Source Of Acetylcholinesterase Research Articles

Analysis of pesticides based on immobilized housefly head acetylcholinesterase reactor with choline oxidase and horseradish peroxidase carbon paste electrode

AbstractAn automatic flow injection system (FIA) using the acetylcholinesterase (AChE) reactor connected with the choline oxidase (ChOx) and horseradish peroxidase (HRP) carbon paste electrode for organophosphorous (ORPs) and carbamate pesticides determination is exploited. The ORPs are measured based on the inhibition of AChE. Two different sources of AChE from eel and housefly head are studied, respectively. The detection limit for paraoxon (10 ppb) and carbofuran (23 ppb) is found in the housefly head AChE. The AChE from housefly head shows 16 times higher sensitivity than the enzyme from eel for paraoxon. The linear range of the housefly head AChE biosensor is 8–100 ppb. The throughput of five‐sample hour−1 is obtained with a good reproducibility. The fruit juice and river water samples are studied and show the feasibility for further applications.

Molecular docking analysis of acetylcholinesterase corroborates the protective effect of pralidoxime against chlorpyrifos-induced behavioral and neurochemical impairments in Nauphoeta cinerea

Toxic organophosphates (OPs) are widely used by the man to manage crops and household insects. Here we used the cockroach, Nauphoeta cinerea, as an alternative model to study the effect of chlorpyrifos (CPF) on the acetylcholinesterase (AChE) and its reactivation by pralidoxime (2-PAM). The main objective was to determine the suitability of N. cinerea as a high-throughput animal model to study the behavioral and biochemical effects of CPF. Here, in silico approaches were also used to further refine the interaction of both CPF and 2-PAM with different sources of AChEs (human, mouse, fruit fly, and cockroach). Since data about the distribution of chemicals in N. cinerea are not available in the literature, we used methylene blue as an “indirect indicator” of CPF and 2-PAM distribution in cockroaches. After 40 min of administration, methylene blue was detected in the head region of cockroaches. A single injection of CPF (3.4 nmol in 10 µL) in the dorsal region of N. cinerea caused typical signs of OP poisoning (from 30 to 60 min after CPF injection). The administration of 2-PAM (10 min after the CPF) was able to reverse the behavioral and biochemical (AChE activity) changes induced by CPF. The in silico docking simulations between the cockroach AChE and the chemicals (CPF and 2-PAM) provided insight into the binding pose and molecular interactions of the chemicals with AChE structure. Thus, the N. cinerea can be a potential experimental model and reliable for researchers because the treatments with CPF and 2-PAM on this species had the same pattern as studies realized in the mammalian models.

A newly developed oxime K203 is the most effective reactivator of tabun-inhibited acetylcholinesterase

BackgroundBased on in vitro and in vivo rat experiments, the newly developed acetylcholinesterase (AChE) reactivator, K203, appears to be much more effective in the treatment of tabun poisonings than currently fielded oximes.MethodsTo determine if this reactivating efficacy would extend to humans, studies were conducted in vitro using human brain homogenate as the source of AChE. The efficacy of K203 was compared with commercially available oximes; pralidoxime, obidoxime and asoxime (HI-6).ResultsReactivation studies showed that K203 was the most effective reactivator with a second order kinetic constant (kr) of 2142 min− 1. M− 1, which was 51 times higher than that obtained for obidoxime (kr = 42 min− 1. M− 1). Both pralidoxime and asoxime (HI-6) failed to significantly reactivate tabun-inhibited human AChE.DiscussionAccording to these results and previous studies, using K203, it appears that oxime K203 is the most effective reactivator of tabun-inhibited cholinesterase in several species including humans and should be considered as a possible medical countermeasure to tabun exposure.

Medicinal Plants Anti-cholinesterase Activity and the Potential for Alzheimer’s Disease Treatment

Alzheimer’s disease (AD) is a slow and progressive neurodegenerative disease, leading to a breakdown in memory preservation and absence of cognitive reasoning and language skills. It is the most popular neurodegenerative disease worldwide, with nearly 20 million new incidences recorded every year. The enzymes, acetyl cholinesterase (AChE) and butyryl-cholinesterase (BChE) play important roles in the cholinergic deficit through enhanced degradation of the neurotransmitter, acetylcholine (ACh). In most cases, to improve neurotransmission and to alleviate cholinergic deficit, the focus is mainly on acetyl cholinesterase (AChE) and butyl cholinesterase (BCh). Currently, drugs for the treatment of AD (Rivastigmine, galanthamine, donepezil and Memantine) targets the later stages and implicated in the progression of the disease. Acetyl cholinesterase enzyme (AChE) is especially an attractive target for the rational drug design and for the discovery of mechanism based inhibitors; this is because of its role in the hydrolysis of neurotransmitter, acetylcholine. Many plants serve as potential source of AChE and BChE inhibitors, and useful compounds that could provide templates for the development of drugs for the treatment of AD. This paper provides a review of several medicinal plants tested for anti-AChE and or BChE, many of which contains promising bioactive compounds as potential anti-cholinesterase that could serve as candidates for the development of drugs to treat AD.

Plant based indian traditional medicine for neurodegenerative diseases - a novel Approach to treat alzheimer

As per the earlier reports, the most prevalent disease of Alzheimer’s, a kind of dementia, affects approximately 24 million people worldwide. There may be many invisible reasons for the disease to be caused and the common evidences for the disease is due to a few reasons like aggregation of selfassembled fibrous proteins and variation in the level of Acetyl choline (ACh). Traditional Indian herbs which are rich in natural alkaloids and are an important source of acetyl-cholinesterase (AChE) which can restore the levels of acetylcholine. Bioactive compounds like Coumarins, flavonoids and other indirect sources from herbs can activate the complexes of the deadly disease, so that the derivatives work against the severity of the disease. The natural compounds derived from herbs can act as modulators and inhibitors for amyloid accumulation so that they can also suppress inflammation and neuronal cytotoxicity. These bioactive compounds have the advantage of being multi-targeted and hence are advantageous over the single target drugs in the treatment of such complex diseases. This review suggests the possibilities of using traditional Indian herbs which are a rich source of these compounds to effectively treat Alzheimer’s disease and hence has attracted attention towards research and development of the therapies.

Iminosugars as a new class of cholinesterase inhibitors

To further extend the scope of iminosugar biological activity, a systematic structure–activity relationship investigation has been performed by synthesizing and evaluating as cholinesterase inhibitors a library of twenty-three iminoalditols with different substitutions and stereochemistry patterns. These compounds have been evaluated in vitro for the inhibition of cholinesterases (different sources of acetylcholinesterase and butyrylcholinesterase). Some compounds have IC50 values in the micromolar range and display significant inhibition selectivity for butyrylcholinesterase over acetylcholinesterase. These are the first examples of iminosugar-based inhibitors of cholinesterases.

The evaluation of Periophtalmodon schlosseri as a source of acetylcholinesterase for the detection of insecticides


 
 The aim of this study is to investigate the ability of acetylcholinesterase from mudskipper, Periophtalmodon schlosseri, as an assay to detect various pesticides, especially carbamate and organophosphate, and selected metal ions. The assay is sensitive to several carbamates with IC50of 0.0993, 0.0945, 0.0532 and 0.0488 mg/L for carbaryl, methomyl, propoxur and bendiocarb, respectively. Carbofuran highly inhibited the activity (0.0313) with the limit of detection (LOD) of 0.0176 and the limit of quantitation (LOQ) of 0.0526. The organophosphate group is less inhibiting compared to carbamate group.

Assessment of Acetylcholinesterase (AChE) from Silver Catfish (Pangasius sp.) as an Assay for Organophosphates and Carbamates

An AChE from the brain tissues from Pangasius sp. was studied for its sensitivity towards insecticides. Bendiocarb, carbaryl, carbofuran, methomyl, chlorpyrifos, diazinon, malathion and parathion caused a near complete inhibition to AChE activity at 1 mg/l. the AChE from Pangasius sp. is more sensitive to all of the carbamates and organophosphates tested than the commercial E. electricus with the exception of carbofuran and malathion. This suggests that in overall, Pangasius sp. AChE is more sensitive to insecticides compared to AChEs from E. electricus. Heavy metals such as mercury, chromium, cadmium, silver, lead and copper showed complete inhibition oh AChE activity at 10 mg/l. Xenobiotics such as SDS completely inhibited AChE activity while acrylamide, phenol and diesel inhibited 30, 45 and 76% AChE activity, respectively. The results from this work clearly indicate that an alternative source of AChE from local fish could be used to replace E. electricus for biomonitoring works.

Comparison of human and guinea pig acetylcholinesterase sequences and rates of oxime-assisted reactivation

Poisoning via organophosphorus (OP) nerve agents occurs when the OP binds and inhibits the enzyme acetylcholinesterase (AChE). This enzyme is responsible for the metabolism of the neurotransmitter acetylcholine (ACh) which transmits signals between nerves and several key somatic regions. When AChE is inhibited, the signal initiated by ACh is not properly terminated. Excessive levels of ACh result in a cholinergic crisis, and in severe cases can lead to death. Current treatments for OP poisoning involve the administration of atropine, which blocks ACh receptors, and oximes, which reactivate AChE after inhibition. Efforts to improve the safety, efficacy, and broad spectrum utility of these treatments are ongoing and usually require the use of appropriate animal model systems. For OP poisoning, the guinea pig ( Cavia porcellus) is a commonly used animal model because guinea pigs more closely mirror primate susceptibility to OP poisoning than do other animals such as rats and mice. This is most likely because among rodents and other small mammals, guinea pigs have a very low relative concentration of serum carboxylesterase, an enzyme known to bind OPs in vitro and to act as an endogenous bioscavenger in vivo. Although guinea pigs historically have been used to test OP poisoning therapies, it has been found recently that guinea pig AChE is substantially more resistant to oxime-mediated reactivation than human AChE. To examine the molecular basis for this difference, we reverse transcribed mRNA encoding guinea pig AChE, amplified the resulting cDNA, and sequenced this product. The nucleotide and deduced amino acid sequences of guinea pig AChE were then compared to the human version. Several amino acid differences were noted, and the predicted locations of these differences were mapped onto a structural model of human AChE. To examine directly how these differences affect oxime-mediated reactivation of AChE after inhibition by OPs, human and guinea pig red blood cell ghosts were prepared and used as sources of AChE, and the relative capacity of several different oximes to reactivate each OP-inhibited AChE were determined. The differences we report between human and guinea pig AChE raise additional concerns about the suitability of the guinea pig as an appropriate small animal model to approximate human responses to OP poisoning and therapies.

Active site directed docking studies: Synthesis and pharmacological evaluation of cis-2,6-dimethyl piperidine sulfonamides as inhibitors of acetylcholinesterase

Hypocholinergic function associated with Alzheimer's disease (AD) is well-accepted hypothesis, in this regard, many research attempts have been made to elevate the reduced cholinergic neurotransmission, among them two main treatment strategies were widely explored, namely stimulation of muscarinic receptor 1 and/or reversible inhibition of acetylcholinesterase (AChE) enzyme. In an attempt to improve the efficacy and to minimize general side effects of these AChE inhibitors, many lead molecules are developed in research; one among them is piperidine derivative. Donazepil is a widely prescribed AChE inhibitor which displays a piperidine ring in its structure. In the present study, we have docked cis-2,6-dimethyl piperidine sulfonamides ( 3a– i) on AChE enzyme and synthesized by nucleophilic substitution reaction between cis-2,6-dimethyl piperidine and alkyl/aryl sulfonyl chlorides in the presence of triethylamine. These piperidine sulfonamides were subjected to in vitro AChE enzyme inhibition studies and in vivo antiamnesic study to reverse scopolamine induced memory loss in rats. Two derivatives ( 3a and f) in this class of piperidines ( 3a– i) showed considerable inhibition against different sources of AChE in vitro and reduced average number of mistakes done by wistar rats as compared to scopolamine treated group in vivo (rodent memory evaluation).

Isolation of Acetylcholinesterase Inhibitors from the Flowers of Chrysanthemum indicum Linne

There have been interests in finding new sources of acetylcholinesterase (AChE) inhibitors to treat Alzheimer’s disease, because only few AChE inhibitors such as galanthamine, physostigmine, and tacrine are currently available in clinical use. In the present study we observed that the ethanol extract of the flowers of Chrysanthemum indicum Linne markedly decreased AChE activity. Using a bio-guided fractionation strategy we identified acaciin and acacetin-7-O-β-D-galactopyranoside as active compounds responsible for the AChE inhibition. In addition, we investigated the relationship between structure and activity with five flavonoids (acaciin, acacetin-7-O-β-D-galactopyranoside, luteolin, and two other commercially available flavonoids, i. e. apigenin and acacetin) and observed that the presence of methoxy groups at C-4′ and a sugar at O-7 in ring A was essential for the inhibition of AChE. The present results suggest that acaciin and acacetin-7-O-β-D-galactopyranoside from the flowers of Chrysanthemum indicum Linne have potential as therapeutic agents for Alzheimer’s disease.

Quaternary salts of 4,3′ and 4,4′ bis-pyridinium monooximes. Part 2: Synthesis and biological activity

In continuation of our investigations of unsymmetrical bisquaternary monooximes, we synthesized four new series of compounds bridged by hexyl, heptyl, octyl and nonyl groups. All eight monooximes viz., dibromides of 1-(4-hydroxyiminomethylpyridinium)6-(3/4-carbamoylpyridinium)hexane, 1-(4-hydroxyiminomethylpyridinium)-7-(3/4-carbamoylpyridinium)heptane, 1-(4-hydroxyiminomethylpyridinium)-8-(3/4-carbamoylpyridinium)octane, 1-(4-hydroxyiminomethylpyridinium)-9-(3/4-carbamoylpyridinium)nonane as well as the corresponding bis-oximes were synthesized and characterized by spectral data. Their ability to reactivate tetraethylpyrophosphate (TEPP) inhibited mouse total brain cholinesterase was investigated and compared with the conventional oxime 2-pyridinealdoxime chloride (2-PAM). Mouse brain homogenate was used as the source of acetylcholinesterase. Among all the compounds, tested the compound with the hexylene bridge ( 6b) and a 3-carbamoyl group on the second pyridine ring was found to be the most active acetylcholinesterase reactivator (72%) which is greater than that of 2-PAM (56%). However, the activity was reversed; as the chain length increased from a heptylene to a nonylene bridge, they potentiated the inhibitory effect of TEPP rather than reactivation. It is interesting to note that compound 6b with a carbamoyl group at the 3rd position of the pyridine ring showed dose dependent reactivation whereas the corresponding compound 6a with the carbamoyl group present at the 4th position of the pyridine ring showed reactivation at lower concentration (30 μM) and potentiation of TEPP inhibition at higher concentrations (100 and 300 μM).

A Comparison of the Potency of Newly Developed Oximes (K005, K027, K033, K048) and Currently Used Oximes (Pralidoxime, Obidoxime, HI-6) to Reactivate Sarin-Inhibited Rat Brain Acetylcholinesterase By in Vitro Methods

The potency of newly developed and currently used oximes to reactivate sarin-inhibited acetylcholinesterase was evaluated using in vitro methods. A rat brain homogenate was used as a source of acetylcholinesterase. Significant differences in reactivation potency among all tested oximes were observed. Although the ability of newly developed oximes to reactivate sarin-inhibited acetylcholinesterase does not reach the reactivating potency of the oxime HI-6, the oxime K033 seems to be a more efficacious reactivator of sarin-inhibited acetylcholinesterase than other currently available oximes (pralidoxime, obidoxime) at concentrations (10-5–10−4 M) corresponding to recommended doses in vivo. The results of our study also confirm that the reactivation potency of the tested reactivators depends on many factors, such as (1) the number of pyridinium rings, (2) the number of oxime groups and their position, and (3) the length and the shape of the linkage bridge between pyridinium rings.The authors are grateful to M. Hrabinová for her technical assistance. The study was supported by a grant from the Ministry of Defense, OBVLAJEP20032 and ONVLAJEP20031.

Anticholinesterase activity in an alkaloid extract of Huperzia saururus

Huperzia saururus (Lam.) Trevis. (Lycopodiaceae) is used widely in Argentinian traditional medicine as an aphrodisiac and for memory improvement. An aqueous extract from the aerial parts was obtained by decoction, revealing the presence of alkaloids, among other constituents. By partition with organic solvent in alkaline media, alkaloids were extracted and then purified by gel permeation. We studied the anticholinesterase activity in vitro of the alkaloid extract using erythrocyte membranes and human serum as sources of acetylcholinesterase and pseudocholinesterase, respectively. The results show a marked inhibition of true acetylcholinesterase with an IC50 value of 0.58 microg/ml. Low inhibition of pseudocholinesterase was observed (IC50 value = 191 microg/ml). This shows a selectivity of the extract for the true acetylcholinesterase. Furthermore, chemical study of the bioactive extract was performed by GC-MS, revealing the presence of seven Lycopodium alkaloids, including some not identified previously: sauroxine, 6-hydroxylycopodine, N-acetyllycodine, lycopodine, lycodine, N-methyllycodine, and clavolonine. Further investigations will be undertaken in order to discover which compound/s are responsible for the aqueous extract's acetylcholinesterase activity.

Synthesis of the three monopyridinium oximes and evaluation of their potency to reactivate acetylcholinesterase inhibited by nerve agents

Summary Three potential reactivators of nerve agents-inhibited acetylcholinesterase: 2-[(hydroxyimino)phenylmethyl]-1-methylpyridinium iodide 3a, 2 -[(hydroxyimino)pyridin-2ylmethyl]-1-methylpyridinium iodide 3b and 2-[(1-hydroxyimino) ethyl]-1-methylpyridinium iodide 3c were synthesized. Their reactivation potency was examined using a standard in vitro reactivation test. A rat brain homogenate was used as the source of acetylcholine sterase. Their reactivation potency was compared with a currently used acetylcholinesterase reactivator – 2-PAM (pralidoxime) 4. All tested reactivators were less effective acetylcholinesterase reactivators compared to 2-PAM. In this study, we also tested the reactivation potency of the oxime 2-PAM against inhibition of acetylcholinesterase by sarin, cyclosarin, VX and tabun. Satisfactory results are shown only for the reactivation sarin- and VX-inhibited acetylcholinesterase.

Tissue Carboxylesterases and Chlorpyrifos Toxicity in the Developing Rat

Young animals are more sensitive than adults to the neurotoxic effects of some organophosphorus insecticides. Many investigators attribute this difference in sensitivity to the immaturity of the detoxification capacity of preweanling rats. Chlorpyrifos [O,O-diethylO-(3,5,6-trichloro-2-pyridyl)phosphorothionate] is an organophosphorus insecticide that demonstrates considerable age-related sensitivity. The carboxylesterases are a group of related enzymes that detoxify organophosphorus insecticides by stoichiometrically binding these molecules before they can inhibit acetylcholinesterase. This study presents in vitro and in vivo evidence demonstrating that the carboxylesterases are critical for explaining the age-related sensitivity of chlorpyrifos. The data show that the fetal rat and the postnatal day 17 (PND17) rat pup have fewer molecules of carboxylesterase (less activity), less sensitive molecules of carboxylesterase, and a larger proportion of chlorpyrifos-insensitive molecules of carboxylesterase. An in vitro mixing experiment, using adult striatum as a source of acetylcholinesterase and liver homogenates as a source of carboxylesterase, demonstrates that the adult liver carboxylesterases are superior to the PND17 liver carboxylesterases for detoxifying chlorpyrifos. In the in vivo experiments the time course profiles of carboxylesterase and cholinesterase activity following a maximum tolerated dose of chlorpyrifos also suggest that the carboxylesterases of the PND17 rat were less capable of detoxifying chlorpyrifos. Carboxylesterase activity in the preweanling rat was not as severely inhibited as in the adult, but decrements in cholinesterase activity as a result of chlorpyrifos treatment were comparable. These in vitro and in vivo findings support the previously proffered postulate that the carboxylesterases are critical for determining the age-related sensitivity of chlorpyrifos. In addition, these detailed experiments allow us to propose that the detoxification potential of these enzymes is multifaceted, and depends on the (1) amount of activity (i.e., number of molecules), (2) affinity for the insecticide or metabolite, and (3) amount of carboxylesterase activity that is refractory to inhibition by the insecticide or metabolite.

Laminar organization of acetylcholinesterase and cytochrome oxidase in the lateral geniculate nucleus of prosimians

Hess and Rockland [Hess and Rockland (1983) Brain Res. 289, 322–325] proposed that the distribution of acetylcholinesterase within the lateral geniculate nucleus might correlate with the daily activity patterns shown by primates. In diurnal primates, the magnocellular laminae show a greater acetylcholinesterase reaction product. In nocturnal primates, the parvocellular laminae are more heavily stained. We have examined the laminar distribution of acetylcholinesterase and cytochrome oxidase in the lateral geniculate nucleus of a series of rare prosimian primates. In all prosimians examined, the most dense acetylcholinesterase reaction product is seen in the parvocellular layers of the lateral geniculate nucleus. Heavy cytochrome oxidase activity is seen in both the magnocellular and parvocellular layers, but not the koniocellular layers of the prosimian lateral geniculate nucleus. We have also employed a polyclonal antibody to choline acetyltransferase to examine the laminar organization or cholinergic activity in the Galago (Bushbaby) lateral geniculate nucleus. We report that choline acetyltransferase immunoreactivity does not correlate with acetylcholinesterase activity in the prosimian lateral geniculate nucleus. Although the lateral geniculate nucleus is more immunoreactive than most other thalamic structures and although the intercalated koniocellular laminae demonstrate somewhat lighter choline acetyltransferase immunoreactivity, no great difference in staining intensity is seen between the parvocellular and magnocellular laminae. In addition, we examined the phenotype of known inputs to assess the laminar specificity of cholinergic projections to the bushbaby lateral geniculate nucleus. Layer VI of primary visual cortex, which is known to be a source of acetylcholinesterase in the parvocellular layers, does not contain cholinergic cells, nor does the pretectal nucleus, which projects mainly to the parvocellular layers. The parabigeminal nucleus is cholinergic; however, this nucleus is known to project to the koniocellular layers, along with the noncholinergic superior colliculus. Finally, the pedunculopontine tegmental nucleus, which provides a strong input to many regions of the thalamus, including the lateral geniculate nucleus, is cholinergic. The laminar organization of its input to the lateral geniculate nucleus is not known. Increased acetylcholinesterase reaction product within the parvocellular layers of the lateral geniculate nucleus is common to all strepsirhine primates. The pattern is also seen in the only two nocturnal haplorhine primates, Tarsius and Aotus (owl monkey). The relation of this increased acetylcholinesterase activity to cholinergic function remains unclear. We note exceptions to the suggested correlation between nocturnality and increased density of acetylcholinesterase within the parvocellular layers. We hypothesize that rather than reflecting nocturnal/diurnal lifestyles, the acetylcholinesterase staining pattern may be a primitive feature of geniculate organization in primates.

Differential response of Apis mellifera acetylcholinesterase towards pirimicarb

The kinetic analysis of Apis mellifera acetylcholinesterase inhibition by the carbamate pirimicarb showed that native and detergent-solubilized membrane enzyme exhibited slightly different carbamylation kinetics. The acetylcholinesterase form sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) was carbamylated more rapidly (kapp = 36.4 X 10(-3) min-1) than the PI-PLC-resistant counterpart (kapp = 10.13 X 10(-3) min-1) which had a behavior close to that of the soluble tryptic enzyme (kapp = 11.89 X 10(-3) min-1). A difference in acetylcholinesterase sensitivity towards pirimicarb was also observed between foraging and emerging bees. These results show that the molecular structure, the mode of preparation and the source of acetylcholinesterase from the bee head should be taken into account in accurate toxicological studies.

Toxicity and anticholinesterase activity of organophosphorus compounds in Triatoma infestans

1. A series of diethyl p -substituted phenyl phosphates, phosphorothionates and phosphorothiolates were examined for anticholinesterase activity and toxicity to Triatoma infestans. 2. The isolated central nervous systems (CNS) of T. infestans was used as a source of acetylcholinesterase (AChE) for the determination of bimolecular rate constants ( k i) of enzyme inhibition because histochemical studies did not show any differences between brain and thoracic ganglia as targets of organophosphorus intoxication. 3. A good linear correlation was obtained between phosphates free energy σ and π parameters and CNS AChE inhibition. 4. There was an excellent linear correlation between insect toxicity and insect acetylcholinesterase inhibition.

Procaine as a substrate and possible allosteric effector of cholinesterases

The local anaesthetic procaine showed the properties of an allosteric effector of bovine erythrocyte acetylcholinesterase at low ionic strength; it antagonised inhibition of substrate hydrolysis caused by decamethonium, decreased the rate of ageing of isopropylmethylphosphonyl-acetylcholinesterase, increased the rate of decarbamylation of dimethylcarbamyl-acetylcholinesterase, and interacted synergistically with the nucleophilic alcohol 3,3-dimethyl-1-butanol in the acceleration of decarbamylation. These allosteric effects almost completely disappeared as the ionic strength was increased to a physiological level, and they could not be demonstrated at the physiological ionic strength with membrane-bound human erythrocyte acetylcholinesterase. There was no evidence of significant cooperativity in the binding of procaine to the enzyme, nor in the binding of the substrate acetylthiocholine in the presence of procaine, contrary to reports in the literature for other sources of acetylcholinesterase. Procaine was not hydrolysed by acetylcholinesterase (EC 3.1.1.7) although it is a substrate for serum cholinesterase (EC 3.1.1.8). The possibility that the results at low ionic strength can be explained on the basis of procaine binding to the active site of acetylcholinesterase (at low concentrations) and also to a peripheral allosteric site (at higher concentrations) is discussed. The results confirm the complexity of the kinetics of acetylcholinesterase, and extend the range of compounds with the ability to modify rates of decarbamylation and ageing.