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Abstract A timely and efficient removal of apoptotic cells and their fragments is essential to maintain tissue homeostasis in normal and pathological conditions. Since the removal of apoptotic substrates is executed by the cells endowed with phagocytic activity, the issue of its regulation is of particular interest. In this work, we studied the effect of two biologically active substances, insulin and ganglioside GM1, on phagocytic activity of rat primary astrocytes. We showed that cell incubation with 1 µM insulin significantly decreased the phagocytic activity of astrocytes (58.5% vs. control), whereas the incubation with 10 µM GM1 caused an increase in phagocytic activity (133.4% vs. control). Preincubation of brain astrocytes with GM1 completely blocked the inhibitory effect of insulin. These results can be instrumental in developing novel therapeutic strategies for the treatment of neurodegenerative diseases accompanied by the emergence of apoptotic substrates.

Two cholinesterases were identified in the cotton bollworm Helicoverpa (Heliothis) armigera Hbn., that is, acetylcholinesterase from the nervous chain and butyrylcholinesterase from homogenates of larvae. A comprehensive study of the substrate-inhibitor specificity of cholinesterases (based on the modern concept of inhibition by high substrate concentrations), as well as the investigation of anti-enzyme action of 57 irreversible organophosphorus inhibitors, indicated a peculiarity of the enzymological characteristics of the bollworm cholinesterases compared to cholinesterases of arthropods and some mammals.

A study of substrate–inhibitor specificity of mitochondrial monoamine oxidase (MAO) in the hepatopancreas of the adult Kamchatka crab Paralithodes camtschaticus revealed specific catalytic properties of the enzyme. On the one hand, crab hepatopancreas MAO, like its classical hepatic counterpart, can deaminate tyramine, tryptamine, dopamine, serotonin, noradrenalin, benzylamine, β-phenylethylamine and N-methylhistamine but shows no sensitivity to 10 mM semicarbazide. On the other hand, MAO deaminates histamine but not putrescine, two classical diamine oxidase (DAO) substrates. It was established that MAO activity was several times higher toward benzylamine, β-phenylethylamine and N-methylhistamine than toward serotonin and noradrenalin. MAO was also found to be almost 500 times more sensitive to its selective inhibitor deprenyl than to chlorogilyn. A substrate–inhibitory analysis with the use of deprenyl and chloroginyl provides an indirect evidence for the existence of a sole MAO molecular form in the Kamchatka crab hepatopancreas.

This review summarizes the literature data as well as experimental results obtained at our Institute over a period of 50 years on the substrate specificity of cholinesterases–acetylcholine acetylhydrolases (EC 3.1.1.7) and acylcholine acylhydrolases (EC 3.1.1.8). The parameters of enzymatic hydrolysis of oxo- and thiocholine and β-methylcholine esters in different organs and tissues were analyzed in 66 animal species including 22 chordate, 20 insect, 1 mite, 17 mollusk, 4 nematode, and 2 flatworm species. Our substrate specificity studies and extensive data on the inhibitory specificity obtained using irreversible organophosphorous inhibitors and reversible effectors unequivocally indicate that the cholinesterase family is characterized by a clear-cut species and tissue specificity.

We report a pioneering analysis of the interaction between mammalian cholinesterases and 36 acylates and thioacylates of ammonium alcohols with different structure of an alkyl chain between ammonium and etheric atoms and with different structure of a cyclic ammonium group. Among these ethers, which were both substrates and reversible inhibitors of erythrocyte cholinesterase and serum butyrylcholinesterase, specific effectors of both enzymes were identified.

A comparative analysis of enzymological characteristics of cholinesterase (ChE) and monoamine oxydase (MAO) from the optic ganglia was performed in the Pacific squid Todarodes pacificus and Commander squid Berryteuthis magister caught in their four habitats across the Pacific Northwest. A substrate–inhibitor analysis revealed a homogeneity of T. pacificus and B. magister ChE preparations as well as homogeneity of T. pacificus vs. heterogeneity of B. magister MAO preparations. In case of thiocholine derivatives, the rate of hydrolysis induced by T. pacificus ChE was practically independent of the structure of the acyl group, whereas in case of B. magister ChE it was found to decrease in this substrate series. It is only T. pacificus MAO that was found to be able to deaminate also a diaminooxydase substrate histamine. ChE activity was higher in T. pacificus than in B. magister for the whole substrate series, while for MAO the same activity pattern was observed for tyramine, tryptamine and serotonin. In both squids, the sensitivity of ChE to organophosphorus inhibitors containing the dimethylbutyl group was by several orders of magnitude higher than that in mammals. The sensitivity of ChE to the siloxane reversible inhibitors was lower in T. pacificus ChE and much lower in B. magister than in mammals.

Inorganic tetramethylene bis-onium compounds were studied as reversible inhibitors of various cholinesterases (ChE), i.e., human erythrocyte acetyl-ChE, horse blood serum butyryl-ChE, grass frog Rana temporaria brain ChE, and Pacific squid Todarodes pacificus and commander squid Berryteuthis magister visual ganglia ChE from various habitats in the northwest Pacific Ocean, in order to study the influence of the nature of the onium atom on their anti-ChE activities. bis-Phosphonium inhibitors turned out to be significantly more potent effectors than bis-ammonium compounds, which may actually have been due to the significant increases of size and hydrophobicity of the onium groups. The bis-ammonium organosilicon compound and its monoammonium analog turned out to be equally active as reversible ChE inhibitors in mammals. The bis-phenyliodonium derivative was studied for the first 7time, was characterized by significantly increased hydrophobicity due to introduction of F atoms into the tetramethylene spacer in the onium chain, and exhibited marked anti-ChE activity with respect to mammalian ChE.

To study the effect of the onium atom nature on anticholinesterase efficiency, we tested elementorganic derivatives of tetramethylenbisonium compounds as reversible inhibitors of the following cholinesterases (ChE): acetyl-ChE from human erythrocytes, butyryl-ChE from horse serum, ChE from the brain of the grass frog Rana temporaria, ChEs from visual ganglia of the Pacific squid Todarodes pacificus, and ChE from visual ganglia of the commander squid Berryteuthis magister from different habitats in the Northwestern Pacific Ocean. Bisphosphonium inhibitors were found to be much stronger effectors than bisammonum compounds, although this may be due to a significantly increased size and hydrophobicity of their onium groups. Bisammonium organosilicon compound and its monoammonium analog were equally active as reversible ChE inhibitors in mammals. The first studied bis(phenyliodonium) derivative, which is characterized by a significantly increased hydrophobicity due to the introduction of fluorine atoms to the interonium tetramethylene chain, also exhibited a pronounced anticholinesterase effect on mammalian ChE.

A comparative enzymological investigation of inhibitory specificity of the liver monoamine oxidases (MAO) from the two frog species, lake frog Rana ridibunda and grass frog Rana temporaria, revealed certain interspecies similarities and distinctions of this enzyme. The anti-monoamine oxidase effect of five derivatives of acridine, three derivatives of phenothiazine and one derivative of xanthene (pyronine G) was comparatively analyzed. The tested six-membered tricyclic compounds were shown to exert an irreversible inhibitory effect on the enzyme from both biological sources, displaying the same substrate deamination specificity. Thus, the rate of interaction of acridine and phenothiazine derivatives with the MAO active center in both frog species was considerably higher when activity was determined using noradrenaline versus N-methylhistamine, while that of pyronine G—when activity was determined using N-methylhistamine versus noradrenaline. Interspecies quantitative differences were found in the inhibitory efficacy and degree of selectivity of the tested tricyclic compounds, indicative of the differences in catalytic properties of liver MAO at the interspecies level in the representatives of the genus Rana, family Ranidae. The data of substratespecific inhibitory analysis provide indirect evidence of the existence of two molecular MAO forms in the liver of the studied frog species.

The review presents data on comparative reactivity of 68 cholinesterase preparation from various organs and tissues in a number of vertebrates and invertebrates based on sensitivity to two highly specific and most studied organophosphorus inhibitors--diisopropyl fluorophosphates (DFP) and (2-ethoxymethyl phosphoryl thioethyl) ethyl (methyl) sulphonium sulphomethylat (GD-42). Analysis of these data suggests a great diversity in enzymologic characteristics of cholinesterase preparation in representatives of vertebrates and invertebrates, this variety observed even for closely related enzymes in animals of almost the same level of development.

The substrate and inhibitory specificity of mitochondrial monoamine oxidase (MAO) in the liver of males of the summer form of the chum salmon Oncorhynchus keta was studied. As to the spectrum of deaminated substrates, the hepatic MAO of the chum salmon is similar to MAO of most terrestrial mammals, for eight classical MAO substrates similarity in their substrate characteristics were found. Analysis of the antimonoamine oxidase activity of two derivaties of 2-propinilamine, five derivatives of acridine as well as of pyronine G revealed significant qualitative and quantitative differences as compared to the hepatic enzyme of tuna and whitefish. The compounds tested manifested themselves as irreversible inhibitors of chum salmon's hepatic MAO possessing various efficacy, but lacking the selectivity of action as dependent on the deaminated substrate. The obtained data on the substrate and inhibitory analysis provide an indirect evidence for the presence of a single molecular form of MAO in the chum salmon liver.

A comparative analysis of the effect of the five acridine, three phenothiazine and one xanten (pyronine G) derivatives on the activity of liver mitochondrial monoamine oxidase (MAO) in sexually mature male river lampreys Lampetra fluviatilis has been conducted. Tyramine, dopamine, serotonin, noradrenaline, benzylamine, β-phenylethylamine and N-methylhistamine have been used as substrates for analyzing the monoamine oxidase activity of heterocyclic compounds. The analyzed synthetic hexamerous tricyclic compounds exhibit irreversible inhibition of the enzyme but no specificity depending on the desaminated substrate. The number and identity of heteroatoms in the analyzed heterocyclic compounds have been established to influence their inhibitory efficiency. The data of substrate-inhibitory analysis obtained with the use of the specific substrates provide indirect evidence for the existence of a single MAO form in the lamprey liver.

Specifically synthesized group of benzimid­azole derivatives possessing varying degrees of delocalization of the positive charge in the cation group of the molecule has been studied in order to search for potential cholinergically active compounds and to study the role of the Coulomb interaction in cholinesterase catalysis. These compounds were reversible inhibitors of cholinesterase (ChE) of human erythrocytes, horse serum, brain of the frog Rana temporaria­ and visual ganglia of the Pacific squid Todarodes pacificus in the presence of acetylthio­choline iodide and propionylthiocholine iodide as substrates. The differences in the nature of reversib­le inhibitory effect were observed. The effect of the inhibitor structure and substrate nature, specific for each of the studied inhibitors, on the character of the process of reversible inhibition was found.

The study was performed to check whether the horse blood serum butyrylcholinesterase expresses transferase activity in the presence of several low-molecular aliphatic alcohols, using chromogenic substrate 1-methyl-8-acetoxychinolium iodide whose phenolic hydrolysis product absorbs intensively at 445 nm, whereas the initial ester practically does not absorb within this spectrum area. This allowed measuring simultaneously accumulation of both products of enzymatic hydrolysis: of acetic acid by the potentiometric, while of phenol—by the colorimetric method. Rates of formation of both products of enzymatic hydrolysis are practically equal in experiments with all studied alcohols. This indicates that horse blood serum butyrylcholinesterase under these experimental conditions does not catalyze the transfer of acetyl residue to the studied aliphatic alcohols, i.e. does not have transferase activity.

There was studied action of aliphatic alcohols (ethanol, propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol), and pH on various kinds of serum cholinesterase. At inhibition of the cholinesterase hydrolytic activity under effect of alcohols the key role was played not by the total number of carbon atoms in the alcohol molecule, but by the “efficient length” of the carbohydrate chain. The fact that the presence of alcohols did not affect parameters of reversible inhibition of cholinesterase by onium ions tetramethylammonium and choline allows suggesting the absence of action of solvents on specific sorption of acetylcholine in the enzyme active center. With aid of two sets of hydrophobic organophosphorus inhibitors (OPI) (12 compounds), we have managed to estimate both the degree and the character itself of serum cholinesterase.

Arylsulfoesters and carbonic lupinin esters are studied for the first time as reversible inhibitors of mammalian blood cholinesterases. Studied in detail is sensitivity of cholinesterases to mono- and bilupinin inhibitors in Commander squid individuals from different habitation zones.

We compared and analyzed data on the sensitivity of four preparations of acetylcholinesterases from humans, rabbit, and housefly heads to the action of a specially synthesized group of quinolizidine organophosphorus inhibitors, whose molecules contained both isomeric unbranched and branched alkoxy derivatives of the phosphoryl group and epimeric lupinine and epilupinine derivatives of the cleaved group. The differences in the characteristics of these compounds helped us to find specific inhibitors of the different acetylcholinesterases and the show the difference in the reactivities of acetylcholinesterases from different animals.

The quaternary phosphonium compounds were found to be reversible inhibitors of cholinesterases of different animals and showed species-specificity of action depending on their inhibitor structure. We have revealed difference in the inhibitory specificity of various acetylcholinesterase preparations. A difference has been shown in inhibitory parameters of optic ganglia of individuals of the squid Berryteuthis magister from different habitat areas. For the first time in comparing phosphonium and ammonium isologues-tetrabutyl- and tributylhexyl derivatives, it has been shown that they are agents practically similar by the character of the anticholinesterase action.

The review presents data on cholinesterase effects of 28 specially synthesized organosilicon compounds (monoonium, organoelement, and bisonium derivatives) studied as reversible inhibitors of acetylcholinesterase (acetyl-ChE) of human erythrocytes, butyryl-ChE of horse blood serum, ChE of brain of common frog Rana temporaria, ChE of the optical ganglia tissue of Pacific squid Todarodes pacificus and of individuals of Commandor squid Berryteuthis magister from various habitats in the Northwestern aquatoria of the Pacific ocean. Among the tested compounds, there are revealed highly specific inhibitors of mammalian ChE as well as of ChE of the B. magister individuals from various habitats.

Kinetic analysis of the activating effect of substrate on the cholinesterase catalysis is performed. There are determined values of coefficient of activation A in the pH zone 5 for the process of hydrolysis of acetylcholine, indophenylacetate (IPhA), and 2,6-dichlorophenolindoph enylacetate (DIPhA) by cholinesterase (ChE) of horse blood serum, as well as of IPhA and DIPhA by ChE of optical ganglia of the Pacific squid Todarodes pacificus. The phenomenon of activation has not been revealed at hydrolysis of phenylacetate by the horse blood serum ChE. The conclusion is made that the cause of the activating effect of substrate on the process of enzymatic hydrolysis by ChEs of different origin is the presence of the onium grouping in the structure of substrates.

Summarized are results of the 40-year studies of the Russian biochemists on the comparative-enzymological characteristics of cholinesterase of optic ganglia of the Pacific squid Todarodes pacificus. The review includes comparative evaluation of the cholinesterase activity of various hydrobiont tissues, the proof of enzymatic homogeneity of tissues of the Pacific squid optic ganglia, data on substrate specificity with study of 18 ester substrates as well as detailed study of inhibitory specificity (61 irreversible inhibitors and 49 reversible onium inhibitors). Peculiarity of properties of this enzyme as compared with vertebrate and invertebrate cholinesterases is shown.

Review of the own and literature data on investigation of substrate specificity of different cholinesterases using thiosubstrates is presented. Dependence of cholinesteratic hydrolysis parameters on various elements of their structure—the acyl part, alkyl “bridge” between ester atom and onium group, and the molecule ammonium grouping—is considered using 44 thioesters in total. A comparative enzymological analysis of the substrate specificity is performed with use of thiocholine esters of acetic, propionic, and butyric acids for 40 cholinesterase preparations of mammals, insects, mollusks, and plants.

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.

This review for the first time combines and analyzes in detail the data on comparison of potency of sulfonium and ammonium (and other onium, in some cases) isostructural effectors of cholinesterases (substrates, irreversible organophosphorus inhibitors, and reversible inhibitors). Besides, analysis is performed of specificity of sulfonium ligands of active center towards cholinesterases of animals standing at different stages of evolutionary development.

Comparative study was performed of action of a group of 15 isoquinoline homoproaporphine and homoaporphine alkaloids, 10 tropolone colchicine alkaloids and their lumoderivatives that were isolated from corms of a representative of the lily family, the showy autumn crocus Colchicum speciosum Stev. on activity of mammalian erythrocyte acetylcholinesterase and serum butyrylcholinesterase. The studied compounds have turned out to be moderate-potency reversible inhibitors of the studied cholinesterases and to show to a certain degree some specificity of action towards different enzymes both quantitatively, by the ratio of total inhibitor constant values, and qualitatively, by the type of mechanism of the enzymatic activity inhibition. The overwhelming majority of the studied alkaloids revealed certain specificity towards butyrylcholinesterase. An exception was colchamine.

Interaction with cholinesterases (ChEs) of nine specially synthesized derivatives of dimethylaminoalkyl esters of 2-chloro-and 2,4-dichlorobenzoic acids and their iodoalkylates is studied. Used as enzyme sources were partially purified preparations of acetylcholinesterase (AChE) from human erythrocytes and butyrylcholinesterase (BChE) from horse blood serum, as well as water homogenates of the frog Rana temporaria brain and of the Pacific squid Todarodes pacificus optical ganglia. The studied benzoates failed to be hydrolyzed by the studied ChEs at the enzyme concentrations exceeding 10 times those used for determination of the acetylthiocholine hydrolysis rate. These compounds have turned out to be reversible inhibitors of ChEs of the mixed-noncompetitive type of action. Effects on the anticholinesterase activity of such structural elements of the inhibitors as the acidic part of the benzoate molecule, length of polymethylene chain in the molecule alcoholic part, and the structure of ammonium group are studied. This study has allowed revealing some peculiarities of the reaction capability of vertebrate and invertebrate ChEs.

A comparative study was carried out of action of 18 specifically synthesized anabasine derivatives and their analogs, including diiodomethylates of anabasine acylates and bis-anabasine derivatives of dicarboxylic glutaric, adipic, azelaic, and sebacic acids, on activity of cholinesterase of brain of frog Rana temporaria and visual ganglia of the Pacific squid Todarodes pacificus and Commander squid Berryteuthis magister from different zones of habitations in the northwest part of the Pacific Ocean as well as of erythrocyte acetylcholinesterase and serum butyrylcholinesterase. These compounds were not submitted to cholinesterase hydrolysis and turned out to be efficient reversible inhibitors that have to certain degree specificity toward the studied enzymes both by their potency and by the type of their inhibitory action. Specificity of effects of the key structural fragments of the anabasine grouping on anticholinesterase efficacy was checked. Also performed was analysis of action of 24 reversible and irreversible organophosphorus inhibitors, anabasine derivatives, toward erythrocyte acetylcholinesterase and serum butyrylcholinesterase. The anticholinesterase effects of anabasine-containing inhibitors differing in structure and action mechanism was compared.

A group of organofluorine ammonium compounds, trimethyltrifluoromethylammonium, diethylmethyltrifluoromethylammonium, hexa(difluoromethylene)-bis(trimethylammonium), their non-substituted analogs as well as bis-onium organosilicone, phenyliodonium, and triphenylphosphonium derivatives were tested as reversible inhibitors of acetylcholinesterase of human erythrocytes, butyrylcholinesterase of horse blood serum, cholinesterase of brain of the frog Rana temporaria and cholinesterases of optic ganglion of the Pacific squid Todarodes pacificus. By the method of molecular mechanics, differences were revealed in conformational mobility of interonium chain and in geometric parameters of the studied compounds. It was shown that introduction of fluorine atoms into the inhibitor molecule affected only their interaction with the Pacific squid cholinesterase. It was possible to separate effects of the onium atom nature and of the interonium chain structure in the inhibitor molecule on the anticholinesterase potency.

Comparative study was carried out on action of a group of bis-alkaloid derivatives (on the basis of lupinine, anabasine, and cytosine, and their iodomethylates) of dicarboxylic acids (succinic, glutaric, azelaic, sebacic) on activity of cholinesterase of optical ganglia of the Commander squid Berryteuthis magister from different zones of habitation in the northwest part of the Pacific Ocean as well as human erythrocyte acetylcholinesterase and horse serum butyrylcholinesterase. These compounds turned out to be potent reversible inhibitors with specificity of action with respect both of sensitivity of the studied enzymes to them and of the type of their inhibitory action. The studied inhibitors can be used as tools in biochemical taxonomy to determine populational structure of such a marketing species as the Commander squid Berryteuthis magister.

Two series of lupinine and epilupinine acetates, the free bases and the N-methyl, N-ethyl, and N-propyl derivatives, were studied. Enzymatic hydrolysis of substrates by human erythrocyte acetylcholinesterase and horse blood serum butyrylcholinesterase was investigated in comparison with the corresponding dimethylalkyl derivatives of acetylcholine. The important role of non-productive substrate binding was taken into account in the data analysis. Additional information for a better understanding of the metabolism of lupinine derivatives in vivo in animals was obtained.

Study of spatial structure of biologically active guanidine derivatives by the method of molecular mechanics has shown that in an anticoccidial drug, 1,3-bis ( p-chlorobenzylidenamino)guanidine (Cl-BAG) the most preferable are convolute conformations, in which the chlorine atoms that are distant in the valent chain are approached to each other at a distance of 3.7 A. This indicates predisposition of the optimal conformations to form chelate complexes with ions of metals, which is confirmed by comparative spectrophotometric studies of the second derivative of differential UV-spectra of Cl-BAG in the presence and absence of calcium ions. Its derivative without chlorine (BAG) is unable to bind Ca2+ and has been shown to have no anticoccidial action, which associates the biological potency with the presence of calcium-binding ability of the compounds. The capability of Cl-BAG for chelation depends essentially on nature of the chelated metal ion. The antienzyme testing of inhibiting action of the guanidine derivatives toward cholinesterases of human erythrocytes, horse blood serum, mink brain and serum, optic ganglia of the Pacific squid Todarodes pacificus has revealed difference between the enzymes due to possibility of redistribution of the positive charge between the guanidinium fragment and amino groups and a change of the degree of charge delocalization.

Comparative enzymologic study of catalytic properties of cholinesterase (ChE) in blood serum of the American mink Mustela vison Schr. has revealed several peculiarities of this enzyme. First, using the method of substrate–inhibitor analysis, homogeneity of the ChE preparation has been established, i.e. only one ChE has been found in mink serum. Second, the rate of acetylcholine hydrolysis was higher than of thiocholine substrates, among which propionylthiocholine was hydrolyzed at the highest rate. Third, propionylthiocholine had the highest V/K M value that reflects to a degree affinity of the substrate to enzyme. Fourth, the phenomenon of “substrate inhibition,” which is not inherent for mammalian serum cholinesterases, is revealed and kinetically analyzed. Fifth, study of inhibitory specificity has not revealed differences of the mink serum ChE from other serum ChE.

An analysis of influence of indophenol substrate structure on rate of their enzymatic hydrolysis under action of cholinesterases (ChE) of different animals is carried out for the first time. Study of indophenylacetate (IPhA) and a group of isomeric dichloroderivatives as substrates of erythrocyte acetylcholinesterase, serum butyrylcholinesterase, and ChE from optical ganglia of the Pacific squid Todarodes pacificus allowed us to reveal a role of steric and inductive effects of the substrates molecule in enzymatic catalysis, as well as differences in substrate specificity of the studied ChE. This comparative enzymologic aspect of the work was evident to a greater degree at studying hydrolysis of choline (acetylcholine, acetylthiocholine) and indophenol (IPhA, 2,6-dichloroindophenylacetate, 2,6-dichloro-3´-methyl indophenylacetate) esters under action of mammalian blood ChEs, ChE from hemolymph of the gastropod mollusc Neptunea, and also of ChE from the nervous tissue of different species of Pacific squids and of the cabbage root fly. Differences in values of the kinetic parameters characterizing sorption and catalytic stages of the hydrolysis process are revealed. Comparison of substrate properties of choline and indophenol esters enabled us to compare enzymes in terms of hydrophobic-hydrophilic interactions.

A study is carried out as a development of A.P. Brestkin's concept of mechanism of irreversible inhibition of cholinesterases (ChE) by organophosphorus inhibitors (OPI) with taking into account reversibility of the first stage of this reaction, which has made it possible to determine individual constants of separate stages of the process. For the first time, a comparative study is performed on horse blood serum BuChE, human erythrocyte AChE, and ChE of optical ganglia of Pacific squid Todarodes pacificus. Besides, the OPI set is enlarged essentially due to use of some highly specific inhibitors of each of the enzymes. To evaluate the cholinesterase activity, chromogenic indophenol esters are used as substrates. For each of the studied ChE, differences in sensitivity to the studied OPI are realized only in values of the kinetic constant of formation of the enzyme-inhibitor complex (k 5), whereas the rate constants of dissociation of this complex to initial components (ChE and OPI) (k −5) and of process of its transformation into phosphorylated ChE (k 6) are close to each other by the values, values of these constants k −5 and k 6 for different enzymes also being similar. Some statements about the molecular mechanism of the cholinesterase catalysis are formulated. It is suggested that the revealed elements of similarity of different ChE are realized in the work of the “catalytic machine” of active centers of the enzymes.

Five derivatives of benzimidazole, compounds with delocalized charge in cationic group, are studied and turned out to be reversible inhibitors of hydrolysis of acetylthiocholine under action of acetylcholinesterase from human erythrocytes, butyrylcholinesterase from horse blood serum, and cholinesterases from brain of the brown frog Rana temporaria and from optical ganglion of the Pacific squid Todarodes pacificus. It was only for acetylcholinesterase from erythrocyte as well as (with propyonylthiocholine as substrate) from squid that sensitivity to the studied benzimidazole derivatives correlated with degree of localization of the charge in the cationic group; this confirms the current concepts of functioning of the enzyme active center. A comparative study of 9 ammonium inhibitors with localized cation in their molecules, including the complete sterical analogue of the benzimidazole derivatives, benzimidazolinium iodide, has revealed both quantitative and qualitative differences.

A comparative study of substrate specificity of monoamine oxidase (MAO) in mitochondria of liver of the Pacific squid Todarodes pacificus and of Wistar rats is carried out. It is revealed that the squid liver MAO, unlike the rat liver MAO, is capable of deaminating not only tyramine, serotonin, and benzylamine, but also histamine. The squid liver MAO activity in relation to all studied substrates is approximately 10 times lower, while the sorption ability, several tens times lower, than the rat liver MAO. Semicarbazide, a classic inhibitor of diamine oxidase, at a concentration 1 × 10−2 M did not inhibit the catalytic activity of both studied enzymes. The specificity of action of an irreversible inhibitor, proflavine, is established, which was seen at deamination of various substrates by the squid liver MAO to the greater degree, than by the rat liver MAO. The values of the bimolecular rate constant of the irreversible inhibition (kII) by proflavine were 2.5–20-fold higher (depending on substrate) in the case of the squid liver MAO, than of the rat liver MAO. A suggestion is put forward about the probable presence of several centers of substrate binding in the enzyme of the studied marine invertebrate, like in the mammalian enzyme.

The mechanism of reversible inhibition of human erythrocyte acetylcholinesterase, horse blood serum butyrylcholinesterase, cholinesterase from optical ganglia of the squids, PacificTodarodes pacificus and CommodoreBerryteuthis magister, from different zones of habitation area is studied in the presence of substrates of various structures (acetylcholine, butyrylcholine, acetylthiocholine, butyrylthiocholine, phenylacetate, indophenylacetate, 2,6-dichlorophenylindophenylacetate). Tested as reversible inhibitors were tetramethylammonium iodide, tetraethylammonium iodide, choline iodide, and two derivatives of α,ω-bis(trimethylammoniommethyl)oligodimethylsiloxane dichloride. It has been revealed that the mechanism of the reversible anticholinesterase action depends essentially both on the enzyme nature and on the structures of substrate and inhibitor. The transfer from cation-containing to hydrophobic substrates increased essentially the contribution of uncompetitive component of the inhibitory constant. In the presence of butyric acid esters (butyrylcholine, butyrylthiocholine), the potency of inhibitors was lower than at hydrolysis of the corresponding acetates. The effect of the substrate structure on the mechanism of reversible inhibition was revealed to a greater extent in reactions with participation of squid cholinesterases.

A comparative study was carried out of the substrate and inhibitory specificity of cholinesterase preparations from squids, representatives of 3 genes and 5 species of the Gonatidae family:Berryteuthis (B. magister andB. anonichos),Gonatus (G. kamtschaticus andG. tinro), andGonatipsis (G. borealis), that have overlapping habitation areals in the Bering Sea. As substrates, there were used bromides of acetylthiocholine, propionylthiocholine, and butyrylthiocholine, as organophosphorus inhibitors, diisopropylfluorophosphate, a cation-containing inhibitor, and 4 hydrophobic compounds. The homogeneity of the cholinesterase activity in these preparations has been shown, the intergenus and interspecies differences in the enzyme properties are revealed, and also the peculiarity of properties of enzymes from Gonatidae squids is emphasized in comparison with cholinesterase from the Pacific squidTodarodes paciflcus and “standard” mammalian enzymes (from human erythrocytes and horse blood serum). The revealed interspecies differences are discussed in terms of evolutionary development of the Gonatidae family.

The authors’ own and literature data are summarized on interaction of 17 irreversible organophosphorus inhibitors with different types of cholinesterases (ChE): erythrocyte acetylcholinesterase (AChE), serum butyrylcholinesterase (BuChE), and cholinesterase of the Pacific squidTodarodes pacificus, in the presence of 9 substrates. The kinetic analysis of the “substrate protective effect” based on A.P. Brestkin’s equation is performed, and the current interpretation of individual components of this process is done. An essential effect of the inhibitor structure on individual phases of the reaction is revealed. Among choline substrates, only formylcholine did not show a protective effect. The inability of an uncationic substrate, phenylacetate, to regulate ChE reactivity is confirmed. Among the studied ChE, the highest substrate protective effect was revealed in the Pacific squid ChE.