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)

The susceptibility to autoxidation of red cell lipids was studied before and after transformation of normal red cells to PNH-like erythrocytes. The transformation was effected by treatment of the red cells with the sulfydryl compounds D-penicillamine (DP) and N-acetyl-L-cysteine (NAC). The autoxidation was induced by incubating the cells with H2O2 and was estimated by measuring the generated malonyl dialdehyde. The susceptibility to autoxidation was significantly higher in DP-treated cells, while the opposite was true for NAC-treated cells. However, both DP- and NAC-treated cells showed a similar sensitivity to lysis by acid serum and about the same degree of acetylcholinesterase (AChE) activity decrease, thus indicating that the susceptibility to autoxidation of lipids is not involved in the determination of complement sensitivity or in the AChE activity decrease of the sulfydryl-treated cells. Finally, since, as evidenced from most of the reported cases in the literature, increased susceptibility to autoxidation is a feature of PNH cells, it seems reasonable to suggest that DP-treated cells should be used in preference to NAC-treated cells as a laboratory substitute for PNH cells.
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PMID:Susceptibility to autoxidation of lipids of paroxysmal nocturnal hemoglobinuria (PNH)-like red cells. 40 34

The anticancer drug caracemide, N-acetyl-N,O- di(methylcarbamoyl)hydroxylamine, and one of its degradation products, N-acetyl-O-methylcarbamoyl-hydroxylamine, were found to inhibit the enzyme ribonucleotide reductase of Escherichia coli by specific interaction with its larger component protein R1. No effect on the smaller protein R2 was observed. The effect of the degradation product was about 30 times lower than that of caracemide itself. The caracemide inactivation of R1 is irreversible, with an apparent second-order rate constant of 150 M-1 s-1. The R1R2 holoenzyme was approximately 30 times more sensitive to caracemide inactivation than the isolated R1 protein. The ribonucleotide reductase substrates were potent competitors of the caracemide inhibition, with a Kdiss for GDP binding to R1 of 80 microM. The reducing agent dithiothreitol was also found to be a potent competitor of caracemide inactivation. These results indicate that caracemide inactivates R1 by covalent modification at the substrate-binding site. By analogy with the known interaction between caracemide and acetylcholinesterase or choline acetyltransferase, we propose that the modification of R1 occurs at an activated cysteine or serine residue in the active site of the enzyme.
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PMID:Caracemide, a site-specific irreversible inhibitor of protein R1 of Escherichia coli ribonucleotide reductase. 161 68

Drosophila acetylcholinesterase (EC 3.1.1.7) is a 150-kDa glycoprotein anchored in plasmic membranes via a glycolipid. It is composed of two active subunits which are themselves made of two noncovalently linked polypeptides of 18 and 55 kDa resulting from the proteolysis of a single precursor of 75 kDa. Active Drosophila acetylcholinesterase can be expressed in Xenopus oocytes as an excreted protein. We have identified some of the amino acids essential in post-translational modifications of the protein by site-directed mutagenesis and expression of mutants in this system. The intersubunit disulfide bond involves cysteine at position 615. Cleavage of the 75-kDa precursor, as observed in Drosophila, originates from a hydrophilic peptide (in position 148 to 180) which does not exist in cholinesterase sequences from vertebrates. This cleavage is associated with excretion out of the cell. Drosophila acetylcholinesterase exhibits four effective sites of asparagine-linked glycosylation in positions 126, 174, 331, and 531. We show that glycosylations and dimerization protect the protein against proteolytic digestion. In contrast, none of these post-translational modifications significantly affects the activity of acetylcholinesterase or affinity for its substrate.
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PMID:Post-translational modifications of Drosophila acetylcholinesterase. In vitro mutagenesis and expression in Xenopus oocytes. 173 Jul 12

Tetrameric detergent-soluble bovine caudate nucleus acetylcholinesterase (AChE) was reduced and alkylated under conditions in which at least 95% of initial activity is retained. This treatment alone did not result in monomerization of AChE, nor did it create a hydrophilic enzyme. However, in the presence of SDS the enzyme became monomerized. Incubation of AChE with trypsin in the presence of the reversible inhibitor edrophonium rendered the enzyme hydrophilic and led to catalytically active monomers being produced. SDS/PAGE of this preparation in non-reducing conditions revealed only a small decrease in the subunit molecular mass. N-Terminal sequencing of the enzyme, before and after trypsin treatment, yielded identical N-termini showing that the enzyme was monomerized subsequent to C-terminal tryptic cleavage. From our results, we conclude that the most C-terminal cysteine residue is involved in inter-subunit disulphide bonding as well as in the attachment of AChE to the membrane anchor. Furthermore, the C-terminal region in the primary structure provides an area for hydrophobic contacts between the different subunits and also between the subunits and the membrane anchor.
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PMID:Monomerization of tetrameric bovine caudate nucleus acetylcholinesterase. Implications for hydrophobic assembly and membrane anchor attachment site. 173 64

Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.
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PMID:The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant. 174 70

The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extracellular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that co-expression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.
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PMID:Primary structure of a collagenic tail peptide of Torpedo acetylcholinesterase: co-expression with catalytic subunit induces the production of collagen-tailed forms in transfected cells. 184 May 20

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.
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PMID:Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages. 201 79

The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence of a catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrate-inhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis.
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PMID:Mutagenesis of essential functional residues in acetylcholinesterase. 221 85

RT6 is an unusual cell membrane protein that is expressed exclusively by postthymic T cells. The inherent defect in its expression has been correlated to lymphopenia and genetically determined susceptibility for insulin-dependent diabetes mellitus in the rat. We report here the primary structure of the RT6.2 alloantigen as deduced from the cDNA sequence. The predicted amino acid sequence of RT6.2 begins with a conventional leader of 20 amino acids and ends in a hydrophobic C-terminal extension peptide of 29 amino acids as is common for phosphatidylinositol-anchored proteins. Native RT6.2 is predicted to comprise 226 amino acids, with a calculated Mr of 26,036. Four cysteine residues account for two intrachain disulfide bonds. The sequence lacks potential N-glycosylation sites and contains an excess of positively charged residues. Homology searches in protein sequence data banks suggest that RT6.2 is not encoded by a member of the immunoglobulin supergene family. Moreover, these analyses did not reveal any close homologies of RT6.2 to known proteins: the highest homology found was 21.2% identity in a 52-amino acid overlap to the torpedo acetylcholinesterase precursor. Southern blot analyses indicate that RT6.2 is the product of a single-copy gene and provide evidence for closely related genes in the mouse and other species. The corresponding gene products remain to be identified.
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PMID:Primary structure of rat RT6.2, a nonglycosylated phosphatidylinositol-linked surface marker of postthymic T cells. 230 May 88

We have isolated a COOH-terminal tryptic peptide from the hydrophobic globular (5.6 S) form of Torpedo californica acetylcholinesterase that exhibits divergence in amino acid sequence from the catalytic subunit of the dimensionally asymmetric (17 S + 13 S) enzyme. The divergent peptide could be recovered from the glycophospholipid-modified 5.6 S enzyme only after treatment with phosphatidylinositol-specific phospholipase C. Upon reduction, carboxymethylation with [14C]iodoacetate, and trypsin digestion the resultant peptides were purified by gel filtration followed by high performance liquid chromatography. The high performance liquid chromatography profiles of 14C-labeled cysteine peptides from lipase-treated 5.6 S enzyme revealed unique radioactive peaks which had not been present in digests of the asymmetric form. These peaks all yielded identical amino acid sequences. The difference in chromatographic behavior of the individual peptides most likely reflects heterogeneity in post-translational processing. Gas-phase sequencing and composition analysis are consistent with the sequence: Leu-Leu-Asn-Ala-Thr-Ala-Cys. Composition includes 2-3 mol each of glucosamine and ethanolamine which is indicative of modification by glycophospholipid. Glucosamine is also present in an asparagine-linked oligosaccharide. The two forms of acetylcholinesterase diverge after the threonine residue within this peptide sequence; the hydrophobic form terminates with cysteine whereas the asymmetric form extends for 40 residues beyond the divergence. The locus of divergence and absence of any other amino acid sequence difference suggest that the molecular forms of acetylcholinesterase arise from a single gene by alternative mRNA processing.
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PMID:Divergence in primary structure between the molecular forms of acetylcholinesterase. 333 34


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