Showing papers on "Acetylthiocholine published in 1992"

Ambenonium is a rapidly reversible noncovalent inhibitor of acetylcholinesterase, with one of the highest known affinities.

Abstract: Steady state patterns of inhibition of purified human erythrocyte acetylcholinesterase by three inhibitors were analyzed. Edrophonium acted essentially as a competitive inhibitor, whereas tacrine and ambenonium gave mixed competitive and uncompetitive inhibition with acetylthiocholine as substrate. Inhibition constants for the competitive components were 470 microM for edrophonium, 65 microM for tacrine, and 0.12 nM for ambenonium. The extremely high affinity of ambenonium permitted analysis of the rates of approach to steady state inhibition. These rates were characterized by a single exponential time course with rate constants, kexp, that showed a linear dependence when plotted against ambenonium concentration, at fixed substrate concentration. The intercepts of these plots were independent of the substrate concentration and indicated an ambenonium dissociation rate constant of 0.013 +/- 0.002 sec-1. The slope of the plot at the lowest substrate concentration approximated the ambenonium bimolecular or association rate constant and gave a value of 5.2 +/- 0.6 x 10(7) M-1 sec-1. Three models were examined to account for the nearly linear dependence of the slopes of these plots on the substrate concentration. These models indicated that ambenonium and acetylthiocholine competed for a peripheral anionic site in the acetyl-enzyme intermediate formed during substrate hydrolysis. The apparent equilibrium dissociation constant of acetylthiocholine for this peripheral site (1.2-1.4 mM) was significantly different from that calculated from substrate inhibition data (20.1 +/- 2.8 mM). We propose that acetylthiocholine can interact with the acetyl-enzyme both at the peripheral site and at the active site but that only the latter interaction inhibits substrate hydrolysis.

The effect of pralidoxime chloride in the assay of acetylcholinesterase using 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent).

Abstract: Pralidoxime chloride (PAM) hydrolyzes acetylthiocholine, the substrate used in the assay of red cell cholinesterase. The thiocholine that is produced forms a yellow complex when Ellman's reagent is used in the assay. This was tested in blood samples of patients who were treated with PAM after organophosphorus (OP) poisoning and after the observation of an immediate increase in absorption of light at 412 nm.

Inhibition of acetylcholinesterase of mice erythrocytes & synaptosomes by dimethylsulfoxide.

Abstract: In vitro inhibitory effect of dimethylsulfoxide (DMSo) on acetylcholinesterase of erythrocyte membranes and synaptosomes of mice was observed using acetylthiocholine as subtrate. DMSO inhibited the enzyme from both sources and the effect was concentration dependent. It produced an inhibition of 26 to 28 per cent at a concentration of 0.13 mM and 92 to 95 per cent at a higher concentration of 1.91 mM. The Km of bound and solubilized enzyine of the synaptosomes was 0.2 and 0.15 mM while that of erythrocyte membranes 0.5 and 0.33 mM respectively. The V max was 0.4 and 0.4 (for synaptosomes) and 0.67 and 0.59 (for erythrocyte membranes) Mmoles/mg protein/min, respectively for bound and solubilized forms. The K i of solubilized enzyme of synaptosomes was 0.11 mM. Kinetic studies showed that the inhibition was competitive in nature

A comparative study of acetylcholinesterase activity in bovine (s.cervi) and human (b. malayi, w. bancrofti) filaria

Abstract: Setaria cervi, a bovine filarial parasite, contains a significant amount of acetylcholinesterase (AChE) activity with microfilaria having five to ten times more AChE activity than female and male adult worms, respectively. Because AChE shows substrate specificity and hydrolyzes acetylthiocholine but not butrylthiocholine, this parasitic enzyme is likely a true acetylcholinesterase. The latter also resembles an AChE enzyme in the human filarial parasite B. malayi which hydrolyzes acetylthiocholine iodide three times faster than butrylthiocholine iodide. The S. cervi AChE, like its counterpart, also exhibit inhibition with eserine, a specific inhibitor of this enzyme. Subcellular localization of AChE in adult female worms shows enzyme activity both in the mitochondrial and post-mitochondrial fraction. However, enzyme activity in the soluble fraction is twenty-seven times greater than in the mitochondrial fraction.

Isolation of a tripeptide (Ala-Gly-Ser) exhibiting weak acetylthiocholine hydrolyzing activity from a high-salt soluble form of monkey diaphragm acetylcholinesterase.

Abstract: A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammoniumchloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [3H]Diisopropyl fluorophosphate ([3H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight ∼300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [3H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme.

[Synthetic bioantioxidants--inhibitors of acetylcholinesterase activity].

Abstract: The inhibitory effects of synthetic antioxidants (3-oxypyridine, pyrimidine and hindered phenols) on the enzymic activity of membrane-bound acetylcholinesterase (AChE) was studied. In terms of estimated kinetic characteristics of AChE-reaction (KM, Vmax, KI), the pattern of enzyme inhibition by the hindered phenol compounds was found to be of non-competitive or mixed type depending on the inhibitor structure or on the substrate acetylcholine or acetylthiocholine used. The comparative study of the inhibitory action of water-soluble derivatives of hindered phenols and fatty-soluble ionol made it possible to reveal possible contributions to the inhibition of both direct and mediated (by the membrane microsurroundings) effects on the membrane-bound AChE by the studied synthetic bioantioxidants.