Amphiphile dependency of the monomeric and dimeric forms of acetylcholinesterase from human erythrocyte membrane

Human erythrocyte membrane-bound acetylcholinesterase was converted to a monomeric species by treatment of ghosts with 2-mercaptoethanol and iodoacetic acid. After solubilization with Triton X-100, the reduced and alkylated enzyme was partially purified by affinity chromatography and separated from residual dimeric enzyme by sucrose density gradient centrifugation in a zonal rotor. Monomeric and dimeric acetylcholinesterase showed full enzymatic activity in presence of Triton X-100 whereas in the absence of detergent, activity was decreased to approx. 20% and 15%, respectively. Preformed egg phosphatidylcholine vesicles fully sustained activity of the monomeric species whereas the dimer was only 80% active. The results suggest that a dimeric structure is not required for manifestation of amphiphile dependency of membrane-bound acetylcholinesterase from human erythrocytes. Furthermore, monomeric enzyme appears to be more easily inserted into phospholipid bilayers than the dimeric species.     

The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octyl beta-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70-80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.     

Molecular form of human lymphocyte membrane-bound acetylcholinesterase

The membrane-bound acetylcholinesterase (AchE) from human peripheral blood lymphocyte gives only one symmetrical peak on sucrose density gradient centrifugation in the presence of Triton X-100 detergent, with the calculated sedimentation coefficient of 6.5 S. However, this dimeric form of AchE was converted to a monomeric 3.8 S form when treated with 2-mercaptoethanol and iodoacetic acid. The results are consistent with studies which have shown by sodium dodecyl sulfate gel electrophoresis that the enzyme is built up of two identical monomers inter-linked by disulfide bond(s). Under reducing conditions, revealed a single species of 70,000 molecular weight, whereas under non-reducing conditions, another species of 140,000 molecular weight of the AchE was found. Polyacrylamide gel electrophoresis indicated a single band with AchE activity in the presence of Triton X-100. In contrast, in the absence of the same detergent multiple band pattern could be observed. These results suggest that membrane-bound AchE enzyme is present in homogenous dimeric form on human lymphocyte membrane.     

Thermal stability of molecular forms of acetylcholinesterase from muscle microsomes

To establish if the predominant form of acetylcholinesterase in muscle microsomes (4.8S) corresponded to the monomeric or dimeric form of the enzyme we studied the sensitivity to heating of Triton X-100 solubilized extract and that of 4.8S, 10-11S and 13.5S species of the enzyme. Inactivation of soluble acetylcholinesterase began at 45-47 degrees C and was almost complete at 60 degrees C. Sedimentation analysis revealed that the partial loss of activity was due to inactivation of the 4.8S form, although by heating the 13.5S was converted into the 10S enzyme. Inactivation of the 4.8S form began at 45 degrees C, whereas the larger forms required higher temperature. The 4.8S component follows a time course of inactivation which could be fitted by a double exponential equation (when heated at 52 degrees C, almost 83% of the activity showed a short half-life). The 10-11S species was also inactivated following a two step process while the 13.5S enzyme was fairly stable at 52 degrees C. The results show that the lightest component behaves as a monomeric form of acetylcholinesterase.     

Molecular Forms of Acetylcholinesterase from Human Caudate Nucleus: Comparison of Salt-Soluble and Detergent-Soluble Tetrameric Enzyme Species

Extraction of human caudate nucleus under high-ionic-strength conditions solubilized 20-30% of total acetylcholinesterase (AChE) activity. Density gradient centrifugation revealed monomeric (5.0 S) and tetrameric (11.0 S) enzyme species. The purified, tetrameric salt-soluble (SS) AChE sedimented at 10.6 S and did not bind detergents. It showed an immunochemical reaction of identity with the detergent-soluble (DS) AChE species from human caudate nucleus and human erythrocytes, but did not cross-react with antibodies raised against human serum cholinesterase. The remaining activity was solubilized under low-ionic-strength conditions in the presence of 1.0% Triton X-100. The purified tetrameric, DS-AChE sedimented at 10.0 S as detergent-protein mixed micelle and on extensive removal of the detergent this enzyme formed defined aggregates by self-micellarization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed that the salt-soluble and detergent-soluble tetrameric enzyme species both contained a heavy and a light dimer; under reducing conditions mainly one band corresponding to the light subunit was seen. Molecular weights of 300,000 dalton and 280,000 dalton were calculated for SS-AChE and DS-AChE, respectively. Limited digestion of DS-AChE with proteinase K led to isolation of an enzyme that no longer bound detergents and lacked the intersubunit disulfide bridges.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes.

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface.In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Tetrameric alkaline phosphatase from human liver is converted to dimers by phosphatidylinositol phospholipase C

Membrane-bound human liver alkaline phosphatase solubilized by a non-ionic detergent, Nonidet P-40 (NP-40), has the molecular mass of a tetramer. It can be converted to a dimeric form by treatment with a phosphatidylinositol phospholipase C (PI-PLC) obtained from Bacillus cereus. When human liver plasma membranes were directly treated with PI-PLC, the released alkaline phosphatase was dimeric. Thus, phosphatidylinositol may help maintain the tetrameric quaternary structure of alkaline phosphatase and aid its binding to human liver plasma membranes.     

Purification, characterisation and crystallisation of photosystem II from Thermosynechococcus elongatus cultivated in a new type of photobioreactor

The thermophilic cyanobacterium Thermosynechococcus elongatus was cultivated under controlled growth conditions using a new type of photobioreactor, allowing us to optimise growth conditions and the biomass yield. A fast large-scale purification method for monomeric and dimeric photosystem II (PSII) solubilized from thylakoid membranes of this cyanobacterium was developed using fast protein liquid chromatography (FPLC). The obtained PSII core complexes (PSIIcc) were analysed for their pigment stoichiometry, photochemical and oxygen evolution activities, as well as lipid and detergent composition. Thirty-six chlorophyll a (Chla), 2 pheophytin a (Pheoa), 9+/- 1 beta-carotene (Car), 2.9+/-0.8 plastoquinone 9 (PQ9) and 3.8+/-0.5 Mn were found per active centre. For the monomeric and dimeric PSIIcc, 18 and 20 lipid as well as 145 and 220 detergent molecules were found in the detergent shell, respectively. The monomeric and dimeric complexes showed high oxygen evolution activity with 1/4 O(2) released per 37-38 Chla and flash in the best cases. Crystals were obtained from dimeric PSIIcc by a micro-batch method. They diffract synchrotron X-rays to a maximum resolution of 2.9-A, resulting in complete data sets of 3.2 A resolution.     

Characterization of acetylcholinesterase in Caco-2 cells

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-beta-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.     

Evidence for complex formation between GTP binding protein(Gs) and membrane-associated nucleoside diphosphate kinase

When the Gs in rat liver membranes was prelabeled with [32P]NAD and cholera toxin, solubilized with octylglucoside, and then analyzed by sucrose density gradient centrifugation, it was fractionated into two peaks with approximate molecular sizes of 12-13S and 3-4S. Pretreatment without or with GDP beta S of the labeled membranes resulted in a larger peak in the high molecular weight region, whereas pretreatment with glucagon plus GTP gamma S caused almost equal peaks in both regions. The affinity-purified anti-nucleoside diphosphate (NDP) kinase antibodies only precipitated the Gs in high molecular weight region. Under the same condition, small but significant NDP kinase activity was associated with the high molecular weight Gs region although a large portion of the enzyme activity was recovered in fractions where it alone should appear (6.2S). Both Lubrol-PX and digitonin solubilized the Gs in forms insensitive to immunoprecipitation by anti-NDP kinase antibodies although the latter detergent was able to solubilize the Gs in a high molecular weight form, that is, a ternary glucagon-receptor-G protein complex. These results demonstrate that Gs and membrane-associated NDP kinase may exist in part in a complexed form in membranes. Physiological relevance of the complex formation in membrane signal transduction is discussed.     

Biophysical studies of the membrane-embedded and cytoplasmic forms of the glucose-specific Enzyme II of the E. coli phosphotransferase system (PTS)

The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-II(Glc)-YFP and MBP-II(Glc)-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET(-)) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET(+)). The monomeric species could form a heterodimeric species (FRET(+)) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-II(Glc) activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-II(Glc) retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-II(Glc) indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-II(Glc).     

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.     

Solubilization of Collagen-Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures

We have extracted acetylcholinesterase from young chick retinas by homogenization in different solutions combining high salt concentration, ionic and nonionic detergents, and EDTA, looking for an optimum procedure for the solubilization of collagen-tailed, asymmetric structural forms of the enzyme. High salt and EDTA seem to be the only necessary requirements for the solubilization of acetylcholinesterase as the A12 form (20S), and the presence of detergent in the homogenization medium does not significantly improve the yield of tailed enzyme. Extraction in the absence of detergent has the potential advantage of a threefold enrichment of tailed enzyme, because only about one-third of the total retinal acetylcholinesterase activity is solubilized. Divalent cations, especially Ca2+, seem to be involved in the attachment of the tailed enzyme to the retinal membranes, at the tail level. High salt-EDTA-extracted 20S acetylcholinesterase (without detergent) aggregates in the presence of exogenous Ca2+ and becomes "insoluble." However, the aggregated 20S acetylcholinesterase can be completely recovered and brought back into solution by further addition of EDTA. Besides, the aggregation can be prevented by the inclusion of Triton X-100 in the homogenization buffer or by adding the detergent concurrently with Ca2+. It is postulated that the acetylcholinesterase collagenous tail is coated by acidic lipid molecules hydrophobically bound to the tail protein so that Ca2+ ionic bridges would actually link these lipid molecules (and consequently the tail) to the membrane matrix. Removal of the lipid coat (e.g., by Triton X-100) produces tailed acetylcholinesterase molecules that no longer aggregate in the presence of Ca2+ and are fully accessible to collagenase digestion.     

Soluble form of acetylcholinesterase from rabbit enterocytes: comparison of its molecular properties with those of the plasma membrane species

A soluble form of acetylcholinesterase was shown to be present in rabbit enterocytes. The enzyme was obtained from a high-speed supernatant (105,000 X g centrifugation) after homogenization of intestinal mucosa without detergent. It was shown to possess no obvious hydrophobic character and could be classified as a low-salt-soluble (LSS) acetylcholinesterase. Sucrose gradient centrifugation revealed a single enzyme species with a sedimentation coefficient of 3.9 +/- 0.2S. By gel filtration performed in HPLC the enzyme was eluted as a protein corresponding to an Mr of 72,000 +/- 3,000. It could be precipitated with concanavalin A by affinoelectrophoresis, but the catalytic activity was not affected by the lectin. Our results are consistent with a G1 globular form for this soluble acetylcholinesterase which differs very clearly from detergent-soluble forms also found recently in the plasma membranes of rabbit enterocytes.     

An inhibitory monoclonal antibody to human acetylcholinesterases

The monoclonal antibody AE-2 raised against acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from human erythrocytes is shown to inhibit the enzyme activity. The reaction of the antibody with a structural epitope is investigated further. The epitope resides on monomeric, dimeric and tetrameric species of the enzyme. The rate of phosphorylation of the enzyme by diisopropylfluorophosphate was not affected by the antibody. On the other hand, inhibitors directed towards the anionic site(s) competed with antibody binding, suggesting that one of these is the epitope. The titration with antibody is biphasic and yields about 80% inhibition even in the presence of a large excess of antibody. Inhibition is fully reversible upon dilution, in a time-dependent manner. AE-2 also inhibited human adult and fetal brain acetylcholinesterase (to the same extent). However bovine brain acetylcholinesterase was inhibited to a lesser extent and rat brain acetylcholinesterase did not interact with the antibody. Butyrylcholinesterase (EC 3.1.1.8) also showed no reactivity towards the antibody.     

Polymorphism and immunochemical cross-reactivity of acetylcholinesterases from the brains of human, dog, hog, bovine and horse

In the caudate nucleus of the species tested about 20% of the acetylcholinesterase was salt soluble and sedimented in sucrose density gradient centrifugation as monomeric 5 S and tetrameric 10 S enzyme. About 80% was solubilized by micellar concentrations of Triton X-100 and sedimented as a tetrameric 10 S species in the presence of detergent but formed aggregates in the absence thereof. All the enzyme displayed poor cross-reactivity with a precipitating assay (Ouchterlony) but in a solid phase non-precipitating assay the cross-reactivity could be quantified and ranged from 96 to less than 1% depending on the species.     

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.     

Acetylcholinesterase of Schistosoma mansoni. Molecular forms of the solubilized enzyme

Several molecular forms of acetylcholinesterase were obtained from Schistosoma mansoni homogenates by extraction in either low-salt buffer, high-salt buffer or detergent buffer. The low-salt soluble form amounts to 25% of the total activity. By contrast, the extract obtained in the presence of Triton X-100 possessed almost almost 3-fold higher enzymatic activity, most of it (86%) being retained in the soluble extract (100 000 X g). High-salt concentration (1 M NaCl) also has a solubilizing effect, but to a lesser extent (50%). Acetylcholinesterase can also be solubilized by treatment with a solution of 1% methylmannoside (40%). In the presence of non-ionic detergents, the enzyme behaves as monodisperse 8 S form. In the absence of detergent the low-salt soluble extract is polydisperse: it contains a 10 S and a 32 S component, the latter could represent high polymers. The molecular form released from tissue homogenate by treatment with alpha-methylmannoside is polydisperse: it contains a major 10 S and a minor 32 S component. Differences in sedimentation coefficient were observed among the enzymes extracted with detergent from the various life cycle stages of the parasite. The enzyme from the cercarial stage sediments as a single 8 S peak. The adult worm exhibits an additional acetylcholinesterase peak of 18 S representing approx. 30% of the total enzymatic activity. The molecular weight of the major 8 S species, as determined by gel filtration, is 450 000.     

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme

1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.