Abstract
Acetylcholinesterase (AChE) is responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh) leading to acetate and choline (Ch) release. The inhibition of AChE produces a generalized synaptic collapse that can lead to insect death. Herein we report for the first time the isolation of two AChEs from Atta sexdens which were purified by sulphate ammonium precipitation followed by ion exchange chromatography. AsAChE-A and AsAChE-B enzymes have optimum pH of 9.5 and 9.0 and higher activities in 30/50°C and 20°C, respectively, using acetylthiocholine (ATCh) as substrate. Immobilized capillary enzyme reactors (ICERs) were obtained for both enzymes (AsAChE-A-ICER and AsAChE-B-ICER) and their activities were measured by LC-MS/MS through hydrolysis product quantification of the natural substrate ACh. The comparison of activities by LC-MS/MS of both AChEs using ACh as substrate showed that AsAChE-B (free or immobilized) had the highest affinity. The inverse result was observed when the colorimetric assay (Elman method) was used for ATCh as substrate. Moreover, by mass spectrometry and phylogenetic studies, AsAChE-A and AsAChE-B were classified as belonging to AChE-2 and AChE-1 classes, respectively.
Highlights
The ecological importance of ants is indisputable due to the effect/influence they have on several processes such as aeration, distribution of nutrients, and seed dispersal [1, 2]
It is being responsible for the hydrolysis of the active neurotransmitter acetylcholine (ACh) into the inactive compounds choline (Ch) and acetic acid
The purified enzymes were used to produce immobilized capillary enzyme reactors (ICERs) to prospect inhibitors based on the direct hydrolysis of ACh and quantification of the produced choline by LC-MS/MS
Summary
The ecological importance of ants is indisputable due to the effect/influence they have on several processes such as aeration, distribution of nutrients, and seed dispersal [1, 2]. Acetylcholinesterase (AChE) (EC 3.1.1.7) is a cholinesterase that acts on the central nervous system and plays an important role during neurotransmission in the cholinergic synapses and neuromuscular junctions. It is being responsible for the hydrolysis of the active neurotransmitter acetylcholine (ACh) into the inactive compounds choline (Ch) and acetic acid. It is worth mentioning that organophosphate and carbamate insecticides are highly toxic to animals and humans [28,29,30,31] To contribute to this field, this work reports on the isolation and characterization of two AChEs from Atta sexdens. The purified enzymes were used to produce immobilized capillary enzyme reactors (ICERs) to prospect inhibitors based on the direct hydrolysis of ACh and quantification of the produced choline by LC-MS/MS
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