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.     

Development, validation and application of an enzyme immunoassay (EIA) of atriopeptin

A rapid, convenient, and sensitive enzyme immunoassay (EIA) for atriopeptin (AP) has been developed. The tracer-ligand for the assay is the 24-amino acid peptide, AP24, which has been covalently coupled to the tetrameric form of acetylcholinesterase (AChE) (EC 3.1.1.7). Tracer, unknown, and primary antibody are incubated in a 96-well microtiter plate precoated with secondary antibody. After washing, a colorimetric reaction is used to measure acetylcholinesterase activity. A direct linear correlation was obtained when comparing the conventional radioimmunoassay and the EIA by using the same primary antibody to assay: plasma samples (rat or human), HPLC column fractions, or atrial extracts. Besides being technically much less demanding and not requiring the use of the radioisotopes, the EIA is more sensitive than the radioimmunoassay and thereby lends itself to a "flash" same-day assay of samples.     

A microassay-based procedure for measuring low levels of toxic organophosphorus compounds through acetylcholinesterase inhibition

Using a microtiter plate spectrophotometric system, an assay procedure was developed for the following toxic organophosphorus compounds: 1,2,2-trimethylpropyl ester of methylphosphonofluoridic acid (1, soman); ethyl N,N-dimethylphosphoramidocyanidate (3, tabun); O-ethyl S-[2-[bis(1-methylethyl)amino]ethyl]- methylphosphonothiolate (4, VX); the diethyl 4-nitrophenyl ester of phosphoric acid (5, paraoxon); and bis(1-methylethyl) phosphorofluoridate (6, DFP). The procedure, based on the Ellman assay method, uses inhibition of eel acetylcholinesterase (0.01 unit per well) to carry out the determination of inhibitor concentrations for both a standard curve and the unknown samples on a single 96-well microtiter plate. On a typical plate, samples of both unknowns and standards (a minimum of six concentrations were used per standard curve) were assayed five times per sample, with three control (uninhibited) enzyme activity points included for each sample. The time required for carrying out a single plate was approx 30 min. Sensitivity for the most potent acetylcholinesterase inhibitor tested was 0.4 nM under the conditions used for a typical assay. It should be noted, however, that no attempt was made to optimize the assay procedure for sensitivity.     

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.     

A microtiter assay for determining protein, acetylcholinesterase activity, and G418 (Neomycin) resistance in cultured cells.

The Coomassie brilliant blue assay for the determination of protein has been extended to rapidly and conveniently measure the protein concentration of cells growing in culture in a 96-well microtiter format. Modifications of the standard assay include sodium hydroxide to solubilize the cells and ovalbumin, instead of bovine serum albumin, as a protein standard. The procedure allows a large number of small samples to be assayed simultaneously. Two examples of its use, enzyme-specific activity and drug resistance, are shown. An assay for acetylcholinesterase activity in the same culture plate is demonstrated. G418, an inhibitor of cell protein synthesis, is frequently used to select for cells transfected with the neomycin resistance gene. The required concentration of G418 can be easily determined with this protein assay.     

A pseudo-first-order kinetic approach to measurement of acetylcholine hydrolysis by acetylcholinesterase

A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 3.1.1.7) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H⁺ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88–95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of K₁ for edrophonium, decamethonium and Al³⁺.     

Use of a 96-well microplate reader for measuring routine enzyme activities

A method is described for the routine determination of the rate of colorimetric enzyme reactions using a 96-well microtiter plate reader commonly used in immunoassay. This approach is illustrated by monitoring esterase activity using three common products: release of thiol, release of ethanol, and release of p-nitrophenylate ion. Examples include monitorings of the rate of hydrolysis of acetylthiocholine iodide by eel acetylcholinesterase and the rate of hydrolysis of malathion and nonconventional esters such as O-methyl, O-ethyl, and O-isobutyl carbonates of p-nitrophenol by commercial porcine liver carboxylesterase. Data obtained from the plate reader were compared to those obtained, under similar conditions, in a conventional spectrophotometer. Absorbance measurements made in both machines on the same solution, as well as absorbance changes measured over time, were similar. The use of the 96-well plate format tremendously increased the number of enzyme assays carried out per person and the interface with a personal computer allowed rapid manipulation of the absorbance values to calculate the desired rate data. This approach should be generally applicable to many routine colorimetric assays in the research laboratory.     

A one-step cobalt-ferrocyanide method for histochemical demonstration of acetylcholinesterase activity in central nervous system tissue.

We introduce a one-step histochemical method with cobalt as the precipitating agent for ferrocyanide for the light microscopic demonstration of acetylcholinesterase activity. This method was used to demonstrate acetylcholinesterase in normal cortical fibers and neurons, as well as pathological elements such as plaques and tangles. This procedure can also be easily combined with immunohistochemical methods that use diaminobenzidine as a chromogen.     

An automated method for acetylcholinesterase kinetics

An automated assay for acetylcholinesterase (EC 3.1.1.7.) has been developed based on the manual spectrophotometric method of Ellmanet al. (1). This method was used to determine (a) the enzyme activity of an unknown sample and (b) the dependence of initial rates given by a fixed enzyme concentration on the substrate concentration. Methods to minimize possible enzyme modification by DTNB (2) are described. Finally a modification of the conventional autoanalyser procedure permitted rapid and reproducible enzyme kinetic analysis under various conditions. This helped to minimize the effects of possible enzyme inactivation at high dilutions especially when using crude enzyme preparations.     

Acetylcholinesterase Activity in Regions of the Rat Brain Following a Convulsion

The effects of electroconvulsive shock on the levels of acetylcholinesterase in several brain regions of the rat were studied. Hippocampus, mesencephalon, cortex, and striatum exhibited rapid changes in acetylcholinesterase activity during the first few minutes following the convulsion, whereas brainstem and basal forebrain levels remained unchanged. In both hippocampus and midbrain there was a sustained decrease in activity: the total acetylcholinesterase activity was decreased by up to 40% within 2 min of the convulsion and did not return to control values for another 3 h. Thirty minutes after a flurothyl-induced convulsion there was a similar fall in acetylcholinesterase activity in both these regions, whereas a subconvulsive electric shock produced no change. It is concluded that a convulsion produces significant short-term decreases in acetylcholinesterase activity in areas of the rat brain that are involved in the generation and propagation of seizures, and the question is raised of whether this is related to the increase in seizure threshold that follows a convulsion.     

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.     

Rapid qualitative method for detecting staphylococcal nuclease in foods.

A rapid method for the detection of heat-stable staphylococcal nuclease in foods is described. The procedure consists of an acid precipitation, boiling, and centrifugation followed by enzyme detection in an agar plate containing deoxyribonucleic acid. To test the efficacy of the procedure, purified Staphylococcus aureus nuclease was added to various foods and recovery experiments were performed. Additionally, foods were inoculated and incubated with S. aureus, and the staphylococcal counts were compared with nuclease activity. The results indicate that the procedure possesses merit for a rapid method that can be incorporated into quality control programs. The procedure requires approximately 2.5 h, and it will detect nuclease levels as low as 10 ng/g of food.     

Effect of heat shock on acetylcholinesterase activity in chick muscle cultures

The effect of heat shock was studied on the acetylcholinesterase activity of chick muscle primary cultures. In cultures transferred from 37 degrees C to 45 degrees C, a sharp drop in activity was followed by rapid spontaneous recovery. The time of onset of recovery resembled the time needed for expression of heat shock proteins. In cultures exposed to heat shock at 45 degrees C and allowed to recover at 37 degrees C, reappearance of acetylcholinesterase activity did not involve de novo protein synthesis since it was not prevented by cycloheximide. Our data raise the possibility of a role for heat shock proteins as molecular chaperones in rescuing heat-denaturing acetylcholinesterase.     

Colorimetric microtiter plate assay of glucose and fructose by enzyme-linked formazan production: applicability to the measurement of fructosyl transferase activity in higher plants

A rapid, enzyme-linked colorimetric assay, for the sequential determination of nanomole quantities of glucose and fructose in the same sample, has been developed for the measurement of fructosyl transferase activity in plant extracts. The assay extends the conventional dehydrogenase-linked assay for these sugars by utilizing the intermediary electron carrier, phenazine methosulfate, to couple NADP reduction to the production of a formazan dye from the tetrazolium salt, thiazolyl blue, in a form suitable for measurement using a microtiter plate reader. When the microtiter plate assay was used to measure the activities of yeast invertase and sucrose:sucrose fructosyl transferase from Lolium temulentum, results obtained were very similar to results obtained using the conventional procedure. The rapidity, small scale, and ease of execution of the method offers considerable advantages over the conventional hexose assay and is particularly suitable for screening of large numbers of small samples, exploiting both the speed of the microtiter plate reader and the facility of for microcomputer processing of data. The potential of this method for use with other enzyme systems and other metabolites is discussed.     

Nonradioactive assay of FLAG-tagged MAPK using ANTI-FLAG antibody-coated multiwell plates

We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ANTI-FLAG M2 antibody (ANTI-FLAG M2 plate): (i) the capture of phosphorylated FLAG-tagged MAPK fusion protein (FLAG-MAPK) from phorbol esters-stimulated, FLAG-MAPK-transfected COS-7 cells, coupled with a very sensitive ELISA procedure to quantitate the level of phosphorylation of FLAG-MAPK; (ii) the in vitro kinase reaction of FLAG-MAPK activity with a substrate and ATP in the same well used to captured the phosphorylated FLAG-MAPK; and (iii) the in vitro kinase reaction of captured non-activated FLAG-MAPK by its upstream kinase from phorbol 12-myristate 13-acetate (PMA)-stimulated COS-7 cells. These results demonstrate that the ANTI-FLAG M2 plate allows for the rapid and quantitative determination of phosphorylation of FLAG-MAPK directly from stimulated, transfected cell lysate. Captured, phosphorylated FLAG-MAPK retains catalytic activity as demonstrated by the phosphorylation of Elk-1 in the same well. Furthermore, phosphorylation of captured FLAG-MAPK by the upstream kinases can be observed directly on the plate. These assays are sensitive, specific, and suitable for handling multiple samples. Thus, the ANTI-FLAG M2 plate forms the basis of a high-throughput screening platform in kinase analysis.     

Thermal gels: A procedure for determination of heat-inactivation temperature of enzymes

A facile procedure for the study of inactivation temperatures of enzymes that could be stained for activity after electrophoresis is described. This is particularly suitable for enzymes that have multiple electrophoretic forms. The results of studies on Drosophila dehydrogenases and acetylcholinesterase are presented. The procedure may be useful in the study of genetic variants.     

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.     

The separation of oligonucleotides of baker''s-yeast value transfer ribonucleic acid 2b by high-voltage electrophoresis on DEAE-paper and by thin-layer chromatography.

A modified procedure for the separation of oligoribonucleotides is described that is based on the combination of t.l.c. on cellulose and electrophoresis on DEAE-paper at 4000 V on a cooling plate. The technique is relatively rapid and allows the analysis of larger quantities than is possible by electrophoresis on cellulose acetate.     

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.     

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.