoked by acetylcholine (ACh), nicotine, 56 mM K⁺, and veratridine from bovine adrenal chromaffin cells in culture. The anti-ChEs chosen were eserine (an inhibitor of both acetylcholinesterase and pseudocholinesterase), 1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) (a specific acetylcholinesterase (AChE) inhibitor), and tetraisopropylprophos-phoramide (iso-OMPA) (a specific pseudocholinesterase inhibitor). Acetylcholinesterase (AChE) activity increased in the cells with time in culture beginning at day 4 and reaching a plateau at day 10. In 9–11-day cultures, both eserine and BW284C51 acted biphasically to increase ACh-induced [³H]NA release from the cells at concentrations of 10^?6M or less whereas higher concentrations reduced or abolished the ACh-induced release. However, in earlier cultures (days 3–5), when AChE activity of the cells was low, both eserine and BW284C51 produced only a monophasic dose-dependent inhibition of ACh-evoked [³H]NA release at high concentrations. When the cells were stimulated with nicotine, an agonist not metabolized by cholinesterase a similar monophasic inhibitory response on the [³H]NA release was elicited by eserine and BW284C51, regardless of the age of the cultured cells. When 56 mM K⁺ or veratridine was used to depolarize the cells, neither eserine nor BW284C51 affected the [³H]NA release from the cells. Unlike eserine and BW284C51, iso-OMPA did not enhance ACh-evoked release in older cultures and at high concentrations (> 10 ⁴M) it produced an inhibition of the [³H]NA release evoked by ACh, nicotine, 56 mM K⁺, and veratridine. The present results suggest that the stimulatory effect on ACh response by low concentrations of eserine and BW284C51 can be attributed to the protection of ACh against enzymatic hydrolysis, whereas the inhibitory effects produced by higher concentrations of eserine and BW284C51 are thought to be due to an interaction with the nicotinic acetylcholine receptor-ionophore complex.     

Evaluation of mechanisms of azinphos-methyl resistance in the codling moth Cydia pomonella (L.)

Resistance of the codling moth Cydia pomonella (L.) to azinphos-methyl is not based on enhanced detoxifying enzymes like oxidation mediated by mixed function oxidases or by glutathione S-transferases. Synergism by S,S,S-tributylphosphoro-trithioate was evident, but the overall activity of general esterases using p-nitrophenyl acetate as the substrate was similar in resistant and susceptible insects. In comparison to acetylcholinesterase (AChE) from susceptible adult codling moth, the enzyme of insects resistant to azinphos-methyl has low affinities (higher K(m) values) to the substrates acetylthiocholine (ATCh) and propionylthiocholine. This difference indicates a possible amino acid alteration at the catalytic or anionic binding sites of the resistant enzyme. Inhibition studies revealed no apparent differences in sensitivity of AChE enzymes from resistant and susceptible moths to organophosphorus compounds (OPs), carbamate insecticides and quaternary ammonium ligands. MEPQ (7-Methylethoxyphosphinyloxy)-1-methylquinolinium) is the most powerful OP inhibitor acting at a nM range, while chlopyrifos oxon, azinphos-methyl oxon and paraoxon are less inhibitory by 22.9, 82.3 and 475 fold, respectively. The codling moth AChE is a typical enzyme that displays substrate inhibition by ATCh, negligible hydrolysis of butyrylthiocholine, very high sensitivity to the bisquaternary ammonium compound BW284c51 and it is not inhibited by the powerful butyrylcholinesterase inhibitor iso-OMPA. Of the three carbamates examined, only carbaryl was inhibitory at the mM range while pirimicarb and aldicarb were inactive. Of the quaternary ammonium ligands (except for the powerful BW284c51), edrophonium and decamethonium displayed appreciable inhibition rates, while d-tubocuraine was practically inactive.     

Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae

Acetylcholinesterases (AChEs) and their genes from susceptible and resistant insects have been extensively studied to understand the molecular basis of target site insensitivity. Due to the existence of other resistance mechanisms, however, it can be problematic to correlate directly a mutation with the resistant phenotype. An alternative approach involves recombinant expression and characterization of highly purified wild-type and mutant AChEs, which serves as a reliable platform for studying structure–function relationships. We expressed the catalytic domain of Anopheles gambiae AChE1 (r-AgAChE1) using the baculovirus system and purified it 2,500-fold from the conditioned medium to near homogeneity. While K_M's of r-AgAChE1 were comparable for ATC, AβMTC, PTC, and BTC, V_max's were substantially different. The IC₅₀'s for eserine, carbaryl, paraoxon, BW284C51, malaoxon, and ethopropazine were 8.3, 72.5, 83.6, 199, 328, and 6.59 × 10⁴ nM, respectively. We determined kinetic constants for inhibition of r-AgAChE1 by four of these compounds. The enzyme bound eserine or paraoxon stronger than carbaryl or malaoxon. Because the covalent modification of r-AgAChE1 by eserine occurred faster than that by the other compounds, eserine is more potent than paraoxon, carbaryl, and malaoxon. Furthermore, we found that choline inhibited r-AgAChE1, a phenomenon related to the enzyme activity decrease at high concentrations of acetylcholine.     

Acetylcholinesterase and butyrylcholinesterase activity in the atria of the heart of adult albino rats

In experiments on adult albino rats the authors used the substances BW 284 C51 (1.5-bis(allyldimethylammoniumphenyl)-pentane-3-one-dibromide) as a specific inhibitor of acetylcholinesterase (AChE) and ethopropazine (10-(2-diethylaminopropyl) phenothiazine hydrochloride) as a specific inhibitor of butyrylcholinesterase (BuChE) to determine the two enzyme activities in atrial homogenates and to investigate changes after AChE or BuChE inhibition of the negative chronotropic effect of acetylcholine (ACh) on atria incubated in vitro. AChE accounted for only 12% and BuChE for 88% of the total ability of atrial homogenates to hydrolyse acetylcholine. The concentration of exogenous ACh needed to reduce the spontaneous frequency of contractions of the isolated right atrium by 30, 60, or 90/min fell by 78%, 79% and 84% respectively after BW 284 C51 inhibition of AChE and by 95%, 94% and 94% after simultaneous inhibition of AChE and BuChE. The significance of AChE in control of the negative chronotropic effect of ACh is thus evidently significantly greater than would correspond to the percentual proportion of AChE in cholinesterase activities in the atria of the rat heart. In can be assumed that AChE is functionally associated with parasympathetic innervation of the heart and that it is probably present in a high concentration in the primary pacemaker region.     

Secretion of acetylcholinesterase by a mouse hepatocyte X rat liver cell hybrid culture

Summary A hybrid cell line (E-2) that secretes the enzyme acetylcholinesterase (AChE) has been prepared. The E-2 cell was the product of a fusion between primary mouse hepatocytes and a chemically transformed rat liver cell line (FRL), neither of which expresses AChE activity. The enzyme was determined to be AChE on the basis of its susceptibility to inhibition by BW284c51 but not by iso-OMPA, as well as its substrate specificity. Although the secreted enzyme was salt soluble and its activity not modified by the addition of the nonionic detergent, Triton X-100, the activity of the cellular enzyme (derived from homogenates of E-2 cells) was greatly enhanced in the presence of the detergent. This work was supported by funds from the Chemical Research and Development Center, Aberdeen Proving Ground, MD. The opinions and assertions are the private ones of the authors and are not to be construed as official. These experiments were conducted according to the principles set forth in the “Guide for the Care and Use of Experimental Animals,” DHEW Publ. No. (NIH) 78-23.     

Developmental changes and acetylcholinesterase activity in the metamorphosing brain of Tenebrio molitor: Correlation to ecdysteroid titers

The brain of Tenebrio molitor exhibited marked fluctuations in acetylcholinesterase (AChE) activity throughout metamorphosis. This was true AChE activity, since it was inhibited by high substrate concentrations and by 10 μM of the specific AChE inhibitor BW284C51 [(1,5-bis'4-allyldimethylammoniumphenyl)-pentan-3-one dibromide] but not by iso-OMPA (tetraisopropylpyrophosphoramide), a cholinesterase (but not AChE) inhibitor. The histochemical AChE activity was localized in the neuropile and the nuclear envelope of neurons and glial cells. The enzyme extracted from brains with 1% Triton X-100 and 1 M NaCl sedimented as a single peak in a sucrose density gradient, with a sedimentation coefficient of 5.4S. This single AChE sedimentation peak was mainly due to an amphiphilic dimeric form. AChE activity per brain increased in newly ecdysed pupa. AChE activity per milligram of protein exhibited a peak in the mid-pupa which could be correlated to the increase in ecdysteroid titers. © 1994 Wiley-Liss, Inc.     

Characterization of a pseudocholinesterase purified from surgeonfish tissues confirms the atypical nature of this enzyme

The sialated, presumed-globular form of an atypical pseudocholinesterase (pseudo-ChE) previously described from surgeonfish tissues (Leibel: Comparative Biochemistry and Physiology 1988) has been purified to apparent homogeneity using a combination of salt fractionation along with ion-exchange and concanavalin A-Sepharose affinity chromatographic techniques. An overall 1,400-fold purification has been achieved with a 24% final yield of a cholinesterase (ChE) whose final specific activity is 50 mumol/min-mg. The purified enzyme was subjected to detailed biochemical and physical analysis. The purified pseudo-ChE is a sialated, globular, tetrameric enzyme with an apparent sedimentation coefficient of 11.5 S (+/- 0.5 S) and a molecular weight of 250 kilodaltons. The monomers are apparently not secured by disulfide bridges. The enzyme preferentially hydrolyzes acetyl(thio)choline but also hydrolyzes propionyl(thio)choline at reduced but comparable rates along with a wide variety of other noncholine esters. As such, it demonstrates the relative nonspecificity associated with classical pseudo-ChEs. However, the enzyme exhibits limited, but real, substrate inhibition with all choline esters as does true acetylcholinesterase (AChE). The enzyme is insensitive to the AChE inhibitor BW 284C51, sensitive to one (RO2-0683) of two (RO2-1250) pseudo-ChE inhibitors, and particularly sensitive to paraoxon inhibition (10(3)-10(4)-fold more so than AChE). It exhibits the short thermal half-life characteristic of pseudo-ChEs but not the expected ionic activation/inhibition profile. It is clear from this and other studies of atypical extrasynaptic cholinesterase activities occurring in other vertebrates that the orthodox categorization of cholinesterase as either "true" ("specific"; E.C. 3.1.1.7) or "pseudo" ("nonspecific"; E.C. 3.1.1.8) is inadequate to accommodate the increasing instances of ChE activities that exhibit atypical, intermediate properties.     

Soluble and membrane-bound acetylcholinesterases in Mytilus galloprovincialis (Pelecypoda: Filibranchia) from the northern Adriatic sea

Three forms of acetylcholinesterase (AChE) were detected in samples of the bivalve mollusc Mytilus galloprovincialis collected in sites of the Adriatic sea. Apart from the origin of the mussels, two spontaneously soluble (SS) AChE occur in the hemolymph and represent about 80% of total activity, perhaps hydrolyzing metabolism-borne choline esters. These hydrophilic enzymes (forms A and B) copurified by affinity chromatography (procainamide-Sepharose gel) and were separated by sucrose gradient centrifugation. They are, respectively, a globular tetramer (11.0-12.0 S) and a dimer (6.0-7.0 S) of catalytic subunits. The third form, also purified from tissue extracts by the same affinity matrix, proved to be an amphiphilic globular dimer (7.0 S) with a phosphatidylinositol tail giving cell membrane insertion, detergent (Triton X-100, Brij 96) interaction and self-aggregation. Such an AChE is likely functional in cholinergic synapses. All three AChE forms show a good substrate specificity and are inactive on butyrylthiocholine. Studies with inhibitors showed low inhibition by eserine and paraoxon, especially on SS forms, high sensitivity to 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284c51) and no inhibition with propoxur and diisopropylfluorophosphate (DFP). The ChE forms in M. galloprovincialis are possibly encoded by different genes. Some kinetic features of these enzymes suggest a genetic polymorphism.     

Cholinergic development in chick optic tectum and retina reaggregated cell cultures

The developmental profiles of acetylcholinesterase and choline acetyltransferase in chick optic tectum and retina cell aggregates, over a 30-day period, have been determined and compared with the corresponding developmental curves obtained in vivo. Both acetylcholinesterase and choline acetyltransferase activities in retina cell aggregates and the acetylcholinesterase activity in optic tectum cell aggregates usually lie between 40 and 90% of the values measured in vivo for the same cell (tissue) type and developmental age. However, the choline acetyltransferase activity in tectum aggregates increases only during the first 7 days of culture, and then decreases to reach a low value of 8% of that measured in vivo, by day 24. This fact, which is associated with widespread degeneration and cell death, could be attributed to the condition of natural deafferentiation occurring in a tectum cell aggregate. A parallel has been drawn between this behavior of a tectum cell aggregate and the effect of early embryonic eye removal on the development of the contralateral optic tectum in vivo. Thus, the tectum may have a biphasic pattern of development, with an autonomous period of growth of about 2 wk, followed by an afference-dependent phase, while the retina behaves, from a cholinergic point of view, as a relatively self-sufficient structure.Abbreviations AChE acetylcholinesterase - ChAT choline acetyltransferase - ACh acetylcholine - BW284 C51 dibromide 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide     

Cellular expression patterns of acetylcholinesterase activity during grasshopper development

We examined the expression of acetylcholinesterase (AChE) in the nervous system and epidermal body structures during embryonic and larval development of two grasshopper species: Locusta migratoria and Schistocerca americana. Histochemical labelling was blocked by the enzyme inhibitors eserine and BW284c51, but not by iso-OMPA, showing that the staining reflected true AChE activity. The majority of staining was localized on the cell surface but granular intracellular staining was also visible in many cell bodies. In both species, the cellular expression of AChE followed a similar but complex spatiotemporal staining pattern. Initially, mainly epidermal tissue structures were stained in the various body appendages (stages 25%–30%). Labelling subsequently appeared in outgrowing neurons of the central nervous system (CNS) and in the nerves innervating the limbs and dorsal body wall (stages 30%–40%). The latter staining originated in motoneurons of the ventral nerve cord. In a third phase (after 45%), the somata of certain identified mechanosensory neurons started to express AChE activity, presumably reflecting cholinergic differentiation. Staining was also found in repo-positive glial cells of the CNS, longitudinal glia of connectives, glia of the stomatogastric nervous system and glial cells ensheathing peripheral nerves. Glial cells remained AChE-positive during larval to adult development, whereas motoneurons lost their AChE expression. The expression pattern in non-neuronal cells and glutamatergic motoneurons and the developmental appearance of AChE prior to synaptogenesis in the CNS suggest non-cholinergic functions of AChE during grasshopper embryogenesis. Financial support was provided by the Deutsche Forschungsgemeinschaft (Bi 262/7-1 and 262/11-1)     

Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.)

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.     

Determinants of substrate specificity of a second non-neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis.

We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met). A recombinant form of AChE C was highly expressed by Pichia pastoris. The enzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate. A double mutation (W302F/W345F, corresponding to positions 290 and 331 in Torpedo) rendered the enzyme 10-fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the 'peripheral site' inhibitor propidium iodide. In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine, while remaining insensitive to the butyrylcholinesterase-specific inhibitor iso-OMPA and displaying a similar profile of excess substrate inhibition as the double mutant. These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEs.     

Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of organophosphorous pesticides by a novel biosensor based on localized surface plasmon resonance

Liquid and gas chromatography are commonly used to measure organophosphorus pesticides. However, these methods are relatively time consuming and require a tedious sample pretreatment. Here, we applied the localized surface plasmon resonance (LSPR) of gold nanoparticles covalently coupled with acetylcholinesterase (AChE) to create a biosensor for detecting an example of serial signals responding to paraoxon in the range of 1-100 ppb by an AChE modified LSPR sensor immersing in a 0.05 mM ACh solution. The underlying mechanism is that paraoxon prevents acetylcholine chloride (ACh) reacting with AChE by destroying the OH bond of serine in AChE. We found that the AChE modified LSPR sensors prepared by incubation with 12.5 mU/mL of AChE in phosphate buffer solution at pH 8.5 room temperature for 14 h have the best linear inhibition response with a 0.234 ppb limit of paraoxon detection. A 14% of inhibition on the sensor corresponds to the change of paraoxon concentration from 1 to 100 ppb. The sensor remained 94% of its original activity after six cycles of inhibition with 500 ppb paraoxon followed with reactivation of AChE by 0.5 mM 2-pyriding-aldoxime methoiodide (2-PAM). In addition, the sensor retains activity and gives reproducible results after storage in dry state at 4 degrees C for 60 days. In conclusion, we demonstrated that the AChE modified LSPR sensors can be used to determine the concentration of paraoxon biosensor with high sensitive and stable characteristics.     

Assembly of monomeric acetylcholinesterase into tetrameric and asymmetric forms

A pulse-chase experiment was performed in embryonic rat myotube cultures to examine possible precursor-product relationships among the various molecular forms of acetylcholinesterase (AChE). AChE was labeled with paraoxon, a compound which diethylphosphorylates AChE at its active site. Diethylphosphorylated (labeled) AChE is inactive but can be reactivated by treatment with 1-methyl-2-hydroxyiminomethyl-pyridinium. Thus labeled enzyme could be followed as AChE that regained activity following treatment with 1-methyl-2-hydroxyiminomethylpyridium. To selectively label monomeric AChE (the hypothesized precursor form), cultures were treated with methanesulfonylfluoride which irreversibly inactivated more than 97% of total cellular AChE. Methylsulfonylfluoride was then washed from the cultures, and they were labeled with paraoxon during a 40-55-min recovery period. AChE appearing in the cultures during this recovery period is newly synthesized and consists almost entirely (92%) of the monomeric form. Immediately and 120-130 min after labeling, cultures were subjected to a sequential extraction procedure to separate globular from asymmetric forms. Individual forms were then separated by velocity sedimentation on sucrose gradients. In our first series of experiments, we observed a 55% decrease in labeled monomers during the chase, a 36% increase in labeled tetramers, and a 36% increase in labeled asymmetric forms. In a second series of experiments focused on individual asymmetric forms, we observed a 55% decrease in labeled monomers, a 58% increase in labeled tetramers, an overall increase of 81% in labeled asymmetric forms, and a 380% increase in labeled A12 AChE. These data provide the first uniequivocal proof that complex forms of AChE are assembled from active monomeric precursors.     

The effect of high and low dosages of paraoxon in β-naphthoflavone-treated rats

Aliesterases (carboxylesterases) are serine esterases that can serve a protective role for the target acetylcholinesterase (AChE) during organophosphorus insecticide intoxication because the former esterases are alternate phosphorylation sites. The levels of aliesterase activity in liver and plasma and AChE activity in brain regions were investigated after the intravenous administration of paraoxon (P = O) into female rats. The rats were pretreated intraperitoneally with β-naphthoflavone (BNF), which decreases hepatic aliesterase activity following a 3 day in vivo treatment, and/or tri-o-totyl phosphate (TOTP) to inhibit aliesterases. The liver aliesterases were inhibited less by P = O in BNF-treated rats than in control rats, which suggests that either BNF exposure may have resulted in aliesterases that are less sensitive to P = O inhibition or BNF may have altered P = O's availability. The BNF treatment did not seem to alter the degree of inhibition of the brain AChE activity following the low dosage of paraoxon (0.04 mg/kg). However, the brain AChE activity in the P = O/TOTP/BNF-treated rats was lower than that in the P = O/TOTP-treated rats, suggesting that BNF also caused changes in systems affecting the disposition of P = O in addition to the changes in the hepatic aliesterases. At the high dosage of paraoxon (0.12 mg/kg), the AChE and aliesterase activities showed a pattern similar to that of the low dosage. This suggests that the aliesterases, as altered by BNF exposure, even when nearly completely inhibited, did not alter the response of the target enzyme, AChE, and, therefore, the magnitude of the toxic response. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 263–268, 1997.