Acetylcholine esterase assay

The mode of action of the isobutylamides is unknown, although Miyakado et al. found that several isobutylamides (i.e. pipercide and related compounds) caused repetitive discharge when the nerve cord of the cockroach, Periplaneta americana was stimulated. We found that fagaramide was inactive as an acetylcholine esterase inhibitor in an m vitro assay(unpublished dat a). 

Assays of acetyl- and butyrylcholine esterases inhibition, as well as of modulation of calcium channels and nicotinic receptors have been conducted in vivo. Moreover, their interaction with the active center of acetylcholine esterase has been simulated by molecular dynamics. For synthesized compounds the IC50 of acetylcholine esterase inhibition was about 9 X M, and for the most active the value was four to five times... 

Enzymatic techniques have also been employed in the analysis of these compounds. The toxicity of carbamate insecticides is due to the inhibition of the enzyme acetylcholine esterase, so the determination of these compounds can be achieved by enzyme inhibition (2,83,119), bioassay (118,167), or enzyme-linked immunosorbent assay (ELISA) (168-171). In the detection of carbamates by fluorimetric enzyme inhibition, the effluent from a reversed-phase chromatographic column was incubated with cholinesterase, which was introduced via a postcolumn reagent delivery pump. Then, the resulting partially inhibited cholinesterase was reacted with N-methyl indoyl acetate to produce a fluorophore and a reduction in the baseline fluorescence (172). 

Since MGL is a serine hydrolase, its sensitivity to many of the available serine hydrolase inhibitors has been explored (Table 3). The results support the hypothesis that MGL can be inhibited by compounds that interact with its reactive serine. On the other hand, the potencies of the inhibitors are quite variable in some cases, this likely reflects differences in assay methodology (i.e., substrate concentration, pH, form of the enzyme). However, in a few cases, the same assay conditions revealed very different inhibitory potencies (e.g., compare the platelet and macrophage membrane studies by Di Marzo et al. 1999). In any event, studies of these compounds are not likely to yield selective inhibitors of MGL. All of these compounds are inhibitors of FAAH (see above) and many are also inhibitors of PLA2, diacylglycerol lipase, and acetylcholine esterase, among other hydrolases. By analogy to the development of the URB series of FAAH inhibitors (Kathuria et al. 2003), it is likely that selective inhibitors of MGL will come from other synthetic avenues. 

Anatoxin-a(s) has been detected in toxic blooms in lakes in Canada [4], Denmark [123, 124], and Brazil [125, 126]. So far, only a few cyanobacterial strains producing anatoxin-a(s) have been characterized Anahaena flos-aquae [4], Anabaena crassa [126], and Anabaena lemmermannii [124]. The detection methods used to assay anatoxin-a(s) rely on the colorimetric assay for acetylcholine esterase [5], or on an electrochemical biosensor based on acetylcholine esterase activity [127,128], or more recently on the use of LC-MS [129]. 

Figure 8.14 Colourimetric assay. Acetyl Choline Esterase (AChE) assay system involving thio-acetylcholine that is hydrolyzed to thio-choline. This in turn combines with colourless reagent 5,5 -dithio-bis(nitrobenzoic acid) (DTNB) to form yellow coloured 5-thio-2-nitro-benzoic acid (TNB).

Potentiometric devices have a relatively high sensitivity and selectivity and abroad response range. They are also simple, rehable, and inexpensive and use commonly available instrumentation. Potentiometric enzyme-based biosensor systems have been recently reviewed. The most general approach for the determination of OP compounds (OPCs) is based on their inhibition of the activity of choline esterase enzymes. The presence of low concentrations of inhibitors strongly and specifically affects the enzyme activity. Therefore, by measuring the enzyme activity, the concentration of the OPCs can be assayed. Different enzymes, such as acetylcholine (ACh), BuCh, and, most often, pH sensors, were used as transducers in potentiometric biosensors. 

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