Study of the interaction of main potato glycoalkaloids in inhibition of immobilized butyryl cholinesterase

The interaction of main potato glycoalkaloids alpha-solanine and alpha-chaconine in inhibition of horse serum butyryl cholinesterases immobilized on the pH-sensitive field-effect transistors has been investigated. The method of isobol diagram of Loewe and Muishnek has been used for interpretation of results. It has been shown the alpha-chaconine inhibits the immobilized bytyryl cholinesterases more strongly than alpha-solanine, and their mixture has the addition effect.     

Development and optimisation of biosensors based on pH-sensitive field effect transistors and cholinesterases for sensitive detection of solanaceous glycoalkaloids

Highly sensitive biosensors based on pH-sensitive field effect transistors and cholinesterases for detection of solanaceous glycoalkaloids have been developed, characterised and optimised. The main analytical characteristics of the biosensors developed have been studied under different conditions and an optimal experimental protocol for glycoalkaloids determination in model solution has been proposed. Using such a biosensor and an enzyme reversible inhibition effect, the total potato glycoalkaloids content can be determined within the range of 0.2-100 microM depending on the type of alkaloid, with lowest detection limits of 0.2 microM for alpha-chaconine, 0.5 microM for alpha-solanine and 1 microM for solanidine. The dynamic ranges for the compounds examined show that such biosensors are suitable for a quantitative detection of glycoalkaloids in real potato samples. High reproducibility, operational and storage stability of the biosensor developed have been shown.     

Reactivation of phosphorylated cholinesterase immobilized in a gelatin membrane

The soluble and immobilized cholinesterases (acetyl cholinesterase of human blood erythrocytes (EC 3.1.1.7) and butyryl cholinesterase of equine blood serum, (EC 3.1.1.8] were inactivated by such irreversible inhibitors as diisopropyl fluorophosphate (DFP), O,O-dimethyl-O-(2,2)-dichlorovinyl) phosphate (DDVP), paraoxone, armine. The inactivated enzymes were reactivated under the effect of TMB-4 (1,1'-trimethylene-bis)-4-formyl-pyridine bromide (dioxime). The values of the reactivation rate constants proved to be equal both for the soluble and immobilized cholinesterases inactivated by the same irreversible inhibitor. The immobilized enzyme is simpler and more correct to study the reactivating action than the soluble one.     

Enzyme Catalyzed Degradation and Formation of Peroxycarboxylic Acids

The ability of hydrolases to catalyze perhydrolysis, i.e. lysis of acyl substrates with hydrogen peroxide to form peroxycarboxylic acids, has been investigated. Lipases, esterases and cholinesterases were found to catalyze perhydrolysis but the preference of the enzymes for hydrogen peroxide relative to water as nucleophile was only 10-100 fold, even in the best cases. Hence, perhydrolysis proceeds with a very low efficiency in aqueous systems. Furthermore, all lipases, esterases and cholinesterases tested degrade peroxycarboxylic acids to the corresponding carboxylic acid and hydrogen peroxide. This reaction is most pronounced in the case of lipases while less so for cholinesterases. Consequently, cholinesterases are superior to the other hydrolases studied in catalyzing net formation of peracids in aqueous systems. In organic solvents, immobilized lipases efficiently catalyze formation of peracids from either triglycerides or the parent carboxylic acid. Proteases and phospholipase A-2 were found to neither degrade peracids nor catalyze perhydrolysis of carboxylic esters or phospholipids, respectively.     

Reversible inhibition of cholinesterases by salts of pyrilium, thiopyrilium and selenopyrilium derivatives

Salts of pyrilium, thiopyrilium and selenopyrilium derivatives at pH 7.5 and temperature of 25 degrees C are studied for their effect on the catalytic activity of acetyl cholinesterase (EC 3.1.1.7) of human blood erythrocytes and butyryl cholinesterase (EC 3.1.1.8) of horse blood serum which is measured by the method of potentiometric titration. All enumerated salts are established to be strong reversible inhibitors of mixed-type cholinesterases, that is testified by small values of the inhibitory constants: competitive Ki, noncompetitive K'i and generalized K epsilon. Pyrilium and selenopyrilium salts inhibit acetyl cholinesterase of human blood erythrocytes to a higher extent than butyryl cholinesterase of horse blood serum, and thiopyrilium salts inhibit the latter to the highest extent. By the value of the inhibitory effect on acetyl cholinesterase of human blood erythrocytes thiopyrilium salts exceed the analogous pyrilium salts, whereas in experiments with butyl cholinesterase of horse blood serum there is an opposite dependence.     

Synthesis,pharmacology and molecular docking on multifunctional tacrine-ferulic acid hybrids as cholinesterase inhibitors against Alzheimer’s disease

The cholinergic hypothesis has long been a “polar star” in drug discovery for Alzheimer’s disease (AD), resulting in many small molecules and biological drug candidates. Most of the drugs marketed for AD are cholinergic. Herein, we report our efforts in the discovery of cholinesterases inhibitors (ChEIs) as multi-target-directed ligands. A series of tacrine-ferulic acid hybrids have been designed and synthesised. All these compounds showed potent acetyl-(AChE) and butyryl cholinesterase(BuChE) inhibition. Among them, the optimal compound 10g, was the most potent inhibitor against AChE (electrophorus electricus (eeAChE) half maximal inhibitory concentration (IC₅₀)?=?37.02?nM), it was also a strong inhibitor against BuChE (equine serum (eqBuChE) IC₅₀?=?101.40?nM). Besides, it inhibited amyloid β-protein self-aggregation by 65.49% at 25?μM. In subsequent in vivo scopolamine-induced AD models, compound 10g obviously ameliorated the cognition impairment and showed preliminary safety in hepatotoxicity evaluation. These data suggest compound 10g as a promising multifunctional agent in the drug discovery process against AD.     

Convenient and rapid detection of pesticides in extracts of sheep wool

A relatively simple, rapid extraction technique based on acetonitrile was combined with the use of screen-printed electrodes bearing cholinesterases to detect organo-phosphate pesticides from an otherwise intractable matrix, sheep wool. It proved possible to separate, for convenience, the exposure of the electrodes from measurement of their (inhibited) activity. The electrodes were used once and then discarded. Estimation of the extent of inhibition is dependent on reference to the activity of control electrodes. The presence of pesticides in the extracts could be detected with any of three commonly available cholinesterases but the most sensitive enzyme was butyryl cholinesterase from horse serum.     

Effect of cetyltrimethylammonium on the human blood cholinesterases activity

The influence of cationic detergent cetyltrimethylammonium on the human blood cholinesterases activity (erythrocyte acetylcholinesterase and plasma butyrylcholinesterase) in reactions of hydrolysis of alpha-thionaphthylacetat and acetylthiocholine is studied. It is shown, that cetyltrimethylammonium is reversible effector for both cholinesterases. This compound competitively inhibited enzymatic hydrolysis of acetylthiocholine by both cholinesterases, and in the reactions of enzymatic hydrolysis alpha-thionaphthylacetat display as the synergistic activator--in experiments with butyrylcholinesterase, and as the reversible inhibitor--in experiments with acetylcholinesterase. Kinetic constants in reaction of acetylcholinesterase inhibition by cetyltrimethylammonium defined by means of different substrates--alpha-thionaphthylacetat and acetylthiocholin. They are close among themselves and amount (2.5 +/- 0.3) x 10(-5) and (2.8 +/- 0.3) x 10(-5) M, accordingly. Butyrylcholinesterase was more sensitive to influence of cetyltrimethylammonium. The kinetic constants defined for this enzyme by the effect of inhibition of acetylthiocholin hydrolysis or activation of alpha-thionaphthylatcetat hydrolysis, are also close among themselves and amount (3.9 +/- 0.4) x 10(-6) and (4.4 +/- 0.4) x 10(-6) M, accordingly.     

Enzymatic detection of mercuric ions in ground-water from vegetable wastes by immobilizing pumpkin (Cucumis melo) urease in calcium alginate beads

Present report describes a quick and simple test based on enzyme inhibition for the detection of mercury in aqueous medium by urease immobilized in alginate beads. Urease was extracted from the discarded seeds of pumpkin (Cucumis melo) and was purified to apparent homogeneity (5.2-fold) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation (Sp activity 353 U/mg protein, A(280)/A(260)=1.12) was immobilized in 3.5% alginate leading to 86% immobilization. Effect of mercuric ion on the activity of soluble as well as immobilized enzyme was investigated. Hg(2+) exhibited a concentration-dependent inhibition both in the presence and absence of the substrate. The alginate immobilized enzyme showed less inhibition. There was no leaching of the enzyme over a period of 15 days at 4 degrees C. The inhibition was non-competitive and the K(i) was found to be 1.26x10(-1)microM. Time-dependent interaction of urease with Hg(2+) exhibited a biphasic inhibition behavior in which approximately half of the initial activity was lost rapidly (within 10 min) and reminder in a slow phase. Binding of Hg(2+) with the enzyme was largely irreversible, as the activity could not be restored by dialysis. The significance of the observations is discussed.     

O,O-dialkyl-S-bromomethylthiophosphates--inhibitors of mammalian choline- and carboxyl esterases: structure-activity relationship

The interaction kinetics of potential pesticides, O,O-dialkyl S-bromomethylthiophosphates (RO)2P(O) SCH2Br (R = Et, i-Pr, n-Pr, n-Bu, or n-Am) with acetylcholinesterase, butyryl cholinesterase, and carboxyl esterase from warm-blooded animals was studied. All the compounds irreversibly inhibit these esterases, with k1 (M-1 min-1) being 1.8 x 10(4) - 1.9 x 10(6) for acetylcholinesterase, 2.0 x 10(6) - 4.1 x 10(7) for the more sensitive butyryl cholinesterase, and 2.3 x 10(7) - 2.3 x 10(8) and higher for the most sensitive carboxyl esterase. By using the Hansch and Kubinyi technique of multiple regression analysis, we quantitatively analyzed the relationship between the structure and inhibiting activity of these substances toward acetylcholinesterase and butyryl cholinesterase. Hydrophobic interactions were found to be important for the inhibition of both enzymes but are more pronounced in the case of butyryl cholinesterase. On the other hand, steric factors were much more significant in the inhibition of acetylcholinesterase. For both enzymes, the steric hindrances affect the phosphorylation stage of the enzyme.     

Sensitivity of blood-clotting factors and digestive enzymes to inhibition by organophosphorus pesticides

Organophosphorus pesticide toxicology is normally evaluated in relation to inhibition of cholinesterases (acetyl and butyryl), neuropathy target esterase, and carboxylesterases, with less attention given to other physiologically important hydrolases. This study considers the relative organophosphate sensitivities of the aforementioned serine hydrolases compared with purified blood-clotting factors (thrombin, plasmin, and kallikrein) and digestive enzymes (alpha-chymotrypsin, trypsin, and elastase), assayed under similar conditions. Inhibitors that we examined are organophosphorus insecticides or their activated metabolites (paraoxon, chlorpyrifos oxon, and profenofos) and other toxicants (phenyl saligenin cyclic phosphonate and tribufos) for comparison with values that are found in the literature for the fluorophosphonates (isoflurophate and sarin). Thrombin is the most sensitive blood-clotting factor with IC-50 values of 19 to 160 microM for tribufos, the cyclic phosphonate, isoflurophate, and profenofos; plasmin and kallikrein are less affected (IC-50 >100 microM). Alpha-Chymotrypsin, trypsin, and elastase are most sensitive to the cyclic phosphonate (IC-50 1.3-15 microM) and less so to isoflurophate, sarin, and profenofos (IC-50 values from 3.6 to greater than 100 microM). The cholinesterases, carboxylesterase, and neuropathy target esterase are the most sensitive to inhibition with IC-50 values for the insecticides of less than 0.001 to 0.6, 0.002 to 0.009, and 0.15 to 100 microM, respectively. The generally low potency of these organophosphates for blood-clotting factors and digestive enzymes suggests that associated toxic effects are unlikely at sublethal doses.     

Effect of LSD-25 on cholinesterases of the rat brain]

The indications for investigation on the LSD-25 "in vivo" activity on brain cholinesterases of the rat, have been considered. Indications of materials and method used have been supplied too. From the results obtained it emerged no clear evidence of a statistically significant inhibition of cholinesterases due to LSD-25. The results have been discussed and it has been evidenced that the lack of action of the psychotomimetic substance on cholinesterases could be only apparent or that the discrepancy between the LSD-25 anticholinesterase "in vivo" and "in vitro" action should be ascribed to the too scarce tissue levels which can be reached with the does utilized. Apart from the hypothesis, tending to explain the results obtained, it has been considered that the LSD-25 behavioural action, is not probably carried out thanks to a cholinesterase activity.     

Characterization of esterases involved in the stereoselective hydrolysis of ester‐type prodrugs of propranolol in rat liver and plasma

An inhibition study showed that the stereoselective hydrolysis of butyryl propranolol (butyryl PL) in rat liver microsomes and plasma involves carboxylesterase. The hydrolysis of (S)‐butyryl PL in plasma was specifically inhibited by eserine and bis‐nitrophenyl phosphate (BNPP), compared to the (R)‐isomer, despite the non‐stereoselective hydrolysis of butyryl PL in plasma. In addition, inhibition of hydroloysis by eserine and BNPP showed little stereoselectivity for butyryl PL in liver, although liver microsomes showed an (S)‐preferential hydrolysis for butyryl PL (R/S ratio of Vmax/Km: 2.1 ± 0.2). The hydrolysis of butyryl PL was not inhibited by a polyclonal antibody against a high affinity carboxylesterase (hydrolase A, RH1). Moreover, the high Km value and the high IC₅₀ for phenylmethylsulfonyl fluoride (PMSF) against the hydrolysis of butyryl PL in rat liver microsomes suggest that a low affinity carboxylesterase (perhaps hydrolase B) might be involved in this hydrolysis in rat liver. Chirality 11:10–13, 1999. © 1999 Wiley‐Liss, Inc.     

Studies on the interaction of papain with human placental cystatin by UV, fluorescence and CD spectroscopy

The interaction of activated papain with low molecular weight cystatin (Mr 12500) purified from human placenta has been studied. Analysis of inhibition of caesinolytic activity of papain by cystatin showed stoichiometry of 1:1. Kinetic studies gave an inhibition constant (K(i)) value of 5.5 x 10(-8) M and association rate constant (K(+1)) value of 3.4 x 10(4) (M(-1) s(-1)). All spectroscopic studies showed conformational changes in both papain and cystatin on formation of complex. The data suggest perturbation of environment of aromatic residues and change of their native structure and conformation thereby shedding light on the behaviour of cystatins, especially interaction of placental cystatin with thiol protease inhibitors.     

Kinetic analysis of the influence of inverse effectors (inhibitors and activators) on enzymatic (transport) activity of proteins

The method is proposed for calculation of the most important parameters of the two-stage enzymatic or transport process--modification factors alpha and beta (which characterize the effector action mechanism) as well as the inhibition constant K(i) or activation constant K(a) (characterize the effector affinity for protein). The method was derived as based on the analysis of kinetic regularities of the action of reversible effectors (inhibitors and activators) on the catalytic (transport) activity of proteins. The method is based on the titration of enzymatic (transport) protein by the substrate with the absence and with presence of the effector taken in one of concentrations as well as determination (under the fixed substrate concentration) of the inhibition coefficient i(0.5) (in case of the inhibitor action) or the activation coefficient a(0.5) (in case of the activator action). Practical use of the method has been demonstrated on the example of reversible inhibition to eosine Y (2', 4', 5', 7' - tetrabromofluorescein) ofthe reaction of enzymatic hydrolysis of ATP catalyzed by highly purified transport Ca2+, Mg(2+)-ATPase isolated from the smooth-muscle sarcolemma. In this case the inhibitory effect is characterized by the following parameters: alpha = 6-8 > 1; beta = 0.50-0.53 < 1; inhibition constant K(i) = 10(-9) - 10(-8) M. Consequently, judging from the values of alpha and beta, the eosine Y effect on the analyzed Ca2+, Mg(2+)-dependent ATP-hydrolase enzymatic reaction is based on the mechanism of the mixed inhibition (one can observe the inhibition of the both stages of enzymatic transformation--the substrate binding with the enzyme and decomposition of "Michaelis complex" in the direction of formation of the reaction products). The inhibitor itself, in correspondence with K(ij) values is characterized by rather high affinity for Ca2+, Mg(2+)-ATPase. It is supposed that the proposed approach can be useful when identifying the type of the reversible effector action on the enzymatic (transport) activity of proteins, estimation of real affinity of the inhibitors and activators for the latter.     

Inhibition of Jack Bean Urease by 1,4-benzoquinone and 2,5-dimethyl-1,4-benzoquinone. Evaluation of the Inhibition Mechanism

1,4-benzoquinone (BQ) and 2,5-dimethyl-1,4-benzoquinone (DMBQ) were studied as inhibitors of jack bean urease in 50 mM phosphate buffer, pH 7.0. The mechanisms of inhibition were evaluated by progress curves studies and steady-state approach to data achieved by preincubation of the enzyme with the inhibitor. The obtained reaction progress curves were time-dependent and characteristic of slow-binding inhibition. The effects of different concentrations of BQ and DMBQ on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. The rapid formation of an initial BQ-urease complex with an inhibition constant of K i =0.031 mM was followed by a slow isomerization into the final BQ-urease complex with the overall inhibition constant of K*_i=4.5 × 10 ^?5 mM. The respective inhibition constants for DMBQ were K i =0.42 mM, K*_i =1.2 × 10 ^?3 mM. The rate constants of the inhibitor-urease isomerization indicated that forward processes were rapid in contrast to slow reverse reactions. The overall inhibition constants obtained by the steady-state analysis were found to be 5.1 × 10 ^?5 mM for BQ and 0.98 × 10 ^?3 mM for DMBQ. BQ was found to be a much stronger inhibitor of urease than DMBQ. A test, based on reaction with L-cysteine, confirmed the essential role of the sulfhydryl group in the inhibition of urease by BQ and DMBQ.     

Immobilization of Aspergillus beta-glucosidase on chitosan.

beta-Glucosidase of Aspergillus phoenicis QM 329 was immobilized on chitosan, using the bifunctional agent glutaraldehyde. The most active preparation based on the amount of support contained a 1:2.5 enzyme-to-chitosan ratio (wt/wt). However, the specific activity of the bound enzyme decreased from 10 to 1% with increasing enzyme-to-chitosan ratio. Compared with free beta-glucosidase, the immobilized enzyme exhibited: (i) a similar pH optimum but more activity at lower pH values; (ii) improved thermal stability; (iii) a similar response to inhibition by glucose; and (iv) mass transfer limitations as reflected by higher apparent Km and lower energy of activation.     

Immobilization of Aspergillus beta-glucosidase on chitosan.

beta-Glucosidase of Aspergillus phoenicis QM 329 was immobilized on chitosan, using the bifunctional agent glutaraldehyde. The most active preparation based on the amount of support contained a 1:2.5 enzyme-to-chitosan ratio (wt/wt). However, the specific activity of the bound enzyme decreased from 10 to 1% with increasing enzyme-to-chitosan ratio. Compared with free beta-glucosidase, the immobilized enzyme exhibited: (i) a similar pH optimum but more activity at lower pH values; (ii) improved thermal stability; (iii) a similar response to inhibition by glucose; and (iv) mass transfer limitations as reflected by higher apparent Km and lower energy of activation.     

Inhibition of enzymes by metal ion-chelating reagents. The action of copper-chelating reagents on diamine oxidase

1. The inhibition of diamine oxidase has been studied by using the following copper-chelating reagents: 1,10-phenanthroline; 2,2'-bipyridyl; 8-hydroxyquinoline (oxine); diethyldithiocarbamate and dithio-oxamide (rubeanic acid). 2. Addition of chelating reagent caused a rapid inhibition of enzyme to a degree dependent solely on the final inhibitor concentration. Addition of substrate gave linear initial rates of reaction showing that under these conditions the inhibition was not being rapidly reversed. 3. The inhibition has been investigated by using new graphical methods and has been found in all cases to involve the chelating agents completely removing two Cu(2+) ions from the enzyme. An alternative possibility, involving ligand substitution, was eliminated. 4. A value of K=8.0x10(-33)m(-2) has been found for the enzyme in equilibrium with 2 Cu(2+) ions (i.e. beta(2), the stability constant for diamine oxidase/two Cu(2+), is 32.1).     

Mechanisms of cholinesterase inhibition by inorganic mercury

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.