Competitive Inhibitor Of Acetylcholinesterase Research Articles

The association of the serotonin-sensitive aryl acylamidase with acetylcholinesterase in the monkey brain.

The serotonin‐sensitive aryl acylamidase was partially purified from monkey brain. The aryl acylamidase activity was inhibited by serotonin (Ki= 0.425 mM) and tryptamine (Ki= 3.6 mM) but not by a number of other amines. It was also inhibited by acetylcholine (Ki= 2 mM) and its analogues and homologues. The relationship of aryl acylamidase to acetylcholinesterase was examined. The ratios of specific activities of aryl acylamidase and acetylcholinesterase in the different steps of purification were approximately constant and the percentage recoveries of both enzyme activities were comparable. Elution profiles of both enzyme activities from concanavalin‐A‐Sepharose, Sephadex G‐200 and DEAE‐Sephadex A‐25 columns were similar. Both enzyme activities migrated in a similar fashion on gel electrophoresis in different percentage gels for different time intervals. Both enzymes showed similar distribution in the various anatomical regions and in the different subcellular fractions of monkey brain.Eserine and neostigmine, potent competitive inhibitors of acetylcholinesterase also potently inhibited aryl acylamidase in a non‐competitive manner. Inhibition of both enzymes was 100% at 10 μM of both the inhibitors. Tetraisopropylpyrophosphoramide, a selective inhibitor of pseudo‐cholinesterase, did not inhibit either the brain acetylcholinesterase or aryl acylamidase at 10μM. Serotonin inhibited acetylcholinesterase only at concentrations above 10 mM. Dixon plots of one inhibitor in the presence of additional inhibitors indicated that serotonin, butyrylcholine, acetylcholine, neostigmine and eserine (all non‐competitive inhibitors of aryl acylamidase) acted at the same site as inhibitors of aryl acylamidase.The serotonin‐insensitive aryl acylamidase of monkey liver was also insensitive to acetylcholine and eserine and was not found associated with acetylcholinesterase [A. Oommen and A. S. Balasubramanian (1978) Biochem. Pharmacol. 27, 891–895]. Erythrocyte membrane known to contain true cholinesterase had aryl acylamidase activity sensitive to serotonin, acetylcholine and eserine. All these considerations suggest that the serotonin‐sensitive aryl acylamidase activity is a property of true cholinesterase. Although some of the experimental results would suggest that in the monkey brain the two activities may be associated with the same protein with two different active sites further experiments are needed to confirm this.

Aryl acylamidase of monkey brain and liver: Response to inhibitors and relationship to acetylcholinesterase

The serotonin sensitive aryl acylamidase (aryl acylamide amidohydrolase EC 3.5.1.13) of monkey brain was compared with the liver enzyme. Although the two enzymes showed some similarities in their properties such as pH optima, the effect of metal ions and thiol agents, they significantly differed in their mol. wt and response to inhibitors. The liver enzyme had a higher mol. wt as observed by gel filtration on Sepharose 6B and a greater heat stability. The brain enzyme was inhibited specifically by the amines serotonin and tryptamine as well as by acetylcholine and its analogues and homologues in a non-competitive manner. The liver enzyme was unaffected by the above mentioned amines or acetylcholine but it was non-competitively inhibited by indole-3-acetic acid and indole-3-propionic acid both compounds having no effect on the brain enzyme. Eserine, a strong competitive inhibitor of acetylcholinesterase, at 10 −7M inhibited the brain aryl acylamidase to 75 per cent leaving the liver enzyme unaffected. Eserine inhibition of the brain enzyme was non-competitive. From Dixon plots serotonin, acetylcholine and eserine were shown to act at the same site on brain aryl acylamidase. The inhibition of the brain enzyme by eserine and acetylcholine, the elution of both aryl acylamidase and acetylcholinesterase activities in the same fractions during gel filtration and the regional distribution of aryl acylamidase in the brain suggested the association of aryl acylamidase with acetylcholinesterase in the brain though not in the liver.

The inactivation of acetylcholinesterase by trimethyloxonium ion, an active-site-directed methylating agent

Trimethyloxonium ion inactivates acetylcholinesterase from the electric eel and acetylcholinesterase on the surface of human red blood cells. Tetramethylammonium ion, which is a competitive inhibitor of acetylcholinesterase, protects against this inactivation. Trimethyloxonium ion does not inactivate the system that transports choline into the red blood cell. We conclude that trimethyloxonium ion is an affinity-labeling reagent for acetylcholinesterase and that red blood cell acetylcholinesterase is probably not a component of the choline transport system.

Cholinergic Anionic Receptors I: Steric Requirements for Quaternary Ammonium Inhibitors of Acetylcholinesterase

As an initial effort to discern some of the fine features of cholinergic anionic receptors, a study was made on inhibitors of acetylcholinesterase which used inhibitors that are relatively free from conformational variation. The inhibitors used were quaternized 1-azabicyclo (2.2.2)octanes. The bicyclic parent amine was prepared by several conventional procedures. The salts were evaluated as competitive inhibitors of acetylcholinesterase by a titrimetric procedure. The affinity of these compounds for the anionic site of this enzyme was compared to the affinity of a series of related salts subject to conformational variation. These comparisons suggest that there are stereochemical requirements for the anionic site of this cholinergic receptor more specific than heretofore suspected.

Competitive inhibition of acetylcholinesterase by several thiazolines and oxazolines

A number of relatively simple thiazolines and oxazolines substituted in the 2-position are shown to be competitive inhibitors of acetylcholinesterase. Inhibition constants at pH 7.4 and 38 ° for these compounds, calculated on the basis of total concentration, range from 10 −2 to 10 −4 M, the lower values being associated with the more basic thiazolines and oxazolines. Inhibition constants calculated on the basis of cation concentration are all of the same order of magnitude, namely, 10 −4 M. These latter values are similar to those found for alkyl ammonium ions suggesting the inhibitors herein studied reversibly combine with the anionic site of the enzyme.