Hydrolysis of low concentrations of the acetylthiocholine analogs acetyl(homo)thiocholine and acetyl(nor)thiocholine by acetylcholinesterase may be limited by selective gating at the enzyme peripheral site. (25/38)


Molecular and kinetic properties of two acetylcholinesterases from the western honey bee, Apis mellifera. (26/38)


Critical evaluation of acetylthiocholine iodide and acetylthiocholine chloride as substrates for amperometric biosensors based on acetylcholinesterase. (27/38)


An enzymatic method for erythrocyte acetylcholinesterase. (28/38)

The acetylcholinesterase (EC in 50 microL of a 61-fold dilution of erythrocytes in water hydrolyzes acetylcholine during a timed 20-min reaction at 37 degrees C. The resulting choline is measured by use of choline oxidase coupled to peroxidase, with phenol and aminoantipyrene to give a pink product that absorbs maximally at 500 nm. For calibration, a choline iodide standard is included in each batch of up to 19 samples. Accuracy was assessed by using specific inhibitors and measuring choline in the presence of excess erythrocyte solution. The standard curve for the assay is linear to threefold the normal enzyme activity. Between-batch precision was 0.40 kU/L at a mean of 11.5 kU/L (CV 3.5%), and comparison with an acetylthiocholine procedure (x) gave a good correlation: y = 1.02x - 0.27 kU/L (r = 0.991). Long-term precision (10 months), assessed from three sets of assays of samples from 17 individuals, was 0.71 kU/L at a mean of 11.7 kU/L (CV 6.1%).  (+info)

Acetylcholinesterase: inhibition by tetranitromethane and arsenite. Binding of arsenite by tyrosine residues. (29/38)

Tetranitromethane inhibits acetylcholinesterase with respect to the hydrolysis of both acetylthiocholine and indophenyl acetate. The loss of activity with indophenyl acetate, a poor substrate, is preceded by an increase in enzyme activity. Only 12 of the 21 tyrosine residues/monomer of enzyme are susceptible to nitration. Loss of activity with respect to indophenyl acetate occurs well after no further nitration of tyrosines occurs and must be due to the modification of other residues. Incubation of the enzyme with arsenite before nitration results in the nitration of only 10 tyrosines. This experiment reveals that the structural basis for the binding of arsenite is the formation of a diester with two tyrosine residues.  (+info)

Formation of an unstable covalent intermediate during the inhibition of electric-eel acetylcholinesterase with 1,3,2-dioxaphosphorinane 2-oxides. (30/38)

The kinetics of interaction of eel acetylcholinesterase (EC with 1,3,2-dioxaphosphorinane 2-oxides were investigated. It was demonstrated that the rate of spontaneous re-activation as well as the re-activation profile in the presence of 2-pyridine aldoxime methiodide of the inhibited enzyme are irrespective of the leaving group of three inhibitors and exhibit the same values. The dissociation constant of the corresponding Michaelis complex was evaluated by two independent methods and the results were found to be in close agreement. It was shown that the active site is essential for interaction between the enzyme and the various dioxaphosphorinanes. The mixed anhydride of diethyl phosphoric acid and 2-hydroxy-1,3,2-dioxaphosphorinane 2-oxide behaves exactly as would be predicted from a typical diethyl phosphate inhibitor. Enxyme that was incubated with the cyclic acid or the corresponding methyl ester recovered immediately upon extensive dilution. Inhibition of enzyme in the presence of high concentratasions of the corresponding 2-chloro and 2-fluoro derivatives decreased the regeneration rates as well as the maximal amount of the re-activated enzyme. This observation could not be explained in terms of a classical aging process. On the basis of the kinetics observations it is suggested that an unstable covalent phospho-enzyme intermediate is formed during the reaction between acetylcholinesterase and 1,3,2-dioxaphosphorinane 2-oxides.  (+info)

Direct determination of acetyl-enzyme intermediate in the acetylcholinesterase-catalyzed hydrolysis of acetylcholine and acetylthiocholine. (31/38)

Acetylcholinesterase from Electrophorus electricus was acetylated during the hydrolysis of [3H]acetylcholine and [3H]acetylthiocholine. The steady state levels of [3H]acetyl-enzyme were measured at different pH and different concentrations of substrate. The maximum acetylation fraction [S)----infinity) at pH 7.0 in 0.5 M salt was 0.65 with acetylcholine as substrate and 0.57 with acetylthiocholine as substrate. Acetylation is faster than deacetylation. The fraction of acetyl-enzyme was not affected by pH which indicates that acetylation and deacetylation are equally affected by changes in pH. This results supports the concept that acetylation and deacetylation involve similar mechanisms.  (+info)

Nature of the cysteinyl residues in lipophilin from human myelin. (32/38)

Ellman's reagent was used to investigate the status and exposure of the cysteinyl residues in lipophilin, a proteolipid apoprotein from human myelin. The hydrolyzed protein contained 3.5 to 4.5 cysteines per molecule, which increased to 11 after complete reduction. The native protein was thought to contain three disulfide bonds and five free sulfhydryl groups, which undergo partial oxidation during purification. Exposure of -SH groups in the aqueous protein was minimal, even in the presence of 6 M guanidinium chloride, suggesting a location in hydrophobic domains not disrupted by this reagent. In the helicogenic solvent 2-chloroethanol, the full complement of -SH groups could not be revealed, even with the addition of sodium dodecyl sulfate; a difference of two sulfhydryls between intact and hydrolyzed protein was consistently observed. Similar sulfhydryl reactivity toward iodoacetamide was also established in this solvent. Sulfhydryl assays on whole myelin in 2-chloroethanol indicated that the occurrence of -SH groups in the proteolipid component was at least as high as in the purified apoprotein. Lipophilin was reduced and alkylated with 4-vinylpyridine at 10 of its cysteinyl residues. The modified protein adopted a beta structure under conditions where lipophilin is normally highly alpha-helical, and was also less helical than usual in 2-chloroethanol; however, it was still abnormally resistant to denaturation by guanidinium chloride. Modified lipophilin contained as many ester groups as the intact protein; thus, it appeared unlikely that the long chain fatty acids associated with the protein were attached to cysteine residues.  (+info)