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PubMed1991

Immunocytochemical demonstration of axonal and perikaryal acetylcholinesterase in human cerebral cortex.

The adult human neocortex contains a dense net of axons and perikarya which yield an acetylcholinesterase-rich enzymatic reaction pattern in histochemical experiments. We employed a monoclonal antibody to human acetylcholinesterase and a method for the concurrent visualization of histochemical and immunohistochemical reaction-products to explore the relationship between immunological and enzymatic markers of acetylcholinesterase. We observed that the cortical axons and perikarya with a histochemically determined acetylcholinesterase-rich enzymatic activity also contain acetylcholinesterase-like immunoreactivity. This was especially informative for the intracortical acetylcholinesterase-rich perikarya of layers III and V since these neurons require prolonged incubations for histochemical detection and since they are not conspicuous in other animal species. The availability of a reliable immunohistochemical method makes it possible to investigate the distribution of the acetylcholinesterase enzyme molecule independent of its enzymatic activity.

Acetylcholinesterase↗

PubMed1986

6-Hydroxydopamine-induced turning behaviour in the rat: the significance of acetylcholinesterase in cerebrospinal fluid.

In the substantia nigra, acetylcholinesterase may have a novel function related not to cholinergic transmission, but to the homeostasis of dopaminergic nigrostriatal neurones. The initial aim of this study was thus to see whether, in the rat, release of the enzyme into cerebrospinal fluid would reflect turning behaviour following unilateral 6-hydroxydopamine lesions of varying severity. It was found that acetylcholinesterase levels, lower than those in the cerebrospinal fluid of control rats, were accompanied by marginal circling behaviour and a small loss of striatal dopamine: on the other hand, elevated acetylcholinesterase activity was observed in the cerebrospinal fluid of rats displaying vigorous turning behaviour and with large depletion of striatal dopamine. It has already been demonstrated that exogenous acetylcholinesterase, applied locally to nigral neurones, has both electrophysiological and behavioural effects reminiscent of dopamine agonists. Hence it is possible that exogenous acetylcholinesterase could modify turning behaviour resulting from unilateral striatal dopamine depletion. Purified acetylcholinesterase, administered by cisternal puncture, attenuated circling behaviour for up to 7 days. The possible mechanisms are discussed by which endogenous acetylcholinesterase in cerebrospinal fluid could serve as an index of dopamine depletion in the nigrostriatal pathway and how exogenous enzymes might alleviate the accompanying motor dysfunction.

Acetylcholinesterase↗

PubMed1976

Regulation of acetylcholinesterase in chick muscle cultures after treatment with diisopropylphosphorofluoridate: ribonucleic acid and protein synthesis.

Previous studies have established that short treatments with diisoprophylphosphorofluoridate of chick embryo muscle cultures irreversibly inhibit the enzyme acetylcholinesterase and that the enzyme's levels in the cells rapidly recover due to synthesis of new protein. In addition, it has been shown that acetylcholine, acetyl-beta-methylcholine and choline increase the acetylcholinesterase content of the cultures. In the experiments presented here, actinomycin D and cycloheximide were used to study the relationships between the synthesis of ribonucleic acid and of protein, the recovery of acetylcholinesterase levels after diisopropylphosphorofluoridate treatment and the increase of acetylcholinesterase levels evoked by choline and its esters. Both recovery of acetylcholinesterase and the increase in its activity with acetyl-beta-methylcholine occurred in the absence of ribonucleic acid synthesis but required protein synthesis. The results suggest that transcription of desoxyribonucleic acid and a change in the degradation rate of acetylcholinesterase are not involved. An additional finding was that the level of newly synthesized acetylcholinesterase activity continued to increase for a short period of time after synthesis of protein had ceased, as if some previously synthesized protein was being transformed into active enzyme.

Acetylcholinesterase↗

PubMed1983

Co-localization of acetylcholinesterase and choline acetyltransferase in the rat cerebrum.

Acetylcholinesterase-histochemistry has been widely used for localizing cholinergic neurons despite specificity problems. The distribution of cells stained with this method has never been directly compared on a histochemical level with the specific cholinergic marker, choline acetyltransferase. We recently reported the immunohistochemical localization of choline acetyltransferase using monoclonal antibodies [Levey A. I., Armstrong D., Atweh S. F., Terry R. D. & Wainer B. H. (1983) J. Neurosci 3, 1-9]. Here we report the development of a combined histochemical and immunohistochemical method for the co-localization of the 2 cholinergic markers, and their comparison in the rat cerebrum. Although the precise relationship between the markers was complex, the important results were: (1) all neurons which contained choline acetyltransferase also contained some acetylcholinesterase; (2) many acetylcholinesterase-containing neurons did not contain any demonstrable choline acetyltransferase; (3) all neurons which stained intensely for acetylcholinesterase in the neostriatum and basal forebrain also contained choline acetyltransferase; and (4) many choline acetyltransferase-containing neurons did not stain intensely for acetylcholinesterase. The results corroborate the assumption that choline acetyltransferase is a more specific marker for cholinergic neurons than acetylcholinesterase. Intense staining for acetylcholinesterase can be reliably used in some regions of the cerebrum for identifying cholinergic neurons, however, it should be recognized that this criterion s not essential for all cholinergic neurons.

Acetylcholinesterase↗

PubMed1984

The section-Golgi-impregnation procedure--3. Combination of Golgi-impregnation with enzyme histochemistry and electron microscopy to characterize acetylcholinesterase-containing neurons in the rat neostriatum.

Three morphologically distinct types of neuron that contain acetylcholinesterase have been distinguished by Golgi-impregnation of sections of the rat neostriatum that had been incubated to reveal acetylcholinesterase activity. The neuron that stained most intensely for acetylcholinesterase was a large cell, with smooth or sparsely spiny dendrites; the axon of one these neurons was partially impregnated by the Golgi stain and had local axon collaterals (type 1). Another acetylcholinesterase-containing neuron had a small to medium-size cell body with long sparsely spiny dendrites emerging from opposite poles (type 2). The third type of neuron that contained acetylcholinesterase was medium to large size and had many primary, sparsely spiny dendrites that branched frequently (type 3). Examination of the same Golgi-impregnated, acetylcholinesterase-stained neurons that had been studied in the light microscope by electron microscopy allowed us to distinguish several other differences between the three types of neuron. Whereas all three types had acetylcholinesterase reaction product in the endoplasmic reticulum and along the nuclear envelope, only neurons of type 1 displayed reaction product in the Golgi apparatus. All three types of neuron received synaptic input, mainly along their dendrites. It is concluded that the combination of Golgi-impregnation with histochemical procedures that demonstrate endogenous enzyme activity can be applied to reveal the morphological characteristics, synaptic input and local synaptic output of neurons with specific biochemical properties.

Acetylcholinesterase↗

PubMed1984

Choline acetyltransferase-immunoreactive neurons intrinsic to rodent cortex and distinction from acetylcholinesterase-positive neurons.

Cholinergic neurons intrinsic to rat cortex were studied using a sensitive method for the localization of choline acetyltransferase immunoreactivity, acetylcholinesterase histochemistry, combined localization of choline acetyltransferase and acetylcholinesterase, and combined localization of choline acetyltransferase and retrogradely transported horseradish peroxidase-wheat germ agglutinin. Choline acetyltransferase immunoreactivity was localized predominantly in small bipolar cortical neurons within the upper layers of isocortex, while small multipolar neurons were the predominantly stained cell type in allocortical regions. Acetylcholinesterase histochemistry demonstrated mainly small polymorphic cells scattered throughout all cellular layers in all cortices. Combined staining for choline acetyltransferase and acetylcholinesterase resulted in localization of the markers in different cell populations; choline acetyltransferase-immunoreactive neurons did not contain detectable acetylcholinesterase and acetylcholinesterase-positive neurons did not contain detectable immunoreactivity to choline acetyltransferase. Some possible connections of the cortical choline acetyltransferase-immunoreactive cells were studied in rats which had received injections of horseradish peroxidase-wheat germ agglutinin into either cortex or brainstem. The choline acetyltransferase-immunoreactive cells were frequently admixed with cells labeled with the retrograde marker; however, no double-labeled cells were observed. It was concluded that cortical cholinergic cells are not visualized by acetylcholinesterase histochemistry, and are likely to be involved in local circuitry.

Acetylcholinesterase↗

PubMed1985

Convergence of afferents from frontal cortex and substantia nigra onto acetylcholinesterase-rich patches of the cat's superior colliculus.

The patterns of distribution of frontotectal and nigrotectal fibers were studied with the anterograde horseradish peroxidase method in the cat. Direct serial-section comparisons were made between the afferent-fiber patterns and the compartmentalized arrangements of acetylcholinesterase staining within the intermediate and deep collicular layers. Many of the patches of high acetylcholinesterase activity in the intermediate gray layer proved to be zones in which labeled frontotectal and nigrotectal fibers converged. These acetylcholinesterase-rich patches may thus represent sites at which functional influences from the basal ganglia and frontal cortex are coordinated. In the deeper tiers of the intermediate gray layer and layers ventral to it, there were also zones of heightened and diminished acetylcholinesterase staining. Much of this histochemical patterning was reflected in the arrangement of fibers labeled by large rostromedial frontal injections, but these deeper tiers were not strongly labeled after more lateral frontal injections or after injections placed in the substantia nigra. The deeper parts of the acetylcholinesterase-positive gridwork in the superior colliculus are thus distinct from its upper tier of acetylcholinesterase-positive patches. We conclude that the compartmentalized patterning of dense acetylcholinesterase staining in the intermediate and deep collicular layers represents a mosaic architecture to which collicular afferent circuitry is tightly related. This gridwork may serve to set up functional domains within which different aspects of collicular processing are accommodated.

Acetylcholinesterase↗

PubMed1987

Acetylcholinesterase in the rat neurohypophysis is decreased after dehydration and released by stimulation of the pituitary stalk.

Specific acetylcholinesterase and non-specific cholinesterases are present in all three lobes of the rat pituitary gland. This paper describes two new observations on hypophyseal acetylcholinesterase. Firstly, a prolonged increase of neurohormone secretion evoked by dehydration and sodium loading was accompanied by a decrease in the acetylcholinesterase activity localized to the neural lobe, where acetylcholinesterase has previously been demonstrated in fine nerve fibres. Secondly, electrical stimulation of the pituitary stalk in vitro elicited the release of acetylcholinesterase and non-specific cholinesterases from the combined neural and intermediate lobe indicating that the enzyme can be released from nerve endings in the hypophysis by action potentials. The observed loss of enzyme activity during dehydration may be the consequence of a prolonged activation of cholinergic nerves in the gland, leading to an increased release of acetylcholinesterase, which is not immediately replaced by fresh enzyme. The decrease in acetylcholinesterase in the neural lobe during dehydration may also be connected with its peptidase function and thus with the previously observed loss of substance P from the neural lobe during dehydration [Holzbauer et al. (1984) Neurosci. Lett. 47, 23-28].

Acetylcholinesterase↗

PubMed1990

Ontogeny of acetylcholinesterase, substance P and calcitonin gene-related peptide-like immunoreactivity in chick dorsal root ganglia.

The distribution of acetylcholinesterase and of two neuropeptide (substance P and calcitonin gene-related peptide) immunoreactivities has been investigated in sensory neurons of lumbosacral dorsal root ganglia during chick embryo development, combining immunolocalization of neuropeptides with simultaneous histochemical detection of acetylcholinesterase, in order to study co-localization of the two peptides and their relations with acetylcholinesterase. Acetylcholinesterase at E7 of development appears in only a few neurons, usually the larger ones located in the lateroventral region of the ganglia. As development proceeds the number of neurons and intensity of staining increase. Until E12-13 acetylcholinesterase positivity is limited to the region of the ganglion containing larger neurons. At later stages (E20) it spreads progressively, leading to staining of cells over the whole ganglion. Substance P-like immunoreactivity appears at E6 and for calcitonin gene-related peptide at E7. These immunoreactivities progressively increase with development, remaining limited to the small neuron compartment of the dorsomedial region of the ganglion. Immunoreactivity for both neuropeptides reaches a maximum around E10-13 and then declines. Using simultaneous double immunostaining, calcitonin gene-related peptide and substance P-like immunoreactivities are largely co-localized, although their distribution is not completely coincident. Neuropeptide-positive cells are usually devoid of any acetylcholinesterase activity until E15. They become positive for the enzyme at later stages. The significance of acetylcholinesterase expression in sensory neurons and the possible relation of its appearance and neuron size is discussed.

Acetylcholinesterase↗

PubMed1993

Effects of antibodies against acetylcholinesterase on the expression of peptides and catecholamine synthesizing enzymes in the rat adrenal gland.

In the rat, systemic administration of murine monoclonal antibodies against acetylcholinesterase caused rapid piloerection and ptosis (within 30-60 min after the injection). Using indirect immunohistochemistry the effect of these antibodies on peptides and enzyme expression was studied in the rat adrenal gland. Four days after antibody administration a total disappearance of acetylcholinesterase-immunoreactive fibers was observed. However, groups of acetylcholinesterase-immunoreactive chromaffin cells and intramedullary ganglion cells, both cell types showing acetylcholinesterase immunoreactivity also in the control adrenal medulla, expressed increased immunoreactivity. Analysis revealed that the acetylcholinesterase-immunoreactive chromaffin cell groups lacked phenylethanolamine-N-methyltransferase staining both in controls and treated rats. Antibody administration also affected levels of several peptides present in nerve fibers and chromaffin cells. Thus, the number of cells expressing enkephalin, calcitonin gene-related peptide and galanin was dramatically increased compared to the very few cells observed containing these three peptides in the normal gland. The majority of cells expressing enkephalin after antibody treatment also showed phenylethanolamine-N-methyltransferase immunoreactivity. In contrast, the few chromaffin cells expressing strong enkephalin-like immunoreactivity in controls were phenylethanolamine-N-methyltransferase negative. The sparse networks of calcitonin gene-related peptide- and galanin-positive fibers found in control adrenals were unchanged after the antibody treatment. However, the dense network of enkephalin varicose fibers totally disappeared after the antibody injection. A few substance P- and somatostatin-immunoreactive cells, not present in the normal gland, appeared after administration of the antibodies, whereas no changes were encountered with regard to immunoreactive nerve fibers. No clear differences between normal and treated animals could be observed in chromaffin cells with regard to immunoreactivity for neuropeptide Y or any of the four catecholamine-synthesizing enzymes, tyrosine hydroxylase, aromatic 1-amino acid decarboxylase, dopamine beta-hydroxylase or phenylethanolamine-N-methyltransferase. The present findings demonstrating a disappearance of acetylcholinesterase- and enkephalin-immunoreactive nerve fibers in the adrenal gland after intravenous injection of acetylcholinesterase antibodies support earlier reports showing that these antibodies cause degeneration of preganglionic fibers, and that neuronal decentralization of the adrenal gland induces marked increases in the levels of several peptides in chromaffin cells.

Acetylcholinesterase↗

PubMed1989

Acetylcholinesterase in the development of chick dorsal root ganglia.

Acetylcholinesterase is expressed in chick dorsal root ganglia neurons very early in development. Since the physiological role of the enzyme in these cells is still obscure, it appeared of interest to investigate its modifications in the course of development. The specific activity of acetylcholinesterase in chick dorsal root ganglia increases, during in ovo development, from day E5 to day E13; after day E13 there is a decrease. Conversely, when acetylcholinesterase activity was expressed on a per ganglion basis, a continuous increase in the level of the enzyme until day E20 was observed. Acetylcholinesterase is a polymorphic enzyme and its molecular forms have different cellular localizations. Two globular forms, a tetramer (G4) and a dimer (G2), are present in the ganglia, as in chick brain. G4 is the major form at day E5, where it represents about 85% of the activity. This form shows a progressive decrease since day E8, and at day E20 exhibits activity levels similar to those of G2. It is known that acetylcholinesterase-producing cells are also able to release the enzyme in the extracellular space. We determined the release of acetylcholinesterase by cultured dorsal root ganglia neurons at various developmental stages: acetylcholinesterase release is significantly increased at day E20, as compared to younger stages, and 90% of the enzyme released is G4.

Acetylcholinesterase↗

PubMed2006

Organophosphate-resistant forms of acetylcholinesterases in two scallops--the Antarctic Adamussium colbecki and the Mediterranean Pecten jacobaeus.

We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G(2), G(4)) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G(2)); a further amphiphilic-low-salt-soluble G(2) acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G(4) and an amphiphilic G(2) acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G(2)) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G(2) forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.

Acetylcholinesterase↗

PubMed1991

Inhibition of acetylcholinesterase and butyrylcholinesterase by the organophosphorus insecticide methylparathion in the central nervous system of the golden hamster (Mesocricetus auratus).

The toxic effects of the organophosphorus pesticide methylparathion are primarily caused by the inhibition of acetylcholinesterase activity in the central nervous system, whereas the relationship between butyrylcholinesterase and poisoning symptoms is unclear. The presumed different effects of methylparathion on acetylcholinesterase in various regions of brain and spinal cord suggest differences in the distribution of molecular enzyme forms. In the present work, the in vitro and in vivo effects of methylparathion on acetylcholinesterase and butyrylcholinesterase were studied in whole brain homogenates of golden hamsters with biochemical methods. Furthermore, acetylcholinesterase activity was determined in regions of the nervous system by quantitative histochemistry (microdensitometry). Biochemically, very low IC50 values of the hydrophilic and lipophilic fractions of both enzymes were measured. Analysis of the time course of enzyme inhibition revealed maximum inhibition 45 min after methylparathion application. Using microdensitometry different degrees of acetylcholinesterase inhibition were found in various areas of the brain. The highest inactivation was observed in the Substantia nigra and in thalamic nuclei; in several regions of the cerebellum, the inhibition rate was comparatively lower. In conclusion, methylparathion acts as an potent inhibitor of acetylcholinesterase and butyrylcholinesterase in the hamster nervous system. The region-specific different inactivation of acetylcholinesterase might be caused by the existence of multiple forms of the enzyme in various brain regions.

Acetylcholinesterase↗

PubMed1999

Rat locomotion and release of acetylcholinesterase.

In the substantia nigra acetylcholinesterase is released from the dopamine cells of the pars compacta independent of cholinergic transmission. In this study the effects of local and systemic amphetamine treatment were compared on acetylcholinesterase release in the rat substantia nigra in relation to concomitant behavior. Acetylcholinesterase release, measured "on-line" with a sensitive chemiluminescent system, was enhanced by amphetamine stimulation administered locally and could not be dissociated from simultaneous amphetamine-induced circling behavior. On the other hand, amphetamine administered systemically resulted in a general increase in locomotor behavior followed by a subsequent increase in acetylcholinesterase release. The alternative scenario of an initial rise in acetylcholinesterase release, subsequently followed by enhanced movement, was never seen. Hence, movement can enhance release of acetylcholinesterase from the substantia nigra, whereas "upstream" local nigral events can affect acetylcholinesterase release and movement simultaneously.

Acetylcholinesterase↗

PubMed2002

A non-cholinergic, trophic action of acetylcholinesterase on hippocampal neurones in vitro: molecular mechanisms.

In this study neurite outgrowth from cultured hippocampal neurones was increased by addition of acetylcholinesterase acting in a non-cholinergic manner. Only monomeric acetylcholinesterase, a form of acetylcholinesterase dominant in development, increased neurite outgrowth (3-10 U/ml); moreover this effect was not blocked by active site blockers (echothiophate and galanthamine) but was sensitive to the addition of peripheral site blockers (fasciculin and BW284c51). It appears therefore that acetylcholinesterase has alternative, non-cholinergic functions, one of which could be in development, via a peripheral site. The possibility of a causal relationship between neurite outgrowth and calcium influx was explored using a spectrum of acetylcholinesterase variants, inhibitors and calcium channel blockers. Acetylcholinesterase regulation of outgrowth was shown to depend on an influx of extracellular calcium specifically via the L-type voltage-gated calcium channel. In summary, we propose that, independent of its catalytic activity, a selective form of acetylcholinesterase has a role in the development of hippocampal neurones via a selective voltage-gated calcium channel.

Acetylcholinesterase↗

PubMed2001

Association of the HNK-1 epitope with the detergent-soluble G4 isoform of acetylcholinesterase from human neuroblastoma cells.

The HNK-1 carbohydrate epitope is expressed in neural and natural killer cells and is a mediator of cell adhesion. It is well documented that acetylcholinesterase has a secondary function in cell adhesion and differentiation. The presence of HNK-1 on isoforms of Torpedo and Electrophorus acetylcholinesterase, as well as isoforms from the bovine central nervous system has been described. In this paper, we have investigated the association of the epitope with acetylcholinesterase from human neuroblastoma cells. Acetylcholinesterase was extracted, with or without detergent, purified on immunoaffinity columns and the isoforms separated by sucrose density gradient sedimentation. Secreted acetylcholinesterase, from spent serum-free culture medium, was similarly treated. The presence of the HNK-1 epitope was determined by ELISA using the anti-HNK-1 and Elec 39 monoclonal antibodies. The epitope was found to be associated with the detergent-soluble G4 isoform, but not with the hydrophilic G1 nor the secreted hydrophilic G4 isoforms. Likewise, no HNK-1 was observed associated with human erythrocyte acetylcholinesterase. These results indicate that acetylcholinesterase-G4, anchored in the extracellular membrane, is capable of mediating cell-substrate adhesion through HNK-1.

Acetylcholinesterase↗

PubMedDate not supplied

Interaction of acetylcholinesterase with the enantiomers of malaoxon and isomalathion.

The biomolecular reaction constants (ki), dissociation constants (Kd), and phosphorylation constants (kp) were determined for the enantiomers of malaoxon against rat brain acetylcholinesterase, and for the stereoisomers of isomalathion against rat brain acetylcholinesterase and electric eel acetylcholinesterase. (R)-Malaoxon was an 8.6-fold more potent anti-cholinesterase than (S)-malaoxon. Isomalathion stereoisomers with the R configuration at carbon were 3-13-fold stronger inhibitors than those with the S configuration. The isomalathion stereoisomers with the R configuration at phosphorus were 4.3-8.8-fold stronger inhibitors of rat brain acetylcholinesterase, yet 3.4-5.8-fold weaker inhibitors of electric eel acetylcholinesterase, than the isomalathion stereoisomers with the S configuration at phosphorus. The rat brain acetylcholinesterase spontaneous (k0 = approximately 13.0 x 10(-3) min-1) and oxime-mediated (koxime) = 51.0 x 10(-3) min-1) reactivation rate constants following inhibition by isomalathion stereoisomers with the R configuration at phosphorus were comparable to spontaneous (11.3 x 10(-3) min-1) and oxime-mediated (50.2 x 10(-3) min-1) reactivation rates obtained for (S)-isoparathion methyl. These data support a common phosphorylation mechanism, namely, the displacement of the thiosuccinyl moiety from isomalathion stereoisomers with the R configuration at phosphorus, and displacement of the p-nitrophenoxy ligand from (S)-isoparathion methyl to form the same O,S-dimethyl phosphorothiolated enzyme. Rat brain acetylcholinesterase inhibited by the isomalathion stereoisomers with the S configuration at phosphorus were refractory to reactivation, suggesting an alternate mechanism of inhibition, i.e., the loss of the methylthio ligand. Several mechanisms are proposed to account for the subsequent nonreactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

PubMed1976

Active-site determinations on forms of mammalian brain and eel acetylcholinesterase.

Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.

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