Characterization of Salt-Soluble Forms of Acetylcholinesterase from Bovine Brain

Abstract: The hydrophilic, salt-soluble (SS) form of acetylcholinesterase (AChE) from bovine brain caudate nucleus exists mainly as a tetramer sedimenting at 10.3S (∼40%), and a monomer sedimenting at 3.4S (∼60%). The enzyme is N -glycosylated and contains similar HNK-1 carbohydrates as detergent-soluble (DS) AChE. No O-linked carbohydrates could be detected. Amino acid sequencing showed that the N terminus of SS-AChE is identical to that of DS-AChE. In tetrameric SS-AChE, two pairs of disulfide-linked dimers are associated by hydrophobic forces located in the C terminus. Antibodies were raised against a peptide identical to the last 10 amino acid residues of bovine brain DS-AChE. The peptide included the sequence of residues 574–583 (H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser-Asp-Leu-OH) of the enzyme. The antibodies cross-reacted with tetrameric, but not with monomeric, SS-AChE, showing that in the latter form, the C terminus is truncated. Limited proteolysis of tetrameric SS-AChE at the C terminus led to the formation of an enzymatically active monomer, which did not react with anti-C-terminal antibody. Although the DS form of AChE contains a structural subunit that serves as membrane anchor, no anchor was detected in SS-AChE. Enzyme antigen immunoassays showed that SS-AChE reacted with all monoclonal antibodies directed against the catalytic subunit of DS-AChE, but not with monoclonal antibodies targeting the membrane-anchored subunits. From our results, we conclude that SS-AChE utilizes the same alternative splicing pattern as DS-AChE, leading to tetrameric SS-AChE devoid of the membrane anchor. The active monomer of SS-AChE is most likely derived from tetrameric forms by limited postsynthetic proteolysis.     

Are soluble and membrane-bound rat brain acetylcholinesterase different?

Salt-soluble and detergent-soluble acetylcholinesterases (AChE) from adult rat brain were purified to homogeneity and studied with the aim to establish the differences existing between these two forms. It was found that the enzymatic activities of the purified salt-soluble AChE as well as the detergent-soluble AChE were dependent on the Triton X-100 concentration. Moreover, the interaction of salt-soluble AChE with liposomes suggests amphiphilic behaviour of this enzyme. Serum cholinesterase (ChE) did not bind to liposomes but its activity was also detergent-dependent. Detergent-soluble AChE remained in solution below critical micellar concentrations of Triton X-100. SDS polyacrylamide gel electrophoresis of purified, Biobeads-treated and iodinated detergent-soluble 11 S AChE showed, under non reducing conditions, bands of 69 kD, 130 kD and >250 kD corresponding, respectively, to monomers, dimers and probably tetramers of the same polypeptide chain. Under reducing conditions, only a 69 kD band was detected. It is proposed that an amphiphilic environment stabilizes the salt-soluble forms of AChE in the brain in vivo and that detergent-soluble Biobeads-treated 11 S AchE possess hydrophobic domain(s) different from the 20 kD peptide already described.Abbreviations used AChE acetylcholinesterase - BSA bovine serum albumin - ChE serum (butyryl) cholinesterase - ConA-Sepharose concanavalin A-Sepharose 4B - DMAEBA-Sepharose dimethylaminoethylbenzoic acid-Sepharose 4B - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMA tetramethylammonium chloride     

The membrane form of acetylcholinesterase from rat brain contains a 20 kDa hydrophobic anchor

Rat brain acetylcholinesterase (AChE, EC 3.1.1.7) consists of about 80% amphiphilic detergent-soluble (DS-) AChE and 20% hydrophilic salt-soluble (SS-) AChE. DS-AChE contains about 65% tetrameric, 20% dimeric and 10% monomeric, SS-AChE about 40% tetrameric and 60% monomeric forms. N-terminal sequencing of DS- and SS-AChE gave identical N-termini corresponding to the published cDNA sequence of the mature enzyme. The band pattern on SDS-gels is similar to that of AChE from human and bovine brain. SDS-PAGE of hydrophobically labeled DS-AChE revealed the presence of a disulfide bonded hydrophobic membrane anchor of about 20 kDa. Monoclonal antibodies (mAbs) recognizing the anchor-containing subunits of mammalian brain DS-AChE, crossreacted with rat brain DS-AChE but not with SS-AChE. DS- and SS-AChE also reacted with antibodies raised against a peptide comprising the last 10 amino acids of the sequence of bovine brain AChE. Our results led us to conclude that both DS- and SS-AChE from rat brain contain T-type catalytic subunits, and DS-AChE in addition a P-type hydrophobic anchor similar to other mammalian brain DS-AChE.     

Acetylcholinesterase in Mouse Neuroblastoma NB2A Cells: Analysis of Production, Secretion, and Molecular Forms

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.     

Acetylcholinesterases of the nematode Steinernema carpocapsae. Characterization of two types of amphiphilic forms differing in their mode of membrane association.

We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae. Two major AChEs are involved in acetylcholine hydrolysis. The first class of AChE is highly sensitive to eserine (IC50 = 0.05 microM). The corresponding molecular forms are: an amphiphilic 14S form converted into a hydrophilic 14.5S form by mild proteolysis and two hydrophilic 12S and 7S forms. Reduction of the amphiphilic 14S form with 10 mM dithiothreitol produces hydrophilic 7S and 4S forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex. Sedimentation coefficients suggest that 4S, 7S, 12S forms correspond to hydrophilic monomer, dimer, tetramer and that the 14S form is also a tetramer linked to one structural element. The second class of AChE is less sensitive to eserine (IC50 = 0.1 mM). Corresponding molecular forms are hydrophilic and amphiphilic 4S forms (monomers) and a major amphiphilic 7S form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. This amphiphilic 7S form thus possesses a glycolipid anchor. It appears that Steinernema (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.     

Native Molecular Forms of Head Acetylcholinesterase from Adult Drosophila melanogaster: Quaternary Structure and Hydrophobic Character

The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100, 10-oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt-soluble fraction contained the same molecular forms as the low salt-soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.     

A unique hydrophobic domain of rat brain globular acetylcholinesterase for binding to cell membranes

Both salt-soluble and detergent-soluble rat brain globular acetylcholinesterases (SS- and DS- AChE EC 3.1.1.7) are amphiphiles, as shown by detergent dependency of enzymatic activity and binding to liposomes. Proteinase K and papain treatment transformed SS-AChE and DS-AChE into forms that, in absence of detergent, no longer aggregated nor bound to liposomes. In contrast, phosphatidylinositol-specific phospholipase C had no effect on these properties. Labeling DS-AChE with 3-(trifluoromethyl)-3-(m-(¹²⁵I)-iodophenyl) diazirine ([¹²⁵I]TID) revealed, by polyacrylamide gel electrophoresis under reducing conditions, one single band of 69 kD apparent molecular mass. The same pattern was previously obtained with Bolton and Hunter reagent-labeled enzyme (1). Proteinase K treatment transformed the 11 S [¹²⁵I]TID labeled AChE into a 4 S form which no longer showed¹²⁵I-radioactivity and was unable to bind to liposomes. These results are compatible with the existence of a hydrophobic segment present both on salt-soluble and detergent-soluble 11 S AChE as well as on the minor forms 4 S and 7 S. This segment is not linked to the catalytic subunits by disulfide bounds in contrast to the 20 kD non-catalytic subunit described by Inestrosa et al. (2).Abbreviations used AChE acetylcholinesterase - SS-AChE salt-soluble AChE - DS-AChE detergent-soluble AChE - BSA bovine serum albumin - ChE serum (butyryl) cholinesterase - ConA-Sepharose concanavalin A-Sepharose 4B - DMAEBA-Sepharose dimethylaminoethylbenzoic acid-Sepharose 4B - PC-Chol-SA liposomes phosphatidylcholine-cholesterol-stearylamine liposomes - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - ¹²⁵I-TID 3-(trifluoromethyl)-3-(m-(¹²⁵I)-iodophenyl) diazirine     

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.     

Amphiphilic Detergent-Soluble Acetylcholinesterase from Torpedo marmorata: Characterization and Conversion by Proteolysis to a Hydrophilic Form

The membrane-bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was solubilized by Triton X-100 or by treatment with proteinase K and purified to apparent homogeneity by affinity chromatography. Although the two forms differed only slightly in their subunit molecular weight (66,000 and 65,000 daltons, respectively), considerable differences existed between native and digested detergent-soluble AChE. The native enzyme sedimented at 6.5 S in the presence of Triton X-100 and formed aggregates in the absence of detergent. The digested enzyme sedimented at 7.5 S in the absence and in the presence of detergent. In contrast to the detergent-solubilized AChE, the proteolytically derived form neither bound detergent nor required amphiphilic molecules for the expression of catalytic activity. This led to the conclusion that limited digestion of detergent-soluble AChE results in the removal of a small hydrophobic peptide which in vivo is responsible for anchoring the protein to the lipid bilayer.     

Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.     

Separation in a single step by affinity chromatography of cholinesterases differing in subunit number.

We describe an affinity chromatography method in which dimethylaminoethylbenzoic acid-Sepharose 4B is used, making it possible to separate in one step the molecular forms of globular acetylcholinesterase (AChE, EC 3.1.1.7) or butyrylcholinesterase (ChE, EC 3.1.1.8). A crude extract containing these enzymes was deposited onto the chromatography gel, washed, and eluted by a linear gradient of tetramethylammonium chloride (0-0.3 M). With rat brain AChE, two well-separated peaks were eluted in the presence of 1% Triton X-100; the first peak corresponded to 4 S forms and the second to 11 S forms. This separation was very efficient for salt-soluble activity and less efficient for the detergent-soluble AChE. In this case, the 4 S peak represented only 6.5% of total detergent-soluble activity and was cross-contaminated by the 11 S form. Rat serum ChE was efficiently separated into two peaks of 7 S and 11 S. This method could potentially be adapted to separate other multimeric proteins with varying numbers of affinity sites.     

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.     

Amphiphilic and Nonamphiphilic Forms of Bovine and Human Dopamine β-Hydroxylase

We show that human and bovine dopamine beta-hydroxylases (DBH) exist under three main molecular forms: a soluble nonamphiphilic form and two amphiphilic forms. Sedimentation in sucrose gradients and electrophoresis under nondenaturing conditions, by comparison with acetylcholinesterase (AChE), suggest that the three forms are tetramers of the DBH catalytic subunit and bind either no detergent, one detergent micelle, or two detergent micelles. By analogy with the Gna4 and Ga4 AChE forms, we propose to call the nonamphiphilic tetramer Dna4 and the amphiphilic tetramers Da4I and Da4II. In addition to the major tetrameric forms, DBH dimers occur as very minor species, both amphiphilic and nonamphiphilic. Reduction under nondenaturing conditions leads to a partial dissociation of tetramers into dimers, retaining their amphiphilic character. This suggests that the hydrophobic domain is not linked to the subunits through disulfide bonds. The two amphiphilic tetramers are insensitive to phosphatidylinositol phospholipase C, but may be converted into soluble DBH by proteolysis in a stepwise manner; Da4II----Da4I----Dna4. Incubation of soluble DBH with various phospholipids did not produce any amphiphilic form. Several bands corresponding to the catalytic subunits of bovine DBH were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but this multiplicity was not simply correlated with the amphiphilic character of the enzyme. In the case of human DBH, we observed two bands of 78 and 84 kDa. As previously reported by others, the presence of the heavy subunit characterizes the amphiphilic forms of the enzyme. We discuss the nature of the hydrophobic domain, which could be an uncleaved signal peptide, and the organization of the different amphiphilic and nonamphiphilic DBH forms. We present two models in which dimers may possess either one hydrophobic domain or two domains belonging to each subunit; in both cases, a single detergent micelle would be bound per dimer.     

Tetrameric (G4) Acetylcholinesterase: Structure, Localization, and Physiological Regulation

Abstract: Acetylcholinesterase (AChE), a highly conserved enzyme in the animal kingdom, is distributed throughout a wide range of vertebrate tissues where it is expressed as multiple molecular forms comprising different arrangements of catalytic and structural subunits. The major AChE form in the CNS is an amphiphilic globular tetramer (G₄ AChE) consisting of four identical catalytic subunits attached to cellular membranes by a hydrophobic noncatalytic subunit (P-subunit). This study focuses primarily on current data involving the structure of the G₄ AChE P-subunit, the expression and regulation of G₄ AChE during development and adulthood, and its role(s) in certain neurological disorders including Alzheimer's disease.     

Purification and properties of the membrane-bound form of acetylcholinesterase from chicken brain. Evidence for two distinct polypeptide chains

The major molecular form of acetylcholinesterase (AChE) from chicken brain is a membrane-bound glycoprotein with an apparent sedimentation coefficient of 11.4 S. Analysis of the purified protein by gel filtration, velocity sedimentation, and sodium dodecyl sulfate-gel electrophoresis shows that the solubilized enzyme is a globular tetramer with an apparent Mr = 420,000. This membrane-bound form of AChE is hydrophobic and readily aggregates in the absence of detergent. These aggregates are concentration-dependent, relatively stable in the presence of high salt concentrations, yet readily dissociate upon addition of detergent to the 11.4 S form, indicating that the interactions are hydrophobic. Polyclonal and monoclonal antibodies raised against chicken brain AChE purified by ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis precipitate AChE enzyme activity. However, these antibodies do not cross-react with the enzyme from chicken muscle which preferentially hydrolyses butyrylcholine. Immunoprecipitation of isotopically labeled enzyme molecules from tissue cultured brain cells and analysis by sodium dodecyl sulfate-gel electrophoresis shows that AChE consists of two polypeptide chains with apparent Mr = 105,000 (alpha) and 100,000 (beta) in a 1:1 ratio. Immunoblotting of brain AChE with either the polyclonal or monoclonal antibodies indicates that the alpha and beta chains share antigenic determinants. Furthermore, both polypeptide chains can be labeled with [3H]diisopropyl fluorophosphate, indicating that they each contain a catalytic site. This is the first indication that globular forms of AChE may consist of multiple polypeptide chains.     

Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes

Differences in the glycosylation of acetylcholinesterase (AChE) subunits which form the dimers of mouse erythrocyte and a suitable procedure to purify the enzyme by affinity chromatography in edrophonium-Sepharose are described. AChE was extracted ( approximately 80%) from erythrocytes with Triton X-100 and sedimentation analyses showed the existence of amphiphilic AChE dimers in the extract. The AChE dimers were converted into monomers by reducing the disulfide bond which links the enzyme subunits. Lectin interaction studies revealed that most of the dimers were bound by concanavalin A (Con A) (90-95%), Lens culinaris agglutinin (LCA) (90-95%), and wheat germ (Triticum vulgaris) agglutinin (WGA) (70-75%), and a small fraction by Ricinus communis agglutinin (RCA(120)) (25-30%). The lower level of binding of the AChE monomers with WGA (55-60%), and especially with RCA (10-15%), with respect to the dimers, reflected heterogeneity in the sugar composition of the glycans linked to each AChE subunit in dimers. Forty per cent of the amphiphilic AChE dimers lost the glycosylphosphatidylinositol (GPI) and, therefore, were converted into hydrophilic forms, by incubation with phosphatidylinositol-specific phospholipase C (PIPLC), which permitted their separation from the amphiphilic variants in octyl-Sepharose. Only the hydrophilic dimers, either isolated or mixed with the amphiphilic forms, were bound by edrophonium-Sepharose, which allowed their purification (4800-fold) with a specific activity of 7700 U/mg protein. The identification of a single protein band of 66 kDa in gel electrophoresis demonstrates that the procedure can be used for the purification of GPI-anchored AChE, providing that the attached glycolipid domain is susceptible to PIPLC.     

Cholinesterases from flounder muscle. Purification and characterization of glycosyl-phosphatidylinositol-anchored and collagen-tailed forms differing in substrate specificity

Flounder (Platichthys flesus) muscle contains two types of cholinesterases, that differ in molecular form and in substrate specificity. Both enzymes were purified by affinity chromatography. About 8% of cholinesterase activity could be attributed to collagen-tailed asymmetric acetylcholinesterase sedimenting at 17S, 13S and 9S, which showed catalytic properties of a true acetylcholinesterase. 92% of cholinesterase activity corresponded to an amphiphilic dimeric enzyme sedimenting at 6S in the presence of Triton X-100. Treatment with phospholipase C yielded a hydrophilic form and uncovered an epitope called the cross-reacting determinant, which is found in the hydrophilic form of a number of glycosyl-phosphatidylinositol-anchored proteins. This enzyme showed catalytic properties intermediate to those of acetylcholinesterase and butyrylcholinesterase. It hydrolyzed acetylthiocholine, propionylthiocholine, butyrylthiocholine and benzoylthiocholine. The Km and the maximal velocity decreased with the length and hydrophobicity of the acyl chain. At high substrate concentrations the enzyme was inhibited. The p(IC50) values for BW284C51 and ethopropazine were between those found for acetylcholinesterase and butylcholinesterase. For purified detergent-soluble cholinesterase a specific activity of 8000 IU/mg protein, a turnover number of 2.8 x 10(7) h-1, and 1 active site/subunit were determined.     

Monoclonal Antibodies to Rabbit Brain Acetylcholinesterase: Selective Enzyme Inhibition, Differential Affinity for Enzyme Forms, and Cross-Reactivity with Other Mammalian Cholinesterases

Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two-step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross-reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50 = 10(-8) M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergent-free brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the "soluble" AChE, was suspected of lacking affinity for the hydrophilic enzyme form.     

Existence of Two Acetylcholinesterases in the Mosquito Culex pipiens (Diptera: Culicidae)

Abstract: Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens . The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol-specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide-susceptible strain S-LAB, AChE1 is inhibited by 5 × 10⁻⁴ M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide-resistant strain, MSE, there is no mortality after exposure to 5 × 10⁻⁴ M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 × 10⁻⁴ M propoxur. The possibility that AChE1 and AChE2 are products of tissue-specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.