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)

We have studied the behavior of a mammalian brain tetrameric acetylcholinesterase (AChE) form released by proteinase K from a crude membrane fraction of bovine caudate nucleus. The solubilization of active AChE indicated the presence of a protease-sensitive site in the anchored protein. Unexpectedly, the solubilized AChE maintained its capacity to form aggregates in detergent-free gradients. We demonstrate here that this property was due neither to the presence of the hydrophobic membrane-anchoring domain still linked to the enzyme, nor to the presence of AChE activity trapped in small plasma membrane vesicles. Moreover, we found that the proteinase K-treated extract, devoid of AChE activity, induced the aggregation of purified hydrophilic AChE which usually does not form aggregates. Our results suggest the presence of an AChE aggregating factor in bovine brain extracts prepared in the presence of proteinase K. It is possible that this aggregation may reflect a process of AChE clustering on neurons.
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PMID:Amphiphilic behavior of a brain tetrameric acetylcholinesterase form lacking the plasma membrane anchoring domain. 150 88

The orientation of the enzyme Mg(2+)-ATPase (EC 3.6.1.3) in the transverse tubule (TT) membranes of skeletal muscle was investigated using highly purified chicken and rabbit TT vesicles. The percentage of sealed vesicles present in these preparations averaged 88 and 78%, respectively, as calculated from the detergent-induced increase in ouabain-sensitive (Na+, K+)-ATPase activity, ATP-dependent ouabain binding, and lactate dehydrogenase activity (sarcoplasmic enzyme trapped in the TT vesicles). Sidedness of the sealed vesicles, estimated from latency of 5'-nucleotidase, acetylcholinesterase, and adenylate cyclase, was predominantly right-side out (69-76%, chicken TT and 62-70%, rabbit TT). In both chicken and rabbit native vesicles, high Mg(2+)-ATPase activity was detected by addition of ATP to the extravesicular medium; this activity was increased 14-12% by alamethicin pointing to the external localization of the active site. Furthermore, the enzymatic activity resulted partially inhibited by treatment of the chicken TT vesicles with proteinase K or p-hydroxymercuribenzoate. Concanavalin A stimulated 4-fold the chicken TT Mg(2+)-ATPase activity, an effect not potentiated by detergent permeabilization of the intact vesicles, indicating that lectin-binding sites were also solvent accessible. This stimulatory effect was not observed in native or permeabilized rabbit TT vesicles. From these results we conclude that the TT Mg(2+)-ATPase is an ectoenzyme with its nucleotide-hydrolyzing site and glycosylated regions facing the extracellular space. Inhibitors of ion-motive ATPases did not modify the enzyme activity, suggesting a different physiological role for the TT Mg(2+)-ATPase which may be involved in the regulation of muscle fiber functions affected by extracellular ATP levels.
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PMID:Transverse tubule Mg(2+)-ATPase of skeletal muscle. Evidence for extracellular orientation of the chicken and rabbit enzymes. 166 Apr 76

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.
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PMID:Acetylcholinesterase in mouse neuroblastoma NB2A cells: analysis of production, secretion, and molecular forms. 292 96

Purified tetrameric detergent-soluble acetylcholinesterase (DS-AChE) from human caudate nucleus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence as well as in presence of a reducing agent. Staining for protein revealed a main band at 66,000 daltons (light monomer) with additional bands at 78,000 daltons (heavy monomer) as well as 130,000 and 150,000 daltons (light and heavy dimers). The same four polypeptides were also detected by Western blotting and by autoradiography of [3H]diisopropylphosphoryl enzyme. Labeling of the enzyme with 3-trifluoromethyl-3-(m-[125I]-iodophenyl)diazirine showed that the heavy monomer contained the hydrophobic anchor of the enzyme, whereas the light monomer was practically not labeled. The hydrophobic anchor was susceptible to proteolytic degradation by proteinase K. The functional molarity of DS-AChE was determined by two independent methods. Four active sites for the tetrameric enzyme were estimated. The turnover number per site was 1.7 X 10(7) mol of acetylthiocholine iodide hydrolyzed X h-1.
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PMID:Tetrameric detergent-soluble acetylcholinesterase from human caudate nucleus: subunit composition and number of active sites. 358 24

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.
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PMID:Amphiphilic detergent-soluble acetylcholinesterase from Torpedo marmorata: characterization and conversion by proteolysis to a hydrophilic form. 388 May 82

Skeletal muscle extract contains a previously undocumented 1300- to 1500-Da neurotrophic factor. Incubation of ventral spinal cord neurons in the presence of this factor enhances the rate of de novo acetylcholine synthesis two- to threefold over control cells, after 6 days in culture. This effect on cholinergic activity appears to be selective, since incubation with the factor results in only slight elevations of lactate dehydrogenase activity and DNA content, and no increase in the acetylcholinesterase activity. The 1300- to 1500-Da factor is acid-stable and partially sensitive to proteolysis by proteinase K, Staphylococcus aureus V8 protease, and subtilisin, but insensitive to trypsin. These results indicate that the active moiety is a peptide. The importance of peptides as neurotransmitters or neuromodulators is well accepted, but their role in the regulation of neuronal development is not widely appreciated. The present cholinergic neurotrophic peptide is distinct from previously characterized cholinergic trophic factors and represents the first example of a small, target-derived peptide which influences cholinergic development.
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PMID:Low-molecular-weight peptide stimulates cholinergic development in ventral spinal cord cultures. 390 37

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.
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PMID:Molecular forms of acetylcholinesterase from human caudate nucleus: comparison of salt-soluble and detergent-soluble tetrameric enzyme species. 397 87

Membrane-bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was labeled with the hydrophobic photoactivatable reagent 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ( [125I]TID). Labeling with [125I]TID was restricted to the membranous polypeptide segment of AChE as shown upon conversion of the amphiphilic form to the hydrophilic one by limited digestion with proteinase K. The labeled membranous segment, which has an Mr of approx. 3000 was isolated by gel filtration on Sephadex LH-60 in ethanol/formic acid.
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PMID:Hydrophobic labeling of the membrane binding domain of acetylcholinesterase from Torpedo marmorata. 637 63

We have distinguished three fractions of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo marmorata electric organs, according to their solubilization characteristics. The low-salt-aggregating collagen-tailed forms are soluble in high-salt buffers; their hydrodynamic properties ae not modified in the presence of detergents. They constitute the A fraction, which amounts to about a third of the tissue's AcChoE activity. The low-salt-soluble (LSS) and detergent-soluble (DS) fractions are not sensitive to ionic strength and collagenase. In the presence of nonionic detergents or bile salts, both fractions behave as a monodisperse "6.3S" form, the properties of which have been investigated mostly in the case of Triton X-100. Disulfide bond reduction dissociates the detergent form into a smaller "5S" form. These two forms are thought to be, respectively, detergent-associated dimers and monomers. In the absence of detergent, the LSS fraction is polydisperse: it contains a major 8S component, 11S and 14S components, and faster-sedimenting aggregates, which appear to represent dimers, tetramers, and higher polymers. The heterogeneity of the 8S component in gel filtration suggests that it also contains variable noncatalytic elements. Upon removal of the detergent the DS fraction forms ill-defined aggregates. Trypsin induces quaternary rearrangements of part of the 8S component into 11S and 14S components, which are still convertible into the detergent form; therefore trypsin probably digests noncatalytic elements. Pronase and proteinase K, on the other hand, convert the enzyme into a dimeric form, G2, that does not interact with detergents, probably by cleaving a minor fragment of the subunit that is involved in hydrophobic interactions.
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PMID:Collagen-tailed and hydrophobic components of acetylcholinesterase in Torpedo marmorata electric organ. 693 97

Monoclonal antibodies were raised against amphiphilic detergent-soluble (DS) acetylcholinesterase (AChE) from human brain caudate nucleus. Three mAb, 132-4 (IgG1), 132-5 (IgG1) and 132-6 (IgG3), specific for brain DS-AChE were selected and subcloned. These mAb reacted with native as well as heat-denatured and SDS-denatured DS-AChE, indicating that the epitopes to which mAb bound are continuous determinants. The mAb cross-reacted with DS-AChE from bovine and mouse brain and with brain DS-AChE from river trout (Salmo trutta forma fario) and lake trout (Salmo trutta forma lacustris). No cross-reaction was detected with the following antigens: salt-soluble (SS) AChE from bovine brain, glycophospholipid-anchored AChE from human and bovine erythrocytes, DS-butyrylcholinesterase and SS-butyrylcholinesterase (BtChE) from the brains of human and bovine, DS-BtChE from chicken and BtChE from human serum. Deglycosylation of brain DS-AChE with N-glycosidase F did not abolish the binding of mAb to DS-AChE. After reduction of brain DS-AChE by dithiothreitol, the mAb no longer reacted with the antigen, indicating that a disulfide bridge is important for the epitope. Monomerization of brain DS-AChE by trypsin and limited proteinase K treatment also abolished the binding of mAb to DS-AChE. Sucrose-density-gradient centrifugation showed that mAb reacted only with native tetrameric forms, but not with dimeric and monomeric forms. Western blot, after SDS/PAGE under non-reducing conditions, showed that mAb reacted with those subunits carrying the hydrophobic anchor (i.e. tetramers, trimers and heavy dimers) but not with those devoid of it (light dimers or monomers). Since mAb 132-4, 132-5 and 132-6 recognized DS-AChE from fish up to mammalian brain in the evolutionary tree, it is concluded that the epitope to which these mAb bind, is conserved in nature.
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PMID:Monoclonal antibodies against brain acetylcholinesterases which recognize the subunits bearing the hydrophobic anchor. 768 3


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