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)

1. Polyacrylamide gel electrophoresis in Tris/glycine buffer (pH 8.3) revealed five forms of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in the 100 000 X g, 1-h supernatants of aqueous fly-head extracts from the DDT/S strain. Five other housefly strains (CSMA, Bayer 21/199, Cradson/P, Malathion/R and DDT/R)were shown qualitatively to have the same soluble forms of the enzyme. 2. Plots of the electrophoretic mobility versus polyacrylamide concentration indicated that the multiple forms constituted a size isomer family. From the retardation coefficients derived from these plots, molecular weight estimates were obtained; these suggested that the smallest active component was a form of approx. 80 000 daltons. The higher aggregates, however, did not appear as simple oligomers of this component. 3. Density gradient sedimentation supported the electrophoretic findings. The smallest active component, with a sedimentation coefficient of 5.3 S, was confirmed as a molecular species of acetylcholinesterase that has not previously been obtained from house-flies; higher aggregates gave sedimentation coefficients of 7.4, 7.8. 8.1, and 11.8 S. 4. Gel-filtration on calibrated Sephadex G-150 columns provided further evidence that the smallest active component was a form of about 80 000 daltons. 5. Autolysis converted much of the particulate enzyme and all of the soluble forms into a species of approx. 160 000 daltons indistinguishable from the native 7.4-S form. Both the autolysed enzyme and the native 7.4-S form were susceptible to cleavage by disulphide reducing agents, and released catalytically active subunits that corresponded to the 5.3-S form of 80 000 daltons. The data were compatible with a monomer-dimer relationship between the 5.3-S and 7.4-S forms. 6. The possibility is suggested that a form of molecular weight approx. 80 000 constitutes the "fundamental unit" of insect cholinesterase.
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PMID:Acetylcholinesterase from the house-fly head. Molecular properties of soluble forms. 95 30

We report Raman spectra of various cholinesterases: lytic tetrameric forms (G4) obtained by tryptic digestion of asymmetric acetylcholinesterase (AChE) from Torpedo californica and Electrophorus electricus, a PI-PLC-treated dimeric form (G2) of AChE from T marmorata, and the soluble tetrameric form (G4) of butyrylcholinesterase (BuChE) from human plasma. The contribution of different types of secondary structure was estimated by analyzing the amide I band, using the method of Williams. The spectra of cholinesterases in 10 mM Tris-HCl (pH 7.0) indicate the presence of both alpha-helices (about 50%) and beta-sheets (about 25%), together with 15% turns and 10% undefined structures. In 20 mM phosphate buffer (pH 7.0), the spectra indicated a smaller contribution of alpha-helical structure (about 35%) and an increased beta-sheet content (from 25 to 35%). This shows that the ionic milieu profoundly affects either the conformation of the protein (AChE activity is known to be sensitive to ionic strength), or the evaluation of secondary structure, or both. In addition, we analyzed vibrations corresponding to the side chains of aromatic and aliphatic amino acids. In particular, the analyses of the tyrosine doublet (830-850 cm-1) and of the tryptophan vibration at 880 cm-1 indicated that these residues are predominantly 'exposed' on the surface of the molecules.
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PMID:A comparative Raman spectroscopic study of cholinesterases. 179 30

The ligand binding and kinetic behaviour of butyrylcholinesterase (EC 3.1.1.8, acylcholine acylhydrolase) from human plasma was studied at 35 degrees C under high hydrostatic pressure. The binding of phenyltrimethylammonium was studied by affinity electrophoresis at various pressures ranging from 10(-3) to 2 kbar. The kinetics of enzyme carbamylation with N-methyl(7-dimethylcarbamoxy)quinolinium iodide was studied in single-turnover conditions up to 1.2 kbar using a high-pressure stopped-flow fluorimeter. Experiments were carried out in different media: 1 mM Tris-HCl (pH 8) with water, water containing 0.1 M lithium chloride and deuterium oxide as solvents. The volume changes (delta V and delta V++) associated with each process were determined from the pressure-dependence of the binding and kinetic constants. Kinetic data show that the binding of substrate to the enzyme leads to a pressure-sensitive enzyme conformational state which cannot accomplish the catalytic act. The pressure-induced inhibitory effect is highly cooperative; it depends on both the nature (charged or neutral) and the concentration of the substrate. Also, large solvent effects indicate that enzyme sensitivity to pressure depends on the solvent structure. This findings suggests that the substrate-dependent pressure effect is modulated by the solvation state of the enzyme.
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PMID:Conformational plasticity of butyrylcholinesterase as revealed by high pressure experiments. 226 67

Octadecyl-bonded silica, commonly used for reverse-phase high-pressure liquid chromatography, was modified using surfactants bearing ionizable groups and the modified packing used in ion-exchange chromatography of proteins. The surfactants 2-(n-hexadecylheptaethoxy)acetic acid, 1-(n-hexadecyloctaethoxy)ethylene-diamine, and N-(n-hexadecyloctaethoxy)pyridinium were adsorbed onto test columns packed with octadecyl-bonded silica particles. The proteins lysozyme, bovine serum albumin, trypsin, horse serum cholinesterase, and bovine liver carboxylesterase were used to study the ion-exchange characteristics of the modified packings. The retention order of the proteins on the surfactant-modified stationary phases were as predicted by the isoelectric point of each protein. In addition, the interaction of enzymes with the packings did not result in significant loss of enzymatic activity. Surfactant removal was possible with the use of organic solvents and this allowed the octadecyl-bonded surface to be used again in the reverse-phase mode. During the course of the experiments, no degradation in the packing's performance was observed due to loss of adsorbed surfactant, even after over 85,000 column volumes of sodium chloride and Tris-HCl buffers were circulated through the column.
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PMID:Reversible conversion of octadecyl-bonded silica to ion-exchange surfaces for protein separations. 254 Jun 75

We have studied the ionic and pharmacological properties of an inward current elicited by edrophonium, a cholinesterase inhibitor, on physically isolated and internally perfused Aplysia neurons using the voltage clamp, internal perfusion and rapid external perfusion techniques. The current amplitude was dependent on the external Na concentration [(Na)o] in an almost linear manner. However, complete replacement of (Na)o with Tris or sucrose failed to abolish the current. Internal application of Na [increased (Na)i] reduced the current amplitude. In normal (Na)o, changing (Ca)o (both increases and decreases in (Ca)o) reduced the current amplitude. In the sucrose-substituted (Na)o-free condition, edrophonium still could cause a small current (less than 5% of the control). However, an increased (Ca)o did not augment this residual current. Cs and Li carried the edrophonium-activated current when substituted for (Na)o. With sucrose-substituted Na-free sea water outside, edrophonium elicited an outward current when the neuron was internally perfused with Cs, but not when the neuron was internally perfused with K. Therefore, it is unlikely that K is permeant. External application of tetrodotoxin, a blocker of voltage-dependent Na channels, external application of Cd and internal application of F did not affect the current. The edrophonium response was most sensitive to strychnine, which was about 10 times more potent than D-tubocurarine. Hexamethonium, however, had no effect. The local anesthetics, lidocaine and procaine, inhibited the response over the same concentration range as D-tubocurarine. We conclude that edrophonium opens a monocationic channel (presumably a type of Na channel) which is sensitive to (Ca)o.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of an inward current elicited by edrophonium in physically isolated and internally perfused Aplysia neurons. 319 1

An antigenic secretory protein with cholinesterase activity was isolated from the excretory gland cells of Stephanurus dentatus and was purified by gel filtration and ion exchange chromatography. The antigenicity of the cholinesterase was demonstrated by an esterase-active immunoprecipitate formed with S. dentatus antiserum and by the ability of the antiserum to protect the enzyme from heat inactivation. The enzyme was found to be secreted by the adult nematodes during in vitro cultivation. The level of cholinesterase activity and its release from the excretory gland cells of the parasite were 27-fold greater in the male than in the female. Ninety per cent of the enzyme activity was localized in the soluble fraction of the gland cells. The molecular weight of the enzyme, estimated by sucrose density gradient centrifugation, was 100,000. Two molecular forms were separated by isoelectrofocusing, with isoelectric points of 7.0 and 6.9. At optimum substrate concentrations, the rate of hydrolysis of acetylthiocholine was 8 times greater than that of butyrylthiocholine; the Michaelis constants were 560 microM and 81 microM for acetylthiocholine and butyrylthiocholine, respectively. The enzyme exhibited substrate inhibition at substrate concentrations greater than 10 mM and was inhibited by eserine sulfate, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, Tris, and acetone. The enzyme was highly unstable in dilute protein solutions.
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PMID:Cholinesterase in the parasitic nematode, Stephanurus dentatus. Characterization and sex dependence of a secretory cholinesterase. 679 May 50

The 2-chloro-12-(2-piperidinoethyl)-dibenzo[d,g] (1,3,6)-dioxazocine . HCl (EGYT-2347), a new specific inhibitor of butyrylcholinesterase inhibits reversibly and non-competitively the enzymatic hydrolysis of butyrylthiocholine iodide (Ki = 0.15 microM, at 37 degrees C in 0.1 M Tris/HCl, pH 7.5). The theoretical progress curve of product accumulation has been developed for the case when a non-competitive reversible inhibitor (EGYT-2347) and an active-site-directed irreversible inhibitor (diisopropylfluorophosphate) act simultaneously. By the aid of this approach it was concluded that the butyrylcholinesterase--EGYT-2347 binary complex does not react with diisopropylfluorophosphate either because of a structural change caused by binding or by the direct steric hindrance of EGYT-2347.
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PMID:Change in the reactivity of the active-site serine OH of butyrylcholinesterase caused by a new reversible inhibitor. 688 55

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

Raman spectra of human butyrylcholinesterase (BuChE; E.C. 3.1.1.8) were analyzed in the native state and after conjugation with organophosphates (soman, DFP and paraoxon). The secondary structure of the native BuChE in Tris-HCl buffer (pH 7.5), determined from analysis of the amide I polypeptide vibration band, indicates 47% alpha-helices, 26% beta-sheets, 16% turns and 12% undefined structure. We obtained the same values for paraoxon-phosphorylated BuChE, but 39% helical structure, 31% beta-sheets, 17% turns and 13% undefined structure for 'aged' DFP-BuChE conjugates and 36% helical structure, 34% beta-sheets, 20% turns and 10% undefined structure for 'aged' soman-BuChE conjugates. The approximately 10% decrease of alpha-helical structure observed upon phosphorylation by DFP and phosphonylation by soman, probably corresponds to the 'aging' process, which does not take place in the case of paraoxon. Considerable differences have been observed between native, paraoxon inhibited and 'aged' BuChE in aromatic ring vibrations, suggesting that the dealkylation of organophosphate conjugates modifies the environment or the interactions of aromatic amino-acid residues. In the aliphatic side chains an increase of the number of gauche configurations has been observed in 'aged' DFP-BuChE and soman-BuChE.
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PMID:Raman spectroscopic study of conjugates of butyrylcholinesterase with organophosphates. 776 82

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


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