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)

An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.
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PMID:Enzyme immunometric assay for leukotriene C4. 793 Jun 43

A new enzyme immunometric assay of small haptens containing primary amino groups (thyroxine, MW 777; substance P, MW 1347; endothelin, MW 2492) is described. The procedure involves different sequential steps: (1) immunocapture of the haptens (standard or sample) by monoclonal anti-hapten antibodies coated on 96-well microtiter plates; (2) cross-linking of haptens via their amino groups to the wells using homobifunctional reagents (glutaraldehyde or disuccinimidyl suberate); (3) denaturing treatments (HCl or methanol); (4) measurement of linked epitope using the same monoclonal anti-hapten antibodies labeled with acetylcholinesterase. A minimal detectable concentration in the 4-10 fmoL/mL range was observed. Each assay appeared to be 70-200 times more sensitive than conventional competitive enzyme immunoassay using the same monoclonal antibody-coated plate technology and acetylcholinesterase-hapten conjugates as enzymatic tracers. Precision and specificity were very satisfying. Good correlation was noted between this assay and the competitive assays performed for different biological samples (plasma, tissues, or supernatant cell culture).
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PMID:Immunometric assay of low molecular weight haptens containing primary amino groups. 811 75

Organophosphate-inhibited cholinesterases may become progressively refractory to reactivation by nucleophilic compounds due to the dealkylation of an alkoxy group from the covalently bound phosphonate ester. This process is termed "aging". It has been found that "aged" cholinesterases are more resistant to protein unfolding than the non-inhibited ones. The pressure-induced denaturation of the native (non-inhibited) and "aged" tetrameric form of human plasma butyrylcholinesterase was investigated in the presence and absence of a denaturing agent (propylene carbonate). This study was undertaken to determine whether the stability of aged butyrylcholinesterase varies with the structure of the alkyl/aryl (R2) group remaining attached to the phosphorus atom of the organophosphoryl moiety. "Aged" organophosphoryl-cholinesterase conjugates were formed by reacting the enzyme with organophosphates: soman (trimethylpropylmethyl-phosphonofluoridate), sarin (isopropylmethyl-phosphonofluoridate), tabun (ethyl-N-dimethyl-phosphoramidocyanidate), DFP (diisopropyl phosphorofluoridate) and PBPDC (pyrenebutyl-phosphorodichloridate). The dual effects of hydrostatic pressure up to 3.5 kbar and propylene carbonate up to 1.2 M were investigated in 10 mM Tris.HCl (pH 7.0). Non-inhibited and aged enzymes were subjected to pressure/propylene carbonate for 12 hours at 20 degrees C. The perturbing effects of this treatment upon cholinesterase structure were analyzed after pressure release by non-denaturing electrophoresis. Pressure and propylene carbonate induced progressive inactivation of the native enzyme. The loss in activity was correlated with irreversible denaturation of the tetramer and its subsequent aggregation. Similarly, pressure and propylene carbonate induced the formation of irreversibly denatured forms of aged butyrylcholinesterase. These denatured forms are partially unfolded enzyme conformations. The native enzyme was found to be more susceptible to denaturation than aged enzymes, with the exception of the PBPDC-aged enzyme. Methyl phosphono adducts, i.e. soman or sarin-aged conjugates were found to be the most stable aged species. Phenomenological analysis of the pressure/propylene carbonate denaturation maps at half-way of the denaturation process indicated that denaturation is a multistep process. The lowest stability of tabun-aged and DFP-aged conjugates suggested that the size, the orientation and the hydrophobicity of the remaining alkyl/aryl chain (R2) of the organophosphoryl moiety play a role in determining the overall stability of aged enzymes. Molecular modelling of aged adducts shed light on steric constraints exerted by the R2 chain on the salt bridge formed between the negatively charged P-O- of the dealkylated organophosphoryl moiety and protonated His438 N epsilon.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Pressure and propylene carbonate denaturation of native and "aged" phosphorylated cholinesterase. 817 37

Asymmetric forms of acetylcholinesterase (AChE) are thought to be the predominant forms of this enzyme at vertebrate neuromuscular junctions where they attach to the synaptic basal lamina via a collagen-like tail. High salt and heparin-containing buffers are capable of solubilizing asymmetric AChE molecules from skeletal muscle; however, detachment of AChE specifically from synaptic basal lamina using these procedures has not been demonstrated. To determine whether AChE can be solubilized from mature neuromuscular junctions, adult quail muscle fibers were extracted with buffered detergent solutions containing either 0.05 M NaCl, 1 m NaCl, 0.5-2 mg/ml heparin, 8 M urea, or 4 m guanidine HCl, and the remaining AChE molecules were localized by indirect immunofluorescence. Analysis of extracted AChE oligomeric forms showed that low salt buffers containing heparin and high salt buffers were capable of solubilizing substantial amounts of catalytically active collagen-tailed AChE, whereas none of these buffers were capable of detaching AChE from synaptic basal lamina. In contrast, digestion with purified collagenase detached asymmetric forms from the non-extractable fraction and removed the AChE from the neuromuscular junctions. Parallel experiments using rat gastrocnemius muscle and enzyme histochemistry to detect AChE gave similar results. These studies indicate that the junctional AChE molecules are firmly attached to the extracellular matrix and that all the conventional extraction buffers used to solubilize the asymmetric collagen-tailed forms of AChE are incapable of detaching this enzyme from the synaptic basal lamina.
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PMID:Localization of "non-extractable" acetylcholinesterase to the vertebrate neuromuscular junction. 836 Jan 97

Three-dimensional structures of the nerves of the guinea-pig gallbladder, after histochemical demonstration of the acetylcholinesterase activity and HCl hydrolysis-collagenase digestion, were examined by scanning electron microscope. HCl-collagenase digestion facilitated easy identification of silver- and gold-intensified acetylcholinesterase-positive nerve fibers at a high accelerating voltage (25 kV), due to their strong reflection image. Ganglia were either triangular or ovoidal in shape. Dense para- and peri-vascular nerve fibers occurred around the cystic artery. There were a few intramuscular nerve fibers with varicosity-like structures among smooth muscle bundles. Dense branched and tapering nerve fibers with varicosities in the lamina propria mucosae were closely attached to epithelial cells. The acetylcholinesterase-positive fibers in the lamina propria and peri- and para-vascular nerves, and fewer positive fibers in the smooth muscle layer probably represent cholinergic nerves involved in the concentration of biliary compounds and lesser in the motor function of the smooth muscle.
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PMID:Scanning electron microscopic observations of nerves in the guinea-pig gallbladder after an acetylcholinesterase histochemistry. 858 3

The human erythrocyte membrane-bound acetylcholinesterase was characterized with respect to optimal assay conditions for its kinetic properties. It was found that 40.0 micrograms protein and 5.0 min incubation time were the suitable concentration of AChE protein and reaction time for the linearity of AChE activity at 25 degrees C. The Vmax for the AChE was 35% higher in the presence of 25 mM sodium phosphate buffer than 25 mM Tris-HCl buffer. The AChE activity was assayed at various strengths of sodium phosphate buffer (0.0125-0.20 M), and the optimum strength was found to be 0.05 M. The optimum substrate (ASCh) concentration was found to be 5.0 mM whereas at higher substrate concentrations, the AChE activity declined. The ASCh concentration ranges for different orders of the reactions were determined and kinetic parameters (Km, Vmax, Kcat and Ksp) were established at each order of the reaction.
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PMID:Optimization and kinetic studies of human erythrocyte membrane-bound acetylcholinesterase. 882 18

Although the underlying pathogenesis of Alzheimer's disease (AD) is not fully understood, one of its key features is the widespread loss of central cholinergic innervation, known to be fundamental for cognitive processes. This finding led to the hypothesis that pharmacological enhancement of acetylcholine (ACh) neurotransmission may alleviate the symptoms of AD. Currently, cholinergic therapy, particularly cholinesterase (ChE) inhibition, represents the most realistic approach to the symptomatic treatment of AD. Donepezil HCl, for example, is a piperidine-based, reversible acetylcholinesterase (AChE) inhibitor, chemically distinct from other ChE inhibitors and rationally designed for the symptomatic treatment of AD. It is highly selective for centrally acting AChE, with little or no affinity for butyrylcholinesterase, present predominantly in the periphery. Phase I and II clinical trials demonstrated donepezil's favourable pharmacokinetic, pharmacodynamic and safety profile with no requirement for dose modification in the elderly or in patients with renal or hepatic impairment. Furthermore, its long half-life supports a simple and convenient once-daily dosing regimen. Subsequent to encouraging phase II clinical trial results, two pivotal, randomized, double-blind phase III trials (of 15 and 30 weeks' duration) demonstrated highly significant improvements in cognition and global function in mild to moderately severe AD patients treated with either 5 or 10 mg/day donepezil compared with placebo. Adverse events in the phase II and III trials, primarily cholinergic in nature, were transient and generally mild in severity and resolved during continued donepezil administration. Thus, the donepezil clinical trials programme has shown that this drug is a clinically effective and well-tolerated, once-daily treatment for the symptoms of mild to moderately severe AD.
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PMID:Clinical profile of donepezil in the treatment of Alzheimer's disease. 987 15

Nizatidine, a histamine H(2)-antagonist, is known to inhibit acetylcholinesterase (AChE) activity and is used clinically as a gastroprokinetic agent as well as the anti-ulcer agent. We examined whether or not nizatidine stimulates duodenal HCO(3)(-) secretion in rats through vagal-cholinergic mechanisms by inhibiting AChE activity. Under pentobarbital anesthesia, a proximal duodenal loop was perfused with saline, and the HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Nizatidine, neostigmine, carbachol, famotidine or ranitidine was administered i.v. as a single injection. Intravenous administration of nizatidine (3-30 mg/kg) dose-dependently increased the HCO(3)(-) secretion, and the effect at 10 mg/kg was equivalent to that obtained by carbachol at 0.01 mg/kg. The HCO(3)(-) stimulatory action of nizatidine was observed at the doses that inhibited the histamine-induced acid secretion and enhanced gastric motility. This effect was mimicked by neostigmine (0.03 mg/kg) and significantly attenuated by bilateral vagotomy and pretreatment with atropine but not indomethacin. The IC(50) of nizatidine for AChE of rat erythrocytes was 1.4 x 10(-6) M, about 12 times higher than that of neostigmine. Ranitidine showed the anti-AchE activity and increased duodenal HCO(3)(-) secretion, similar to nizatidine, whereas famotidine had any influence on neither AChE activity nor the HCO(3)(-) secretion. On the other hand, duodenal damage induced by acid perfusion (100 mM HCl for 4 h) in the presence of indomethacin was significantly prevented by nizatidine and neostigmine, at the doses that increased the HCO(3)(-) secretion. These results suggest that nizatidine increases HCO(3)(-) secretion in the rat duodenum, mediated by vagal-cholinergic mechanism, the action being associated with the anti-AChE activity of this agent.
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PMID:Bicarbonate stimulatory action of nizatidine, a histamine H(2)-receptor antagonist, in rat duodenums. 1159 32

Recent evidence indicates that stimulation of postsynaptic 5-HT(1A) receptors abates excitotoxic neuronal death. Here we investigated whether oral post-lesion administration of the 5-HT(1A) receptor agonist (-)-(R)-2-[4-[[(3,4-dihydro-2H-1-benzopyran-2-yl)methyl]amino]butyl]-1,2-benzisothiazol-3(2H)-one 1,1-dioxide monohydrochloride (Repinotan HCl) attenuates N-methyl-D-aspartate (NMDA) excitotoxicity (60 nmol/microl) in the rat magnocellular nucleus basalis. Repinotan HCl (1 mg/kg) was administered from day 1, 2, 3, or 6 post-surgery twice daily for five consecutive days. This delayed drug administration protocol was employed to investigate the initiation period during which 5-HT(1A) receptor agonists may significantly influence ongoing neurodegeneration processes. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 1 mg/kg) served as reference compound. Twenty-four hours after drug delivery a small open-field test, while on day 14 post-surgery a passive avoidance test was performed. Effects of Repinotan HCl treatment on the survival of cholinergic magnocellular nucleus basalis neurons and their cortical projections were determined by quantitative acetylcholinesterase (AChE) and choline-acetyltransferase (ChAT) histochemistry. Moreover, AChE and ChAT activities were biochemically measured both in the cerebral cortex and in the magnocellular nucleus basalis. Repinotan HCl treatment markedly increased spontaneous activities in the small open-field at any time-point investigated. Improved memory performance was only demonstrated when Repinotan HCl was administered from day 1 post-lesion on wards. Repinotan HCl treatment from day 2 and 3 post-lesion on markedly attenuated both histochemical and neurochemical characteristics of NMDA excitotoxicity on cholinergic magnocellular nucleus basalis neurons and on their cortical projections. Whereas the neuroprotective profile of Repinotan HCl was superior to that of 8-OH-DPAT, oral administration of both 5-HT(1A) receptor agonists yielded largely equivalent behavioral recovery after NMDA infusion in the magnocellular nucleus basalis. In conclusion, the present data indicate the potent neuroprotective action of the 5-HT(1A) receptor agonist Repinotan HCl with a peak efficacy of delayed (2-3 day) post-lesion drug treatment in vivo. Post-lesion treatment with 5-HT(1A) receptor agonists may therefore be of significance in the intervention of neuronal damage associated with acute excitotoxic conditions.
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PMID:Oral post-lesion administration of 5-HT(1A) receptor agonist repinotan hydrochloride (BAY x 3702) attenuates NMDA-induced delayed neuronal death in rat magnocellular nucleus basalis. 1173 99

In a dialysis procedure not requiring perfusate addition of acetylcholinesterase inhibitors to "boost" basal levels of acetylcholine (ACh), the influence of the antiparkinson agent piribedil upon levels of ACh in frontal cortex and dorsal hippocampus of freely moving rats was compared with those of other antiparkinson drugs and selective ligands at alpha(2)-adrenoceptors (ARs). Suggesting a tonic, inhibitory influence of alpha(2A)-ARs upon cholinergic transmission, the alpha(2)-AR agonist 5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline tartrate (UK14,304), and the preferential alpha(2A)-AR agonist guanabenz reduced levels of ACh. They were elevated by the antagonists 2(2-methoxy-1,4 benzodioxan-2-yl)-2-imidazoline HCl (RX821002) and atipamezole and by the preferential alpha(2A)-AR antagonist 2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidazole (BRL44008). In contrast, trans-2,3,9,13b-tetrahydro-1,2-dimethyl-1H-dibenz[c,f]imidazo[1,5-a]azepine (BRL41992) and prazosin, preferential alpha(2B/2C)-AR antagonists, were inactive. The dopaminergic agonist and antiparkinson agent piribedil, which behaves as an antagonist at alpha(2)-ARs, dose dependently increased extracellular levels of ACh. This action was absent upon pretreatment with a maximally effective dose of RX821002. On the other hand, a further dopaminergic agonist and antiparkinson agent, talipexole, which possesses agonist properties at alpha(2)-ARs, dose dependently reduced levels of ACh. This action was also blocked by RX821002. In contrast to piribedil and talipexole, quinelorane, which interacts with dopaminergic receptors but not alpha(2)-ARs, failed to affect ACh levels. Finally, in analogy to the frontal cortex, piribedil likewise elicited a dose-dependent increase in extracellular levels of ACh in the dorsal hippocampus. In conclusion, in distinction to talipexole and quinelorane, and reflecting its antagonist properties at alpha(2A)-ARs, piribedil reinforces cholinergic transmission in the frontal cortex and dorsal hippocampus of freely moving rats. These actions may be related to its facilitatory influence upon cognitive function.
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PMID:Piribedil enhances frontocortical and hippocampal release of acetylcholine in freely moving rats by blockade of alpha 2A-adrenoceptors: a dialysis comparison to talipexole and quinelorane in the absence of acetylcholinesterase inhibitors. 1264 87


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