Horse Serum Cholinesterase Research Articles

Comparison of chemical composition and biological activities of Seseli rigidum fruit essential oils from Serbia

Abstract Plants from genus Seseli, have been widely used in European traditional medicine, exhibiting antibacterial, antifungal, insect repellent, emmenogogue, antiflatulence, anti-inflammatory, antinociceptive, anti-tumor, antirheumatic activities and protective effect on human lymphocytes DNA. They usually grow on mountain rocky terrains. Part of their habitat on Vidlic Mountain, located in South-east Serbia, was struck with a large wildfire. Seseli rigidum fruit essential oils (from post fire and control areas) compositions were analyzed by GC and GC-MS, identifying monoterpenes α-pinene and sabinene as most abundant. Statistical tests showed a non-significant difference in chemical composition of these two oils, but a significant difference in comparison with the herb from a geographically different origin. Antimicrobial tests showed strong activities of the oils against tested bacteria, thus confirming its administration in various inflammation processes as a quite effective remedy. Applying DPPH. and ABTS+. radical scavenging and total reducing Fe(III) to Fe(II) power assays, antioxidant characteristics of both studied essential oils were estimated as weak, though of close values. Seseli rigidum fruit essential oil was proven as a potent inhibitor of human and horse serum cholinesterase, recognizing its possible application as neural protective agent

Ionization constants, octanol partition coefficients and cholinesterase inhibitor constants for chlorpromazine and its metabolites.

The relationship between the lipophilic character of chlorpromazine and seven of its metabolites and their ability to inhibit horse serum cholinesterase (Ki) has been investigated. Log(1/Ki) values were correlated with log P octanol partition coefficients (r = 0.88, P less than 0.01, n = 8). The inhibitor values ranged from 2.7 x 10(-6) M for chlorpromazine to 48.6 x 10(-6) M for monodesmethylchlorpromazine sulphoxide. Ionization constants were determined by limiting solubility, spectrophotometry and pH-partition characteristics. Demethylated metabolites were more basic than the tertiary amines and the sulphoxides were slightly less basic than the corresponding sulphides.

Cholinesterase-inhibiting Potentials of Amberlite XAD-2 Resin Extracts Collected from River and Drinking Waters in Northwest District of Chiba Prefecture, Japan

Amberlite XAD-2 resin extracts of river and drinking water sampled in each month during the period from January to December 2008 from the Northwest district of Chiba Prefecture were investigated to characterize and determine their cholinesterase (ChE)-inhibiting potentials and pesticide levels. The XAD-2 extracts from river water collected during the mid-spring to mid-summer periods exhibited strong inhibition effect to horse serum ChE, reflecting the application of organophosphorus and carbamate pesticides to paddy fields. Gas chromatographicmass spectrometric (GC/MS) determinations of the XAD-2 extracts of the river water collected during spring to summer periods also showed to be comparatively high levels of agricultural chemicals, such as herbicides, insecticides and fungicides, as compared with those detected in the drinking water. Although a considerable reduction in the ChE-inhibiting potentials and in the GC/MS detectable compound levels was observed for the river water samples, it is particularly interest that ChE-inhibiting potentials still remained in the drinking water.

How thionphosphonates inhibit activity of different cholinesterases

Analysis of mechanism of reversible inhibition of human erythrocyte acetylcholinesterase (AChE), of horse serum cholinesterase (ChE), and ChE of optical ganglia tissue of individuals of the Commander squid Berryleuthis magister from various habitat zones was studied under effect of thionphosphonates (P=S), derivatives of piperidine, morpholine, perhydroazepine as well as several heterocyclic model compounds. Data of comparative inhibitory specificity have allowed us to suggest that thionphosphonates are sorbed in the area of cholinesterase esterase center through the phosphoryl part of the inhibitor molecule, rather than through its heterocyclic grouping. An advantage in the antienzyme efficiency of thionphosphonates (P=S) over phosphonates (P=O) is revealed. Effect of the ion strength is used for analysis of contribution of the hydrophobic—hydrophilic interaction in the enzyme—inhibitor system.

Inhibition of serum cholinesterase by trialkylphosphorothiolates.

The kinetics of inhibition of horse serum cholinesterase (EC 3.1.1.8) by six trialkylphosphorothiolates was studies (25 degrees C, pH 7.4). The compounds were : OOS-trimethylphosphorothiolate (OOS-Me), OSS-trimethylphosphorodithiolate (OSS-Me), SSS-trimethylphosphorotrithiolate (SSS-Me) and their corresponding ethyl analogues (OOS-Et, OSS-Et, SSS-Et). The second order rate constants of inhibition ranged from 7.2 to 2128 mol-1 1 min-1, of inhibition ranged from 7.2 to 2128 mol-1 1 min-1, and the enzyme/inhibitor dissociation constants from 0.079 to 1.5 mM. The ethyl esters were better inhibitors than their methyl analogues and the OSS-compounds were better inhibitors than the OOS- or SSS-compounds. The same structure-activity relationship is known to hold for the reaction of the compounds with acetylcholinesterase (EC 3.1.1.7).

Degradation by rat tissues in vitro of organophosphorus esters which inhibit cholinesterase

Hydrolytic “A”-esterase activities of various tissues of rat (plasma, liver, kidney, brain and intestinal mucosa) against selected OP esters of diverse structure as potential substrates (paraoxon, di- n-propyl paraoxon, di- n-butyl paraoxon, chlorpyrifos oxon, di-(4-phenyl butyl) phosphorofluoridate and the chiral isomers of ethyl 4-nitrophenyl phenylphosphonate) were studied. We have developed a sensitive and widely applicable assay depending on measuring decline in residual inhibitory power of any chosen OP against horse serum cholinesterase: for seven compounds examined so far I 50s against BuChE ranged from 0.07 to 70 nM, and it is easy to monitor loss of OP starting from an initial 25 μM concentration. Progressive destruction rates were always highest in liver and plasma with activity sometimes detectable in kidney, brain but not in intestinal mucosa, but the ratios of activity between tissues differed for different substrates. At 25 μM/37°/pH 7.2 hydrolysis rates ranged from 8500 nmol/min/g liver for di-(4-phenylbutyl) phosphorofluoridate down to 0.8 nmol/min for the butyl analogue of paraoxon; the rate for l(-) isomer of EPN oxon (23 nmol/min/g liver) was >2× that for the d(+) isomer and for paraoxon. From our data we conclude that several OP hydrolases exist whose identity may be further characterised by use of selective substrates.

Reversible conversion of octadecyl-bonded silica to ion-exchange surfaces for protein separations

Octadecyl-bonded silica, commonly used for reversephase high-pressure liquid chromatography, was modified using surfactants bearing ionizable groups and the modified packing used in ion-exchange chromatography of proteins. The surfactants 2-( n-hexadecylheptaethoxy)acetic acid, 1-( n-hexadecyloctaethoxy)ethylenediamine, and N-( n-hexadecyloctaethoxy)pyridinium were adsorbed onto test columns packed with octadecyl-bonded silica particles. The proteins lysozyme, bovine serum albumin, trypsin, horse serum cholinesterase, and bovine liver carboxylesterase were used to study the ion-exchange characteristics of the modified packings. The retention order of the proteins on the surfactant-modified stationary phases were as predicted by the isoelectric point of each protein. In addition, the interaction of enzymes with the packings did not result in significant loss of enzymatic activity. Surfactant removal was possible with the use of organic solvents and this allowed the octadecyl-bonded surface to be used again in the reverse-phase mode. During the course of the experiments, no degradation in the packing's performance was observed due to loss of adsorbed surfactant, even after over 85,000 column volumes of sodium chloride and Tris-HCl buffers were circulated through the column.

N alpha-dansyl-L-arginine 4-phenylpiperidine amide. A potent and selective inhibitor of horse serum cholinesterase.

The relationship between chemical modifications of arginine derivatives and inhibitory activity to horse serum cholinesterase (BuChE) was investigated. It provided a new insight into the topography of the active site of BuChE. 1) BuChE has the hydrophobic binding pocket, the depth of which corresponds to the length of ethylpiperidine. 2) In the opposite side to the hydrophobic binding pocket, BuChE has a certain entity which repulses carboxyl group at the 2-position of piperidine of L-arginine piperidine amide. 3) The P site of BuChE can allow 4-propyl and 4-phenyl group attached to piperidine. Comparison of the results with those of thrombin and trypsin clearly revealed similarities and dissimilarities among BuChE, trypsin, and thrombin in the active site topography, and hence, we introduce a new selective inhibitor for BuChE, N alpha-dansyl-L-arginine 4-phenylpiperidine amide. It inhibits BuChE strongly (Ki = 0.016 microM), whereas it inhibits trypsin, thrombin, plasmin, and glandular kallikrein only weakly and shows actually no inhibition on acetylcholinesterase from the human erythrocyte. In addition, the new inhibitor becomes highly fluorescent when bound with BuChE, indicating that the compound is an ideal probe of the interactions of BuChE as well as a titrant of it.

Enzymatic method for the estimation of residual organophosphorus compounds in water after treatment with chlorine-containing oxidant

A sensitive method for the estimation of residual organophosphorus compounds in water after treatment with chlorine-containing oxidants has been developed. The method is based on the inhibiting action of organophosphorus compounds towards the enzyme-catalyzed hydrolysis of esters. After the oxidants have been quantitatively destroyed by a mixture of NaHCO3 and NaNO2 small amounts of O-ethyl S-(N, N-diisopropylamino)ethyl methylphosphonothioate (MESP), dichlorvos (DDVP) and dimethoate are determined by using horse-serum cholinesterase with 2,6-dichloroindophenyl acetate as the substrate. A linear response range of 2 of 3 orders of magnitude and recovery within 100 ± 5% has been generally obtained.

Affinity surfactants as reversibly bound ligands for high-performance affinity chromatography

Pyridine was coupled covalently to a nonionic ethoxylated alcohol: octaethylene glycol n-hexadecyl ether. This modified surfactant was found to be a reversible, competitive inhibitor of horse serum cholinesterase. The surfactant bound irreversibly, in aqueous media, to octadecylbonded reverse phase silica particles commonly used for high-performance liquid chromatography. The amount of ligand bound was found to be 550 μmol/ml of packing, a concentration that is over 100 times higher than what can be normally bound to agarose affinity chromatography supports. With this packing, a 280-fold purification of cholinesterase from horse serum and a 79-fold purification of human serum cholinesterase were accomplished, with yields greater than 80%, using a 2-cm-long column and a 7-min elution time. The affinity surfactant could be eluted from the column using a 6:4 (v/v) mixture of methanol and isopropanol. This technique should be generally applicable in the development of biospecific supports for high-performance affinity chromatography.

Isoproterenol Inhibition of Horse Serum Cholinesterase is Connected with Subunit Dissociation

Tetrameric cholinesterase from horse serum undergoes concentration-dependent dissociation. The dimer is highly stable so that even on SDS polyacrylamide gels subunit dissociation to the 80-kDa polypeptide chains is incomplete. Glutaraldehyde cross-linking confirms this finding, giving rise to a tetramer: dimer ratio of approximately 1:1. The beta-adrenergic agent isoproterenol acts as an inhibitor of the enzyme with respect to butyrylthiocholine hydrolysis; inhibition kinetics point to a dissociative effect of the ligand as the underlying mechanism (Söylemez, Z. & Ozer, I. (1985) Comp. Biochem. Physiol. 81c, 433-437). Evidence from sedimentation analysis confirms this hypothetical mechanism: the sedimentation coefficient in the presence of saturating concentrations of both the substrate butyrylthiocholine and the inhibitor isoproterenol shows a 35 +/- 5% decrease; in high speed sedimentation equilibria the weight average molecular mass is shifted from the tetramer (Mr = 312 +/- 12 kDa) to the dimer (Mr = 160 +/- 10 kDa). The transition is complete at isoproterenol concentrations below saturation. Applying glutaraldehyde cross-linking to monitor the particle distribution at varying isoproterenol concentrations confirms the change in quaternary structure in a qualitative way. Enzyme concentrations applied in the present experiments are in the range of the concentration of cholinesterase in horse serum. Therefore the dissociative mechanism of isoproterenol on the enzyme may be of biological significance.

Serum cholinesterase inhibition by boronic acid

Horse serum cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) was reversibly inhibited by a variety of alkyl- and areneboronic acids with K i values ranging from 6.2 mM (methaneboronic acid) to 3.1 μM (diphenylboric acid). Binding to the enzyme was apparently at the active center, because inhibition obeyed competitive kinetics and because boronic acids protected the enzyme from inactivation by phenylmethane-sulfonyl fluoride. Boronic acids should prove useful in probing the active center of serum cholinesterase.

ChemInform Abstract: SYNTHESIS OF 5′‐THYMIDINYL BIS(1‐AZIRIDINYL)PHOSPHINATES AS ANTINEOPLASTIC AGENTS

AbstractDas Acetyl‐thymidin (I) wird über die Stufe des Phosphorsäureesterdichlorids (II) in die Aziridin‐Derivate (IV) übergeführt, aus denen die recht instabilen freien Hydroxy‐ Verbindungen (V) entstehen.

Synthesis of 5'-thymidinyl bis(1-aziridinyl)phosphinates as antineoplastic agents.

Reaction of 3'-acetylthymidine with phosphorus oxychloride in trimethyl phosphate yielded the phosphorodichloridate 5, which was subsequently reacted with aziridine, or 2,2-dimethylaziridine to give compounds 6 and 7, respectively. The 2,2-dimethylaziridine derivative 7 was considerably more active than 6 against leukemia L1210 and P-388 in mice but less active than the previously synthesized, simpler phosphinate derivatives 2 and 3. It appears that the thymidine moiety did not enable these compounds to use the nucleoside transport mechanism of the cells and also failed to increase the selectivity of the 2,2-dimethylaziridine analogues by interference with their binding to cholinesterase. Compound 7 strongly inhibited horse serum cholinesterase, while 6 was inactive.

Fluorescence and N.M.R. studies of the binding of cholinergic fluorescent probes to horse serum cholinesterase.

The interaction of the cholinergic fluorescent probes, 1-(5-dimethyl-aminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminoaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like carbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.

Aryl N-methoxy- N-methylcarbamates: Potent competitive reversible inhibitors of cholinesterases

A series of 27 substituted aryl N-methoxy- N-methylcarbamates were synthesized and their ability to reversibly inhibit house fly-head and bovine-erythrocyte acetylcholinesterase and horse-serum cholinesterase was determined. These compounds were all competitive, reversible inhibitors of bovine erythrocyte acetylcholinesterase but some of them showed mixed competitive inhibition against the house fly-head and horse-serum enzymes. Dissociation constants ( K i ) as small as 9.9 × 10 −9 M and as large as 1.4 × 10 −4 M were observed. A highly satisfactory correlation between log K i for the inhibition of fly-head acetylcholinesterase by the N-methoxy- N-methylcarbamates and −log I 50 for the inhibition of the same enzyme by the corresponding methylcarbamates was noted. Analysis of the anticholinesterase data by multiple regression showed -log K i to be related to Hansch's π constant and ring position terms. The results indicate that reversible binding of these compounds to acetylcholinesterase occurs by hydrophobic bonding.

INHIBITION OF RAT CHOLINESTERASE ISOENZYMES IN VITRO AND IN VIVO BY THE POTATO ALKALOID, ?-CHACONINE2

The inhibition of purified bovine erythrocyte acetylcholinesterase (AchE) and horse serum cholinesterase (ChE) by α-chaconine was found to be a mixed-type with inhibition constants (Ki) for both enzymes of 8.3 × 10-6 M and 4.0 × 10-4 M, respectively. Kinetic constants for AchE obtained for the hydrolysis of acetylthiocholine iodide averaged 7.2 × 10-5 moles/liter/min (Vmax) and 6.2 × 10-5 M (Km) whereas for ChE these were 3.8 × 10-4 moles/liter/min (Vmax) and 1.3 × 10-4 M (Km) for the hydrolysis of butyrylthiocholine iodide. Subcellular acetylcholinesterase activity in nuclear-free rat brain homogenate showed the highest activity to be equally distributed between the mitochondrial and microsomal fractions, with only little activity in the nuclear fraction. Inhibition of the rat brain subcellular enzyme activity in vitro by 0.016 M α-chaconine was 43% for whole homogenate, 55% for nuclear fraction, 35% for mitochondria, and 33% for microsomes. In vivo administration of α-chaconine, (10, 30 and 60 mg/kg) to adult male rats caused a reduction of brain AchE activity to 79, 55 and 18% of the control activity for the respective doses. Heart and plasma AchE activity of treated animals did not show a dose-related inhibition similar to that of brain. Inhibition of heart AchE was 61%, while that of plasma was 51% for rats administered to 10 mg/kg and no further inhibition for rats given 30 mg/kg α-chaconine was noted. Isoenzymes of rat brain cholinesterases, separated by acrylamide gel electrophoresis, were inhibited by 30 and 60 mg/kg α-chaconine administered intraperitoneally. Electrophoretic separation of plasma from the treated animals resulted in five anodally migrating zones that hydrolyzed acetylthiocholine and α-naphthylacetate. Inhibition of the activity of isoenzyme bands 1 and 2 was 40 and 77% respectively for animals given 10 mg/kg α-chaconine, while rats given 30 mg/kg showed inhibition of 100 and 75%, respectively. Isoenzyme bands 3 and 4 were completely inhibited in the plasma of alkaloid treated animals. In vitro inhibition of rat plasma, erythrocyte and brain esterase isoenzymes was estimated by incubating gels with 10-4 M α-chaconine after electrophoretic separations. The slower-moving isoenzyme bands were inhibited completely, while the faster migrating isoenzyme bands in plasma and erythrocytes were least affected by the concentration of α-chaconine used.

Relationships between Chemical Structure and Inhibition of Human Placental Choline Acetyltransferase by Keto Analogs of Acetylcholine

Seven keto analogs of acetylcholine were synthesized and evaluated as inhihitors of human placental choline acetyltransferase. Their potencies for inhibition of horse serum cholinesterase and stimulation of cholinergic receptors in the longitudinal ileal muscle of the guinea pig were investigated. The most potent and selective inhibitor of choline acetyltransferase was (2-benzoylethyl)trimethylammonium chloride with an I50 of 3 × 10–6M. It exhibited considerably low activities at muscarinic and nicotinic receptors and cholinesterases. Its high potency for inhibiting choline acetyltransferase was attributed to: (a) its cationic terminal, a site for an electron acceptor interaction; (b) an aryl moiety for hydrophobic and electron donor contributions; and (c) a positive charge on the carbon atom adjacent to the benzene ring due to the presence of the carbonyl group, which interacts with the nucleophilic residue on the enzyme.

Studies on the chiral isomers of fonofos and fonofos oxon: I. Toxicity and antiesterase activities

The toxicity of the ( R) P and ( S) P chiral isomers and racemates of fonofos and fonofos oxon to insects and white mice were determined. ( R) P-Fonofos and ( S) P-fonofos oxon were 2- to 12-fold more toxic to house flies, mosquito larvae, and mice than were the corresponding enantiomers. The racemates were intermediate in toxicity. Stereoselectivity also was observed in the in vitro inhibition of house fly-head and bovine erythrocyte acetylcholinesterase, horse serum cholinesterase, chymotrypsin, trypsin, and a variety of esterases. In all cases the ( S) P-oxon was a more potent inhibitor than the ( R) P-oxon with k 1 ratios of (S) P (R) P ranging from 4- to 60-fold. Further, differences in levels of house fly-head, mouse brain, and blood cholinesterase obtained from house flies and mice treated with the enantiomers and racemates of fonofos and fonofos oxon were observed. Differences in toxicity of the enantiomers and racemates to house flies and mice were more closely related to in vivo than to in vitro cholinesterase inhibition.

Competition between substrates for acetylcholinesterase and cholinesterase

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.