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Pesticide exposure has been associated with different adverse health effects which may be modulated to some extent by paraoxonase-1 (PON1) activity and genetic polymorphisms. This study assessed seasonal variations in PON1 activity (using paraoxon –POase–, phenylacetate –AREase–, diazoxon –DZOase– and dihydrocoumarin –DHCase– as substrates), erythrocyte acetylcholinesterase (AChE) and plasma cholinesterase (using butyrylthiocholine –BuChE– and benzoylcholine –BeChE– as substrates. The study population consisted of intensive agriculture workers regularly exposed to pesticides other than organophosphates and non-exposed controls from Almeria (Southeastern Spain). The effect of common genetic polymorphisms of PON1 and BCHE on paraoxonase-1 and cholinesterase activities toward different substrates was also assessed. Linear mixed models were used to compare esterase activities in agricultural workers and control subjects over the two study periods (high and low exposure to pesticides). The significant decrease in AChE and increase in BuChE and BeChE activities observed in workers with respect to control subjects was attributed to pesticide exposure. Workers also had higher levels of AREase, DZOase and, to a lesser extent, of POase, but showed decreased DHCase activity. While PON1 Q192R and PON1 -108C/T gene polymorphisms were significantly associated with all PON1 activities, PON1 L55M showed a significant association with AREase, DZOase and DHCase. BCHE-K (Karlow variant) was significantly associated with lower BeChE activity (but not with BuChE) and BCHE-A (atypical variant) showed no significant association with any cholinesterase activity. These findings suggest that increased PON1, BuChE and BeChE activities in exposed workers might result from an adaptive response against pesticide exposure to compensate for adverse effects at the biochemical level. This response appears to be modulated by PON1 and BCHE gene polymorphisms.

Human butyrylcholinesterase (hBChE) is a highly glycosylated protein present in human plasma. The enzyme hydrolyses choline esters, for example benzoylcholine, butyrylthiocholine and acetylthiocholine as well as noncholine esters like heroin and aspirin. hBChE is primarily involved in neuronal transmission and is a potential bioscavenger of toxic organophosphates to protect acetylcholinesterase. A prerequisite for the therapeutic use of hBChE is a detailed characterization of this glycoprotein purified from human plasma. In this study, MS/MS could confirm most of the protein backbone, including the N- and the C-terminus. Site-specific analysis of all nine potential N-glycosylation sites revealed mainly mono- and disialylated N-glycans to be present on this glycoprotein. Sialic acids (Neu5Ac) are mainly alpha2,6-linked, however a fraction of the N-glycans contained Neu5Ac also in alpha2,3 linkage. On monosialylated N-glycans, sialic acid is exclusively located on the 3-arm and in alpha2,6 linkage, as verified by 2D-HPLC and exoglycosidase digests of 2-aminopyridine (PA)-labelled N-glycans. This first comprehensive glycoproteomic analysis of the important human plasma glycoprotein BChE did not give any indication of O-glycosylation or any other kind of PTMs as previously postulated.

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 K(M)=0.40 mmol/L, maximal reaction rate V(max)=52.2 micromol/L min, inhibition constant K(i)=0,56 micromol/L and IC(50)=1.31 micromol/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 micromol/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.

A special component is isolated from Semen sinapis Albae (white mustard seed), a traditional Chinese medicine. According to the physical and chemical investigation and spectroscopic identification, this component can be known as p-hydroxybenzoylcholine bisulfate, a choline base. This component in the drug is also determined by RP-HPLC. A reversed-phase C(18) column is used to separate the p-hydroxybenzoylcholine with an eluent of methanol-0.05 mol/L monopotassium phosphate solution (30:70) (adjusted by phosphoric acid to pH 3.6) at the flow rate of 0.5 mL/min. Detection is carried out with a UV detector operated at 285 nm, and the column temperature is 25 degrees C. It reveals that there is 0.021% (w/w) of p-hydroxybenzoylcholine bisulfate in Semen sinapis Albae and 0.037% (w/w) in stir-baked Semen sinapis Albae.

Both enantiomers of quinuclidin-3-yl benzoate (RQBz and SQBz) were synthesized in order to examine the stereoselectivity of the hydrolysis of these esters catalyzed by horse serum butyrylcholinesterase (BChE). The hydrolysis of benzoylcholine (BzCh) was also studied in order to determine the influence of the alcohol part of ­the esters upon the kinetics. The kcat value for the substrates decreased in order BzCh > RQBz (4-fold slower) ≫­SQBz (76-fold slower reaction). KM values determined for quinuclidinium substrates revealed that the binding affinity of RQBz (0.28 mM) is approximately 2-fold lower than that of SQBz (0.13 mM) towards BChE. From the ratio of the enantiomeric kcat/KM values, an enantiomeric excess of 78% was calculated, indicating that the resolution of racemic quinuclidin-3-yl benzoate can be achieved by hydrolysis with BChE. The orientations of all the studied benzoate esters and butyrylcholine (BuCh) in the active site of human BChE were proposed by flexible ligand docking with AutoDock 3.0. Analyses of the Michaelis complexes obtained revealed that there are numerous similar close contacts in the active site. The main difference in binding of quinuclidinium and choline esters was found in the ammonium electrostatic region which includes cation–π interaction of the ammonium moiety of substrates with the indole ring ­of Trp84. The important cation–π interaction with Trp84 was lowest in the case of the S-enantiomer of QBz, which might be the main explanation for the slowest rate of hydrolysis of that compound. Copyright © 2002 John Wiley & Sons, Ltd.

The rat is the model animal for toxicity studies. Butyrylcholinesterase (BChE), being sensitive to inhibition by some organophosphorus and carbamate pesticides, is a biomarker of toxic exposure. The goal of this work was to characterize the purified rat BChE enzyme. The cDNA sequence showed eight amino acid differences between the active site gorge of rat and human BChE, six clustered around the acyl binding pocket and two below the active site serine. A prominent difference in rat was the substitution of arginine for leucine at position 286 in the acyl pocket. Wild-type rat BChE, the mutant R286L, wild-type human BChE, and the mutant L286R were expressed in CHO cells and purified. Arg286 was found responsible for the resistance of rat BChE to inhibition by Triton X-100. Replacement of Arg286 with leucine caused the affinity for Triton X-100 to increase 20-fold, making it as sensitive as human BChE to inhibition by Triton X-100. Wild-type rat BChE had an 8- to 9-fold higher Km for the positively charged substrates butyrylthiocholine, acetylthiocholine, propionylthiocholine, benzoylcholine, and cocaine compared with wild-type human BChE. Wild-type rat BChE catalyzed turnover 2- to 7-fold more rapidly than human BChE, showing the highest turnover with propionylthiocholine (201,000 min−1). Human BChE does not reactivate spontaneously after inhibition by echothiophate, but rat BChE reactivates with a half-life of 4.3 hr. Human serum contains 5 mg/L of BChE and 0.01 mg/L of AChE. Male rat serum contains 0.2 mg/L of BChE and approximately 0.2 mg/L of AChE.

In a previous study, we showed that purified commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)-H(2)O(2) system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPB-serum-HRP-H(2)O(2) system consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at < or =4 degrees C. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O(2) uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-beta-methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyrylcholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific chemiluminescent assay for this enzyme.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

The glycoside 2-(2-azidoethoxy)ethyl 2-O-benzyl-4,6-O-benzylidene-β-d-glucopyranoside was prepared and then reacted, first with methyl phosphonyl dichloride, then with dimethylaminoethanol, to give the methylphosphonyl diester 9. N-Methylation of 9 with methyl iodide and subsequent catalytic hydrogenation gave the methylphosphonyl diester 11. The corresponding phenylphosphonyl diester 14 was also prepared in a similar way. The phosphonyl diesters 11 and 14 are analogs of the transition states for 3-O-acetylation and 3-O-benzoylation of β-d-glucopyranose derivatives with acetyl- and benzoylcholine, respectively. They will be used for generation of catalytic antibodies.

Secondary metabolites derived from benzoic acid (BA) are of central importance in the interactions of plants with pests, pathogens, and symbionts and are potentially important in plant development. Peroxisomal β-oxidation has recently been shown to contribute to BA biosynthesis in plants, but not all of the enzymes involved have been defined. In this report, we demonstrate that the peroxisomal ATP-binding cassette transporter COMATOSE is required for the accumulation of benzoylated secondary metabolites in Arabidopsis (Arabidopsis thaliana) seeds, including benzoylated glucosinolates and substituted hydroxybenzoylcholines. The ABNORMAL INFLORESCENCE MERISTEM protein, one of two multifunctional proteins encoded by Arabidopsis, is essential for the accumulation of these compounds, and MULTIFUNCTIONAL PROTEIN2 contributes to the synthesis of substituted hydroxybenzoylcholines. Of the two major 3-ketoacyl coenzyme A thiolases, KAT2 plays the primary role in BA synthesis. Thus, BA biosynthesis in Arabidopsis employs the same core set of β-oxidation enzymes as in the synthesis of indole-3-acetic acid from indole-3-butyric acid.

A benzoate-selective membrane electrode made with heptyl-4-trifluoroacetylbenzoate as a neutral carrier was successfully used to determine cholinesterase activity in blood serum, by detecting benzoic acid formed by the enzymatic hydrolysis of benzoylcholine with cholinesterase. The electrode decreased interference by chloride much more effectively than a previous benzoate electrode made with the ion exchanger methyltridodecylammonium chloride, and facilitated the sensitive assay of benzoate (0.2–50 mM) in the presence of serum. The detection limit of benzoate in physiological saline was 100 μM. The present procedure compares favorably with an established colorimetric determination of cholinesterase, being simple, rapid and economical.

The orientations of chiral quinuclidin-3-ol esters and benzoylcholine in the active site of horse butyrylcholinesterase have been investigated by flexible ligand docking. Change of the esters' acyl moiety as well as the substituent at the quinuclidinium nitrogen atom affected the activity and stereoselectivity of the biotransformations. Analysis of interactions in the active site revealed the most important binding pat-terns for enantiomers, which define their reactivity. Calculated Gibbs energies of binding obtained by mo-lecular docking simulations were well correlated to the experimentally determined binding affinities of the investigated chiral esters. (doi: 10.5562/cca2060)

The mechanisms of acetylcholinesterase (AChE)-catalyzed hydrolyses of four choline esters, (acetylthio)choline, (propanoylthio)choline, (butanoylthio)choline, and benzoylcholine, have been compared by measuring relative activities and pH-rate effects. The reactivity of Electrophoris electricus AChE toward these substrates decreases in the above order and spans a 1500-fold range of k cat , and a 2300-fold range of k cat /K m . The pH-V/K profile for (acetylthio)choline hydrolysis shows that activity depends on the basic form of an active site residue, probably H440, that has a pK a =6.22±0.03 (Rao, M.; et al. J. Am. Chem. Soc. 1993, 115, in press; ref 23)

Pseudocholinesterase (ChE) (acylcholineacylhydrolase, EC 3.1.1.8) has been partially purified (about 270-fold) from sheep brain. The procedure included ammonium sulfate fractionation (20-80%), DEAE-Trisacryl M chromatography and procainamide-Sepharose 4B affinity chromatography. The molecular weight of purified ChE was found to be 290,000 by gel filtration. Kinetic properties of the enzyme have been studied using the substrate analogues choline, succinylcholine and benzoylcholine. It was shown that the inhibition was partially competitive.

A SPECIFIC choline esterase is an enzyme which hydrolyses acetylcholine at a higher rate than any other esters1. Richards and Cutkomp2, however, have found that choline esterase from insect nerves is more active on acetyl-s-methylcholine than on acetylcholine. I have also arrived at similar results with other invertebrates3. Thus the whole animal of Patella vulgata, and the ampulla and podia of Asterias rubens from the west coast of Sweden, split acetyl-I-methylcholine at a higher rate than acetylcholine. Benzoylcholine is very little split. In contrast to this, Terebratulina caput serpentis hydrolyses acetylcholine but neither acetyl-I-methylcholine nor benzoylcholine.

The behaviors of the enantiomers of cocaine (benzoylecgonine methyl ester) and related compounds with butyrylcholinesterase (BChE; EC 3.1.1.8) were investigated spectrophotometrically at 235 nm. The unnatural enantiomer, (+)-cocaine, was hydrolyzed by BChE (extinction coefficient 6.7 L.mmol-1.cm-1) at about half the rate of benzoylcholine, but over 2000 times faster than naturally occurring (-)-cocaine. This rapid hydrolysis of (+)-cocaine may account, in part, for its pharmacological inactivity. (+)-Norcocaine, (+)-benzoylecgonine, (-)-psi-cocaine and tropacocaine were also substrates for BChE. Hydrolysis of (+)-cocaine was sensitive to several standard inhibitors of BChE, including those of competitive, carbamate and organophosphorus classes. Although (-)-cocaine was a poor substrate for debenzoylation, it was a fairly good competitive inhibitor (Ki approximately 10 microM) of the hydrolysis of other substrates. The cocaine metabolites (-)-norcocaine, (-)-benzoylecgonine and (-)-ecgonine methyl ester inhibited BChE with Ki values of 15, 76 and 1300 microM, respectively. (+)-psi-Cocaine had Ki = 3 microM, p-Nitro and p-fluoro derivatives of cocaine and analogs with phenyl and p-fluorophenyl groups in place of the benzoyl ester linkage (WIN 35,065-2 and WIN 35,428) inhibited BChE comparably to (-)-cocaine itself. Both cocaine enantiomers were weak inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7) from human erythrocytes with similar Ki values (160-170 microM). Although it is unlikely that the inhibition of BChE is an important factor in the subjective effects of cocaine, it may have implications for the toxicity of cocaine to the fetus, since BChE appears in the development of the central nervous system before AChE, and has been suggested to function as an embryonic acetylcholinesterase.

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 1.3 min and 5.5 min, respectively. By the peak areas of A233 signals, the linear dynamic ranges for both analytes were 0.01–50.0 mM, and the relative standard deviations of 1.0 mM 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 5.0 mM (r = 0.999). The relative standard deviations for 1.0 mM 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).

The 5000-g supernatant fraction of whole-bulb-mite homogenates was shown to possess a cholinesterase (ChE) that hydrolyzed acylcholine esters in the order acetyl>propionyl>butyryl. Acetyl-β-methylcholine, but not benzoylcholine, also was hydrolyzed as were acetylthiocholine and acetyl-β-methylthiocholine. No inhibition by excess substrate was observed at cholinester concentrations as high as 30mM. Cholinesterase activity was markedly insensitive to eserine and to certain other carbamates and organophosphates. Only organophosphates of the dimethylphosphate type generally were active ChE inhibitors. It was concluded that the inability of carbamates such as eserine, and organophosphates such as those with alkyl groups larger than dimethyl, to inhibit the bulb-mite ChE was probably a consequence of the nature of the esteratic site. The data suggested that ChE inhibition was likely involved in the toxicity to bulb mites of some of the toxic carbamates and organophosphates, but that it might not be the only mechanism involved, at least with several of the compounds.

Acetylcholinesterases (AChEs) have been estimated in the infective juveniles (IJs) of eight different strains of heterorhabditid nematodes. The enzyme content ranged from 45.6 to 421.3 units/10 5 IJs with specific activity 34.0 to 82.6 units/mg protein. The isoenzyme patterns revealed the existence of two-slow-moving isoforms. Heterorhabditis bacteriophora AChE1A has been purified from the IJs of the heterorhabditid nematode strain of the highest enzymatic activity to homogeneity by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and DEAE-Sepharose. The specific activity of the purified enzyme was 1378.1 units/mg protein with purification fold 17.5 over crude extract. The enzyme has a pH optimum at 7.5. The optimum temperature for enzyme activity and stability was 35 °C. The activation energy was calculated to be 9.0 kcal/mol. The enzyme hydrolyzes acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), S -butyrylthiocholine (BuSCh) and benzoylthiocholine (BzSCh) iodides with relative rate 100, 74.6, 41.7 and 22.2%, respectively. It displayed an apparent Michaelis–Menten behavior in the concentration range from 0.1 to 2 mM for the three former substrates with K m values 0.27, 0.42 and 0.59 mM, respectively. H. bacteriophora ChE1A is an AChE since it hydrolyzed AcSChI at higher rate than the other substrates and displayed excess substrate inhibition with AcSChI at concentrations over 2 mM. It was inhibited by eserine and BW284C51, but not by iso -OMPA. Its biochemical properties were compared with those reported for different species of insects as target hosts for heterorhabditid nematodes and animal parasitic nematodes.

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 K(m) 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 k(cat) 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 V(max). This was due to the loss of substrate availability. The best substrates for rat butyrylcholinesterase were short alkyl homologues, BzCh - BCH4.

Catalytic parameters of human butyrylcholinesterase (BuChE) for hydrolysis of homologous pairs of oxo-esters and thio-esters were compared. Substrates were positively charged (benzoylcholine versus benzoylthiocholine) and neutral (phenylacetate versus phenylthioacetate). In addition to wild-type BuChE, enzymes containing mutations were used. Single mutants at positions: G117, a key residue in the oxyanion hole, and D70, the main component of the peripheral anionic site were tested. Double mutants containing G117H and mutations on residues of the oxyanion hole (G115, A199), or the π-cation binding site (W82), or residue E197 that is involved in stabilization of tetrahedral intermediates were also studied. A mathematical analysis was used to compare data for BuChE-catalyzed hydrolysis of various pairs of oxo-esters and thio-esters and to determine the rate-limiting step of catalysis for each substrate. The interest and limitation of this method is discussed. Molecular docking was used to analyze how the mutations could have altered the binding of the oxo-ester or the thio-ester. Results indicate that substitution of the ethereal oxygen for sulfur in substrates may alter the adjustment of substrate in the active site and stabilization of the transition-state for acylation. This affects the k2/k3 ratio and, in turn, controls the rate-limiting step of the hydrolytic reaction. Stabilization of the transition state is modulated both by the alcohol and acyl moieties of substrate. Interaction of these groups with the ethereal hetero-atom can have a neutral, an additive or an antagonistic effect on transition state stabilization, depending on their molecular structure, size and enantiomeric configuration.

Publisher Summary Two structurally and functionally very similar, yet distinct enzymes form the family of cholinesterases (ChEs). Acetylcholinesterase and butyrylcholinesterase both catalyze acetylcholine (ACh) hydrolysis with similarly high efficiency, and only differ in efficiency to catalyze the hydrolysis of carboxylic acid esters of larger acyl group size, such as butyrylcholine or benzoylcholine. Larger substrates are hydrolyzed much better by BuChE because of small but significant differences in their structure that also allows BuChE to bind generally bulky ligands much better, including covalent inhibitors iso-OMPA and bambuterol, and reversible inhibitor ethopropazine, all well-known specific inhibitors of ChEs. The chapter summarizes the latest structural information on the cholinesterase family of enzymes and discusses their catalytic properties. Alignment of 125 full-length or nearly full-length ChE sequences demonstrates a high degree of similarity conserved in the family from fungal and viral enzymes to man. Sixty-one reported three-dimensional structures are analyzed, illustrating the high degree of similarity in protein-fold conformation, irrespective of the presence of bound ligands, and this argues for the absence of large conformational changes in the protein interaction with both inhibitors and substrates. At the same time, a plentitude of small-amplitude backbone and selected side chain movements illustrate that rapid fluctuations in conformational states of ChEs prevail in solution, and ligands have the capacity to select those conferring higher affinity and lower energy of the respective complex.

The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) 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 BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2)k(3) 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.

Benzoic acid, formed by the enzymatic hydrolysis of benzoylcholine with cholinesterase, is detected with a benzoate-sensitive membrane electrode made with tridodecylmethylammonium chloride as ion-exchanger and o -nitrophenyl octyl ether as plasticizer. The detection limits were 0.02 mM benzoate in phosphate buffer at pH 7.4 and 0.1 mM in the presence of blood serum. Cholinesterase in blood serum was determined successfully by using this electrode.

A 64-year-old man was admitted to our hospital because of possible liver cirrhosis. His serum cholinesterase was anomalously low with a delta pH of 0.1 (normal range; 0.8-1.1). His enzyme was more heat-labile than the normal controls. Km value of his enzyme for benzoylcholine was 1.1 x 10(-5) mol/l, while that for normal controls was 2.3 x 10(-6) mol/l. In addition, isozymic alteration of his enzyme was observed. Sequencing of the white blood cell DNA of the patient showed a point mutation at nucleotide 1093 (GGA to CGA), which changes codon 365 from glycine to arginine.

The inhibition of the human serum cholinesterase phenotypes, usual (U), atypical (A) and heterozygous (UA), by the dimethylcarbamate of (2-hydroxy-5-phenylbenzyl)-trimethylammonium bromide (Ro 02-0683), was followed with benzoylcholine, acetyl-, butyryl- and propionyl-thiocholine as substrates. The first-order rate constants were calculated from the linear part of the inhibition curves and were independent of the substrate used for measuring the enzyme activity. The second-order rate constants for the U, UA and A phenotypes were 8.3 × 106, 6.1 × 106 and 0.05 × 106 M^− 1 min^− 1, respectively. The constant of the enzyme-inhibitor complex for the atypical serum was 7.7 μ M, and the rate of carbamylation of the enzyme was 0.386 min^− 1. The rate of reactivation of carbamylated usual and atypical enzyme was found to be same ; the half-time of reactivation was about 3.5 hr. The deviation from the linearity of the inhibition course was explained by spontaneous reactivation of the inhibited enzyme ; the theoretical inhibition curves were in good agreement with the experimentally obtained values. The three phenotypes could be distinguished by the rate of inhibition by the dimethylcarbamate, Ro 02-0683, in the progressive phase of inhibition or by the degree of inhibition in the apparent steadystate.

A fluorometric assay using 3-(p-hydroxyphenyl) propionic acid (HPPA) was conducted to determine the activity of pseudocholinesterase (ChE) [Enzyme Commission (EC) No. 3.1.1.8] in postmortem blood samples so as to test for organophosphate poisoning. By the enzymatic reaction of ChE, its substrate, benzoylcholine, produces choline, which is oxidized by choline oxidase to generate hydrogen peroxide. HPPA is oxidized by hydrogen peroxide and peroxidase to become the fluorogenic dimer whose concentration is measured fluorometrically at an excitation emission wavelength of 320 nm and an elimination emission wavelength of 404 nm. The selectivity and sensitivity of the present method were found to be superior to those of conventional pH and spectrophotometric methods.

Effects of rat and human calcitonin gene-related peptide (r alpha CGRP and h beta CGRP, respectively) upon uterine contractile force were investigated using uterine horns from nonpregnant rats, r alpha CGRP and h beta CGRP were equipotent (pD2 = 8.85-9.09) in inhibiting spontaneous and electrically evoked uterine contractions. r alpha CGRP was relatively ineffective in inhibiting potassium-induced contractures of preparations from stilbestrol-pretreated rats. The use of selective antagonists established that r alpha CGRP did not release prostanoids, or release or act at receptors for catecholamines and histamine. The effects of the peptides were not significantly modulated by estrogen levels since pD2 values were similar (8.56-8.86) in field-stimulated preparations from rats in proestrus/estrus or metestrus/diestrus.

A new method was developed using flow injection analysis (FIA) with amperometric detection for determining the activity of esterases, such as lipase and cholinesterase. Such esterases catalyze hydrolysis reactions of the substrate esters to release constituent acids. The released acids were determined based on current height of the reduction prepeak of quinone. Olive oil, as a substrate, produces higher fatty acids through lipase enzyme reaction. Benzoylcholine or acetylcholine, as a substrate, produces benzoic acid or acetic acid through cholinesterase enzyme reactions. Samples containing the enzymes were incubated with a substrate at 37°C, and test solutions were prepared. The released acids in the test solutions were determined by FIA. Enzyme activity in the samples was subsequently calculated. The relative standard deviation of activity values in human serum were less than 3% (n=5) in all cases. Good correlation was observed for the present and conventional methods in lipase and cholinesterase activity determinations, respectively.

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.

A method that overcomes the difficulties of the 240-nm benzoylcholine method for phenotyping plasma cholinesterases has been developed. After a timed reaction, under the same reaction conditions as in the classic procedure, choline is detected at 500 nm by use of choline oxidase coupled with the peroxidase/phenol/aminoantipyrine system. Cholinesterase activity measurements, calibrated by use of choline iodide as standard, are linearly related to results obtained with propionylthiocholine as substrate at 25 degrees C (y = 0.14x + 0.17, n = 30, r = 0.98). Results of differential inhibition with dibucaine and fluoride are virtually identical with those obtained by the ultraviolet method (y = 0.97x + 4.3, r = 0.995, and y = 0.93x - 0.5, r = 0.987, respectively) and give the same classification of homo- and heterozygotes for the usual, atypical, and fluoride-resistant variants. The new method has substantial advantages in that it eliminates the difficulties associated with measuring small changes in high absorbances at a suboptimal wavelength on a steep portion of the absorption curve.

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.

An enzyme that degrades atropine is found in varying frequency in the sera and tissues of rabbits. Its presence or absence can readily be detected by the rate of hydrolysis of α-naphthyl acetate (α-NA). The frequency distributions of the plasma enzyme(s) hydrolyzing α-NA, atropine, procaine and benzoylcholine were markedly similar, raising questions regarding the genetic inheritance and whether these esters were hydrolyzed by 1 enzyme, by a group of closely related esterases or by distinctly different proteins. Techniques including electrophoresis, titrimetric and spectrophotometric analysis of substrate specificity and inhibition sensitivity were employed to characterize the plasma enzyme in adult male and female albino New Zealand rabbits. Atropinesterase was identified as a nonspecific carboxyl-esterase, occurring with a 50% frequency in either sex. Electrophoretically, 2 distinct phenotypes could be detected though the rabbit population could be divided into 3 phenotypes on the basis of rates of α-NA hydrolysis. Inhibition studies suggested that the hydrolysis of the ester-type drugs was the function of 1 genetically determined enzyme existing in multiple forms. The importance of this enzyme in toxicologic studies was confirmed by a study of atropine-induced mydriasis and procaine-induced convulsions in animals known to possess or be deficient in this plasma carboxylesterase.

1. Except for pH optimum, reactivation and “aging”, the results indicate that the ChE of Eisenia foetida is distinct from acetylcholinesterase and butyrylcholinesterase. 2. Propionylcholine was found to be a better substrate than acetylcholine, while butyrylcholine, benzoylcholine and acetyl-beta-methylcholine were found to be poor substrates for this ChE. 3. Little or no tendency towards substrate inhibition was seen. 4. The rate constants of inhibition by alkyl phosphates and carbamates favour the concept of this enzyme being a B-esterase, but different from the two main classes of ChE. 5. The buffer-soluble and membrane-bound ChE showed no differences in substrate specificity and Michaelis-Menten constants. 6. These enzymes separately also showed little or no tendency towards substrate inhibition. 7. Crude homogenate, membrane-bound, buffer-soluble and Triton X-100 solubilized ChE, as well as fractions from isoelectric focusing are acted upon by inhibitors at rather similar rates. 8. The presence of only one type of enzyme is therefore indicated. 9. The existence of isoenzymes is not clearly indicated either by isoelectric focusing, by gel electrophoresis technique or density gradient centrifugation. 10. The latter method indicated the molecular weight to be 108,000 ± 7000.

The kinetics of competition of pairs of two substrates for bovine erythrocyte acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and horse serum cholinesterase (acylcholine acyl-hydrolase, EC 3.1.1.8) was studied so that the hydrolysis of only one substrate was measured at a time. The substrates were acetylthiocholine, phenylacetate and benzoylcholine; the same compounds, and also acetylcholine, were used as competiting substrates i.e. inhibitors. The substrate inhibition constants ( K ss ) and Michaelis constants for the reaction of a single substrate were also determined. It was concluded that the substrate inhibition site in the enzyme does not show up in the competition between two substrates.

A butyrylcholine-hydrolyzing enzyme (EC 3.1.1.-) of Pseudomonas polycolor IFO 3918 was purified approximately 9270-fold with a recovery of 9.9% by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoretic and ultracentrifugational analyses. The molecular weight was determined as approximately 59 000 by gel filtration. Isoelectric focusing electrophoresis revealed that the enzyme had an isoelectric point around pH 5.1. The enzyme catalyzed the hydrolysis of butyrylcholine with the maximum activity among various esters tested, and split benzoylcholine, propionylcholine and some aliphatic esters, but did not attack acetylcholine. The estimated value of Km at pH 7.5 and 25°C was 7 · 10−4 M for butyrylcholine. The enzyme was irreversibly inhibited by organophosphorus compounds and carbamates, such as diisopropylphosphofluoridate and eserine. The enzyme was inhibited by some compounds, such as atropine and quinidine. Quaternary ammonium salts showed an inhibitory effect on the enzyme resemblng co-operation inhibition.

Flounder (Platichthys flesus) muscle contains two types of cholinesterases, that differ in molecular form and in substrate specificity. Both enzymes were purified by affinity chromatography. About 8% of cholinesterase activity could be attributed to collagen-tailed asymmetric acetylcholinesterase sedimenting at 17S, 13S and 9S, which showed catalytic properties of a true acetylcholinesterase. 92% of cholinesterase activity corresponded to an amphiphilic dimeric enzyme sedimenting at 6S in the presence of Triton X-100. Treatment with phospholipase C yielded a hydrophilic form and uncovered an epitope called the cross-reacting determinant, which is found in the hydrophilic form of a number of glycosyl-phosphatidylinositol-anchored proteins. This enzyme showed catalytic properties intermediate to those of acetylcholinesterase and butyrylcholinesterase. It hydrolyzed acetylthiocholine, propionylthiocholine, butyrylthiocholine and benzoylthiocholine. The Km and the maximal velocity decreased with the length and hydrophobicity of the acyl chain. At high substrate concentrations the enzyme was inhibited. The p(IC50) values for BW284C51 and ethopropazine were between those found for acetylcholinesterase and butylcholinesterase. For purified detergent-soluble cholinesterase a specific activity of 8000 IU/mg protein, a turnover number of 2.8 x 10(7) h-1, and 1 active site/subunit were determined.

Esmolol, a new cardioselective beta adrenergic blocker inhibits plasmacholinesterase activity in vitro. The concentration of esmolol hydrochloride that inhibits by 50 per cent the hydrolysis of 50.0 mumol.L-1 benzoylcholine hydrochloride by 1:200 diluted, heparinized pooled plasma of six healthy volunteers at 37 degrees C and 240 nm, determined by the ultraviolet spectrophotometric method of Kalow, was 50 mumol.L-1. Esmolol's primary metabolite, 3-[4-(2-hydroxy-3-(isopropylamino)propoxy)-phenyl]propionic acid, had an I50 = 190 mumol.L-1. The benzoylcholine hydrolysis rates in the plasma of ten patients who received an esmolol infusion of 500 micrograms.kg-1.min-1 for 4 minutes were 58.6 +/- 6.2 mumol.hr-1.ml-1 (mean +/- SE) before and 55.1 +/- 6.6 mumol.hr-1.ml-1 after the infusion. The benzoylcholine hydrolysis rates in the plasma of ten patients who received an esmolol infusion of 500 micrograms.kg-1.min-1 for two minutes and 200 micrograms.kg-1.min-1 for an additional two minutes were 70.2 +/- 8.9 mumol.hr-1.ml-1 before and 69.1 +/- 9.5 mumol.hr-1.ml-1 after the infusion. The pre- and post-infusion plasmacholinesterase activities were not significantly different. Since plasmacholinesterase is responsible for the hydrolysis of succinylcholine and that of the ester-type local anaesthetics this lack of in vivo interaction of esmolol with the hydrolysis of these drugs should be further confirmed by experiments with these combinations in man.

Four methods for measuring serum cholinesterase activity have been applied to sera of normal individuals and of patients shown to be sensitive to short-acting muscle relaxants of the succinyldicholine type. They have been assessed according to their ability to differentiate between sensitive and insensitive individuals on the basis of enzyme activity measurements alone. The method described, based upon that of Dietz et al. [Clin. Chem. 19, 1309 (1973)], in which propionylthiocholine is used as substrate, is best for this purpose, being capable of identifying over 90% of affected individuals with no false positives. Acetylcholine and butyrylthiocholine are slightly inferior substrates in this respect, and benzoylcholine gives little useful information.

Five differential inhibitors of plasma cholinesterase have been compared using benzoylcholine as substrate. None of the inhibitors (dibucaine, NaF, NaBr, NaCl, or pancuronium dibutyryloxy bromide) could be used singly to resolve all the variants. Better resolution was obtained when two inhibitors were used in conjunction. Clear differentiation of all six genotypes was obtained only with the combined use of pancuronium dibutyryloxy bromide and sodium fluoride. The limitations of some of the parameters are discussed.

From the upper parts of SPIGELIA ANTHELMIA L. besides some widely distributed phenolcarboxylic acids and flavonoids two volatile alkaloids have been isolated and identified as isoquinoline and an iridoid compound of the actinidine type. In the water extract choline, benzoylcholine and 3,3-dimethyl-acryloylcholine could be identified. Preliminary pharmacological investigations revealed that the alkaloids are involved in the cardiotonic activity of the plant.

We report the evaluation of a new commercially available assay system for determination of the catalytic activity of serum cholinesterase (EC 3.1.1.8) and application of the method to a centrifugal fast analyzer. Serum cholinesterase activity is determined at 30 degrees C using para-hydroxybenzoylcholine as substrate. This reaction is coupled to a second reaction using para-hydroxybenzoate hydroxylase (EC 1.14.13.2) as coupling enzyme. Enzyme activity is measured kinetically by monitoring the decrease in absorbance at 340 nm of NADPH in the second reaction. The procedure is precise and the results obtained from normal and pathological sera show good correlation with those obtained by the alternative procedures employing propionylthiocholine, butyrylthiocholine and benzoylcholine as substrates. The reference range for 700 healthy subjects was estimated to be 140-345 U/L (95% central range, determined non-parametrically), with significant difference between males and females (155-353 U/L for men and 134-323 U/L for women, p less than 0.001).

Highly purified human serum cholinesterase (EC 3.1.1.8, also known as pseudocholinesterase and butyrylcholinesterase) had peptidase activity toward substance P. Digestion of substance P was monitored by high performance liquid chromatography, which separated three product peptides. The cleavages occurred sequentially. The first peptide to appear was Arg1-Pro2. The Km for this hydrolysis was 0.3 mM; maximum activity was 7.9 nmol min−1 mg−1 of protein, which corresponded to a turnover number of 0.6 min−1. A second cleavage yielded Lys3-Pro4. A third cleavage occurred at the C-terminal, where the amide was removed from Met11 to yield a peptide containing residues 5–11. Both the peptidase and esterase activities of the enzyme were completely inhibited by the anticholinesterase agent, diisopropyl-fluorophosphate. Substance P inhibited the hydrolysis of benzoylcholine (a good ester substrate) with a KI of 0.17 mM, indicating that substance P interacted with cholinesterase rather than with a trace contaminant. Peptidase and amidase activities for serum cholinesterase are novel activities for this enzyme. It was demonstrated previously that the related enzyme acetylcholinesterase (EC 3.1.1.7) catalyzed the hydrolysis of substance P, but at entirely different cleavage sites from those reported in the present work. Since butyrylcholinesterase is present in brain and muscle, as well as in serum, it may be involved in the physiological regulation of substance P.

For assaying plasma cholinesterase (EC 3.1.1.8) activity and phenotyping by means of dibucaine inhibition, we have compared a commercially available kit, in which butyrylthiocholine is used as substrate, with two reference methods, one using benzoylcholine and the other propionylthiocholine. With 50 different samples of three of the most common genetic variants, we could clearly differentiate the variants with benzoylcholine and dibucaine, whereas there was some overlap of the E1uE1u and E1uE1a phenotypes with the other two substrates at 30 degrees C. The phenotypes were better differentiated at 25 degrees C, and in our hands the use of butyrylthiocholine was preferable to propionylthiocholine for phenotyping with dibucaine. The affinity of the usual and atypical homozygotes for fluoride with butyrylthiocholine gave an inverted response to the affinity of these variants for the anion with benzoylcholine. We suggest that this may be explained by the role of the chromogen or its products in the assay procedure with the thiocholine substrate.

A new enzymatic method for the determination of serum pseudo-cholinesterase activity is described. Choline, which is liberated from benzoylcholine as substrate by cholinesterase, is oxidized by choline oxidase to betaine with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to yield a chromogen with maximal absorbance at 500 nm. The calibration curve is linear up to 1500 units per liter of serum. The method is reproducible, and the results correlate well with those obtained by the method using butyrylthiocholine as substrate and 5,5'-dithiobis-(2-nitrobenzoic acid) as color reagent.

Several alkylating agents (bromobenzylcyanide, chloroacetophenon, and xylyl bromide) were found to inhibit cholisterase from human plasma. Less reactive alkylating compounds such as bromoacetone, chloroacetate, and iodoacetate did not inhibit the enzyme. Chloropicrin, which is a powerful oxidizing agent, was not found to inhibit either. Several mercaptide-forming agents, like p- chloromercuribenzoic acid, diphenylaminechloroarsine, and ethyl-dichloroarsine, were effective inhibitors. w- Chloroacetophenon was found to be an instantaneous and noncompetitive inhibitor. The inhibition was reversible by dilution or dialysis. The degree of inhibition was higher in an aqueous medium that when a less polar solvent was used. These findings suggest that a sulfhydryl group is not involved during the inhibition of cholinesterase by alkylating agents. The possibility of a reaction on the histidine or methionine groups of the enzyme is discussed. The inhibition also was dependent on the substrate employed during the activity assay. The order of percentage of inhibition was found to be acetylcholine > butyrylcholine > benzoylcholine. These data are used to offer an interpretation for some aspects of the lachrymatory action of these agents.

Although the nature of the cholinesterases in brain, red blood cells and plasma has been studied extensively, little comparative information is available on the distribution and characteristics of the types of cholinesterase present in other issues, and I would like to describe very briefly some of the results which Miss M. G. Ord and I have been obtaining in the course of a survey of different mammalian issues (Ord & Thompson 1950 a ). Owing to the species differences in blood esterases we have limited our work to a study of rat and human tissues. In order to obtain some initial information about the distribution of the so-called ‘true’ and ‘pseudo-’ cholinesterases we began by comparing the relative rates of hydrolysis of acetylcholine (ACh) and of acetyl- β -methylcholine (MCh) and benzoylcholine (BCh); that is to say, we used the physiological substrate and also the selective substrates for the true and pseudo-cholinesterases introduced by Mendel, Mundell & Rudney (1943). Apart from a few scattered observations on a small number of tissues from different animal species no attempt to compare systematically the distribution of these esterases in different tissues of animals of the same species was made until Sawye & Everett (1947) showed that the liver, the uterus and a number of glandula tissues of the rat were able to hydrolyze benzoylcholine more rapidly than acetyl β -methylcholine.

1. 1. The acetylcholinesterase (acetylcholine acetyl hydrolase, E.C. 3.1.1.7) levels in erythrocytes and benzoyl cholinesterase (benzoylcholine hydrolase, E.C. 3.1.1.9) levels in plasma of rats subjected to portacaval anastomosis were found to be significantly depressed to the subnormal range for many weeks after the operation. 2. 2. Depression of the plasma enzyme is probably due to decreased synthesis as a result of liver dysfunction, while the erythrocyte depression is due to inhibition. 3. 3. Dilution and dialysis restored the erythrocyte enzyme activity to normal, showing that the inhibitor is reversible. 4. 4. The haematocrit reading in shunted rats showed a slight but significant decrease post-operatively. 5. 5. Recovery of the enzyme levels to the normal range parallels total body weight gain.