Acetylcholine assay

Dlprenorphlne (XlXa) Is a pure morphine antagonist (3-5 times naloxone) pharmacologically, but shows some agonist activity In the acetylcholine assay. In this series, structure around C-I9 is critical, e.g. XlXb and c are not antagonists. ... 

The use of mutant 34486 of Neurospora crassa for the microbiological assay of ch oline has been described (8). A physiological method has also been used in which the ch oline is extracted after hydrolysis from a sample of biological material and acetylated. The acetylcholine is then assayed by a kymographic procedure, in which its effect in causing contraction of a piece of isolated rabbit intestine is measured (33). 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

CBs, like OPs, act as inhibitors of ChE. They are treated as substrates by the enzyme and carbamylate the serine of the active site (Figure 10.8). Speaking generally, car-bamylated AChE reactivates more rapidly than phosphorylated AChE. After aging has occurred, phosphorylation of the enzyme is effectively irreversible (see Section 10.2.4). Carbamylated AChE reactivates when preparations are diluted with water, a process that is accelerated in the presence of acetylcholine, which competes as a substrate. Thus, the measurement of AChE inhibition is complicated by the fact that reactivation occurs during the course of the assay. Carbamylated AChE is not reactivated by PAM and related compounds that are used as antidotes to OP poisoning (see Box 10.1). 

Salminen, O., Whiteaker, P., Grady, S.R., Collins, A.C., McIntosh, J.M., Marks, M.J. The subunit composition and pharmacology of alpha-Conotoxin Mil-binding nicotinic acetylcholine receptors studied by a novel membrane-binding assay. Neuropharmacology. 48 696, 2005. 

The cholinesterase-inhibiting activity of the phosphorofluoridates was compared quantitatively with that of eserine sulphate thus. To 0-2 ml. of heparinized human plasma was added 05 ml. of a solution containing either eserine or the phosphorofluoridate in varying concentrations then the mixture was kept at room temperature for 10 min. before 1 /tg. of acetylcholine in 1 c.c. saline solution was added. After 5 min. at room temperature, the mixture was made up to 10 ml. with frog saline containing eserine 1/100,000, which at once stopped the action of any cholinesterase not yet inactivated. The solution was then assayed for acetylcholine on the frog rectus-muscle preparation. 

Compound 10 was evaluated for anti-bacterial activity and acetylchohnesterase (AChE) inhibitory activities. This compound was foimd to be inactive in antibacterial assay and exhibited AChE inhibitory activity with an ICj, value of 67 pM. Acetylcholine serves as a neurotransmitter in the central and peripheral nervous system. Acetylcholinesterase (AChE) stops the function of acetylcholine by its... 

Nimmo lA, Mabood SF. 1979. The nature of the random experimental error encountered when acetylcholine hydrolase and alcohol dehydrogenase are assayed. Anal Biochem... 

High-performance liquid chromatography/tandem mass spectrometry assay for the rapid high sensitivity measurement of basal acetylcholine from microdialysates. J Neurosci Methods 121 33. 

To illustrate the principles of an equilibrium dialysis experiment, we will describe the binding of [ H] acetylcholine to the nicotinic acetylcholine receptor (nAChR) in native membranes from Torpedo electro-plax. The data obtained from such an experiment are shown in Figure 10-7 where they are compared with data obtained from a centrifugation assay described below (Protocol 4.2). 

Anderson DJ, Puttfarcken PS, Jacobs 1, Faltynek C (2000) Assessment of nicotinic acetylcholine receptor-mediated release of [ H]-norepinephrine from rat brain slices using a new 96-well format assay. Neuropharmacology 39 2663-2672 Anney RJ, Olsson CA, Lotfi-Miri M, Patton GC, Williamson R (2004) Nicotine dependence in a prospective population-based study of adolescents the protective role of a functional tyrosine hydroxylase polymorphism. Pharmacogenetics 14 73-81 Auerbach A, Akk G (1998) Desensitization of mouse nicotinic acetylcholine receptor channels. 

In the following example we describe the implementation of a mass spectromet-ric assay for acetylcholinesterase (AChE) [22]. AChE plays an important role in the nervous system. This enzyme rapidly hydrolyzes the active neurotransmitter acetylcholine into the inactive compounds choline and acetic acid. Amongst others, low levels of acetylcholine in the synaptic cleft are associated with Alzheimer s disease [23, 24]. Patients afflicted by this disease may benefit from inhibition of AChE activity thereby increasing ACh level. 

Fig. 5.8 Analysis of a Narcissus extract by HPLC coupled to the MS-based AChE assay. MS instrument Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source, (a) Total ion current (TIC) (b) product trace (choline) m/z 104 (c) substrate trace (acetylcholine) m/z 146 (d)...

G. A. Development of a nonisotopic acetylcholine receptor assay for the investigation of cholinergic ligands. Anal. Chim. Acta 1992, 267, 55-62. 

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... 

Schmidt J, Rafrery MA (1973) A simple assay for the study of solubilized acetylcholine receptors. Anal Biochem 52 349-354... 

Rao, T. S., Correa, L. D., Reid, R. T., Lloyd, G. K. Evaluation of anti-nociceptive effects of neuronal nicotinic acetylcholine receptor (nAChR) ligands in the rat tail-flick assay, Neuropharmacology 1996, 35, 393-405. 

Sullivan, J. P., Briggs, C. A., Donnelly-Roberts, D., Brioni, J. D., Radek, R. J., McKenna, D. G., Campbell, J. E., Arneric, S. P., Decker, M. W. (+)-Epibatidine can differentially evoke responses mediated by putative subtypes of nicotinic acetylcholine receptors (nAChR) ligands in the rat tail-flick assay, Med. Chem. Res. 1994, 4, 502-516. 

In a typical analysis, 15 mL of an aqueous solution containing an unknown amount of acetylcholine had a pH of 7.65. When incubated with acetylcholinesterase, the pH of the solution decreased to 6.87. Assuming that there was no buffer in the assay mixture, determine the number of moles of acetylcholine in the 15 mL sample. 

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