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A portable amperometric ion sensor was fabricated by integrating silica isoporous membrane (SIM) and organogel composed of polyvinyl chloride and 1,2-dichloroethane (PVC-DCE) on a 3D-printed polymer chip. The detection of ionic species in aqueous samples could be accomplished by adding a microliter of sample droplet to the sensor and by identifying the ion-transfer potential and current magnitude at the water/organogel interface array templated by SIM. Thanks to the ultrasmall channel size (2–3 nm in diameter), high channel density (4 × 108 μm–2), and ultrathin thickness (80 nm) of SIM, the ensemble of nanoscopic water/organogel (nano-W/Gel) interface array behaved like a microinterface with two back-to-back hemispherical mass diffusion zones. So, the heterogeneous ion-transfer across the nano-W/Gel interface array generated a steady-state sigmoidal current wave. The detection of choline (Ch) and its derivatives, including acetylcholine (ACh), benzoylcholine (BCh), and atropine (AP), in aqueous samples wa...

Semiempirical molecular orbital calculations have been used to investigate the properties of certain sulfur and selenium analogs of pharmacologically active agents. Calculations have been performed for acetylthiocholine and acetylselenocholine with and without extravalent d orbitals. The study reveals that octet expansion in the ester fragment of the acetylcholine congeners is not necessary to explain the bonding. Calculations were extended to model compounds where the ether and acyl oxygen are sequentially replaced by sulfur and selenium in an attempt to obtain structure-activity relationships for similar benzoylcholine congeners.

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.

Butyrylcholinesterase (BuChE) occurs in a multiple molecular forms whose catalytic activity depends on tissue distribution and species. The hypothesis led us to the study of BuChE catalytic properties focused on the inter-tissue and inter-species level with benzoylcholine and N-alkyl derivates of benzoylcholine (BCHn) as substrates. These compounds are soft disinfectants easily biodegradable to biologically inactive hydrolytic products, substituted choline and benzoic acid. Different sources of BuChE were used: rabbit and rat liver microsomal fraction (membrane-anchored enzyme) and serum (soluble form). Hydrolytic activity of both these BuChE forms was compared to rat recombinant BuChE (rBuChE). Hydrolytic product (benzoic acid) formation was recorded as function of time, and hydrolytic rate was determined. Tissue distribution of BuChE plays an important role in hydrolysis of BCHn. High BuChE activity was observed in a serum of both studied species rat and rabbit and was significantly dependent on a structure of substrates. Activity of soluble serum forms was the same as that for the rBuChE. Significant change of BuChE activity was recorded on the inter-species level in the microsomal fractions. It is because the rabbit microsomal BuChE activity had absolutely different course for all substrates as compared to rat microsomes. Inhibitory studies of BCHn enzymatic hydrolysis of all BuChE forms were performed to determine the level of BuChE participation in BCHn hydrolysis. It can be concluded that short-chain BCHn substrates are exclusively hydrolyzed by BuChE from all studied sources except for the rabbit liver microsomal fraction. Rabbit seems to have different enzymes involved in the hydrolysis of all studied BCHn compounds.

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.

Butyrylcholinesterase (BChE) displays hysteretic behavior with certain neutral and charged substrates in the approach to steady state. Previous studies led us to interpret this phenomenon in terms of slow transitions between two enzyme conformers E and E'. This kinetic peculiarity is observed in human, horse and rat BChE. Oscillations that superimpose on the hysteretic lag are observed when benzoylcholine and N-alkyl derivatives of benzoylcholine are used as substrate. Hysteresis of BChE can be modulated by medium parameters (pH, salts, temperature, and pressure). Though mutant enzymes show different hysteretic behavior, so far attempts to provide a molecular mechanism of BChE hysteresis from mutagenesis studies have been unproductive. However, the substrate dependence of the hysteretic induction times, using wild-type BChE and several mutants, allowed us to build a general, mechanistic model for the hysteresis. In this model, substrate can bind to E, E', or both conformers, and ES and/or E'S can be catalytically active. The exact pathway followed depends on both the nature of the substrate and the structure of the BChE mutant under study. We propose that oscillations develop when substrate exists in different, slowly interconvertible, conformational and/or aggregation forms, of which only the minor form is capable of reacting with BChE. In support of this proposal, NMR studies have provided direct evidence for slow equilibria between monomeric and micellar forms of long-chain, alkyl derivatives of benzoyl-(N-substituted) choline. There is no direct evidence that hysteresis plays a role in BChE function(s). However, the "new view" of protein dynamics proposes that proteins are normally in equilibrium between pre-existing, functional and non-functional conformers; and that binding a ligand to the functional form shifts that equilibrium towards the functional conformation. Therefore, a physiological or toxicological relevance for the hysteresis in BChE cannot be ruled out.

The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (K(eq) = 12 microM) for the two agonist binding sites. Upon UV irradiation at 254 nm, [3H]APFBzcholine was photoincorporated into the nAChR alpha, gamma, and delta subunits in an agonist-inhibitable manner. Photolabeled amino acids in the agonist binding sites were identified by Edman degradation of isolated, labeled subunit fragments. [3H]APFBzcholine photolabeled gammaLeu-109/deltaLeu-111, gammaTyr-111, and gammaTyr-117 in binding site segment E as well as alphaTyr-198 in alpha subunit binding site segment C. The observed pattern of photolabeling is examined in relation to the predicted orientation of the azide when APFBzcholine is docked in the agonist binding site of a homology model of the nAChR extracellular domain based upon the structure of the snail acetylcholine binding protein.

Butyrylcholinesterase (BChE; E.C. 3.1.1.8.) was 260-fold purified from soluble fraction of rat intestine. The enzyme was composed of tetrameric globular form by nonreducing electrophoresis. Optimum pH value was determined as 7.2 after zero buffer extrapolation. Optimum temperature was examined as 37 degrees C after zero time extrapolation. The enzyme showed marked substrate activation with positively charged, acyl-choline substrates. As a measure of catalytic efficiency, kcat/Km values were determined as 16,210, 25,650, and 46,150 for acetylthiocholine (ATCh), propionylthiocholine (PTCh), and butyrylthiocholine (BTCh), respectively. When the catalytic efficiencies are compared, soluble isoform of rat intestinal BChE became increasingly efficient as the size of the acyl portion of the substrate increases; BTCh > PTCh > ATCh. Differently, the enzyme showed substrate inhibition with benzoylcholine (BzCh) and a kcat/Km value of 21,190 was found. Triton X-100 inhibited more efficiently the rat intestinal BChE soluble isoform than it did the human serum BChE.

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.

Butyrylcholinesterase purified from human serum as 6600-fold was heated at 37 degrees, 40 degrees, 45 degrees, and 50 degrees C for 24 hr. It was observed that the enzyme heated at 45 degrees 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 degrees C for 12 hr shown negative cooperativity. On the contrary to the heated enzyme at 40 degrees C for 24 hr, the heated enzyme at 45 degrees C for 24 hr could not be reactivated when it was kept at 4 degrees 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 degrees C for 24 hr competitively, whereas succinylcholine was the partial competitive inhibitor of native enzyme but the pure competitive inhibitor of the heated enzyme.

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.

The G117H mutant of human butyrylcholinesterase (EC 3.1.1.8) was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.

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.

The optimized molecular structures and vibrational frequencies and also gauge including atomic orbital (GIAO)1H and13C NMR shift values of benzoylcholine chloride [(2-benzoyloxyethyl) trimethyl ammonium chloride] have been calculated using density functional theory (B3LYP) method with 6-31++G(d) basis set. The comparison of the experimental and calculated infrared (IR), Raman, and nuclear magnetic resonance (NMR) spectra has indicated that the experimental spectra are formed from the superposition of the spectra of two lowest energy conformers of the compound. So, it was concluded that the compound simultaneously exists in two optimized conformers in the ground state. Also the natural bond orbital (NBO) analysis has supported the simultaneous exiting of two conformers in the ground state. The calculated optimized geometric parameters (bond lengths and bond angles) and vibrational frequencies for both the lowest energy conformers were seen to be in a well agreement with the corresponding experimental data.

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).

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.

Benzoylcholine Iodide and Chloride reactant: 80.5 g. (66ml., 1 mole) of redistilled ethylene chlorohydrin intermediate: 2-chloroethyl benzoate intermediate: 194 g. (0.70 mole) of 2-iodoethyl benzoate reactant: 270 ml. of a 19.5% solution of trimethylamine product: 210 g. (0.63 mole) of benzoylcholine iodide product: Benzoylcholine chloride Keywords: acylation; alkylation, N-alkylation; esterification, unsubstituted monobasic acids; replacement reactions, halogen by halogen; replacement reactions, miscellaneous; methyl ethyl ketone; silver chloride, preparation of; trimethylamine, generation of

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.

[(3)H]4-Benzoylbenzoylcholine (Bz(2)choline) was synthesized as a photoaffinity probe for the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Bz(2)choline acts as an nAChR competitive antagonist and binds at equilibrium with the same affinity (K(D) = 1.4 microm) to both agonist sites. Irradiation at 320 nm of nAChR-rich membranes equilibrated with [(3)H]Bz(2)choline results in the covalent incorporation of [(3)H]Bz(2)choline into the nAChR gamma- and delta-subunits that is inhibitable by agonist, with little specific incorporation in the alpha-subunits. To identify the sites of photoincorporation, gamma- and delta-subunits, isolated from nAChR-rich membranes photolabeled with [(3)H]Bz(2)choline, were digested enzymatically, and the labeled fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. For the gamma-subunit, Staphylococcus aureus V8 protease produced a specifically labeled peptide beginning at gammaVal-102, whereas for the delta-subunit, endoproteinase Asp-N produced a labeled peptide beginning at deltaAsp-99. Amino-terminal sequence analysis identified the homologous residues gammaLeu-109 and deltaLeu-111 as the primary sites of [(3)H]Bz(2)choline photoincorporation. This is the first identification by affinity labeling of non-reactive amino acids within the acetylcholine-binding sites, and these results establish that when choline esters of benzoic acid are bound to the nAChR agonist sites, the para substituent is selectively oriented toward and in proximity to amino acids gammaLeu-109/deltaLeu-111.

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.

To the Editor: The magnitude of sevoflurane’s (and enflurane’s) effect on fluoride inhibition of pseudocholinesterase (PCHE) deserves attention in modern anesthetic practice. Nearly all studies dealing with this topic1–3 refer to the study of Kambam et al.4 who reported an inhibition of PCHE between 16 to 73% with 10 to 100 μmol·L–1 fluoride, the concentrations usually obtained during sevoflurane (and enflurane) anesthesia. Since we could not confirm this inhibition with mivacurium and butyrylthiocholine as substrates and at temperatures between 28 to 37°C3,5 we investigated fluoride inhibition of PCHE with benzoylcholine at 25°C, the substrate and temperature Kambam et al. used in their study, and at 37°C, the temperature currently recommended for determination of enzymatic activity. After approval by our Local Ethics Committee and with written informed consent, serum samples of eight healthy volunteers were obtained, spiked with fluoride (0, 10, 50 and 100 μmol·L–1) and adjusted to 25°C and 37°C, respectively. Thereafter, 50 μmol·L–1 benzoylcholine were added, and at timed interval aliquots were withdrawn to measure the concentrations of benzoylcholine by high-performance liquid chromatography. After calculating the half-lives with KINETICA 2000 (InnaPhase Corp. Philadelphia, PA, USA) a one-factor (fluoride concentration) analysis of variance with a post hoc Dunnett test was performed at each temperature, using the fluoride-free sample as the control category. The results are presented in the Figure. At 25°C there was an increase of the benzoylcholine half-time of 9.3% with 10 μmol·L–1 fluoride, and a 36% increase with 50 μmol·L–1 fluoride. The 68% increase in benzoylcholine half-time was significant (P < 0.05) at a concentration of 100 μmol·L–1. In contrast, no change was observed over the same range of fluoride concentrations at 37°C. We thus confirm one aspect of the study of Kambam et al.4 who measured PCHE activity at 25°C with a photometric assay. In contrast, when using a high-performance liquid chromatography assay at clinically relevant body temperature (37°C) and with fluoride concentrations up to 100 μmol·L–1, fluoride inhibition of PCHE is not apparent, whether using the substrates mivacurium and butyrylthiocholine3,5 or benzoylcholine, as reported here. In conclusion, the influence of substrate and temperature on fluoride inhibition of PCHE must be taken into consideration when interpreting the results of investigations with sevoflurane and enflurane.

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.

Using quin2, the effects of aliphatic hydrocarbons on the system of Ca(2+)-induced Ca2+ release in isolated membranes of rabbit skeletal muscle terminal cisterns have been studied. The hydrocarbons were inserted into the membranes by means of hydrocarbon-containing liposomes. 2,2,4-Trimethylpentane (isooctane) caused a rapid release of 70-75% of Ca2+ taken up by the terminal cistern vesicles during the Ca(2+)-pump operation. This effect was inhibited by the caffeine-induced Ca2+ release blockers--Mg2+, ruthenium red and tetracaine. The same was observed with a decrease in the concentration of ATP that is known to activate the terminal cistern Ca2+ channels. The effect of 2,2,4-trimethylpentane on the longitudinal cistern fractions practically devoid of Ca(2+)-channels was insignificant. Heptane, hexane and octane caused a slow release of 5-10% of the accumulated Ca2+ from the terminal cistern vesicles; no such effect was induced by decane.

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 initial rate of incorporation of 14C or 3H-labeled choline into Ehrlich-Lettre ascites cells of the glycogen-free strain seven days after inoculation was investigated in vitro. 1. At choline concentrations in the medium between 6 to 30 muM and 100 to 500 muM the choline uptake by the cells followed Michaelis-Menton Kinetics with V values between 31 to 100 and 59 to 500 pmol per minute at a given cell density, and average Q10-values of 2.1 at the high and of 2.4 at the low choline molarity. The K-m-values increased from 27 muM to 58.8 muM at low and from 0.11 mM to 0.22 mM at high choline concentrations over a temperature range between 15 degrees C and 37 degrees C. Arrhenius plot of the V values gave two lines, one with a transition temperature at 25 degrees C at low and one straight line at high choline concentrations, from which the energy of activation for choline uptake was determined to be 16 kcal/mol. 2. It is assumed that two systems exist for the choline uptake by the ascites cells. One, operative at low substrate concentrations, which is saturable and probably is to be classified as a carrier-mediated facilitated diffusion process, can be strongly inhibited by deoxyglucose or 2,4-dinitrophenol and also by substrate analogues such as chlorocholine or benzoylcholine. Ouabain affects this system to a lesser extent. The other system functioning at high choline concentrations may be a simple diffusion process, which is little inhibited by substrate analogues, ouabain and deoxyglucose; however, it is also inhibited by 2,4-dinitrophenol and p-chloromercuribenzoate. 3. Choline incorporation into the acid-insoluble material (lecithin) gave linear Michaelis-Menton kinetics at the low and the high substrate concentration respectively. K-m-values decreased with an increase in temperature at low and increased with rising temperature at high substrate concentrations thus reflecting a close relationship between choline uptake and its metabolism. Labeling of lecithin choline in the various subcellular fractions under the conditions of the functioning of a carrier-mediated process was in the order: mitochondria (50%) greater than plasma membranes (25%) greater nuclei (14%) greater than microsomes (9%) greater than supernatant (1.5%). 4. Treatment of the cells with p-chloromercuribenzoate or heat shock at 50 degrees C markedly reduced the cholinee uptake and concomitantly its conversion into lecithin. Kinetic analysis revealed that the inhibitory effect of p-chloromercuribenzoate was competitive and that of the heat shock non-competitive in nature. Further the choline uptake by the cells was found to be the rate-limiting step, since the rate of choline phosphorylation was determined by the extracellular choline concentration. Pulse chase experiments showed a rapid turnover of the choline moiety with a concomitant increase in activity of the lecithin fraction and little change within the choline phosphate pool.

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.

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.