Stability Of Acetylcholinesterase Research Articles

Acetylcholinesterase entrapment onto carboxyl-modified single-walled carbon nanotubes and poly (3,4-ethylenedioxythiophene) nanocomposite, film electrosynthesis characterization, and sensor application for dichlorvos detection in apple juice

Carboxylic acid functionalized single-walled carbon nanotubes and poly (3,4-ethylenedioxythiophene), C-SWCNT:PEDOT, were homogenously electrodeposited onto working electrodes (WE) in order to develop an acetylcholinesterase (AChE)-based biosensor. RAMAN, FTIR, SEM, EDX and electrochemical methods provided evidence for the enzyme immobilization and the conducting composite formation. The SWCNT dissolved in poly (sodium 4-styrenesulphonate) (PSS), and 3,4-ethylenedioxythiophene (EDOT) dissolved in polyethyleneglicol (PEG) were used for composite electrodeposition to provide a homogenous layer and a compatible environment around the AChE. The composite C-SWCNT:PEDOT has proved to be highly efficient for retaining the activity and stability of AChE and also for promoting the electron transfer reaction to the gold electrode. Under optimized experimental conditions, the inhibition of dichlorvos (DVSO) on AChE showed a proportional inhibition current signal with increasing concentrations of DVSO in the range 1 ppb to 600 ppb. The correlation coefficient obtained with spectrophotometer enzyme assay method versus a biosensor was 0.989. It showed high accuracy, precision. The detection limits were 5.54 ppb using the CA method and 0.447 ppb using differential pulse voltammetry (DPV). Both values are below the permitted limit for DVSO in fruit and vegetables. The sensor showed stability and could be used as a disposable amperometric sensor with potential application for monitoring organophosphorus pesticides.

MODELING AND STRUCTURAL ANALYSIS OF ACETYLCHOLINESTERASE ENZYME OF FISHES

Objective: Computational studies on fish brain acetylcholinesterase were conducted, expanding our views and for a deeper understanding of the activity of the fish acetylcholinesterase enzyme.
 Methods: Physico-chemical properties of the fish acetylcholinesterase enzyme were studied. Homology model of the acetylcholinesterase enzyme was predicted, validated its quality and active sites were predicted. The amino acid frequency in the active sites was also compared. Similarly, the secondary structure of the sequences was predicted and compared. Phylogenetic analysis was performed by the neighbour joining tree method.
 Results: Among the selected fish species stability of acetylcholinesterase was found in fish species namely Esox lucius. The negative GRAVY score value of enzyme in all the fish species ensured better interaction and activity in the aqueous phase. It was found that the molecular weight of the acetylcholinesterase enzyme ranged between 9113 and 15991 Da. Iso-electric (pI) of acetylcholinesterase was found to be acidic in nature. GOR IV was used to predict the secondary structure of acetylcholinesterase, which showed that random coil was dominated. Neighbor joining tree of the enzyme showed that fish species named Amphiprion ocellaris as the most divergent species, while the species Oreochromis niloticus is the most primitive one.
 Conclusion: Acetlycholinesterase enzyme of Esox lucius was found to be the best compared to the other species, which possess a high number of active sites with Ile, Set and Glu rich active sites.

An electrochemical sensor modified with poly(3,4-ethylenedioxythiophene)-wrapped multi-walled carbon nanotubes for enzyme inhibition-based determination of organophosphates

We report on an acetylcholinesterase (AChE) based sensor for the determination of organophosphates (OPs). A nanocomposite consisting of poly(3,4-ethylenedioxythiophene) (PEDOT; a conducting polymer) and functionalized multi-walled carbon nanotubes (f-MWCNTs) was used as a host matrix for covalent immobilization of AChE. The unique properties of PEDOT and f-MWCNTs proved to be highly efficient for retaining the activity and stability of AChE. The effects of pH value of the phosphate buffer, concentration of enzyme, substrate concentration and incubation time were optimized using chlorpyrifos-methyl as a model OP as it exerts a strong inhibitory effect on AChE. Differential pulse voltammetry (DPV) analysis performed at +0.15 V revealed a linear relation of current in respect to chlorpyrifos-methyl concentration range (0.001 to 50 ppb) with 0.001 ppb as a detection limit. The sensor can be regenerated to 97% of its activity by applying 2-pyridine aldoxime methiodide (2-PAM) as a regenerating agent. It can be re-used up to six times and stored up to one month. The sensor was applied to the determination of chlorpyrifos-methyl in spiked lettuce where it gave recoveries ranging between 95 and 97%.

Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits

The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.

Acetylcholinesterase immobilized onto PEI-coated silica nanoparticles

Polyethyleneimine (PEI) coated-silica nanoparticles were prepared by the Stöber method. The formation and the structure of the nanoparticles were characterized by ATR-FT-IR spectroscopy and transmission electron microscopy (TEM). TEM images of the silica and PEI-coated nanoparticles revealed that they were well dispersed and that there was no agglomeration. The acetylcholineesterase enzyme was immobilized onto these nanoparticles. The effects of pH and temperature on the storage stability of the free and immobilized enzyme were investigated. The optimum pHs for free and immobilized enzymes were determined as 7.0 and 8.0, respectively. The optimum temperatures for free and immobilized enzymes were found to be 30.0 and 35.0°C, respectively. The maximum reaction rate (V max) and the Michaelis-Menten constant (K m) were investigated for the free and immobilized enzyme. The storage stability of acetylcholinesterase was increased when immobilized onto the novel PEI-coated silica nanoparticles. The reuse numbers of immobilized enzyme were also studied. These hybrid nanoparticles are desirable as carriers for biomedical applications.

Acetylcholinesterase Mobility and Stability at the Neuromuscular Junction of Living Mice

Acetylcholinesterase (AChE) is an enzyme that terminates acetylcholine neurotransmitter function at the synaptic cleft of cholinergic synapses. However, the mechanism by which AChE number and density are maintained at the synaptic cleft is poorly understood. In this work, we used fluorescence recovery after photobleaching, photo-unbinding, and quantitative fluorescence imaging to investigate the surface mobility and stability of AChE at the adult innervated neuromuscular junction of living mice. In wild-type synapses, we found that nonsynaptic (perisynaptic and extrasynaptic) AChEs are mobile and gradually recruited into synaptic sites and that most of the trapped AChEs come from the perijunctional pool. Selective labeling of a subset of synaptic AChEs within the synapse by using sequential unbinding and relabeling with different colors of streptavidin followed by time-lapse imaging showed that synaptic AChEs are nearly immobile. At neuromuscular junctions of mice deficient in alpha-dystrobrevin, a component of the dystrophin glycoprotein complex, we found that the density and distribution of synaptic AChEs are profoundly altered and that the loss rate of AChE significantly increased. These results demonstrate that nonsynaptic AChEs are mobile, whereas synaptic AChEs are more stable, and that alpha-dystrobrevin is important for controlling the density and stability of AChEs at neuromuscular synapses.

Acetylcholinesterase alterations reveal the fitness cost of mutations conferring insecticide resistance

BackgroundInsecticide resistance is now common in insects due to the frequent use of chemicals to control them, which provides a useful tool to study the adaptation of eukaryotic genome to new environments. Although numerous potential mutations may provide high level of resistance, only few alleles are found in insect natural populations. Then, we hypothesized that only alleles linked to the highest fitness in the absence of insecticide are selected.ResultsTo obtain information on the origin of the fitness of resistant alleles, we studied Drosophila melanogaster acetylcholinesterase, the target of organophosphate and carbamate insecticides. We produced in vitro 15 possible proteins resulting from the combination of the four most frequent mutations and we tested their catalytic activity and enzymatic stability. Mutations affected deacetylation of the enzyme, decreasing or increasing its catalytic efficiency and all mutations diminished the stability of the enzyme. Combination of mutations result to an additive alteration.ConclusionOur findings suggest that the alteration of activity and stability of acetylcholinesterase are at the origin of the fitness cost associated with mutations providing resistance. Magnitude of the alterations was related to the allelic frequency in Drosophila populations suggesting that the fitness cost is the main driving force for the maintenance of resistant alleles in insecticide free conditions.

Development of sensors for direct detection of organophosphates. Part I: immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (<1 mm 2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol–gel process to create highly porous and stable silica matrices. Surface porosity of sol–gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol–gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-γ-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol–gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.

Effect of mutations within the peripheral anionic site on the stability of acetylcholinesterase.

Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the "peripheral" anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4'-dithiodipyridine. The wild-type and mutated enzymes could be either protected or sensitized. In some cases, opposite effects of the same ligand were observed for chemical modification and thermal denaturation. The mutated residues are within a conserved loop, W279-S291, at the top of the active-site gorge, that contributes to the peripheral anionic site. Theoretical analysis showed that Torpedo acetylcholinesterase consists of two structural domains, each comprising one contiguous polypeptide segment. The W279-S291 loop, located in the first domain, makes multiple contacts with the second domain across the active-site gorge. We postulate that the mutations to residues with smaller side chains destabilize the conserved loop, thus disrupting cross-gorge interactions and, ultimately, the entire structure.

The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes.

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octyl beta-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70-80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.

Stability of acetylcholinesterase in guanidine hydrochloride solution.

The irreversible unfolding of acetylcholinesterase (acetylcholine hydrolyase, EC 3.1.1.7) by guanidine hydrochloride was studied by difference spectral, circular dichroic, and enzyme activity measurements. At pH 7.0 and in 1.1 M denaturant solution, a conformational state in which enzyme is completely inactive was detected. It is identical to the native enzyme as far as sedimentation coefficient and molecular weight are concerned, but differs from the native molecule by a slight loss in secondary structure and by a small perturbation of aromatic residues. Acetylcholinesterase in concentrated guanidine hydrochloride solution containing beta-mercaptoethanol dissociates and exists as a random coil of molecular weight 68 000.