Targeting of acetylcholinesterase to lipid rafts of muscle.

Despite the great progress made in setting the basis for the molecular diversity of acetylcholinesterase (AChE), an explanation for the existence of two types of amphiphilic subunits, with and without glicosylphosphatidylinositol (GPI) (Types I and II), has not been provided yet. In searching whether, as for the deficiency of dystrophin, that of merosin (laminin-alpha2 chain) alters the number of caveolae in muscle, a high increase in caveolin-3 (Cav3) was observed in the Triton X-100-resistant membranes (TRM) isolated from muscle of merosin-deficient dystrophic mice (Lama2dy). The rise in Cav3 was accompanied by that of non-caveolar lipid rafts, as showed by the greater ecto-5'-nucleotidase (eNT) activity, a marker of non-caveolar rafts, in TRM of dystrophic muscle. The observation of AChE activity in TRM, the increased levels of rafts and raft-bound AChE activity in merosin-deficient muscle and the presence of phospholipase C-sensitive AChE dimers in TRM supported targeting of glypiated AChE to rafts. This issue and the involvement of TRM in conveying nicotinic receptors to the neuromuscular junction and particular muscarinic receptors to cardiac sarcolemma strongly support a role for lipid rafts in targeting ACh receptors and glypiated AChE. Their nearby location in the surface membrane may provide cells with a fine tuning for regulating cholinergic responses.

The level of aryl acylamidase activity displayed by human butyrylcholinesterase depends on its molecular distribution.

Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) display both esterase and aryl acylamidase (AAA) activities. Their AAA activity can be measured using o-nitroacetanilide (ONA). In human samples depleted of acetylcholinesterase, we noticed that the ratio of amidase to esterase activities varied depending on the source, despite both activities being due to BuChE. Searching for an explanation, we compared the activities of BuChE molecular forms in samples of human colon, kidney and serum, and observed that BuChE monomers (G(1)) hydrolyzed o-nitroacetanilide much faster than tetramers (G(4)). This fact suggested that association might cause differences in the AAA site between single and polymerized subunits. This and other post-translational modifications in BuChE subunits probably determine their level of AAA activity. The higher amidase activity of monomers could justify the presence of single BuChE subunits in cells as a way to preserve the AAA activity of BuChE, which could be lost by oligomerization.

Cholinergic system and cell proliferation.

The cholinergic system, comprising acetylcholine, the proteins responsible for acetylcholine synthesis and release, acetylcholine receptors and cholinesterases, is expressed by most human cell types. Acetylcholine is a neurotransmitter, but also a local signalling molecule which regulates basic cell functions, and cholinergic responses are involved in cell proliferation and apoptosis. So, activation of nicotinic and muscarinic receptors has a proliferative and anti-apoptotic effect in many cells. The content of choline acetyltransferase, acetylcholine receptors and cholinesterases is altered in many tumours, and cholinesterase content correlates with patient survival in some cancers. During apoptosis, acetylcholinesterase is induced and appears in the nuclei. Acetylcholinesterase participates in the regulation of cell proliferation and apoptosis through hydrolysis of acetylcholine and by other catalytic and non catalytic mechanisms, in a variant-specific manner. This review gathers information on the role of cholinergic system and specially acetylcholinesterase in cell proliferation and apoptosis.

Biochemical properties of acetyl- and butyrylcholinesterase in human meningioma.

The structural properties of acetyl-(AChE) and butyrylcholinesterase (BuChE) in meningioma and the possible relationship with brain and plasma were investigated. Meningioma ChEs were extracted with saline and saline-Triton X-100 buffers. The tumor ChE forms were identified by sedimentation analysis, and their amphiphilic/hydrophilic behaviour was assessed by Triton X-114 phase-partitioning and hydrophobic chromatography. Meningioma contained amphiphilic globular AChE dimers (G2A) and monomers (G1A), and hydrophilic BuChE tetramers (G4H). The conversion of G2A into G1A AChE by reduction confirmed their structures. In contrast to the meningioma species, brain G1A AChE forms remained amphiphilic after incubation with alkaline hydroxylamine and phosphatidylinositol-specific phospholipase C (PIPLC). Meningioma G1A and PIPLC-converted G1H, and brain G1A AChE showed similar rate constants for thermal inactivation, and this suggested that the thermal stability of AChE subunits was unaffected by the presence or not of phosphatidylinositol residues. AChE in meningioma and brain did not differ in the interaction with the lectins Con A, LCA, WGA and RCA. BuChE in meningioma and brain bound to a similar extent to Con A, LCA and WGA-Agarose, whereas one-half of BuChE in the tumor, all in plasma and little in brain was fixed by RCA. Therefore, meningioma possesses RCA(+)- and RCA(-)-BuChE, the former predominating in brain and the latter in plasma. It remains to be clarified whether the tumor RCA(+)-BuChE is intrinsic or derived from plasma.

Acetylcholinesterase is orientated facing the cytoplasmic side in membranes derived from sarcoplasmic reticulum.

(1) Microsomal membranes from white rabbit muscle enriched in sarcoplasmic reticulum (SR) were used to investigate the preferential localization of acetylcholinesterase (AChE) in these membranes. (2) Integrity and orientation of the vesicles was assessed by measuring the inulin-inaccessible space of the vesicles and its calcium-loading capacity. (3) Treatment of the membranes with diisopropyl phosphorofluoridate (DFP), an irreversible inhibitor which is free soluble in lipid, produced an almost complete inactivation of AChE. The inhibition was prevented in assays performed with the non-permeant reversible inhibitor BW 284c51 (BW). (4) Similar results were obtained if echothiophate iodide (ECHO), an irreversible and poorly permeant inhibitor, instead of DFP was used. (5) Sedimentation profiles of enzyme solubilized with Triton X-100 from membranes inhibited by DFP after protection with BW showed a minor reduction in the relative proportion of a 4.5 S (G1) form. (6) Treatment of intact or saponin-permeabilized membranes with concanavalin A (ConA) produced enzyme-lectin complexes. In both cases, most of the enzyme was recovered in the sedimented complexes after centrifugation of the Triton-solubilized membranes. (7) Incubation of intact membranes with the antibody AE1 led to the formation of immuno complexes. Sedimentation analyses of the molecular forms of AChE revealed a shift in the sedimentation coefficients, whether the antibody was added before or after solubilization of the enzyme. (8) These results firmly establish an external localization of AChE in SR, most of the protein backbone facing the cytoplasmic side of the membrane.

Alkaline treatment of muscle microsomes releases amphiphilic and hydrophilic forms of acetylcholinesterase.

To obtain information about the mode of attachment of amphiphilic monomers of acetylcholinesterase (AChE) in sarcoplasmic reticulum (SR) of skeletal muscle, attempts were made to release the enzyme by alkaline hydroxylamine. About half of the activity measured in microsomes preincubated with 0.5% (w/v) Triton X-100 is detached by incubation of SR with bicarbonate buffer (pH 10.5). Addition of 1 M hydroxylamine to the alkaline buffer did not improve enzyme solubilization. Molecular forms of 16S (A12), 10.5S (G4) and 4.0S (G1) are separated by sedimentation analyses of Triton X-100 or bicarbonate-solubilized AChE. Monomeric AChE, released under alkaline conditions (G1A), displays amphiphilic properties. G1A, but not G4 and A12, forms are retained in a phenyl-Sepharose column and this allows its separation from hydrophilic forms. Isolated monomers extracted with Triton X-100 (G1D) or alkaline buffer showed identical kinetic behaviour. The two forms reacted with lectins in a similar manner. However, thermal inactivation experiments revealed that about 90 and 40% of the activity in the G1D and G1A forms were lost by heating at 50 degrees C, following the same rate constant (k = 0.130 min-1). Addition of Triton X-100 to the G1A form leads to an increase of its thermal sensitivity, the enzyme being fully inactivated very rapidly (k = 0.230 min-1). The results suggest that the hydrophobic moiety of the enzyme might be exposed or hidden depending on the environmental hydrophobicity. Changes in the composition of the solvent will determine the final conformational state of the protein.

Biochemical properties of 5'-nucleotidase from mouse skeletal muscle.

Ecto-5'-nucleotidase (eNT) from mouse muscle has been purified after extraction with detergent followed by chromatography on concanavalin A- and AMP-Sepharose. Three fractions were recovered: UF was NT non-retained in immobilised AMP; F-I was bound enzyme eluted with beta-glycerophosphate, and F-II was bound NT released with AMP. eNT was 80000-fold purified in F-II, this fraction showing proteins of 74, 68 and 51 kDa after immunoblotting. NT in UF migrated at 6.7S after centrifugation in sucrose gradients with Triton X-100, the peak being split into two of 6.7S and 4.4S in gradients with Brij 96. Ecto-NT in F-I or F-II migrated at 5.8S in Triton X-100-, or 4.4S in Brij 96-containing gradients. The hydrodynamic behaviour, concentration in Triton X-114, binding to phenyl-agarose, and sensitivity to phosphatidylinositol-specific phospholipase C revealed that enzyme forms in F-I or F-II were amphiphilic dimers with linked phosphatidylinositol residues, whilst most of NT forms in UF were hydrophilic dimers. A zinc/protein molar ratio of 2.2 was determined for eNT in F-II. NT activity was decreased in assays made in imidazole buffer, and was partly restored with 10 microM Zn2+ or 100 microM Mn2+. In assays with Tris buffer, NT showed a Km for AMP of 12 microM, and was competitively inhibited by ATP or ADP.

G4 forms of acetylcholinesterase and butyrylcholinesterase in normal and dystrophic mouse muscle differ in their interaction with Ricinus communis agglutinin.

Differences in glycosylation between molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in muscle and serum of normal and dystrophic mice have been studied by means of their adsorption to immobilized lectins. Application of a two-step extraction procedure, first with saline buffer, and second with saline buffer and Triton X-100, brought into solution most of the muscle AChE and BuChE activities. The AChE activity was five times greater than that of BuChE in normal (NM) and dystrophic muscle (DM). The AChE activity in the serum of dystrophic mice was twice that measured in control animals, but the BuChE activity remained almost unchanged. Both AChE and BuChE in muscle and serum bound completely to concanavalin A (Con A) and Lens culinaris agglutinin (LCA). A12, A8 and G4 AChE, but not the light G2 and G1 AChE forms, in NM and DM were completely adsorbed to wheat germ agglutinin (WGA). Similarly, G4 BuChE, but not the G2 and G1 forms, were associated to WGA. A high proportion of G4 and G1 AChE and G4 BuChE forms in mouse serum were fixed to WGA. Asymmetric AChE in NM and DM reacted with Ricinus communis agglutinin (RCA) but the light AChE and BuChE forms in muscle and serum did not bind to the lectin. G4 AChE and G4 BuChE in NM were not recognized by RCA, but the isoforms in DM bound fully to the lectin. Serum G4 AChE from control or dystrophic mice did not react with RCA, but G4 BuChE was fixed to the lectin. Since RCA is specific for galactose, the results suggest that in dystrophic muscle galactose is incorporated early in G4 AChE and this affects the level of the functional tetramers destined for insertion in the plasma membrane.

Amphiphilic and hydrophilic molecular forms of acetylcholinesterase in membranes derived from sarcoplasmic reticulum of skeletal muscle.

Native molecular forms of acetylcholinesterase (AChE) present in a microsomal fraction enriched in SR of rabbit skeletal muscle were characterized by sedimentation analysis in sucrose gradients and by digestion with phospholipases and proteinases. The hydrophobic properties of AChE forms were studied by phase-partition of Triton X-114 and Triton X-100-solubilized enzyme and by comparing their migration in sucrose gradient containing either Triton X-100 or Brij 96. We found that in the microsomal preparation two hydrophilic 13.5 S and 10.5 S forms and an amphiphilic 4.5 S form exist. The 13.5 S is an asymmetric molecule which by incubation with collagenase and trypsin is converted into a 'lytic' 10.5 S form. The hydrophobic 4.5 S form is the predominant one in extracts prepared with Triton X-100. Proteolytic digestion of the membranes with trypsin brought into solution a significant portion of the total activity. Incubation of the membranes with phospholipase C failed to solubilize the enzyme. The sedimentation coefficient of the amphiphilic 4.5 S form remained unchanged after partial reduction, thus confirming its monomeric structure. Conversion of the monomeric amphiphilic form into a monomeric hydrophilic molecule was performed by incubating the 4.5 S AChE with trypsin. This conversion was not produced by phospholipase treatment.

The ecto-5'-nucleotidase subunits in dimers are not linked by disulfide bridges but by non-covalent bonds.

It has long been considered that ecto-5'-nucleotidase (eNT) dimers consist of subunits linked by disulfide bonds. Hydrophilic (6.7S) and amphiphilic (4.0S) dimers were separated by sedimentation analysis of eNT purified from bull seminal plasma. Hydrophilic (4. 2S) and amphiphilic (2.6S) eNT monomers were obtained after reduction of disulfide bonds in dimers. The amphiphilic eNT dimers or monomers were converted into their hydrophilic variants with phosphatidylinositol-specific phospholipase C. SDS-PAGE plus Western blot showed 68 kDa subunits, regardless of the addition of beta-mercaptoethanol to the SDS mixture. Active eNT monomers were obtained by addition of 1 M guanidinium chloride (Gdn) to dimers, and unfolded subunits by addition of 4 M Gdn. The results unambiguously demonstrate that the subunits in eNT dimers are not linked by disulfide bridges, but by non-covalent bonds, and that dissociation precedes inactivation and unfolding.

Muscular dystrophy with laminin deficiency decreases acetylcholinesterase activity in thymus of dystrophic Lama2dy mice.

We have studied the effect of muscular dystrophy by merosin deficiency on mouse thymus acetyl- (AChE) and butyrylcholinesterase (BuChE). The organ contains AChE and BuChE activities. Merosin deficiency causes an important decrease (46%) in AChE specific activity. Thymus produces dimers, monomers and tetramers of AChE, and the three kinds of AChE mRNAs. The drop in AChE activity in dystrophic animals could affect the amount of ACh reaching cholinergic receptors in cells of lymphoid organs.

Identification of hybrid cholinesterase forms consisting of acetyl- and butyrylcholinesterase subunits in human glioma.

Brain and non-brain tumors contain acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) transcripts and enzyme activity. AChE and BuChE occur in tissues as a set of molecular components, whose distribution in a cyst fluid from a human astrocytoma we investigated. The fluid displayed high BuChE and low AChE activities. Three types of cholinesterase (ChE) tetramers were identified in the fluid by means of sedimentation analyses and assays with specific inhibitors, and their sedimentation coefficients were 11.7S (ChE-I), 11.1S (ChE-II), and 10.5S (ChE-III). ChE-I was unretained, ChE-II was weakly retained and ChE-III was adsorbed to edrophonium-agarose, confirming the AChE nature of the latter. ChE-I and ChE-II tetramers contained BuChE subunits as shown by their binding with an antiserum against BuChE. The ChE activity of the immunocomplexes made with ChE-II and anti-BuChE antibodies decreased with the AChE inhibitor BW284c51, revealing that ChE-II was made of AChE and BuChE subunits, in contrast to ChE-I, which only contained BuChE subunits. The binding of an anti-AChE antibody (AE1) to ChE-II and ChE-III, but not to ChE-I, demonstrated the hybrid composition of ChE-II. A substantial fraction of the AChE tetramers and dimers of astrocytomas and oligodendrogliomas bound both to anti-AChE and anti-BuChE antibodies, which revealed a mixed composition of AChE and BuChE subunits in them. The AChE components of brain, meningiomas and neurinomas were only recognized by AE1. In conclusion, our results demonstrate that aberrant ChE oligomers consisting of AChE and BuChE subunits are generated in astrocytomatous cyst and gliomas but not in brain, meningiomas or neurinomas.

Inhibition of human serum and rabbit muscle cholinesterase by local anesthetics.

The effects of tertiary amine local anesthetics (procaine, mepivacaine, lidocaine, tetracaine, dibucaine, and bupivacaine) and chlorpromazine were investigated for rabbit muscle acetylcholinesterase and human serum cholinesterase. The muscle enzyme was poorly inhibited by local anesthetics containing an amide linkage. The serum cholinesterase was inhibited by all those compounds, their relative potencies being proportional to their octanol/water partition coefficients. The dissociation constants of tetracaine and procaine, ester anesthetics, were 1000-fold and 100-fold, respectively, that which would be expected from their partition coefficient basis respective to the other amide anesthetics. Procaine showed competitive inhibition of serum cholinesterase, whereas for most anesthetics a mixed type of inhibition was observed. Procaine probably binds at the main anionic site, while the other positively charged anesthetics bind to either the catalytic centre or to the peripheral or modulator anionic site, modifying the kinetic behaviour of cholinesterase as has been demonstrated by the appearance of negative cooperativity for binding to the substrate.

Interactions between lectins and acetylcholinesterase from the sarcotubular system of skeletal muscle.

The oligosaccharide chains attached to the multiple forms of acetylcholinesterase (AChE) in a fraction enriched in membranes of the sarcoplasmic reticulum and T-tubules of rabbit skeletal muscle were studied using lectins. By sequential extraction of the membranes with Triton X-100, two preparations of soluble enzyme were obtained, S(1) and S(2). Monomeric, tetrameric and asymmetric forms of AChE are present, and all of these bind to concanavalin A, yielding heavy aggregates with no loss of enzyme activity. The interaction of AChE with lectins suggests that the carbohydrate residues are N-linked to the protein backbone. The extent of the association of isolated AChE forms with wheatgerm and Ricinus communis agglutinins was variable, but grew as the molecular complexity increased. The interaction of monomeric AChE, in S(1) and S(2), with concanavalin A and Lens culinaris agglutinin suggests heterogeneity in the oligosaccharide moieties of this single form. Antibodies of the HNK-1 anti-carbohydrate type show no reaction with any of the AChE forms. Sialylation also appeared to be absent. The results overall indicate that some monomeric forms of AChE are fucosylated in the Golgi system to become precursors of tetrameric and asymmetric components of this enzyme in muscle.

Acetylcholinesterase in membrane fractions derived from sarcotubular system of skeletal muscle: presence of monomeric acetylcholinesterase in sarcoplasmic reticulum and transverse tubule membranes.

Microsomes were isolated from white rabbit muscle and separated into several fractions by centrifugation in a discontinuous sucrose density gradient. Four membrane fractions were obtained namely surface membrane, light, intermediate and heavy sarcoplasmic reticulum. The origin of these microsomal vesicles was investigated by studying biochemical markers of sarcoplasmic reticulum and surface and T-tubular membranes. The transverse tubule derived membranes were further purified by using a discontinuous sucrose density gradient after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. All membrane preparations displayed acetylcholinesterase activity (AChE, EC 3.1.1.7), this being relatively more concentrated in T-tubule membranes than in those derived from sarcoplasmic reticulum. The membrane-bound AChE of unfractioned microsomes notably increased its activity by aging, treatment with detergents and low trypsin concentrations indicating that the enzyme is probably attached to the membrane in an occluded form, the unconstrained enzyme displaying higher activity than the vesicular acetylcholinesterase. Sedimentation analysis of Triton-solubilized AChE from different membrane fractions revealed enzymic multiple forms of 13.5S, 9-10S and 4.5-4.8S, the lightest form being the predominant one in all membrane preparations. Therefore, in both sarcoplasmic reticulum and T-tubule membrane the major component of AChE appears to be a membrane-bound component, probably a G(1) form.

The effect of temperature on the activity of acetylcholinesterase preparations from rat brain.

Homogenization of rat brain with dilute buffer shows that about 15% of the acetylcholinesterase is soluble while the remaining 85% is present in a membrane-bound form which can be brought into solution by extraction with Triton X-100. The effect of temperature on the values of V(max) and K(m) of the buffer-soluble, the membrane-bound and the Triton-soluble forms of acetylcholinesterase have been compared and the results discussed in terms of possible changes in the conformation, dissociation or aggregation of the enzyme molecule. Gradient-gel electrophoresis of the soluble preparations carried out at 4 degrees C or 37 degrees C suggest that the normal tetrameric structure present at 4 degrees C dissociates into monomers and forms some higher molecular weight species at 37 degrees C. The effect of prior storage of the brains in toluene on these properties is also considered.

The effect of solubilization on the properties and molecular forms of rat brain acetylcholinesterase.

Attempts were made to solubilize acetylcholinesterase from Wistar rat brains by extraction with dilute buffer, Triton X-100 and proteolytic digestion. About 13% of the total enzyme activity could be solubilized with 30 mM sodium phosphate buffer (pH 7) and the remainder brought into solution with 1% w/v Triton X-100. Storage of the brains in dry toluene for 3-6 months followed by extraction did not improve the yield and resulted in the loss of about half of the enzyme activity. Digestion with trypsin or collagenase was totally ineffective in solubilizing the enzyme from fresh or toluene-stored brains. The enzyme in the buffer and detergent extracts of fresh and toluene-stored brains was very stable when stored at -20 degrees C for several months although some activity was found in a 100,000 g pellet obtained by centrifugation of the thawed extracts. All enzyme preparations showed inhibition by excess substrate and an optimum substrate concentration of 2 mM acetylcholine. The Km of the crude tissue suspension was 80 microM acetylcholine while that of the buffer-soluble enzyme was 91 microM and that of the detergent-solubilized enzyme was 250 microM. Storage of the brains in toluene had little effect on these values. Starch-block electrophoresis and polyacrylamide gel electrophoresis showed up to five bands with different net charge while gradient gel electrophoresis revealed up to eleven forms with molecular weights ranging from 39,000 to 450,000. The electrophoretic pattern obtained depended on the preparation and extraction of the tissue as well as the temperature and the presence of salt, mercaptoethanol and inhibitors. Storage of the tissue in toluene does affect the yield and the properties of acetylcholinesterase obtained from rat brain thus emphasising the need to clearly define the methods and conditions of solubilization when reporting the presence of multiple molecular forms of acetylcholinesterase.

Molecular forms of acetylcholinesterase present in the white and grey matter of pig brain.

Extraction of the white matter of pig brain with EDTA, lysolecithin or Triton X-100 gave poor yields of soluble acetylcholinesterase although these agents had proved effective at solubilizing the enzyme in the grey matter. This finding, together with the observation that the strong detergent sodium deoxycholate, was needed to solubilize the enzyme, shows that it is more difficult to remove acetylcholinesterase from the white matter of brain than from the grey. This could mean that the enzyme in the white matter is more firmly bound to the membrane than the enzyme in the grey matter. The difference in binding of the enzyme from the two regions of the brain is also reflected in the affinity chromatography experiments which showed a lower recovery for the acetylcholinesterase of white matter compared with the enzyme from grey matter. Starch-block electrophoresis of acetylcholinesterase showed a single negatively charged peak of activity for both the naturally soluble and the deoxycholate solubilized preparations. The presence of only one form on electrophoresis suggests that the molecular species of acetylcholinesterase do not arise from differences in charge. Sucrose density gradient centrifugation of the two preparations from white matter gave a single peak of activity with a sedimentation constant of about 10 S. This corresponds closely to the major species of molecular weight 260,000 detected by gradient gel electrophoresis. Other forms detected in both enzyme preparations by gradient gel electrophoresis were species with molecular weights of 660,000, 180,000, 130,000 and 115,000. The significance of these species in terms of the formation of oligomers is discussed. A comparison was made with the corresponding preparations of acetylcholinesterase from the grey matter and the results showed that acetylcholinesterase from the white and grey matter of pig brain were very similar. The exception to this was the species with a molecular weight of 68,000 which was present in the grey but not the white matter of pig brain.

Monomers and dimers of acetylcholinesterase in human meningioma are anchored to the membrane by glycosylphosphatidylinositol.

Amphiphilic monomers and dimers of acetylcholinesterase (AChE) and hydrophilic tetramers of butyrylcholinesterase (BuChE) were released by extracting human meningioma with Tris-saline and Tris-saline-Triton X-100 buffers. The amphiphilic or hydrophilic behavior of the AChE and BuChE forms was assessed by sedimentation analysis, hydrophobic chromatography and Triton X-114 phase-partitioning. A significant fraction of the amphiphilic AChE species was converted into hydrophilic components by incubation of the soluble enzyme with phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis, this fraction being increased by a double treatment with PIPLC and alkaline hydroxylamine. A significant amount of the membrane-bound AChE was released by incubation with PIPLC. These results demonstrate that AChE forms in meningioma are attached to the membrane via glycosylphosphatidylinositol, although part of the enzyme forms are resistant to PIPLC.

Molecular forms of acetyl- and butyrylcholinesterase in human glioma.

Specimens of astrocytoma, oligodendroglioma and medulloblastoma were sequentially extracted with saline and saline-Triton X-100 buffers. Acetyl- (AChE) and butyrylcholinesterase (BuChE) activities were assayed in the soluble fractions, these being further analyzed to establish the distribution of molecular forms. All the tumors tested showed AChE and BuChE activities, the measured AChE/BuChE ratios being unrelated to the malignant grading. Hydrophilic and amphiphilic AChE and BuChE tetramers, amphiphilic AChE dimers and monomers, and hydrophilic BuChE monomers were identified in all the tumors analyzed. The amphiphilic behavior of the enzyme forms was assessed by sedimentation analysis and hydrophobic chromatography on phenyl-Agarose. A small fraction of glioma AChE monomers was released as, or transformed into, hydrophilic forms by incubation with phosphatidylinositol-specific phospholipase C (PIPLC). These data suggest that AChE monomers bearing distinct hydrophobic domains coexist in human glioma.