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:3.1.1.8 (cholinesterase)
12,691 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dissociated cells from 13- and 17-day-old embryonic rat mesencephali have grown in primary cultures in order to compare the early and late influences of different agents--insulin, dexamethasone and nerve growth factor (NGF)--on the expression of cholinergic maturation process. We have studied cholin acetyltransferase (ChAT) activity, which is regarded as a specific marker for cholinergic function of the brain, and a widely used differentiation marker, the acetyl-cholinesterase (AchE) enzyme. Biochemical maturation of increasing specific activity of ChAT in both younger and older cells was taken into consideration. During cultivation the AchE activity was slightly increased in younger cells, but a dramatic decrease could be noted in older ones. Insulin in concentration from 10 to 27 micrograms mL-1 causes a significant inhibition in ChAT activity in comparison with the enzyme activity measured in control cultures (insulin ranging from 1 to 100 ng), independently of embryos age. This polypeptide hormone is able to enhance AchE activity in the cultured cells, especially in older ones. With continuous treatment of the culture with dexamethasone, a synthetic glucocorticoid, the ChAT activity in younger cells reaches a maximum curve by day 9 (nine). At this time the AchE activity shows a slighter, no significant increase than at any other time during cultivation. In cell cultures taken from 17-day-old embryos however dexamethasone treatment evoked a significant decrease in ChAT activity with a concomitant increase of AchE activity which was compared to insulin treatment. In spite of the fact that the NGF is able to enhance the ChAT activity, no significant alteration in AchE activity can be measured in younger cell cultures. These results suggest an uneven expression of the enzymes in embryonic rat mesencephali in the presence of above agents depending on the age of cells.
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PMID:Early and late hormonal modulation of cholinergic maturation in culture of embryonic mesencephali. 141 68

Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.
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PMID:Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding. 151 12

The structure and some functional sites of human milk bile salt activated lipase (BAL) were studied by cDNA cloning and chemical analysis of the enzyme. Eighteen cDNA clones of human BAL were identified from lactating human breast cDNA libraries in lambda gt11 and lambda gt10 with antibody and synthetic oligonucleotides as probes. The sequence of four clones was sufficient to construct a 3018-bp BAL cDNA structure. This sequence codes for an open reading frame of 742 amino acid residues. There is a putative signal sequence of 20 residues which is followed by the amino-terminal sequence of BAL, and the mature BAL contains 722 amino acid residues. The cDNA sequence also contains a 678-base 5'-untranslated sequence, a 97-base 3'-untranslated region, and a 14-base poly(A) tail. The sequence of a 1.8-kbp insert of clone G10-4A differs from that of the other cDNA in that it contains a deletion of 198 bases (1966-2163) corresponding to 66 amino acid residues. By use of BAL cDNA as probe, it was found that the major molecular species of BAL mRNA in human mammary gland HBL-100 cells had a size of 2.9 kb and two minor species had sizes of 3.8 and 5.1 kb by Northern blot analyses. The deduced BAL protein structure contains in the carboxyl-terminal region 16 repeating units of 11 amino acids each. The repeating units have the basic structure Pro-Val-Pro-Pro-Thr-Gly-Asp-Ser-Gly-Ala-Pro with only minor substitutions. The amino acid sequence of human BAL is related to that of pancreatic lysophospholipase, cholesterol esterase, cholinesterase, acetylcholinesterase, and thyroglobulin. Ten of the 14 cyanogen bromide fragments of diisopropyl fluorophosphate inhibited human milk BAL were isolated, determined for N-terminal sequences, analyzed for amino sugars, and tested for some functional properties. These chemical studies established that the active site of human milk BAL is located at serine-194, the N-glycosylation site is present at asparagine-187, the O-glycosylation region is in the 16 repeating units near the C-terminus, and the heparin binding domain is in the N-terminal region. We have also determined the location of disulfide bridges as Cys64-Cys80 and Cys246-Cys257. The cyanogen bromide cleavage and the partial sequencing of CNBr peptides also confirmed the location of methionines in the polypeptide chain as well as the deduced cDNA sequence of BAL.
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PMID:Structure of human milk bile salt activated lipase. 198 41

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 greater than 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.
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PMID:Are soluble and membrane-bound rat brain acetylcholinesterase different? 208 66

The aim of our work was to assess the performance of tissue polypeptide antigen in detecting hepatocellular carcinoma in cirrhotic patients, while also checking for any influence of liver dysfunction on the serum level of the marker. One hundred and twenty-five consecutive cirrhotic patients, 35 with and 90 without, hepatocellular carcinoma were studied. Tissue polypeptide antigen had a different distribution in the two groups and the best diagnostic accuracy with 48.6% sensitivity and 85.6% specificity was found at the cut-off value of 240 UL-1. In cirrhotic patients significant linear correlations were found between tissue polypeptide antigen and alanine-transaminase, aspartate-transaminase, G-glutamyl-transpeptidase and alkaline phosphatase; there was no correlation with bilirubin or pseudo-cholinesterase. In patients with hepatocellular carcinoma a significant linear correlation was found only with alanine and aspartate transaminase and G-glutamyl-transpeptidase. The analysis of covariance still showed a significant difference between mean tissue polypeptide antigen levels in the two groups also accounting for covariates. These results suggest that: a) the liver dysfunction may be involved in increasing tissue polypeptide antigen values; b) tissue polypeptide antigen has a different distribution in cirrhotic patients with and without hepatocellular carcinoma also accounting for covariates; these findings further support the specificity of tissue polypeptide antigen.
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PMID:The serum tissue polypeptide antigen in the detection of hepatocellular carcinoma in cirrhotic patients. 217 22

To study the primary structure of human acetylcholinesterase (AcChoEase; EC 3.1.1.7) and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with greater than or equal to 50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase (BtChoEase; EC 3.1.1.8). However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy (-117 kcal/mol) downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the BtChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification.
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PMID:Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure. 226 19

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.
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PMID:Cross-homologies and structural differences between human cholinesterases revealed by antibodies against cDNA-produced human butyrylcholinesterase peptides. 246 May 89

Extracts of the electric organ of Torpedo californica contain a proteinaceous factor that causes the formation of patches on cultured myotubes at which acetylcholine receptors (AChR), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) are concentrated. Results of previous experiments indicate that this factor is similar to the molecules in the synaptic basal lamina that direct the aggregation of AChR and AChE at regenerating neuromuscular junctions in vivo. We have purified the active components in the extracts 9,000-fold. mAbs against four different epitopes on the AChR/AChE/BuChE-aggregating molecules each immunoprecipitated four polypeptides from electric organ extracts, with molecular masses of 150, 135, 95, and 70 kD. Gel filtration chromatography of electric organ extracts revealed two peaks of AChR/AChE/BuChE-aggregation activity; one comigrated with the 150-kD polypeptide, the other with the 95-kD polypeptide. The 135- and 70-kD polypeptides did not cause AChR/AChE/BuChE aggregation. Based on these molecular characteristics and on the pattern of staining seen in sections of muscle labeled with the mAbs, we conclude that the electric organ-aggregating factor is distinct from previously identified molecules, and we have named it "agrin."
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PMID:Identification of agrin, a synaptic organizing protein from Torpedo electric organ. 282 89

Coated vesicles isolated from rat liver perfused with diisopropylfluorophosphate (DFP) to inactivate endogenous cholinesterase contained newly synthesized secretory cholinesterase after a 30 min recovery. The cholinesterase is found in coated vesicles of presumed endocytic origin following DFP treatment and perfusion for 3 min with galactosylated cholinesterase, a ligand for the asialoglycoprotein receptor. Highly enriched populations of endocytic and exocytic coated vesicles can be separated by use of a novel cholinesterase mediated density shift technique. The two coated vesicle classes have very similar polypeptide compositions but differ significantly in the ratio of cholesterol to phospholipid.
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PMID:Separation of endocytic from exocytic coated vesicles using a novel cholinesterase mediated density shift technique. 286 30

CSF neurotransmitter markers may reflect neurochemical alterations in Alzheimer's disease (AD). The best studied neurochemical deficit in AD is that of acetylcholine. Both acetylcholinesterase and butyrylcholinesterase activity have been reported to be reduced in some but not all studies of AD CSF. Studies of monoamine metabolites have also been controversial but most authors have found reduced concentrations of CSF HVA, lesser reductions in HIAA and no change in MHPG. CSF GABA concentrations have been found to be reduced in AD. Studies of CSF neuropeptides in AD have shown reduced concentrations of somatostatin and vasopressin, normal concentrations of vasoactive intestinal polypeptide and either normal or decreased concentrations of beta-endorphin and corticotropin releasing factor. Although no individual CSF neurochemical markers are specific for AD it may be possible to develop a profile of several neurochemical markers which will have enhanced specificity.
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PMID:CSF neurotransmitter markers in Alzheimer's disease. 287 17


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