Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.1.7 (acetylcholinesterase)
28,390 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A dimeric form of acetylcholinesterase from the electric organ of Torpedo californica was solubilized by phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. The solubilized enzyme had a sedimentation coefficient of 7.3S which was not modified by detergents. The high salt-soluble asymmetric forms of acetylcholinesterase were not solubilized by the phospholipase. Our data suggest that the hydrophobic dimer of acetylcholinesterase may be associated with the plasma membrane through a specific interaction involving phosphatidylinositol.
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PMID:A hydrophobic dimer of acetylcholinesterase from Torpedo californica electric organ is solubilized by phosphatidylinositol-specific phospholipase C. 663 70

The role of phosphatidylinositol-specific phospholipase C (PIase C) in a) the enigmatic phosphatidylinositol (PI) turnover and b) in our understanding of membrane enzyme-PI interactions is the subject matter of this article. PIase C is present in both procaryotes and eukaryotes. This enzyme is considered to be involved in the cells PI breakdown which occurs in response to several external stimuli. Recent information on the physical properties, Ca2+ requirement, cellular localization and modulation of the activity of PIase C of mammalian systems can help to evaluate the PI turnover from a new angle. Existing evidence suggests that Ca2+-dependent PI breakdown is probably mediated through the cytosolic and particulate PIase C while a Ca2+ independent pathway is catalyzed by a lysosomal enzyme. Apparently PI turnover may be operating through more than one mechanism. The association of this phenomenon with a membrane receptor event linked with "Ca2+ gating" may have to be reconsidered. Modulation of the PIase C activity by unsaturated amphiphiles or the presence of this enzyme in different physico-chemical forms could be a potential regulatory feature. Hydrolysis of membrane PI of a number of cells and tissues by the bacterial PIase C has been shown to cause substantial release of acetylcholinesterase, alkaline phosphatase and 5'-nucleotidase in free, soluble form. Other membrane enzymes, e.g., alkaline phosphodiesterase I, L-leucyl-beta naphthyl amidase and Ca2+ or Mg2+ ATPase are not affected. These results indicate a specific interaction between PI and certain enzymes in membranes. The chemical nature of this linkage, whether it is covalent or non-covalent, has also been explored and has provided intriguing insight into this phenomenon. New findings also indicate that hydrolysis of PI by PIase C also can cause modifications in membrane-enzyme activities, e.g., adenylate cyclase.
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PMID:Minireview. Phosphatidylinositol specific phospholipases C. 708 67

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.
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PMID:Monomers and dimers of acetylcholinesterase in human meningioma are anchored to the membrane by glycosylphosphatidylinositol. 747 60

Bovine erythrocyte acetylcholinesterase was prepared for tryptic digestion by radiomethylating with [14C]HCHO and NaCNBH3, cleaving with purified bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the glycoinositol phospholipid anchor, and reducing and alkylating the intersubunit disulfide bonds. Two alternative denaturation procedures were then compared prior to incubation with trypsin. In the conventional procedure, acetylcholinesterase was treated with 6 M guanidine hydrochloride for 40 min at room temperature and dialyzed. In a new procedure, acetonitrile (CH3CN) was added to 30% v/v for 10-15 min at room temperature and then removed by vacuum evaporation. The CH3CN concentration during evaporation could be estimated from the apparent pH of the solution (20 mM phosphate buffer), which varied linearly over the range of 0-75% CH3CN. CH3CN was removed in a mixture of constant composition (approximately 11% H2O-89% CH3CN), so that a final CH3CN content of 0-5% could be monitored by solution weight alone. The tryptic digests of the two denatured stocks yielded comparable HPLC profiles for A215 and radioactivity. This new denaturation protocol may be of general utility because of its convenience and gentle conditions.
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PMID:Protein denaturation by addition and removal of acetonitrile: application to tryptic digestion of acetylcholinesterase. 771 Jan 3

The ability of the phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes to hydrolyze glycosyl phosphatidylinositol (GPI)-anchored membrane proteins was compared with the ability of the PI-PLC from Bacillus thuringiensis to hydrolyze such proteins. The L. monocytogenes enzyme produced no detectable release of acetylcholinesterase from bovine, sheep, and human erythrocytes. The cleavage of the GPI anchors of alkaline phosphatase from rat and rabbit kidney slices was less than 10% of the cleavage seen with the PI-PLC from B. thuringiensis. Activity for release of Fc gamma receptor IIIB (CD16) on human granulocytes was also low. Variations in pH and salt concentration had little effect on the release of GPI-anchored proteins. Our data show that L. monocytogenes PI-PLC has low activity on GPI-anchored proteins.
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PMID:Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins. 825 89

Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethylated on its amine groups and incubated with bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fragment that contained the residual GPI glycan was isolated by HPLC. Analysis by electrospray-ionization mass spectrometry revealed a parent ion of m/z 3798. The fragmentation patterns produced by collision-induced dissociation mass spectrometry of the +4 and +5 states of the parent ion indicated a 23-amino acid peptide in amide linkage to ethanolamine-P04-Hex-Hex-Hex(PO4-ethanolamine)(HexNAc)-Hex N(Me)2-inositol phosphate. The glycan structure is completely consistent with that obtained previously for the GPI anchor of human erythrocyte AChE except for the addition of the HexNAc substituent. A nearly complete peptide sequence was deduced from the fragmentation patterns, although four assignments were based only on single fragments of very low abundance. To resolve this uncertainty, a segment of bovine genomic DNA corresponding to the C-terminal AChE sequence was amplified by PCR. DNA sequencing established the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTCSGPAHG, in agreement with the MS data and consistent with results from Edman protein sequencing. Dimerization of AChE polypeptides is mediated by intersubunit disulphide bonding in this C-terminal segment, but the bovine AChE contained two cysteine residues in a ...CTC... motif, in contrast with human AChE which contains only a single cysteine in this segment. Although bovine AChE contained no free thiol groups reactive with iodo[14C]acetamide, partial reduction and alkylation with iodo[14C]acetamide revealed that conversion into monomers occurred with an overall incorporation of only one alkyl group per monomer. An identical level of alkylation was observed when dimeric human AChE was converted into monomers by partial reduction. The question of whether the bovine AChE contains one or two intersubunit disulphide linkages is considered.
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PMID:Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase: analysis by mass spectrometry and by protein and DNA sequencing. 861 75

Despite advances in understanding the cell biology of glycoinositol phospholipid (GPI)-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.
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PMID:Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring. 873 Jan 2

A water-soluble dimeric form of acetylcholinesterase from electric organ tissue of Torpedo californica was obtained by solubilization with phosphatidylinositol-specific phospholipase C of the glycophosphatidylinositol-anchored species, followed by purification by affinity chromatography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head-group region of the bilayer. The effects of tryptic digestion of the bound acetycholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass approximately 5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274-Met 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.
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PMID:Interaction of partially unfolded forms of Torpedo acetylcholinesterase with liposomes. 877 Nov 95

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 x 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 x 10(-4) M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 x 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.
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PMID:Existence of two acetylcholinesterases in the mosquito Culex pipiens (Diptera:Culicidae). 886 21

We have isolated cDNAs coding for the complete amino acid sequences of cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2) from amphioxus. Both ChE transcripts have the characteristics of H-type catalytic subunits, which are inserted in the membrane via an ethanolamine-glycan-phosphatidylinositol anchor. The members of the catalytic triad of ChEs, the three pairs of cysteine residues involved in intrachain disulfide bonding, a cysteine near the carboxy terminal of both sequences, which could mediate interchain disulfide bonding, and 11 of the 14 aromatic amino acids that line the catalytic gorge of AChE are conserved. A remarkable difference between the two enzymes is in the region of the acyl-binding pocket, which plays an important role in determining substrate specificity in cholinesterases. ChE2 contains a sequence that resembles the acyl pocket of invertebrate ChE, while the acyl-binding site of ChE1 is novel. There are also differences between the two enzymes in the peripheral anionic site, which mediates inhibition by certain ligands. In vitro expression in COS-7 cells demonstrates that ChE2 hydrolyzes acetylthiocholine almost exclusively, while ChE1 hydrolyzes both acetylthiocholine and butyrylthiocholine. Both enzymes are inhibited comparably by BW284c51, but ChE1 is considerably more resistant to inhibition by propidium, ethopropazine, and eserine than is ChE2. Velocity sedimentation indicates that ChE1 and ChE2 are present as amphiphilic and nonamphiphilic G2 forms in vivo and in vitro. Another molecular form, which sediments at 17 S, is also present in vivo. Nondenaturing gel electrophoresis in conjunction with digestion by phosphatidylinositol-specific phospholipase C demonstrates that the vast majority of ChE1 and ChE2 is present as ethanolamine-glycan-phosphatidylinositol-anchored G2 forms in vivo. ChE1 also possesses an ethanolamine-glycan-phosphatidylinositol-anchor in vitro; however, ChE2 produced in vitro could not be detected on nondenaturing gels.
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PMID:cDNA cloning, in vitro expression, and biochemical characterization of cholinesterase 1 and cholinesterase 2 from amphioxus--comparison with cholinesterase 1 and cholinesterase 2 produced in vivo. 987 7


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