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)

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

The delayed neurotoxic organophosphate [3H]diisopropylfluorophosphate ([3H]DFP) binds with high affinity to membrane-bound proteins from the chicken spinal cord. The DFP binding proteins were solubilized from membrane preparations, using a detergent (CHAPS). The protein(s) sites that labeled with a low concentration of [3H]DFP, e.g. 10(-10)-10(-9) M, were defined as the high-affinity binding sites. The density (or concentration) of the high-affinity binding sites in protein(s) was determined by the difference between total and non-specific binding. The high-affinity binding sites were saturable, and the maximal amount of binding sites was estimated at 400 fmol/mg protein. [3H]DFP binding to solubilized proteins was not completely reversible. Concentration-dependent curves suggested that the [3H]DFP binding sites differ from the active sites of acetylcholinesterase, butyrylcholinesterase, and neuropathy target esterase, as well as from muscarinic acetylcholine receptors. The amount of DFP binding sites after a neurotoxic dose of tri-o-cresyl phosphate (TOCP) decreased markedly in membrane preparations from the chicken spinal cord. These results indicate that a TOCP metabolite(s) interacts with the DFP binding sites in vivo. Gel filtration chromatography of the solubilized membranes indicated at least two major high-affinity DFP binding proteins with apparent molecular weights of 300 and 110 kDa. The DFP binding sites corresponding to the 110 kDa protein were insensitive to eserine, a potent anti-cholinesterase agent.
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PMID:Characterization of high-affinity binding sites for diisopropylfluorophosphate (DFP) from chicken spinal cord membranes. 780 97

The effects of a subacute intoxication with diisopropyl fluorophosphate (DPF) on total muscarinic acetylcholine receptor sites (mAChRs) and M-1 AChRs were evaluated in the cerebral cortex of young (2-4 months) and aged (22-24 months) Fischer 344 rats. Since M-1 AChRs are coupled to the metabolism of phosphoinositides, carbachol-induced accumulation of inositol phosphates (IP) and its inhibition by glutamate and NMDA was also measured in the cortical slices. DFP treatment caused about 75% inhibition of cholinesterase and 35% down-regulation of mAChRs (measured as [3H]quinuclidinyl benzylate binding) in both young and aged rats. The down-regulation of M-1-ACHRs (measured as [3H]pirenzepine binding) was more pronounced in aged (30%) than in young (17%) DFP-treated rats. There was a significant increase in carbachol-induced IP accumulation in aged, with respect to young, untreated rats. DFP treatment caused a considerable decrease in such IP accumulation in aged but not in young rats. Glutamate and NMDA antagonized carbachol-induced IP accumulation in untreated young and aged rats (and the effects of NMDA were reversed by carboxy-piperazinyl-propyl phosphonic acid). In DFP-treated rats such antagonism was somewhat less pronounced. The data appear of interest in relation to the use of anticholinesterase compounds in the therapy of senile dementia of Alzheimer's type. They suggest that beside their primary action (increasing brain ACh levels) such compounds also act on post-receptor mechanisms and on the interactions between cholinergic and glutamatergic neurotransmitter systems.
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PMID:Carbachol-induced accumulation of inositol phosphates and its modulation by excitatory amino acids in cortical slices of young and aged rats with down-regulation of muscarinic M-1 receptors. 789 49

Administration of the irreversible cholinesterase inhibitor isoflurophate (diisopropylfluorophosphate, DFP) before 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) enhanced the loss in tyrosine hydroxylase activity and dopamine and 3,4-dihydroxy-phenylacetic acid content in the striatum of mice in a dose-dependent manner. The effect of DFP on the MPTP-induced changes of dopaminergic markers was evident 30 days after initiating treatment, suggesting augmented neurotoxicity. Neurotoxicity was also enhanced by prior treatment with nicotine, carbachol or oxotremorine. We conclude that activation of either muscarinic or nicotinic receptors enhances the neurotoxicity of MPTP.
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PMID:Enhanced MPTP neurotoxicity after treatment with isoflurophate or cholinergic agonists. 791 17

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

Some compounds that inhibit acetylcholinesterase (AChE) activity compete directly with quinuclidinyl benzilate (QNB) binding, a muscarinic antagonist which binds to all subtypes equally, and with cis-methyldioxolane (CD), an agonist that binds with high affinity to the M2 subtype of muscarinic receptors. The relationship between inhibition of AChE activity and the capability to affect muscarinic receptors directly has not been systematically explored. The interaction of eight organophosphates with muscarinic receptors was compared to their ability to inhibit AChE activity in vitro in tissue homogenates from rat hippocampus and frontal cortex, two cholinergically enriched areas of the brain. Of the compounds tested only echothiophate competed for [3H]QNB binding and only at concentrations greater than 100 microM. The anticholinesterase compounds were also tested for their ability to compete with a muscarinic receptor agonist, [3H]CD, which binds with high affinity (approximate KD = 3.5 nM) to 10 and 3% of the muscarinic receptors in the frontal cortex and hippocampus, respectively. The anticholinesterase compounds inhibited high-affinity [3H]CD binding up to 80% and the effects were similar in both tissues. Echothiophate and DFP were potent inhibitors of [3H]CD binding, as were the active "oxon" forms of parathion, malathion, and disulfoton. The parent "thio" forms of these insecticides, however, were much less effective in competing for [3H]CD binding. A similar pattern of potency was observed for the inhibition of brain AChE activity. A strong correlation was found between the ability of a compound to inhibit AChE activity and the ability to compete with [3H]CD binding. These data suggest that the biological effects of cholinesterase-inhibiting compounds may be due to more than their ability to inhibit AChE.
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PMID:Correlation of the anticholinesterase activity of a series of organophosphates with their ability to compete with agonist binding to muscarinic receptors. 821 12

31P NMR spectroscopy of butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and chymotrypsin (Cht) inhibited by pinacolyl methylphosphonofluoridate (soman), methylphosphonodifluoridate (MPDF), and diisopropyl phosphorofluoridate (DFP) allowed direct observation of the OP-linked moiety of aged (nonreactivatable) and nonaged organophosphorus (OP)-ChE conjugates. The 31P NMR chemical shifts of OP-ChE conjugates clearly demonstrated insertion of a P-O- bond into the active site of aged OP-ChE adducts. The OP moiety of nonaged OP-ChEs was shown to be uncharged. The OP-bound pinacolyl moiety of soman-inhibited and aged AChE was detached completely, whereas only partial dealkylation of the pinacolyl group was observed for soman-inhibited BChEs. This suggests that the latter enzyme reacted with the less active stereoisomer(s) of soman. In the case of soman-inhibited Cht, no dealkylation could be experimentally detected for any of the four stereoisomers of OP-Cht adducts. Results are consistent with the contention that the phenomenon of enzyme-catalyzed dealkylation of OP adducts of serine hydrolases strongly depends on the orientation of both the catalytic His and the carboxyl side chain of either Glu or Asp positioned next to the catalytic Ser. The denatured protein of aged OP-ChE or OP-Cht is a convenient leaving group in nucleophilic displacements of tetrahedral OP compounds despite the presence of a P-O- bond. This indicates that the unusual resistance to reactivation of the aged enzyme cannot be ascribed to simple electrostatic repulsion of an approaching nucleophile. The broadening of the 31P NMR signal of native OP-ChEs relative to that of OP-Cht is in agreement with the crystal structure of AChE, showing that the active site region of ChEs in solution resides in a deep, narrow gorge.
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PMID:Direct observation and elucidation of the structures of aged and nonaged phosphorylated cholinesterases by 31P NMR spectroscopy. 825 80

The influence of genotypes of the major histocompatibility complex (MHC) on susceptibility to acute and delayed effects of an organophosphorus ester was measured in adult White Leghorn chickens from lines differing in response to sheep red blood cell (SRBC) antigen. Chickens from lines selected for high (HA) or low (LA) antibody response to SRBC and homozygous for B13B13 or B21B21 genotypes at the MHC were administered a single subcutaneous injection of diisopropyl phosphorofluoridate (DFP) at dosages of 0, 0.25, 0.50, or 1.0 mg/kg body weight using corn oil as the carrier. Criteria for toxicological responses included clinical, biochemical, and pathological measures. Clinical signs of acute cholinergic poisoning and delayed neuropathy were dose related. Brain and blood cholinesterase and carboxylesterase activities were more sensitive to inhibition by DFP than were liver cholinesterase and carboxylesterase activities. Cholinergic signs 3 h after administration of DFP were more pronounced in line HA than in line LA chickens. Pathological evidence of delayed neuropathy 2 wk after DFP administration was also more evident in HA than LA chickens. Although less pronounced than that for lines, differences in neurotoxic manifestations following DFP administration were greater for chickens of B21B21 than B13B13 genotypes. Activity of A-esterases, which hydrolyze organophosphorus esters without being inhibited by them, was lower in plasma of line HA than line LA chickens. Differences among the genotypes in activity of other esterases were not found in chickens not receiving DFP. These results indicated that responses of chickens to the neurotoxicant DFP were influenced by the background genome of the chickens.
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PMID:Differences between genetic stocks of chickens in response to acute and delayed effects of an organophosphorus compound. 834 37

Neuroblastoma cell lines were used to examine the differential interspecies response (i.e., species selectivity) to organophosphates (OPs). Baseline activities of the major target esterases, i.e., cholinesterase, carboxylesterase, and neurotoxic esterase, were assayed in mouse and several human neural candidate cell lines. These activities were found to be variable within individual cell lines and among the various tested cell lines. Cytotoxicity data using the neutral red fluorometric assay were collected on both human (SH-SY5Y) and mouse (NB41A3) neuroblastoma clones exposed to a variety of OP insecticides. IC50 data indicated that the tested mouse cell line was consistently more sensitive than the human cell line to equimolar doses of various OP compounds (e.g., mipafox, parathion, paraoxon, DFP, leptophos oxon, fenthion, and fenitrothion). This difference in cytotoxic sensitivity was most pronounced in response to compounds requiring metabolic bioactivation (i.e., protoxicants). Cytotoxicity data also demonstrated that the NB41A3 mouse neuroblastoma cell line was more metabolically competent than the SH-SY5Y human cell line in converting the protoxicant parathion to its neurotoxic metabolite, paraoxon. B-lymphoblastoids, genetically engineered with human P450 cDNAs, demonstrated higher cytotoxic sensitivity to parathion than unengineered cells, indicating that cytochrome P450-associated monooxidase activity could also influence cytotoxic sensitivity to parathion in culture. These data suggest that interspecies-selectivity in response to OP-related cytotoxicity is influenced by intercellular differences in metabolism and baseline esterase activities.
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PMID:Differential cytotoxic sensitivity in mouse and human cell lines exposed to organophosphate insecticides. 851 93

The serum cholinesterase (ChE) isoenzyme in rats shows 6 bands after polyacrylamide gradient gel electrophoresis. The effects of organophosphates (fenthion, chlorpyrifos, diazinon, bromophos, propaphos, haloxon, and DFP) on serum ChE isoenzyme bands were studied in 32 male and 32 female 6-w-old Sprague-Dawley rats. Each organophosphate was randomly administered to 4 male and 4 female rats. Blood samples were collected from the abdominal aorta under halothane anesthesia 6 h after dosing. The isoenzyme patterns were determined simultaneously with erythrocyte and serum ChE activities. Changes were observed in all 6 bands of the serum ChE isoenzymes after administration of fenthion, chlorpyrifos and propaphos. Diazinon had no influence on band 6, and DFP and bromophos had no influence on band 5. Haloxon did not effect any of the serum ChE isoenzyme bands. Serum ChE was most suppressed by fenthion, followed by DFP, bromophos, chlorpyrifos, propaphos and diazinon in that order of effect. Serum ChE activity was not suppressed by haloxon. Erythrocyte ChE activity was suppressed by every organophosphate. This experiment demonstrated a correlation between the organophosphate suppression of serum ChE activity and the concentration of serum ChE isoenzyme band 6.
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PMID:Effects of selected organophosphate insecticides on serum cholinesterase isoenzyme patterns in the rat. 872 19


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