Abstract Acetylcholinesterase Research Articles

Chemical modification of acetylcholinesterase with methoxypolyethylene glycol

Acetylcholinesterase (AChE) (EC 3.1.1.7) was modified with activated monomethoxypolyethylene glycol (mPEG). A decrease of 50% in the catalytic activity was measured during the coupling reaction and the change in the surface properties of AChE was used to separate by hydrophobic interaction chromatography the native and the modified enzyme. The native and the modified enzymes were found to have the same optimalcatalytic conditions. Moreover, the Michaelis constant of both enzymes were similar, whereas theV m and the bimolecular-velocity constant calculated for organophosphorus inhibitors were slightly higher for the modified AChE. Finally, the modification with mPEG did not improve the thermal stability, whereas the stability in a few organic solvents increased.

Asymmetric distribution of acetylcholinesterase in gravistimulated maize seedlings.

Acetylcholinesterase (AChE) activity has previously been studied by this laboratory and shown to occur at the interface between the stele and cortex of the mesocotyl of maize (Zea mays L.) seedlings. In this work we studied the distribution of AChE activity in 5-d-old maize seedlings following a gravity stimulus. After the stimulus, we found an asymmetric distribution of the enzyme in the coleoptile, the coleoptile node, and the mesocotyl of the stimulated seedlings using both histochemical and colorimetric methods for measuring the hydrolysis of acetylthiocholine. The hydrolytic capability of the esterase was greater on the lower side of the horizontally placed seedlings. Using the histochemical method, we localized the hydrolytic capability in the cortical cells around the vascular stele of the tissues. The hydrolytic activity was inhibited 80 to 90% by neostigmine, an inhibitor of AChE. When neostigmine was applied to the corn kernel, the gravity response of the seedling was inhibited and no enzyme-positive spots appeared in the gravity-stimulated seedlings. We believe these results indicate a role for AChE in the gravity response of maize seedlings.

Oligomerization of Chicken Acetylcholinesterase Does Not Require Intersubunit Disulfide Bonds

Acetylcholinesterase (AChE) is secreted from muscle and nerve cells and associates as multimers through intermolecular covalent and noncovalent bonds. The amino acid sequence of the C-terminus is thought to play an important role in these interactions. We generated mutants in the C-terminus of the catalytic T-subunit of chicken AChE to determine the importance of this region to oligomerization and to the amphipathic character of the protein. Wild-type recombinant chicken AChE secreted from human embryonic kidney 293 cells was assembled into dimers and tetramers exclusively. Mutants lacking the C-terminal Cys764, the only cysteine involved in interchain disulfide bonds, showed lower but significant levels of the secreted dimeric and tetrameric forms. A truncated mutant, lacking the C-terminal 39 amino acids, exhibited a severe decrease in content of the multimeric forms, yet small amounts of the dimer were detectable. The amphipathic character was dependent on the state of oligomerization. When analyzed by sucrose gradients, the sedimentation of tetramers was not affected by detergent, but monomers and dimers sedimented more slowly in the presence of detergent. Most of the recombinant wild-type enzyme, shown to be dimeric and tetrameric by sedimentation analysis, was monomeric when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that much of the secreted oligomeric AChE was not disulfide bonded. These data suggest that disulfide bonding of Cys764 is not required for the catalytic subunit of chicken AChE to form oligomers and that regions outside of the C-terminus contribute to the hydrophobic interactions that are important for stabilizing the oligomeric forms.

Acetylcholinesterase from desert cobra (Walterinnesia aegyptia) venom: optimization and kinetics study.

Acetylcholinesterase (AChE) was investigated in Walterinnesia aegyptia venom and characterized with respect to its kinetic properties. It was found that 4.0 micrograms of crude venom protein and an incubation time of 4.0 min were suitable conditions for linearity of AChE activity at 25 degrees C. The optimum strength of the sodium phosphate buffer was 0.05 M, and the optimum pH was 7.75. The optimum temperature was 30 degrees C. The activation energy and the heat of activation were observed to be 6510 and 5922 cal/mole. The AChE was specific for acetylthiocholine but it did not hydrolyse butyrylthiocholine. The optimum substrate concentration was 3.0 mM but at higher substrate concentrations, the AChE activity declined. The ASCh concentration ranges for different orders of the reactions were determined and kinetic parameters (Km, Vmax, Kcat, and Ksp) were established at each order of the reaction.

Purification and biochemical characterization of acetylcholinesterase (AChE) from the excretory/secretory products ofTrichostrongylus colubriformis

Acetylcholinesterase (AChE) has been purified from the excretory/secretory (ES) products of Trichostrongylus colubriformis (using edrophonium chloride linked to epoxy-activated Sepharose) with yields of 40-50%. Purity was confirmed by polyacrylamide gel electrophoresis (using silver [protein] and Karnovsky [activity] stains) and measurement of specific AChE activity. Further analysis of the purified fractions by gel filtration and sucrose density gradient techniques revealed the existence of 2 forms of hydrophilic AChE (M(r) 189 and 80 kDa). From the data we deduce these to be the globular monomer and dimer, G1 and G2 forms of AChE. Inhibition studies using BW284C51, iso-OMPA and excess substrate, along with substrate specificity studies, show both forms to be true acetylcholinesterases. We are currently assessing the protective immunogenicity of purified AChE in sheep.

Acetylcholinesterase from the Horn Fly (Diptera: Muscidae): Distribution and Purification

Acetylcholinesterase (AChE) in newly emerged adults of the horn fly, Haematobia irritans (L.), was distributed mostly in the head (72.9%), less in the thorax (24.4%), and least in the abdomen (2.7%). The major portion of total AChE was membrane-bound and associated primarily with the microsomes. The enzyme was solubilized with phosphate buffer containing 0.5% (vol/vol) of Triton X-100 and purified with affinity chromatography. The extent of purification was about 217-fold for the membranous fraction and 329-fold for the soluble fraction of whole fly homogenates with recoveries of 55% and 79%, respectively. The extent of purification reached 600- and 784-fold for the membranous and the soluble fractions, respectively, in the collections of eluents with peaked activity of AChE. Electrophoresis revealed four bands of AChE activity in the membrane-bound preparation and three bands in the soluble preparation. Silver staining of the electrophoresed gels showed that the major proteins in purified AChE solutions corresponded with AChE bands.

The molecular forms of acetylcholinesterase in cerebrospinal fluid of normal subjects ? effect of aging

Acetylcholinesterase (AChE) activity is increased in human cerebrospinal fluid (CSF) during aging. The present study investigated whether the relative amounts of different molecular forms of CSF-AChE are also affected during aging. Thus, the CSF samples of healthy human subjects (age range 20-79 years, n = 23) were analyzed for sedimentation forms of AChE activity. Five different forms of AChe activity were detected in human CSF. The relative amount of tetrameric and dimeric globular forms, which are the main forms of AChE in CSF, were not related with age. Furthermore, the relative amount of monomeric globular and asymmetric forms which are minor forms of AChE in CSF did not seem to be related to age. Since total CSF-AChE activity is increased during aging, it seems to be due to the increased amounts of the tetrameric and dimeric forms of enzyme activity.

Necator americanus secretory acetylcholinesterase and its purification from excretory‐secretory products by affinity chromatography

Acetylcholinesterase (AChE) secretion by adult N. americanus was enhanced in vitro by incorporating insoluble collagen rafts into culture dishes. Enzyme produced in this way had preferential substrate specificity for acetylthiocholine iodide (ATC), and its activity was inhibited by eserine (1.1 x 10(-8) M). Ancylostoma ceylanicum, another hookworm species, failed to produce comparable amounts of AChE in culture. AChE was efficiently purified from culture medium by affinity chromatography on edrophonium sepharose; 81% of the AChE activity was retained by the affinity matrix, although this fraction contained only 4.3% of the protein loaded. Antisera raised against purified AChE in rabbits immunohistochemically stained the oesophageal glands of the parasite, and reacted with molecules of 32, 60, 80, 140 and 220 kDa in reduced adult ES products on Western blotting, although differential activity was observed against worm homogenates and earlier developmental stages. On IEF, purified AChE resolved predominantly with a pl of 3.55; proteins with a similar pl were recognized by rabbit anti-AChE. IgG preparations of this antiserum inhibited AChE activity in ES products, and inhibited AChE secretion by adult worms in culture. The availability of this immunological probe will allow definitive experiments to be conducted on the role of this enigmatic enzyme in the host-parasite relationship.

Decrease in the rat bronchial acetylcholinesterase activity after toluene diisocyanate inhalation

Acetylcholinesterase (AChE) activity was measured in bronchial tissue homogenate and blood from rats subjected to single and repeated 4-h daily inhalation exposure to toluene diisocyanate (TDI) or control atmospheres. A single 4-h exposure to TDI in the concentration range of 0.7-4.3 ppm did not decrease AChE activity in bronchial tissue but a 4-h exposure to 0.6 ppm TDI, or greater, for two consecutive days did reduce this activity (19% to 33% of the controls). Increasing the level of exposure to TDI for two consecutive days from 0.6 to 4.0 ppm and extending the length of exposure to 1.2 ppm TDI from 2 to 4, 9 or 14 days produced no further decrease in bronchial ACHE activity. Throughout these experiments, blood AChE activity remained unchanged. In rats exposed to 0.3 or 1 ppm TDI for 3 weeks, staining of the bronchial smooth muscle for AChE was reduced (36% of the controls) after exposure to 1 ppm TDI. These results support, for the first time, in vivo and localized anti-AChE activity by TDI.

Cerebrospinal fluid gradients of acetylcholinesterase and butyrylcholinesterase activity in healthy aging

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in 13 sequential 2 ml aliquots of cerebrospinal fluid (CSF) obtained by lumbar puncture from 7 young and 7 elderly healthy normal subjects. The slopes of the rostrocaudal gradients of AChE and BChE were calculated and compared to those of total protein concentration and the major dopaminergic metabolite homovanillic acid (HVA), for which a pronounced rostrocaudal gradient (with highest concentrations of HVA in more rostral CSF) is consistent with HVA originating primarily from the brain. AChE activity was higher in more caudal fractions of young, but not elderly subjects and there was a significant difference between the mean AChE gradient slopes in the young and old groups. These results suggest that the spinal cord makes an important contribution to AChE activity in lumbar CSF. Furthermore, the absence of a negative AChE gradient in elderly subjects may be the result of a greater rate of entry of cerebral AChE into CSF, possibly as a consequence of an increased ventricular surface area and shorter diffusion distances in atrophic elderly brains. In contrast to AChE, BChE activity and total protein concentrations were higher in more caudal CSF fractions of not only young but also old subjects. In addition, there was a significant correlation between the gradient slopes of BChE activity and total protein concentrations, suggesting that the majority of BChE activity in lumbar CSF derives from the same source as the majority of total protein, namely plasma. The diffuse (i.e. brain and spinal cord) origin of AChE in lumbar CSF would explain the relatively modest changes in lumbar CSF AChE activity in diseases involving certain central cholinergic systems, most notably Alzheimer's disease.

Acetylcholinesterase and Nissl staining in the same histological section

Acetylcholinesterase (AChE) enzyme histochemistry and Nissl staining are commonly utilized in neural architectonic studies. However, the opaque reaction deposit produced by the most commonly used AChE histochemical methods is not compatible with satisfactory Nissl staining. As a result, precise correlation of AChE and Nissl staining necessitates time-consuming comparisons of adjacent sections which may have differential shrinkage. Here, we have modified the Koelle-Friedenwald histochemical reaction for AChE by omitting the final intensification steps. The modified reaction yields a non-opaque reaction product that is selectively visualized by darkfield illumination. This non-intensified darkfield AChE (NIDA) reaction allows clear visualization of Nissl staining in the same histological section. This combined AChE-Nissl method greatly facilitates detailed correlation of enzyme and cytoarchitectonic organization.

Ultrastructural changes in acetylcholinesterase activity in the deafferented spinal trigeminal nucleus.

Acetylcholinesterase (AChE) activity was investigated in synaptic areas of the cat spinal trigeminal nucleus (pars interpolaris and pars caudalis) ipsilateral and contralateral to complete retrogasserian rhizotomy. Vibratome sections of tissue taken from animals of 1, 3, 6, 14, and 21 days survival were examined by electron microscopy following a histochemical reaction for AChE activity employing a method based on the Karnovsky-Roots technique for demonstrating reaction product. As degeneration progressed with survival time, enzymatic activity was initially reduced in synaptic clefts of injured afferent terminals and subsequently was enhanced throughout the extracellular space, including within synaptic clefts of possibly reinnervated sites. These changes in enzymatic activity with primary deafferentation are discussed in relation to the process of reinnervation, the development of neuronal hyperactivity, and possible noncholinergic functions of AChE.

Posttranslational modification as a means of anchoring acetylcholinesterase to the cell surface.

Acetylcholinesterase (AChE) exists in multiple molecular forms that differ in their quaternary structure and mode of attachment to the cell surface. Attachment is achieved by posttranslational modification of the catalytic subunits. Two such modifications are described. One modification involves the attachment to subunit tetramers, via disulfide bridges, of a collagen-like fibrous anchor that serves to attach the enzyme, by ionic interactions, to the extracellular matrix. A second modification involves the covalent attachment of a single phosphatidylinositol residue, which serves to anchor the enzyme hydrophobically in the lipid bilayer of the plasma membrane. The detailed molecular structure, assembly, and modes of anchoring of these two classes of AChE are discussed.

Acetylcholinesterase Activity in Rectal Biopsies

Acetylcholinesterase (AChE) activity was measured in rectal suction biopsies from 392 patients investigated for Hirschsprung's disease (HD). Results in HD (2.7-23.7 RU; n = 37) were not clearly differentiated from those in patients without HD (0.6-18.0 RU; n = 355). Sample instability was shown not to be a significant cause of error. From analysis of duplicate biopsies and a consideration of false-negative results, tissue inhomogeneity or incorrect siting of the biopsy appeared likely causes of error. Examination of a number of possible diagnostic decision levels indicated an optimal choice of AChE activity greater than 10 RU with an AChE activity not less than 60% of total cholinesterase activity. At a prevalence of 10%, this decision level resulted in sensitivity of 64.9%, specificity 98.7%, predictive value of positive 85.7%, and predictive value of negative 96.3%. The false positives (n = 4) and false negatives (n = 13) by these criteria were examined to detect possible common features.

Histoarchitectonics of the cholinergic innervation of the frog tongue

Acetylcholinesterase (AChE) activity was studied in dorsal tongue surface structures and in both tongue nerves (hypoglossal and glossopharyngeal) of frogs (Rana temporaria). AChE was found in nerve fibers of fungiform and filiform papillae, blood vessels, glandular ducts of tongue mucosa, both nerve fibers and also in the bodies of cholinergic neurons in subepithelial connective tissue and along the glossopharyngeal nerve. Their parasympathetic origin was suggested. The experiments with butirilthiocholin have revealed no activity of non-specific cholinesterases in the above structures. Possible role of cholinergic system in the regulation of tongue receptor function is discussed.

Acetylcholinesterase levels in skeletal muscle of mice infected with Trypanosoma cruzi.

Acetylcholinesterase (AChE) activity was measured in skeletal muscle from susceptible (A/J) and resistant (C57BL/6) mice infected with the Brazil strain (myotropic) of Trypanosoma cruzi. There was a 60% decrease in activity in skeletal muscle obtained from A/J mice 20 days post-infection as compared to controls. There was no decrease in AChE activity in skeletal muscle obtained from infected C57BL/6 mice 20 and 150 days post-infection. Histologic examination of skeletal muscle from infected A/J mice revealed marked necrosis, pseudocysts, and minimal inflammation. Similar examinations in C57BL/6 mice revealed marked inflammation in the absence of necrosis and parasites. These data provide additional biochemical support that denervation hypersensitivity is an important concomitant of Chagas' disease and that it is already present during the acute stage. Additionally, it may support the notion that the presence of the parasite mediates these abnormalities.

Acetylcholinesterase activity and its fast axonal transport in rabbit sciatic nerves during the recovery phase of experimental allergic neuritis.

Acetylcholinesterase (AChE) activity and its fast axonal transport were studied in rabbit sciatic nerves with a double ligature system during the recovery phase of experimental allergic neuritis (EAN), 6-9 days after the maximal symptoms, in order to obtain biochemical evidence of possible axonal damage in this primary demyelinating disease. The stationary AChE activity was significantly decreased, but the amount of the fast transported enzyme activity remained at the level of the controls. The velocity of the orthograde transport of AChE was slowed by about 15%, but this decrease was not statistically significant. Our results lend further support for the suggested neuronal damage in EAN, which can provide an explanation to the finding that the clinical symptoms in demyelinating diseases of the peripheral nervous system do not always correlate with the state of myelin.

Polymorphism of acetylcholinesterase and identification of new molecular forms after sedimentation analysis.

Acetylcholinesterase (AChE) is composed of several distinct molecular forms, which are identified and partly resolved by velocity sedimentation analysis on sucrose gradients. We made the assumption that each AChE form sediments as a peak of activity with a gaussian shape in the continuous sucrose gradient. We experimentally demonstrate that the complex AChE profiles can be decomposed in gaussian distributions of separate molecular entities. We performed a high salt-detergent extraction of AChE from mouse skeletal muscle and isolated fractions enriched in each particular from. These fractions were then submitted to a second sedimentation, to assess the stability and to further characterize each AChE form. Then, we calculated the statistical significance level of each AChE form and identified up to 9 separate molecular specifies in mouse adult muscle. These forms are the major "4 S", "6.5 S", "10 S", "12 S" and "16 S" and minor molecular active components of AChE. These results suggest complex structural interactions between catalytic and non catalytic subunits of AChE and do not simply fit the tailed asymmetric globular model of AChE with six molecular species.

Topographical distribution of acetylcholinesterase in the subfornical organ of the rat.

Acetylcholinesterase (AChE) activity in the rat subfornical organ (SFO) was determined by the Koelle and Friedenwald method. Coronal sections showed a homogeneous reaction in the rostral region, which adopted a ring-like form in the anteromedial zone and a horseshoe pattern in the posteromedial zone. In the latter, AChE-specific positive neurons had been observed clearly in our sections. In sagittal sections the enzymatic reaction was intense in the anterior subependymal zone and dorsal region, and it continued caudally to the hippocampal area. The enzymatic activity was absent in the central region of the anteromedial zone and the subependymal region of the posteromedial zone. In sagittal sections the reaction was strongly positive in the periphery of the organ and negative in the posteromedial zone, near the choroid plexuses. Thus, we have shown that each region of the SFO presents a particular distribution of the AChE activity, related in some cases to neurons and in others to nerve fibers.

Acetylcholinesterase in developing embryos of Hyalomma rufipes Koch, 1844 (Ixodoidea: Ixodidae).

Acetylcholinesterase (AChE) is demonstrated, apparently for the first time, in developing embryos of the tick Hyalomma rufipes. The enzyme was detected in the neuropile of the oesophageal ganglia and in the nerve endings of the appendages; it was absent from the neurons and neurolemma of the nerve ganglia.