Showing papers on "Acetylthiocholine published in 2019"

Acetylcholinesterase with mesoporous silica: Covalent immobilization, physiochemical characterization, and its application in food for pesticide detection.

Abstract: Toxic contamination of commonly consumed food products and water due to food chain vulnerability via agricultural products and commodities is a serious health hazard. This study reports on Santa Barbara Amorphous (SBA-15), a type of mesoporous silica nanoparticles, for efficient and stable acetylcholinesterase (AChE) adhesion toward detection of toxic pesticides. AChE was immobilized to the inert framework of mesoporous materials viz. SBA-15 with a proficient hydrolytic response toward acetylthiocholine. The immobilized system acts as a biosensor for the detection of pesticides, which are organophosphorus compounds in food. Both the SBA-15 and immobilized SBA-15 were characterized to give an insight on the physiochemical and morphological modification properties. The enzyme activity was accessed by Ellman's spectrophotometric bioassay for bare and enzyme-immobilized SBA-15 that resulted in promising enzymatic activity with the counterpart. Enzyme stability was also studied, which exhibited that immobilized AChE retained its catalytic activity up to 60 days and retained 80% of the hydrolytic activity even at 37°C. On the basis of the success of immobilized enzyme (covalent) being inhibited by acetylthiocholine, the sensor was administered for the inhibition by monocrotophos and dimethoate that are used widely as pesticides in agricultural. The inhibitory concentration (IC50 ) value was found to be 2.5 ppb for monocrotophos and 1.5 ppb for dimethoate inhibiting immobilized AChE. This was verified using cyclic voltammetry, an electrochemical analysis thus proving that the SBA-15@AChE complex could be used as a sensitive and highly stable sensor for detecting the concentration of hazardous pesticide compounds.

A novel cholinesterase assay for the evaluation of neurotoxin poisoning based on the electron-transfer promotion effect of thiocholine on an Au electrode

Abstract: A simple and cost-effective cholinesterase assay is presented. Thiocholine (TCh), which is generated from substrates by the action of cholinesterase, promotes the electron transfer between [Fe(CN)6]3–/4– and Au electrodes. The electric current increases with the TCh concentration; therefore, the current decreases when cholinesterase is inhibited by neurotoxic chemicals, such as organophosphorus and carbamate compounds. First, we monitored the responses of acetylcholinesterase to cholinesterase inhibitors. The substrate acetylthiocholine was digested with acetylcholinesterase in Tris−HCl buffer, then the reaction mixture was combined with acetonitrile. A polished Au electrode was immersed in the mixed solution for 30 s to immobilize the enzymatic hydrolysis product, TCh, on the electrode surface. The electrode was then subjected to differential pulse voltammetry in 0.5 mM [Fe(CN)6]3– in the presence of KCl as a supporting electrolyte, with a concentration as low as 1 mM. The optimum conditions for the enzymatic treatment were for 10 min at 30 °C with 1.25 mU/mL acetylcholinesterase and 100 μM acetylthiocholine as final concentrations. The inhibition of acetylcholinesterase activities from DDVP (organophosphorus) and methomyl (carbamates) could be monitored. This system was applied to human serum samples containing butyrylcholinesterase. The inhibition of butyrylcholinesterase by DDVP and methomyl were successfully monitored even in human serum. Finally, real human blood donated by 10 volunteers were subjected to a cholinesterase assay using this system. The results from the present method showed good correlation with those obtained from the conventional method using 5,5′-dithiobis-(2-nitrobenzoicacid). The present method was considered valid as a novel ChE assay for human blood.

First evidence of cholinesterase-like activity in Basidiomycota

Abstract: Cholinesterases (ChE), the enzymes whose primary function is the hydrolysis of choline esters, are widely expressed throughout the nature. Although they have already been found in plants and microorganisms, including ascomycete fungi, this study is the first report of ChE-like activity in fungi of the phylum Basidiomycota. This activity was detected in almost a quarter of the 45 tested aqueous fungal extracts. The ability of these extracts to hydrolyse acetylthiocholine was about ten times stronger than the hydrolytic activity towards butyrylthiocholine and propionylthiocholine. In-gel detection of ChE-like activity with acetylthiocholine indicated a great variability in the characteristics of these enzymes which are not characterized as vertebrate-like based on (i) differences in inhibition by excess substrate, (ii) susceptibility to different vertebrate acetylcholinesterase and butyrylcholinesterase inhibitors, and (iii) a lack of orthologs using phylogenetic analysis. Limited inhibition by single inhibitors and multiple activity bands using in-gel detection indicate the presence of several ChE-like enzymes in these aqueous extracts. We also observed inhibitory activity of the same aqueous mushroom extracts against insect acetylcholinesterase in 10 of the 45 samples tested; activity was independent of the presence of ChE-like activity in extracts. Both ChE-like activities with different substrates and the ability of extracts to inhibit insect acetylcholinesterase were not restricted to any fungal family but were rather present across all included Basidiomycota families. This study can serve as a platform for further research regarding ChE activity in mushrooms.

Interactions of human butyrylcholinesterase with phenylvalerate and acetylthiocholine as substrates and inhibitors: kinetic and molecular modeling approaches.

Abstract: Phenyl valerate (PV) is a substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Purified human butyrylcholinesterase (hBChE) showed PVase activity with a similar sensitivity to inhibitors as its cholinesterase (ChE) activity. Further kinetic and theoretical molecular simulation studies were performed. The kinetics did not fit classic competition models among substrates. Partially mixed inhibition was the best-fitting model to acetylthiocholine (AtCh) interacting with PVase activity. ChE activity showed substrate activation, and non-competitive inhibition was the best-fitting model to PV interacting with the non-activated enzyme and partial non-competitive inhibition was the best fitted model for PV interacting with the activated enzyme by excess of AtCh. The kinetic results suggest that other sites could be involved in those activities. From the theoretical docking analysis, we deduced other more favorable sites for binding PV related with Asn289 residue, situated far from the catalytic site (“PV-site”). Both substrates acethylcholine (ACh) and PV presented similar docking values in both the PV-site and catalytic site pockets, which explained some of the observed substrate interactions. Molecular dynamic simulations based on the theoretical structure of crystallized hBChE were performed. Molecular modeling studies suggested that PV has a higher potential for non-competitive inhibition, being also able to inhibit the hydrolysis of ACh through interactions with the PV-site. Further theoretical studies also suggested that PV could yet be able to promote competitive inhibition. We concluded that the kinetic and theoretical studies did not fit the simple classic competition among substrates, but were compatible with the interaction with two different binding sites.

Soluble esterases as biomarkers of neurotoxic compounds in the widespread serpulid Ficopomatus enigmaticus (Fauvel, 1923).

Abstract: The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (Km and Vmax) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.

A New Tellurium- and Selenoorganic Compound as an Inhibitor of Acetylcholinesterase in Brain.

Abstract: We studied the effect of a new cyanine dye containing selenium and tellurium on acetylcholinesterase activity in synaptic membrane in rat brain. The cyanine dye dose-dependently inhibits activity of this enzyme, and the concentration of half-maximal inhibition of acetylcholinesterase activity was 20.46 μM. The cyanine dye instantly inhibits the enzyme; the degree of inhibition depends on acetylthiocholine concentration: the lower is acetylthiocholine concentration, the higher is the degree of inhibition. On the Lineweaver-Burk plot, the concentration dependence curves of acetylcholinesterase with and without cyanine dye intersect in one point on the abscissa axis. In this case, the cyanine dye reduces the maximum inhibition rate (Vmax) and does not affect Michaelis constant (Km). The calculated inhibition constant Ki for the cyanine dye is 7.74 μM. Thus, the cyanine dye is a non-competitive inhibitor of acetylcholinesterase.

Disposable, acetylcholinesterase-coated, screen-printed carbon electrodes for the determination of organophosphorus pesticides

Abstract: Because of the bioaccumulation effect, organophosphorus pesticides cause long-term damage to mammals, even at small concentrations. The ability to perturb the phospholipid bilayer structure as well as the overstimulation of cholinergic receptors makes them hazardous to humans. Therefore, there is a need for a quick and inexpensive detection of organophosphorus pesticides for agricultural and household use. As organophosphorus pesticides are acetylcholinesterase (AChE) inhibitors, biosensors using this mechanism hold a great promise to meet these requirements with a fraction of reagents and time used for measurement comparing to laboratory methods. This study aims to manufacture AChE-coated, screen-printed carbon electrodes applicable in such amperometric biosensors.,AChE enzyme, known for catalytic activity for the hydrolysis of acetylthiocholine (ATCh), could be used to obtain electrochemically active thiocholine from acetylthiocholine chloride in aqueous solutions. Using Malathion’s inhibitory effect towards AChE, pesticides’ presence can be detected by reduction of anodic oxidation peaks of thiocholine in cyclic voltammetry.,The conducted research proved that it is possible to detect pesticides using low-cost, simple-to-manufacture screen-printed graphite (GR) electrodes with an enzymatic (AChE) coating. Investigated electrodes displayed significant catalytic activity to the hydrolysis of ATCh. Owing to inhibition effect of the enzyme, amperometric response of the samples decreased in pesticide-spiked solution, allowing determination of organophosphorus pesticides.,Printed electronics has grown significantly in recent years as well as research focused on carbon-based nanocomposites. Yet, the utilization of carbon nanocomposites in screen-printed electronics is still considered a novelty in the market. Biosensors have proved useful not only in laboratory conditions but also in home applications, as glucometers are a superior solution for glucose determination for personal use. Although pesticides could be detected accurately using chromatography, spectroscopy, spectrometry or spectrophotometry, the market lacks low-cost, disposable solutions for pesticide detection applicable for household use. With biosensing techniques and electric paths screen-printed with GR or graphene nanocomposites, this preliminary research focuses on meeting these needs.

Nonenzymatic Hydrolysis of Acetylthiocholine by Silver Nanoparticles

Abstract: Since five decades, acetylthiocholine, an O → S substituted synthetic analogue of the natural neurotransmitter acetylcholine, has become a key element in various assays used for probing the presenc...