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Butyrylcholinesterase (BChE) is associated with many neurodegenerative diseases, especially Alzheimer's disease (AD). Developing specific imaging tools for BChE is now highly pursued because BChE has substantially similar biochemical properties to acetylcholinesterase (AChE) but distinct roles in AD. Inspired by the BChE-catalyzed hydrolysis of specific substrates (benzoylcholine, tetracaine, and cocaine) containing benzoyl group in physiological condition, a bioinspired probe PM1 with high specificity towards BChE was designed. The docking models indicated that PM1 could easily access the active center of BChE rather than AChE, showing the significance of benzoyl group in specific recognition of BChE. The control probes PM2–5 with smaller recognition groups displayed lower specificity to BChE than PM1 , showing the high efficiency of the bioinspired design strategy. The living cells experiments showed that PM1 signals were weakened by BChE inhibitor but not influenced by AChE inhibitor, which was in contrast to the performance of probe PM2–5 , showing the specific recognition of PM1 for BChE in biological system. Moreover, PM1 has been used to study the positive correlation between BChE level and cell apoptosis for the first time, and the further study showed that Aβ deposition could induce the increase in BChE level and accompany the occurrence of cell apoptosis. Notably, PM1 was successfully traced the enhancement of BChE activity in the brain slice of AD mouse model. Such a rational approach may provide a solid basis for designing specific probes with different properties to study BChE in biosystems. [Display omitted] • Inspired by the BChE-catalyzed hydrolysis of specific substrates, a bioinspired probe towards BChE was designed. • Screening probes with different recognition groups and the docking models showed rationality of our probe. • Probe PM1 has been used to study the positive correlation between BChE level and cell apoptosis for the first time. • The study showed that Aβ could induce the increase in BChE level and accompany the occurrence of cell apoptosis. • PM1 was successfully traced the enhancement of BChE activity in the brain slice of AD mouse model. [ABSTRACT FROM AUTHOR]

In this study we studied the kinetic interaction of indole-3- acetic acid with human serum butyrylcholinesterase. Butyrylcholinesterase was purified from human serum and the benzoylcholine kinetics was analyzed. The enzyme had a Km value of 14.22 ± 2.97 μM. The Hill plot was linear with a value of nH = 0.95, displaying that the enzyme follows Michaelis-Menten kinetics with regard to benzoylcholine. Through inhibition studies, indole-3-acetic acid was found to be a noncompetitive inhibitor for human serum butyrylcholinesterase with benzoylcholineas substrate. The Ki value was calculated as 1.86 ± 0.27 mM. In our previous study on human serum butyrylcholinesterase using butyrylthiocholine as substrate, indole-3-acetic acid was found to be a linear-mixed type inhibitor.This difference in kinetic behavior with regards to two different substrates could arise from the tighter binding of the more bulky and hydrophobic benzoyl group to the hydrophobic pocket of the active site gorge rather than the butyryl group and that the binding of this butyryl group could be sterically hindered by the indole-3-acetic acid.

Abstract: A capillary electrophoresis method and a durable choline biosensor were developed for measuring serum cholinesterase (EC 3.1.1.8) activity, a useful clinical index for liver function. The former is based on separation of benzoate and benzoylcholine (the artificial substrate of cholinesterase) in an uncoated fused-silica capillary. The migration time of benzoylcholine and benzoate was 1.3min and 5.5min, respectively. By the peak areas of A 233 signals, the linear dynamic ranges for both analytes were 0.01–50.0mM, and the relative standard deviations of 1.0mM benzoylcholine and benzoate were less than 4% and 6%, respectively. The FIA-choline sensor was constructed with the working electrode of the flow cell covered with a natural chitinous membrane purified from Taiwanese soldier crab, Mictyris brevidactylus. The biomembrane served as the supporting material for enzyme immobilization (choline oxidase, EC 1.1.3.17), and also prevented protein adsorption on the electrode surface. The calibration curve was linear between 0.05 and 5.0mM (r =0.999). The relative standard deviations for 1.0mM choline (n =7) were less than 3%, and the activity of the bioactive membrane lasted for about 2 months. The analytical results of both methods correlated well (r =0.940). [Copyright &y& Elsevier]

Pancuronium bromide (PCBr) inhibition effect on enzyme cholinesterase from pooled human serum (Che, EC 3.1.1.8 acylcholine acylhydrolase) was used for development of a spectrophotometric kinetic method for PCBr determination in human serum and urine. Optimal conditions for the basic and inhibitor reactions were established: pH=7.7 and substrate concentration c(benzoylcholine chloride)=1.33 mmol/L. Kinetic parameters were also determined: Michaelis-Menten’s constant KM=0.40 mmol/L, maximal reaction rate Vmax=52.2 μmol/L min, inhibition constant Ki=0,56 μmol/L and IC50=1.31 μmol/L. Linear dependence between the reaction rate and inhibitor concentration exists in PCBr concentration range 8.20–68.25 nmol/L, which corresponds to the real sample concentrations from 0.328 to 2.730 μmol/L. The method detection and quantification limits were 2.01 nmol/L and 6.67 nmol/L, respectively. Precision of the method was tested for three pancuronium concentrations (10.70, 29.35 and 51.25 nmol/L). Relative standard deviation (RSD) was in the range 0.15–7.45%. Accuracy was examined by standard addition method. Influence of the substances usually present in serum and urine on the reaction rate was tested. The developed method was applied for PCBr content determination in serum model samples, urine model samples and in urine taken during surgery. The method has good sensitivity, accuracy, precision and it is suitable for clinical practice. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The purpose of this work was to study the catalytic properties of rat butyrylcholinesterase with benzoylcholine (BzCh) and N-alkyl derivatives of BzCh (BCHn) as substrates. Complex hysteretic behaviour was observed in the approach to steady-state kinetics for each ester. Hysteresis consisted of a long lag phase with damped oscillation. The presence of a long lag phase, with no oscillations, in substrate hydrolysis by rat butyrylcholinesterase was also observed with N-methylindoxyl acetate as substrate. Hysteretic behaviour was explained by the existence of two interconvertible butyrylcholinesterase forms in slow equilibrium, while just one of them is catalytically active. The damped oscillations were explained by the existence of different substrate conformational states and/or aggregates (micelles) in slow equilibrium. Different substrate conformational states were confirmed by 1H-NMR. The Km values for substrates decreased as the length of the alkyl chain increased. High affinity of the enzyme for the longest alkyl chain length substrates was explained by multiple hydrophobic interactions of the alkyl chain with amino acid residues lining the active site gorge. Molecular modelling studies supported this interpretation; docking energy decreased as the length of the alkyl chain increased. The long-chain substrates had reduced kcat values. Docking studies showed that long-chain substrates were not optimally oriented in the active site for catalysis, thus explaining the slow rate of hydrolysis. The hydrolytic rate of BCH12 and longer alkyl chain esters vs. substrate concentration showed a premature plateau far below Vmax. This was due to the loss of substrate availability. The best substrates for rat butyrylcholinesterase were short alkyl homologues, BzCh – BCH4. [ABSTRACT FROM AUTHOR]

The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcho linesterase (BuChE) is deacylation (k3), whereas it is acylation (k2) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzChbywild-typeandat75 MPaforD70G. For hydrolysis of BzSCh the change occurred at higher pressures because k2 < < k3 at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BuChE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state. [ABSTRACT FROM AUTHOR]

Butyrylcholinesterase purified from human serum as 6600-fold was heated at 37°, 40°, 45°, and 50°C for 24 hr. It was observed that the enzyme heated at 45°C for 24 hr converted to a stabilized form and followed Michaelis-Menten kinetics, whereas the enzyme samples, heated at the other temperatures for 24 hr, shown negative cooperativity with respect to its substrate, butyrylthiocholine. Even the sample heated at 45°C for 12 hr shown negative cooperativity. On the contrary to the heated enzyme at 40°C for 24 hr, the heated enzyme at 45°C for 24 hr could not be reactivated when it was kept at 4°C for 24 hr. In the kinetic studies, it was found that substrate analogs choline and benzoylcholine inhibited both the native enzyme and the enzyme heated at 45°C for 24 hr competitively, whereas succinylcholine was the partial competitive inhibitor of native enzyme but the pure competitive inhibitor of the heated enzyme. [ABSTRACT FROM AUTHOR]

Two substrates, acetyl thiocholine iodide ATCI and benzoylcholine chloride BCC were compared for the determination of plasma cholinesterase ChE levels and after incorporation of dibucaine and sodium fluoride in the assay, their usefulness in determining plasma ChE genetic variability was assessed in 64 healthy subjects. With both substrates, plasma ChE levels were found to be in the reference range. However, ATCI could detect only two variants with the usual phenotype UU in 60 of 64 [93.75%] subjects whereas with BCC 6 different groups could be determined. Though both substrates are of equal value in estimation of plasma ChE levels, BCC is definitely superior in determining its genetic variants.

The measurement of cholinesterase activity in human serum is an important investigation, especially in patients suspected of poisoning with organophosphate insecticides, or those experiencing prolonged paralysis following treatment with the short acting muscle relaxant succinylcholine. Appropriate clinical management of affected individuals can only be instituted if enzyme activity is measured using a method capable of clear interpretation and phenotypic ascription of cholinesterase, ascertained by use of selected enzyme inhibitors, is reliable. This review considers factors which lead to significant cholinesterase abnormalities and advises on the techniques most appropriate to their investigation. It is concluded that no one method is suitable both for measurement of activity and determination of genotype. The use of propionyl thiocholine is recommended for the first procedure and benzoyl choline for the latter. Those laboratories in which a request for the assessment of cholinesterase status is an unusual event should make greater use of assistance from colleagues whose experience with the problem is greater.

Discriminant function analysis has been applied to the results of activity and inhibitor measurements carried out on a series of 229 specimens using benzoylcholine and butyrylthiocholine as substrate. The discriminant function was more effective in differentiating cholinesterase genotypes than either a single test or a combination of two tests.

Cholinesterase (ChE) activity was measured in the central nervous system (CNS) and in the digestive gland of the pestiferous land snail Achatinafulica Bowdich, by the method of Huegra et al. (1952). Acetylcholinesterase (AChE), and benzoylcholinesterase (BeChE) activity was higher in the former than in the latter. The complete inhibition of the enzyme activity with 10(-2) M eserine indicates that the ChE examined is AChE. The Km values of the AChE from the digestive gland and the CNS were 3.1 x 10(-5) and 9.0 x 10(-5) (M), respectively. The enzyme is the most active at pH 8.2 and 37 degrees C up to 60 min.

The in vitro inhibitory effect of glycopyrrolate and atropine on the hydrolysis of benzoylcholine at 50.0 microM concentration was determined in the plasma of 6 healthy volunteers at 37 degrees C and at 240 nm by the ultraviolet spectrophotometric method of Kalow. The 50 per cent inhibitory concentration (I50) of glycopyrrolate was found to be 1.0 mM and the I50 of atropine was 0.9 mM. This inhibitory effect of glycopyrrolate and atropine is moderate but its interaction with the in vivo hydrolysis of succinylcholine or that of ester-type of local anaesthetics in patients should be further studied. Although both glycopyrrolate and atropine inhibit PChE to the same extent, glycopyrrolate should be utilized in patients in whom inhibition of brain PChE or central effects ought to be avoided, since glycopyrrolate does not penetrate the blood-brain barrier.