Why acetylcholinesterase reactivators do not work in butyrylcholinesterase

The pyridinium oxime therapy for treatment of organophosphate poisoning is a well established, but not sufficient method. Recent trends also focus on prophylaxis as a way of preventing even the entrance of organophosphates into the nervous system. One of the possible prophylactic methods is increasing the concentration of butyrylcholinesterase in the blood with the simultaneous administration of butyrylcholinesterase reactivators, when the enzyme is continuously reactivated by oxime. This article summarizes and sets forth the structural differences between butyrylcholinesterase and acetylcholinesterase, essential for the future design of butyrylcholinesterase reactivators. Butyrylcholinesterase lacks the reactivator aromatic binding pocket found in acetylcholinesterase, which is itself a part of the acetylcholinesterase peripheral anionic site. This difference finally renders the current acetylcholinesterase reactivators, when used in butyrylcholinesterase, non-functional.     

A new assay for endocytosis in erythrocyte ghosts based on loss of acetylcholinesterase activity

A new method for assaying endocytosis in erythrocyte ghosts is presented. The method involves measuring the percentage loss of acetylcholinesterase activity which occurs when vacuoles form, making the acetylcholinesterase on the vacuole surface inaccessible. This method is compared to other methods of measuring endocytosis in this system, including phase contrast microscope estimation of vesiculation, stereological analysis of electron micrographs to determine vesiculation and loss of sialic acid accessible to neuraminidase due to endocytosis. Comparison of the percentage loss of acetylcholinesterase activity with the electron micrographic and sialic acid methods showed that all three methods gave a quantitative measure of the percentage of total membrane area taken in as vesicles. Since the acetylcholinesterase method was fast, easy, inexpensive, and quantitative, it was the preferred method for assay of endocytosis. The inhibition of endocytosis by Ca²⁺ was observed with this method; the success of this experiment demonstrated the applicability of the method to the study of inhibitors of endocytosis.     

A silica gel plate-based qualitative assay for acetylcholinesterase activity: a mass method to screen for potential inhibitors.

A procedure for the qualitative assessment of inhibitory activity towards acetylcholinesterase for a given compound is described. Solutions of the compounds of interest are spotted on silica gel TLC plates in a matrix pattern. The silica gel plate is sprayed with a solution of acetylthiocholine iodide and 5,5-dithiobis(2-nitrobenzoic acid) followed by a solution of acetylcholinesterase. The enzyme reaction produces a yellow background color with inhibitor compounds exposed as white zones where color has failed to develop. The results for a test set of compounds were compared to those obtained using the standard Ellman assay procedure and found to agree for virtually all of these compounds. The conditions of silica gel plate thickness, reagent concentration, and enzyme source under which this procedure is suitable were investigated. This represents an extremely rapid method to screen large numbers of compounds to uncover new inhibitors of acetylcholinesterase and potentially other enzymes as well.     

Phenyldichlorophosphate as an aid in studies of decarbamylation of carbamylated acetylcholinesterase

An improved method for assaying carbamylated acetylcholinesterase is described which has substantial benefits over current methods. Acetylcholinesterase was carbamylated with neostigmine and diluted extensively into buffer to allow decarbamylation to occur. At various times, phenyldichlorophosphate was added to the mixture of free and carbamylated enzyme, whereupon two very rapid, simultaneous reactions occurred: near total, and permanent, inactivation of free acetylcholinesterase by the organophosphate, and inactivation of phenyldichlorophosphate by hydrolysis. The carbamylated acetylcholinesterase was allowed to reactivate fully and then assayed for enzyme activity. The assay provided a measure of the amount of carbamylated enzyme present at the time of addition of phenyldichlorophosphate, thereby enabling the first-order rate constant for decarbamylation to be calculated. This new method of studying decarbamylation was applied to two systems of soluble acetylcholinesterase, where the half-life for decarbamylation was approximately 1/2 h or 4 min, respectively, and to membrane-bound acetylcholinesterase. The results agreed well with those determined by a conventional method; moreover, the standard error of the mean was lower for the new method. The advantages of the method using phenyldichlorophosphate over conventional methods are particularly evident when decarbamylation is rapid or when in vivo studies are being performed and it is not practical or desirable to run assays immediately on isolation of the tissue. The new method also has advantages over a published related technique using the organophosphate anticholinesterase soman.     

Effect of membrane fluidity upon binding of Electrophorus acetylcholinesterase to lipid vesicles

A previous report (Watkins, M.S., Hitt, A.S. and Bulger, J.E. (1977) Biochem. Biophys. Res. Commun. 79, 640-647) has indicated that the asymmetric forms of Electrophorus acetylcholinesterase bind exclusively to sphingomyelin vesicles through interaction with the collagen-like 'tail' portion of the enzyme. We report here that acetylcholinesterase also binds to phosphatidylcholine vesicles containing saturated fatty acyl chains and to egg phosphatidylcholine vesicles containing cholesterol. This suggests preferential binding of acetylcholinesterase to membranes of lower fluidity. Surface charge of vesicles and density of zwitterionic lipid headgroups do not significantly affect binding of native acetylcholinesterase. The presence of chondroitin sulfate or hyaluronic acid slightly increases the binding of native acetylcholinesterase to sphingomyelin vesicles, while the presence of 1 M NaCl, bovine serum albumin, or tissue fractions enriched in basement membrane diminish binding. The dissociation constant for native acetylcholinesterase and sphingomyelin vesicles is (1.0-1.5) X 10(-7) M, as measured by a flotation binding assay. The globular, 11S form of acetylcholinesterase also binds to lipid vesicles, although not to the same degree as native acetylcholinesterase. This suggests that the collagen tail of the enzyme enhances binding, but is not essential for binding to occur. These results are consistent with the location of acetylcholinesterase on the surface of the postsynaptic plasma membrane in vivo.     

Pre-conditioning to global cerebral ischemia changes hippocampal acetylcholinesterase in the rat

This study shows the effect of transient global cerebral ischemia (ISC) on hippocampal acetylcholinesterase (AChE) activity. Naive adult Wistar rats received either a brief (2 min) or a long (10 min) ischemic episode by the four-vessel occlusion method. Pre-conditioned rats received double ischemia: a 10 min episode inflicted 24 h after a 2 min event, a condition known to confer cytoprotection to CA1 pyramidal cells of hippocampus. 2 min of ischemia caused an increase in acetylcholinesterase activity both immediately and 30 min after the episode, however enzyme activity was significantly decreased after 24 h of reperfusion. 10 min of ischemia caused an increase in activity both 60 min and 24 h after ischemia. Conversely, pre-conditioned rats displayed lower activity both immediately and 60 min after ischemia. Our results suggest that: a) neuronal death, that follows 10 min of ischemia, is associated to a late increase in acetylcholinesterase activity; b) pre-conditioning is related to diminished acetylcholinesterase activity. This is in agreement with previous evidence that acetylcholinesterase inhibition and maintenance of acetylcholine levels are beneficial for cell surviving after cerebral ischemia.     

Internal standard method for the measurement of choline and acetylcholine by capillary electrophoresis with electrochemical detection

An internal standard method has been developed for the determination of the neurotransmitter acetylcholine and/or its metabolic precursor choline. This approach couples the high separation efficiency of capillary electrophoresis with the sensitivity and selectivity of electrochemical detection at an enzyme-modified electrode. Indirect electrochemical detection is accomplished at a 25 microm platinum electrode modified by cross-linking the enzymes choline oxidase and acetylcholinesterase with glutaraldehyde. Although in this simple form of electrode fabrication there is a gradual loss of response from the electrochemical detector with time, accurate quantitation is achieved by the addition of butyrylcholine, which is also a substrate for acetylcholinesterase, as an internal standard. A linear response is achieved between 0 and 125 microM with a limit of detection of 2 microM (25 fmol). The utility of this method was demonstrated by monitoring the kinetics of choline uptake in synaptosomal preparations.     

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.     

Microtiter assay for acetylcholinesterase

A microtiter plate adaptation of the classical Ellman colorimetric procedure for measurement of acetylcholinesterase activity is described. This method permits use of an enzyme-linked immunosorbent assay plate reader for rapid analysis of multiple samples and is particularly suitable for analysis of acetylcholinesterase activity on sucrose gradients. The novel procedure is rapid and sensitive and does not require use of radioactive material.     

An ultrasensitive electrometric system to measure membrane-bound acetylcholinesterase activity

A very sensitive method for the determination of membrane-bound acetylcholinesterase from sarcoplasmic reticulum is described. The acetic acid which is released by the enzymatic hydrolysis of acetylcholine is measured by means of an electrometric system. Diluted hydrochloric acid is used as the standard to evaluate the amount of H+ produced during the time course of the reaction. With the use of a bucking voltage device the sensitivity of the method permits one to follow changes in H+ concentration below 1 microM. Therefore the enzyme activity can be estimated using a very small amount of sarcoplasmic reticulum protein. This procedure is very simple, accurate and reproducible, and it can be applied to measure membrane-bound acetylcholinesterase where the membrane suspension makes it difficult to employ spectrophotometric techniques.     

The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a DD-carboxypeptidase in vitro

A chemiluminescence method for determining acetylcholinesterase activity is described. It is an adaptation of the chemiluminescence assay of acetylcholine described by Israël & Lesbats [(1981) Neurochem. Int. 3, 81-90; (1981) J. Neurochem. 37, 1475-1483]. The acetylcholinesterase activity is measured by monitoring the increase in light emission produced by the accumulation of choline or by determining the amount of choline generated after a short interval. The assay is rapid and sensitive, and uses the natural substrate of the enzyme. Kinetic data obtained with this procedure for acetylcholinesterase from Torpedo and Electrophorus electric organs were comparable with those obtained by using the method of Ellman, Courtney, Andres & Featherstone [(1961) Biochem. Pharmacol. 7, 88-95]. In addition, it was shown that sodium deoxycholate totally inactivated Torpedo acetylcholinesterase but not the Electrophorus enzyme. Competitive inhibitors of acetylcholinesterase protected the enzyme from inactivation.     

Differential inhibition of soluble and membrane-bound acetylcholinesterase forms from mouse brain by choline esters with an acyl moiety of an intermediate size

Differential inhibitions of soluble and membrane-bound acetylcholinesterase forms purified from mouse brain were examined by the comparison of kinetic constants such as a K _m value, a K_ss value (substrate inhibition constant), and IC₅₀ values of active site-selective ligands including choline esters. Membrane-bound acetylcholinesterase form (solubilized only in the presence of detergent) showed lower K_m and K_ss values than soluble acetylcholinesterase form (easily solubilized without detergent). Edrophonium expressed a slightly but significantly (p<0.01) higher inhibition of detergent-soluble acetylcholinesterase form than aqueous-soluble acetylcholinesterase form, while physostigmine inhibited both forms with a similar potency. A remarkable difference in inhibition was observed using choline esters; although choline esters with acyl chain of a short size (acetyl-to butyrylcholine) or a long size (heptanoyl- to decanoylcholine) showed a similar inhibitory potency for two forms of acetylcholinesterase, pentanoylcholine and hexanoylcholine inhibited more strongly aqueous-soluble acetylcholinesterase than detergent-soluble acetylcholinesterase. Thus, it is suggested that the two forms of AChE may be distinguished kinetically by pentanoyl- or hexanoylcholine.This work was supported in part by Agency for Defense Development.     

Cholinesterase solubilizing factor from Cytophaga sp. is a phosphatidylinositol-specific phospholipase C

In the culture supernatant of Cytophaga sp. we detected an enzyme that converted glycosylphosphatidyl-inositol-anchored acetylcholinesterase to the hydrophilic form. This enzyme had a cleavage specificity of a phospholipase C. It hydrolyzed phosphatidylinositol but did not act on phosphatidylcholine. On gel filtration the enzyme migrated with an apparent molecular mass of about 17 kDa. It displayed maximal activity between pH 6-6.5 and did not require cofactors for the expression of catalytic activity. Mercurials and zinc ions inhibited the enzyme and its activity also decreased with increasing ionic strength in the assay. With acetylcholinesterase as substrate optimal activity was obtained in pure micelles of Triton X-100, whereas in mixed micelles containing Triton X-100 and phosphatidylcholine the activity was reduced. The enzyme from Cytophaga sp. showed little activity towards acetylcholinesterase embedded in intact membranes where more than 1000-times higher concentrations of phosphatidylinositol-specific phospholipase C was necessary to solubilize acetylcholinesterase as compared to acetylcholinesterase in detergent micelles.     

Structures of paraoxon‐inhibited human acetylcholinesterase reveal perturbations of the acyl loop and the dimer interface

Irreversible inhibition of the essential nervous system enzyme acetylcholinesterase by organophosphate nerve agents and pesticides may quickly lead to death. Oxime reactivators currently used as antidotes are generally less effective against pesticide exposure than nerve agent exposure, and pesticide exposure constitutes the majority of cases of organophosphate poisoning in the world. The current lack of published structural data specific to human acetylcholinesterase organophosphate‐inhibited and oxime‐bound states hinders development of effective medical treatments. We have solved structures of human acetylcholinesterase in different states in complex with the organophosphate insecticide, paraoxon, and oximes. Reaction with paraoxon results in a highly perturbed acyl loop that causes a narrowing of the gorge in the peripheral site that may impede entry of reactivators. This appears characteristic of acetylcholinesterase inhibition by organophosphate insecticides but not nerve agents. Additional changes seen at the dimer interface are novel and provide further examples of the disruptive effect of paraoxon. Ternary structures of paraoxon‐inhibited human acetylcholinesterase in complex with the oximes HI6 and 2‐PAM reveals relatively poor positioning for reactivation. This study provides a structural foundation for improved reactivator design for the treatment of organophosphate intoxication. Proteins 2016; 84:1246–1256. © 2016 Wiley Periodicals, Inc.     

Regulation of acetylcholinesterase expression by calcium signaling during calcium ionophore A23187- and thapsigargin-induced apoptosis

We have recently reported that acetylcholinesterase expression was induced during apoptosis in various cell types. In the current study we provide evidence to suggest that the induction of acetylcholinesterase expression during apoptosis is regulated by the mobilization of intracellular Ca(2+). During apoptosis, treatment of HeLa and MDA-MB-435s cells with the calcium ionophore A23187 resulted in a significant increase in acetylcholinesterase mRNA and protein levels. Chelation of intracellular Ca(2+) by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester), an intracellular Ca(2+) chelator, inhibited acetylcholinesterase expression. A23187 also enhanced the stability of acetylcholinesterase mRNA and increased the activity of acetylcholinesterase promoter, effects that were blocked by BAPTA-AM. Perturbations of cellular Ca(2+) homeostasis by thapsigargin resulted in the increase of acetylcholinesterase expression as well as acetylcholinesterase promoter activity during thapsigargin induced apoptosis in HeLa and MDA-MB-435s cells, effects that were also inhibited by BAPTA-AM. We further demonstrated that the transactivation of the human acetylcholinesterase promoter by A23187 and thapsigargin was partially mediated by a CCAAT motif within the -1270 to -1248 fragment of the human acetylcholinesterase promoter. This motif was able to bind to CCAAT binding factor (CBF/NF-Y). These results strongly suggest that cytosolic Ca(2+) plays a key role in acetylcholinesterase regulation during apoptosis induced by A23187 and thapsigargin.     

Functional idiotypic mimicry of an adhesion- and differentiation-promoting site on acetylcholinesterase

Acetylcholinesterase mediates cell adhesion and neurite outgrowth through a site associated with the peripheral anionic site (PAS). Monoclonal antibodies raised to this site block cell adhesion. We have raised anti-idiotypic antibodies to one of these antibodies. The anti-idiotypic antibodies recognized the immunogenic antibody and non-specific mouse IgG, but not acetylcholinesterase. Five antibodies (out of 143 clones, an incidence of 3.5%) were able to promote neurite outgrowth in human neuroblastoma cells in vitro in a similar manner to acetylcholinesterase itself, suggesting that these antibodies carry an internal image of the neuritogenic site. Two of the antibodies were significantly more effective (P < 0.01) than acetylcholinesterase in this regard. The antibodies also bound specifically to mouse laminin-1 and human collagen IV, as does acetylcholinesterase. This binding was displaced by unlabelled antibody, as well as by acetylcholinesterase itself, indicating competition with acetylcholinesterase. We have also investigated the development of anti-anti-idiotypic antibodies in mice in vivo, and have observed that four of these (out of 318 clones, an incidence of 1.26%) mimic the idiotypic antibody and abrogate adhesion in neuroblastoma cells. We have thus demonstrated functional mimicry of the neuritogenic site on acetylcholinesterase in anti-idiotypic antibodies, enhancement of this activity in one antibody, and mimicry of the idiotypic antibody site in anti-anti-idiotypic antibodies. Implications of these findings for differentiation-promoting cancer therapy are discussed.     

A radioactive assay for acetylcholinesterase using anion-exchange disk

A radioactive assay for acetylcholinesterase is described. The assay is based on the separation of [¹⁴C]acetate from [¹⁴C]acetylcholine by differential adsorption of the former on DEAE anion-exchange disks. The procedure is simple and sensitive and eliminates the use of ion-exchange resin columns or organic extractions. Moreover, when unpurified enzyme preparations are assayed, linear steady-state kinetics can be observed with this method as contrasted to the nonlinear colorimetric method using acetylthiocholine and dithiobisnitrobenzoate. This method also permits the detection in biological samples of low levels of acetylcholinesterase activity, which is not detectable by the colorimetric method. Using the present radioactive method, cellular levels of acetylcholinesterase have been surveyed in N4TG1 neuroblastoma cells, NG108-15 neuroblastoma x glioma hybrid cells, H9c2 myoblasts, and 3T3-L1 and 3T3-C2 fibroblasts.     

Enzyme biosensor for studying therapeutics of Alzheimer's disease.

An electrochemical method for the investigation and comparison of anti-Alzheimer medications that is based on the inhibition of the acetylcholinesterase is presented. The developed amperometric biosensor determines the in-vitro inhibition of the acetylcholinesterase that is co-immobilized with choline oxidase on the working electrode surface of a three-electrode system using gel entrapment. The sensor has been applied to determine the IC50 values of two known and one newly developed Alzheimer remedy. A simultaneous measurement with the photometric standard method shows the applicability of our method for fast drug screening.     

Regulatory effects of polyamines on membrane-bound acetylcholinesterase

The effects of putrescene, spermidine and spermine on membrane-bound acetylcholinesterase from human erythrocyte ;ghosts' and the solubilized enzyme of the electric organ of the electric eel were studied by kinetic methods. Measurements were made by using a photometric method which made it possible to record the enzyme reaction in the steady-state phase. Substrate-concentration-dependent activation and inhibition of acetylcholinesterase by polyamines is similar to that by Na(+), K(+), Ca(2+), Mg(2+) and certain quaternary and bisquaternary amines. The kinetics suggest an allosteric reaction mechanism. On the basis of the kinetic results a role for the polyamines as modulators of synaptic acetylcholinesterase is proposed.     

Fluorinated aldehydes and ketones acting as quasi-substrate inhibitors of acetylcholinesterase.

1. The inhibition of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) by compounds containing trifluoromethyl-carbonyl groups was investigated and related to the effects observed with structurally similar, non-fluorinated chemicals. 2. Compounds that in aqueous solution readily form hydrates inhibit acetylcholinesterase in a time-dependent process. On the other hand non-hydrated, carbonyl-containing compounds showed rapid and reversible, time-independent enzyme inactivation when assayed under steady state conditions. 3. m-N,N,N-Trimethylammonium-acetophenone acts as a rapid and reversible, time-independent, linear competitive inhibitor of acetylcholinesterase (Ki = 5.0 . 10(-7) M). 4. The most potent enzyme inhibitor tested in this series was N,N,N,-trimethylammonium-m-trifluoroacetophenone. It gives time-dependent inhibition and the concentration which inactivates eel acetylcholinesterase to 50% of the original activity after 30 min exposure is 1.3 . 10(-8) M. The bimolecular rate constant for this reaction is 1.8 . 10(6) 1 . mol-1 . min-1. The enzyme-inhibitor complex is very stable as the inhibited enzyme after 8 days of dialysis is reactivated to 20% only. This compound represents a quasi-substrate inhibitor of acetylcholinesterase.