Acetylcholinesterase Activity Assay Research Articles

Assay of acetylcholinesterase activity and elemental composition in brain compartments by electron probe microanalysis

Using histochemical techniques the distribution of acetylcholinesterase (AChE) activity in rat brain has been well established [1]. These assays are based on the precipitation of a specific deposit containing copper-ferricyanide [2]. The amount of locally deposited product is proportional to the AChE activity in the examined area of the section. Thus, the amounts of copper or (and) iron as the deposited histochemical labels mark the distribution and magnitude of the enzyme activity. The present protocol describes an approach to obtain by electron probe microanalysis (EPMA) [3]a map of copper deposited in different brain regions via AChE histochemistry. EPMA allows a wide range of chemical elements to be analyzed [4]. Thus, together with the determination of the copper concentration, elemental contents (Na, Cl, K and Ca) are measured as the specific environment for local AChE activity [5].

Quantitative, Video-Based Histochemistry to Measure Regional Effects of Anticholinesterase Pesticides in Rat Brain

Acetylcholinesterase histochemistry was coupled to inexpensive and widely available apparatus for video microscopy and densitometry to study enzyme activity and inhibition in different parts of the rat brain. Quantitative histochemistry, under properly defined conditions, yielded an output that increased linearly with incubation time and section thickness and was a smooth hyperbolic function of substrate concentration. The time-course of staining afterin vivoexposure to eserine revealed no sign that carbamate-induced cholinesterase inhibition was readily reversedin vitro.Brains from rats treated either with a carbamate or an organophosphate anticholinesterase pesticide showed significant regional variation in cholinesterase inhibition. The histochemical data corresponded well with data from biochemical assays of acetylcholinesterase activity (overall correlation coefficient of absolute values,r= 0.95). Also, a comparison of assay types by two-way analysis of variance showed no significant main effect. These results support the conclusion that video-based histochemistry is suitable for detailed studies of developmental and toxicological influences on cholinesterases in multiple microscopic regions of the rat brain.

A microtiter assay for determining protein, acetylcholinesterase activity, and G418 (neomycin) resistance in cultured cells

The Coomassie brilliant blue assay for the determination of protein has been extended to rapidly and conveniently measure the protein concentration of cells growing in culture in a 96-well microtiter format. Modifications of the standard assay include sodium hydroxide to solubilize the cells and ovalbumin, instead of bovine serum albumin, as a protein standard. The procedure allows a large number of small samples to be assayed simultaneously. Two examples of its use, enzyme-specific activity and drug resistance, are shown. An assay for acetylcholinesterase activity in the same culture plate is demonstrated. G418, an inhibitor of cell protein synthesis, is frequently used to select for cells transfected with the neomycin resistance gene. The required concentration of G418 can be easily determined with this protein assay.

A silica gel plate-based qualitative assay for acetylcholinesterase activity: A mass method to screen for potential inhibitors

A procedure for the qualitative assessment of inhibitory activity towards acetylcholinesterase for a given compound is described. Solutions of the compounds of interest are spotted on silica gel TLC plates in a matrix pattern. The silica gel plate is sprayed with a solution of acetylthiocholine iodide and 5,5-dithiobis(2-nitrobenzoic acid) followed by a solution of acetylcholinesterase. The enzyme reaction produces a yellow background color with inhibitor compounds exposed as white zones where color has failed to develop. The results for a test set of compounds were compared to those obtained using the standard Ellman assay procedure and found to agree for virtually all of these compounds. The conditions of silica gel plate thickness, reagent concentration, and enzyme source under which this procedure is suitable were investigated. This represents an extremely rapid method to screen large numbers of compounds to uncover new inhibitors of acetylcholinesterase and potentially other enzymes as well.

Highly sensitive assay for acetylcholinesterase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for acetylcholinesterase (AChE) activity was devised by high-performance liquid chromatography with electrochemical detection. It is based on the separation of acetylcholine and choline on an octadecylsilane reversed-phase column, followed by their enzymatic conversion into hydrogen peroxide through the post-column reaction with immobilized AChE and choline oxidase. The system is highly sensitive, and the relationship between the peak height and the amount of choline is linear over the range 5 pmol to 5 nmol. When homogenate of bovine caudate nucleus was used as enzyme, the Michaelis constant of the enzyme for acetylcholine was 0.4 m M. The regional distribution of AChE activity in rat brain was examined, and the order of the activity from the highest to the lowest agreed with the reported brain distribution of AChE: striatum, thalamus plus hypothalamus, pons plus medulla oblongata, cerebral cortex, olfactory bulb, and cerebellum.

DIAGNOSTIC VALUE OF RECTAL MUCOSAL ACETYLCHOLINESTERASE LEVELS IN HIRSCHSPRUNG'S DISEASE

Acetylcholinesterase (AChE) activity was measured in rectal biopsy specimens obtained from 68 children aged between 2 days and 141/2years in whom Hirschsprung's disease was suspected. The diagnosis was subsequently established in 12; in these, the mean AChE activity was found to be 30·5×10 -7 units/g tissue (range 16·9 to 63·0). The 56 non-Hirschsprung cases had a mean of 5·0×10 -7 units/g tissue (S.D. 2·2), the highest value in this group being 10·9. The results were unaffected by age, sex, nature of biopsy procedure, or the presence of blood. It is suggested that the assay of AChE activity in rectal biopsy material is a simple and quick procedure that is useful in the diagnosis of Hirschsprung's disease.

Radiomimetric assay of acetylcholinesterase activity in submicrogram amounts of tissue

A radiometric procedure for the estimation of cholinesterase activity was described which measures acetate-1- 14C after the selective removal of the unhydrolyzed substrate, acetyl-1- 14C-choline, as an insoluble reinecke salt. Its sensitivity is adequate for the assay of 0.05 μg (protein) brain tissue. The convenience has been illustrated by various kinetic studies, including the inhibition by acetylthiocholine. A detailed comparison with other radiometric methods was presented.

Acetylcholinesterase activity in partially isolated cerebral cortex after prolonged intermittent stimulation

Acetylcholinesterase (AChE) levels were determined in control samples of cat cerebral cortex, in partially neuronally isolated (undercut) cortex, and in undercut or intact cortex that had received long-term intermittent electrical stimulation. Assay of AChE activity was based on an automatic titration method using methalcholine as substrate. Tissues from ten of 15 undercut cerebral cortices showed the expected decrease in AChE. Long-term intermittent electrical stimulation of undercut cortex prevented the expected decrease in AChE in seven of 12 cortices. Previous data showed that similar long-term stimulation could prevent some electrical manifestations of supersensitivity. The present findings implicate, in part, a cholinergic system in the phenomenon of cortical supersensitivity.

Paroxysmal nocturnal hemoglobinuria presenting as aplastic anemia in a child: Case report with evidence of deficient leukocyte acetylcholinesterase activity

A seven year old child is described who presented initially with clinical and laboratory manifestations of aplastic anemia. She gradually improved after prolonged medical treatment, together with splenectomy and infusion of homologous marrow. Subsequently she exhibited the clinical and laboratory findings characteristic of paroxysmal nocturnal hemoglobinuria. Assays of acetylcholinesterase activity indicated that the defect of that enzyme is not limited to the erythrocytes in paroxysmal nocturnal hemoglobinuria but also involves the leukocytes. This finding lends support to previous evidence that paroxysmal nocturnal hemoglobinuria is a defect involving all of the hematopoietic tissue. The parallel pathogenesis and pathophysiology of and possible interrelationship between aplastic anemia and paroxysmal nocturnal hemoglobinuria are discussed. It is proposed that the latter disease may represent a somatic mutation involving the hematopoietic tissue. This may be the result of some insult, perhaps viral or toxic, to the marrow.