Each gradient ramp was made with 2 mL of buffer maintaining 10 mL of buffer between the steps. The separation was carried out at 0. The samples were used for enzymatic characterization as free enzyme and for producing the ICERs. The native—PAGE was accomplished in the absence of denaturing agents 2-mercaptoethanol; and sodium dodecyl sulfate and the samples were not heated prior the run.
After the run, the gel was stained with Coomassie Blue. After gel running, the gel was equilibrated with 0. The gel was incubated for 30 min with 0. The isolated enzymes were identified by mass spectrometry from gel-tryptic digestion. To do this, sample bands were excised from Coomassie stained native-PAGE and were tryptic cleaved [ 34 ]. Databases with different numbers of sequences were used to increase the protein identification confidence.
Phylogenetic trees were constructed by the Phylogeny. The cholinesterase activity was evaluated by the Ellman method [ 32 ] using acetylthiocholine ATCh as substrate. Each sample was analyzed in triplicate. The effect of pH on the activity of AChE was evaluated using three different buffers, with two points intersecting two different buffers McIlvaine buffer pH 5.
Activities were plotted against temperature or pH values, respectively. The kinetic parameters were evaluated in the optimal conditions of pH and the temperature previously determined. The experiments were carried out in triplicate. Michaelis-Menten constants and maximum velocities were estimated through Lineweaver-Burk reciprocal plots using GraphPad Prism 5.
The immobilization was carried out in duplicate to ensure the reproducibility of the produced ICERs. MassLynx 4. The mass spectrometer was operated by selected reaction monitoring SRM in which the protonated molecular ion was isolate and the fragments ions were monitored to the choline and acetylcholine. The capillary voltage was set at 2. The activity and kinetic parameters were evaluated by one SRM transition for each analyte at the following cone voltage CV and collision energy CE : Methanol was used in the combined mode to improve ionization which was delivered by the syringe pump at a flow rate of 0.
The total analysis time was of 8. To evaluate the stability of the ACh solution to spontaneous hydrolysis in the sample injector and in the pretreated capillary, a chromatographic separation under HILIC conditions was used. No Ch production was observed and the ACh maintained the same peak area with a carryover effect of 0. The areas of the Ch peak produced were correlated to the concentrations through the calibration curves.
The values obtained for Ch concentrations were related to substrate concentrations and the best-fit nonlinear regression method using the GraphPad Prism 5. The substrate concentration used was 1. For each AChEIs assay, 0. For each analyzed sample, a negative control absence of ACh and positive control samples absence of tacrine were used.
Two AsChEs were purified from worker heads of A. Medium worker ants were selected considering our previous studies, in which we identified higher AChE expression levels in this developmental stage, compared to larva and pupa [ 40 ].
No surfactant was used to extract both AChEs thus showing their hydrophilic characteristics. Similar results were reported for isolation from other insects of the Hymenoptera order such as Apis mellifera [ 14 ] and Nematoda Heterorhabditis bacteriophora [ 41 ]. To meet this end, the proteins were precipitated with ammonium sulfate.
Table 1 lists the activity recoveries for the purification protocol. The separation of two AChE active fractions was obtained by ion exchange chromatography. Moreover, the presence of these two enzymes was also identified by zymography of the crude ant head extracts, corresponding to the isolated AChEs Figure 1. Purification and activity of AChEs from A.
Elution profile of ion exchange chromatography left. AChE activity of free enzyme is shown with dotted line. The list of identified peptides is summarized in Table 2. These data showed that both isolated enzymes were identified as AchEs.
The identified peptides based on the phylogeny studies were also used to classify the enzymes in accordance with their classes as AChE1 or AChE2. S1 and Fig. The influence of the pH and temperature in the activity of free enzymes was determined using ATCh as substrate.
With this substrate, AChEs usually have optimum pH around 7. The kinetic parameters of the two free enzymes were then determined varying the concentration of ATCh under optimal conditions of pH and temperature. At the analyzed concentration range, typical Michaelian kinetics was observed. At these conditions, their catalytic efficiency was calculated as described by Kim et al. Michaelian kinetic was also observed when ACh was used as substrate and the activities measured by LC-MS; however under these experimental conditions, AsAChE-B emerged as the one with the highest substrate affinity Table 3.
The initial activity assays using an These results showed, as expected, that the immobilized enzymes retained its activity toward its natural substrate, ACh. The kinetic parameters of free and immobilized enzymes should not be directly compared [ 45 ], especially in cases where hydrolysis occurs on flow and that the contact time between the enzyme and the substrate is shorter.
Our results have shown the importance of using the natural substrate. For the inhibition screening assay tacrine was selected as reference inhibitor. Herein, tacrine inhibited only Inferring that despite the established use of AChE from E. The assays described in the literature to determine the activity of AChEs in the insects generally use ATCh as substrate to infer which main enzyme AChE1 or AChE2 is involved in the hydrolysis in the synapses [ 12 , 14 , 52 ].
Due to its higher catalytic activity and affinity for ATCh, AChE1 was inferred as the main enzyme involved in the hydrolysis of ACh in the pest insect Cnaphalocrocis medinalis [ 52 ]. Meanwhile, for Blattella germanica cockroach species , AChE2 was appointed as the main enzyme involved in synapses as it has a greater catalytic efficiency and affinity toward ATCh than AChE1 [ 12 ].
Taking this approach into account, the kinetic data obtained using ATCh as substrate suggests that AsAChE-A is the main cholinergic enzyme in Atta sexdens which is in agreement with the work carried out by Kim and Lee [ 17 ], which shows that all the insects belonging to the order Hymenoptera presented AChE2 as the main enzyme involved in the synapse.
It is important to stress, however, that the functions attributed to each of the AChEs are not completely clear and that different physiological functions have been assumed to either AChE1 or AChE2 [ 20 ].
Studies of the biological functions using RNA interference RNAi and gel electrophoresis followed by the enzyme activity test with ATCh in Tribolium castaneum beetle have suggested that AChE1 is the cholinergic enzyme while AChE2 has been shown to be related to noncholinergic functions, such as embryonic development, growth, and reproduction [ 19 ].
The same was observed for grasshoppers [ 53 ]. A study carried out in Helicoverpa armigera species demonstrated that gene silencing resulted in mortality, developmental inhibition, decreased fecundity, and poor formation [ 18 ]. In this context, future work using A. These experiments are necessary, especially considering the apparent kinetic constant obtained for AsAChEs using ACh as substrate. Dulce Helena F.
Souza, Quezia B. Cass, and Odair C. Bueno designed the research and provided guidance; Adriana M. Fracola carried out the biochemical and kinetic experiments. Fernando G. All authors read and approved the final manuscript. Supporting Information A. Analytical Method Validation. The linearity of the method was evaluated with a standard solution of Ch at 32 mM in water and the working solutions for calibration curves and quality controls QC were prepared from that.
The calibration standards were prepared in duplicate and the calibration curve was constructed by logarithmic nonlinear regression, plotting the peak area as a function of a given concentration of Ch. The analysis of QC samples allowed the determination of intra- and interbatch with precision and accuracy. Five samples of each concentration were prepared in water. The accuracy was calculated for the concentrations examined by the back calculation and expressed as the percentage of deviation between the concentrations found and the nominal concentrations.
Qualification Study. The calibration curves were logarithmic in the concentration ranges studied, with mean correlation coefficients R 2 of 0. The intra- and interlot precision and accuracy of the method were determined by analyzing five replicates of the three quality controls QCs , which led to precision values with RSD between 1.
The accuracy of QCs varied between Supporting Information B. Supplementary Materials. Dos Santos et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Article of the Year Award: Outstanding research contributions of , as selected by our Chief Editors. Read the winning articles. Special Issues. Dos Santos, 1 Ariele C. Fracola, 1 Fernando G. Bueno, 3 Quezia B. Cass , 1 and Dulce Helena F. Academic Editor: Qi-Zhuang Ye. Distribution and subcellular localization in Phaseolus aureus Roxb. Plant Physiol 53 : — Phytochemistry 13 : — Preliminary characterization of enzymes from Solanum melongena L. Biochim Biophys Acta : — Direct evidence for the hydrolysis of choline-auxin conjugates by pea cholinesterase.
Plant Physiol Biochem 38 : — Hartmann E, Kilbinger H Occurrence of light-dependent acetylcholine concentration in higher plants. Experientia 30 : — Hirano H, Watanabe T Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application to an insoluble protein. Electrophoresis 11 : — Life Sci 72 : — J Agric Food Chem 52 : — Genome Res 9 : — Annu Rev Neurosci 2 : — Kishibayashi A, Ishii A, Karasawa A Inhibitory effects of KW, a novel gastroprokinetic agent, on the activity of acetylcholinesterase in guinea pig ileum.
Jpn J Pharmacol 66 : — Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature : — J Exp Bot 29 : — Lucas WJ Plasmodesmata: intercellular channels for macromolecular transport in plants. Curr Opin Cell Biol 7 : — MacIntosh FC Acetylcholine. Little Brown and Co. Momonoki YS Asymmetric distribution of glucose and indole myo -inositol in geostimulated Zea mays seedlings. Plant Physiol 87 : — Momonoki YS Occurrence of acetylcholine-hydrolyzing activity at the stele-cortex interface.
Plant Physiol 99 : — Momonoki YS Asymmetric distribution of acetylcholinesterase in gravistimulated maize seedlings. Plant Physiol : 47 — Asymmetric solute distribution controlled by ACh in gravistimulated maize seedlings.
Plant Prod Sci 1 : 83 — Plant Prod Sci 3 : 17 — Momonoki YS, Momonoki T Changes in acetylcholine levels following leaf wilting and leaf recovery by heat stress in plant cultivars. Jpn J Crop Sci 60 : — Momonoki YS, Momonoki T Changes in acetylcholine-hydrolyzing activity in heat-stressed plant cultivars. Jpn J Crop Sci 62 : — Jpn J Crop Sci 65 : — Acc Chem Res 35 : — Standaert F Neuromuscular physiology.
In R Miller, ed, Anesthesia. Churchill-Livingstone, New York, pp — Eur J Biochem : — Curr Med Chem 1 : — Toutant JP Insect acetylcholinesterase: catalytic properties, tissue distribution and molecular forms.
Prog Neurobiol 32 : — Umbach AL, Siedow JN Covalent and noncovalent dimers of the cyanide-resistant alternative oxidase protein in higher plant mitochondria and their relationship to enzyme activity.
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Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Molecular Characterization of Maize Acetylcholinesterase. Yoshimasa Sagane , Yoshimasa Sagane. Oxford Academic. Google Scholar. Tomoyuki Nakagawa. Kosuke Yamamoto. Soichi Michikawa. Suguru Oguri. Yoshie S. Revision received:. Select Format Select format. Permissions Icon Permissions.
Abstract Acetylcholinesterase AChE has been increasingly recognized in plants by indirect evidence of its activity. Ten batches of the crude extract of the maize seedlings were combined and subjected to the following chromatographic purification procedure. The extract was applied to a Sephadex G gel filtration column 2.
Open in new tab Download slide. Figure 1. Table I. Summary of the purification of maize AChE. Purification Steps. Total Protein. Specific Activity. Open in new tab. Figure 2. The cholinester-hydrolyzing enzymes are usually classified into AChE and butyrylcholinesterase BChE , according to their substrate and inhibitor specificity.
This effectively stops the signal, allowing the pieces to be recycled and rebuilt into new neurotransmitters for the next message. Acetylcholinesterase has one of the fastest reaction rates of any of our enzymes, breaking up each molecule in about 80 microseconds. Acetylcholinesterase was first studied by using the form found in electric fish, such as the torpedo ray. These fish have massive arrays of nerve-like structures in the organs that generate electricity, so acetylcholinesterase is particularly abundant.
The form shown here, from PDB entry 1acj , forms a dimer in the crystal structure. It normally has lipids attached to the protein chains, which anchor the enzyme to the cell membrane. The lipids were removed in the crystal structure, however, to allow crystallization. The active site is found in a deep pocket, just big enough for the acetylcholine to slip down inside.
At the base of the pocket is a triad of three amino acids--serine-histidine-glutamate--that is almost identical to the triad used in the serine proteases like trypsin and chymotrypsin. Acetylcholinesterase top with a snake toxin center and Aricept bottom. Since acetylcholinesterase has an essential function, it is a potential weak point in our nervous system.
Poisons and toxins that attack the enzyme cause acetylcholine to accumulate in the nerve synapse, paralyzing the muscle. Over the years, acetylcholinesterase has been attacked in many ways by natural enemies. For instance, some snake toxins attack acetylcholinesterase. The picture at the top shows a view straight down the active site tunnel, from PDB entry 1b41 , showing the active site serine in red. The middle picture shows how a lethal toxin from the eastern green mamba blocks the active site and poisons the action of the enzyme.
For more information on snake toxins, take a look at the Protein of the Month at the European Bioinformatics Institute. Doctors are now willfully poisoning acetylcholinesterase in an attempt to reverse the symptoms of Alzheimer's disease. People with Alzheimer's disease lose many nerve cells as the disease progresses. By taking a drug that partially blocks acetylcholinesterase, the levels of the neurotransmitter can be raised, strengthening the nerve signals that remain.
One drug being used in the way is shown at the bottom, from PDB entry 1eve. It inserts into the active site pocket and temporarily blocks entry of acetylcholine.
Other poisons, as shown next, take a more permanent approach. The nerve toxin sarin and insecticides such as malathion directly attack the active site machinery of acetylcholinesterase. The structure shown here, from PDB entry 1cfj , shows the active site triad of acetylcholinesterase after being poisoned by sarin.
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