Comparative analysis of sensitivity of cholinesterases of different origin to bis-onium reversible inhibitors

Analytical review of the literature data on interaction constant of cholinesterases from different animal (vertebrates and squids) with 45 bis-onium reversible inhibitors forming homologous series with regularly varied structure has been carried out. Values of the competitive, uncompetitive and generalized inhibitor constants are compared. Interspecies and intraspecies differences in sensitivity of ChE are revealed. Results of conformational analysis of the investigated ligand molecules are presented. The data on population of individual conformations are compared with the data on anticholinesterase potency. Conclusions are made on the action mechanism of the investigated compounds and predominant place of their sorption. The presented data are considered from the point of view of comparative biochemistry and in the light of current information about the active center structure of cholinesterases.     

Comparative analysis of sensitivity of cholinesterase inhibitors by multidimensional statistical methods

Using the methods of factor and cluster analysis, the statistical treatment is performed of data on interaction of 7 cholinesterases (ChE)--human acetylcholinesterase, horse butyrylcholinesterase, cholinesterases of frog brain and of various squid species (Todarodes pacificus and Berrytheutis magister; in the latter case, individuals from three different habitats are compared)--with 141 reversible inhibitors of various structures. Statistically significant differences between ChE of squids and vertebrates are shown. The previously revealed intraspecies peculiarities of ChE in the Commander squid B. magister are statistically confirmed.     

Comparative analysis of sensitivity of cholinesterases to reversible inhibitors by methods of multidimensional statistics

Using the methods of factor and cluster analysis, the statistical treatment is performed of data on interaction of seven cholinesterases (ChE)—human acetylcholinesterase, horse butyrylcholinesterase, cholinesterases of frog brain and of different squid species (Todarodes pacificus and Berrytheutis magister, in the latter case, individuals from three different habitats are compared)—with 141 reversible inhibitors of various structures. Statistically significant differences between ChE of squids and vertebrates are shown. The previously revealed intraspecies peculiarities of ChE in the Commander squid B. magister are statistically confirmed.     

Comparative Sensitivity of Cholinesterases of Different Origin to Some Irreversible Inhibitors

The analytical review is presented of literature data obtained with use of different substrates, on sensitivity of more than 90 preparations of cholinesterases from 65 different animals (vertebrate, insects, molluscs) to the most studied 6 irreversible inhibitors (eserine and 5 organophosphorus compounds). The considered data are discussed from the point of view of comparative biochemistry and in the light of current information about structure of active center of cholinesterases.     

Insect cholinesterases and irreversible inhibitors. Statistical treatment of data

The data on sensitivity of cholinesterases (ChE) of different insects to irreversible inhibitors, as well as the data on physicochemical parameters of amino acids constituting their active centers, were treated by factor analysis and compared. These both characteristics have been shown to be connected with the taxonomical position of the insects. There is revealed the “material substrate” of the factors responsible for the action specificity, which are specific sites in the ChE active center.     

Comparative analysis of sensitivity of cholinesterases of various origin to monoonium reversible inhibitors

Analytical review of literature data has been presented about constants of interaction of cholinesterases of various animals (verterbrates and squids) with 89 onium (ammonium, phosphonium, and sulfonium) reversible inhibitors forming homologous series with regularly changed structure. Values of competitive, uncompetitive, and generalized inhibition constants have been compared. On this basis, conclusions are made about mechanism of action of the studied compounds and the predominant areas of their sorption--in the or peripheral sites of the enzymes. The presented data are discussed from the point of view of comparative biochemistry and in the light of the current information about structure of the cholinesterase active center.     

Kinetic analysis of the “substrate protective effect” in cholinesterases of different origin

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.     

Insect cholinesterases and irreversible inhibitors. Statistical treatment of the data

The data on sensitivity of cholinesterases (ChE) of different insects to reversible inhibitors, as well as the data on physico-chemical parameters of amino acids constituting their active centers, were treated by factor analysis and juxtaposed. It is shown that both these characteristics are related to taxonomical belonging of insects. It is revealed the "material substrate" of the factors determining inhibitor action specificity, which are specific sites in ChE active center.     

Comparative analysis of sensitivity of cholinesterases of different origin to monoonium reversible inhibitors

The analytic review of the literature data on constants of interaction of cholinesterases of different animal (vertebrates and squids) with 89 onium (ammonium, phosphonium, sulfonium) reversible inhibitors constituting homologous series with regularly varied structure is carried out. Values of the competitive, uncompetitive and generalized inhibitor constants are compared. On the basis of that, conclusions about the mechanism of action of the studied compounds and primary place of their sorption—in “anionic” or peripheral “anionic” sites of enzymes—are made. The presented data are considered from the point of view of comparative biochemistry and in light of current concepts of cholinesterase active center structure.     

Comparative Study of Reversible Organofluorine Ammonium Inhibitors of Cholinesterases of Different Animals

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.     

Tetramethonium derivatives as reversible inhibitors of various cholinesterases

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.     

On Mechanism of Interaction of Organophosphorus Inhibitors with Cholinesterases of Different Origin

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.     

Derivatives of lupinin and epilupinin as ligands of various cholinesterases

Literature data are summarized on cholinesterases of some mammals and arthropods with a group of isomer derivatives of alkaloid lupinin and its epimer epilupinin. As substrates of cholinesterases of some mammals there are studied 8 acetates containing in their molecule chinolysidin bicycle with different structure of N-alkyl radical, which showed certain elements of action specificity. For 2 isomer esters that are derivatives of protonated base of lupinin and epilupinin, differences are revealed in their substrate characteristics. Polyenzyme analysis of anticholinesterase efficiency is performed for 30 organophosphorus inhibitors that are dialcoxyphosphorus derivatives of lupinin and epilupinin; as a result, we managed to find out quite a few peculiarities of their action depending on structure. Several tested compounds were revealed to be specific inhibitors of cholinesterases of several mammals and arthropods.     

Derivatives of lupinin and epilupinin as ligands of various cholinesterases

Literature data have been summarized on interaction of cholinesterases of some mammals and arthropods with a group of isomer derivatives of alkaloid lupini and its epimer epilupinin. As substrates of cholinesterases of several mammals there are studied 8 acetates containing in their molecules the chinolysidin bicycle with different structure of N-alkyl radical, which showed certain elements of specificity of action. For 2 isomer esters that are derivatives of the protonated base of the lupinin and epilupinin structures, differences in their substrate characteristics were revealed. The polyenzyme analysis if anticholinesterase efficiency was performed for 30 organophosphorus inhibitors that are dialkoxyphosphorus derivatives of lupinin and epilupinin; as a result, quite a few peculiarities of their action depending on their structure were revealed. Several tested compounds turned out to act as specific inhibitors of cholinesterases of some mammals and arthropods.     

The Correlation between Antiesterase Activities and Chemical Structure of Saligenin Cyclic Phosphates

The effect of substituents on phosphorus of saligenin cyclic phosphorus esters on inhibitory activities toward cholinesterases and ali-esterases in vitro and in vivo was studied. The most insecticidal methyl derivative was the strongest inhibitor of insect cholinesterase both in vitro and in vivo. When the size of the substituent increased, the specificity toward ali-esterase increased, while decreased toward cholinesterase. The inhibitor ratio (I₅₀ ChE/I₅₀ AliE) appears to relate closely with insecticidal activity. Non-insecticidal aryl derivatives are very specific to inhibit insect ali-esterase. They are very weak inhibitors of insect cholinesterase in vivo. Their thiono analogs are stronger in vivo than corresponding oxo analogs.     

Indophenol Chromogenic Substrates of Cholinesterases of Different Origin

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.     

Carboxylic esterases in developing myoneural junctions of rat striated muscle

Summary The distribution of acetylcholinesterase (AChE; E.C. 3.1.1.7), other cholinesterases (ns.ChE; E.C. 3.1.1.8) and eserine-resistant carboxylic esterases (ns. E; E.C. 3.1.1.1) was studied in the developing myoneural junction of the rat tibialis anterior muscle from the 16th intrauterine day onwards. Acetyl-and butyrylthiocholine were used as substrates for AChE and ns.ChE, and -naphthyl acetate for ns. E.Acetylcholinesterase was first visible in 18-day rat embryos, ns.ChE in 21-day embryos and ns. E in 1-day-old postnatal rats and thereafter. Both AChE and ns.ChE activities were localized at the level of the plasma membrane in the middle of the muscle fibres. In the early stages this area of activity had the appearance of a plate-like structure, which deepened to form a depression in the surface of the muscle fibre by the 2nd to the 4th postnatal day. About 5 days later subneural lamellaes appeared in this structure, and ramification and segmentation took place, their extent increasing concomitantly with the increase of enzyme activity. The adult pattern was attained by the age of one month. Precise localization of ns. E was not possible in the immature stages, mainly owing to the granularity of the reaction end-product. After the age of about 10 days, however, the distribution of the reaction end-product suggested a mainly presynaptic location. Other cholinesterases and ns. E, but not AChE, were detected in the neurilemma cells along nerve fibres. This neurilemmal enzyme activity gradually diminished after birth and was lost at about the age of 3 weeks.These observations demonstrate that the formation of the junction between the nerve and the muscle fibre takes place just before the first appearance of AChE activity in a sharply delineated area of the plasma membrane. The structural changes made apparent by the distribution of the reaction end-products are assumed to be linked to the spatial rearrangement of the synaptic membranes, seen in earlier electron microscopic studies.     

Esterases in natural populations of normal and Echinostoma revolutum-infected Helisoma trivolvis (Gastropoda)

Esterases of the digestive gland-gonad (DGG) complex of individual snails from a wild population of Helisoma trivolvis infected with the trematode Echinostoma revolutum were analyzed by vertical slab PAGE and compared to similar DGG homogenates of uninfected conspecifics from the same population. Our analysis indicated that: 1. Four classes of esterases, some atypical, could be resolved using diagnostic inhibitors. 2. Uninfected snails demonstrated polymorphism for two of these four esterase groups, including cholinesterases (CHE), in the 34 individual DGGs analyzed. 3. The rarer of the two ChE phenotypes in the uninfected sample (29.4%) was present in 100% of the 17 infected snails examined. However, no changes in esterase zymograms of infected DGGs due to the parasite were noted. 4. The possibility that the 'rare' ChE phenotype is somehow related to host susceptibility to Echinostoma revolutum is discussed in view of similar apparent linkages in other snail-trematode systems.     

Siliconorganic reversible inhibitors of cholinesterases of various animals

The review present data on cholinesterase effects of 28 specially synthesized siliconorganic compounds (monoonium, clementorganic, 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.     

Cholinesterasen mit Nebenjobs

Moonlighting cholinesterases in non-synaptic cholinergic mechanisms The early phylogenetic and ontogenetic appearance of acetylcholine (ACh) and its cholinergic protein components render their possible functionalities, apart from purely neuronal ones, most likely. The capacities of cholinesterases (ChEs) to form large protein complexes opened wide functional fields for them. Already existent in stem cells, ChEs in cooperation with components of the cell matrix (ECM) promote cell differentiation, whereby their enzymatic activity is (at least partially) dispensable. This is independently supported by effects of inactive AChE protein exerted in non-neuronal cells, as well as the discovery of cholinesterase-like adhesion molecules (CLAMs). Therefore, much evidence supports the conclusion that the original functionalities of cholinesterases, and, more generally of cholinergic systems, are to be sought in cell-cell-communication. Here, these views were exemplified by some in vitro and in vivo model studies. In the vertebrate retina early differentiating amacrine cells co-regulate network formation. Similarly potent are cholinergic mechanisms during skeletogenesis. ACh accelerates bone formation, and ChEs not only regulate its concentration, but exert additional structural functions. As much convincing, a study on tadpoles documented that gut formation in Xenopus laevis depends strictly on the AChE protein, but not on its enzymatic activity. A full elucidation of ChE functionalities is essential, since a multitude of anticholinesterases (ChE inhibitors) are widely applied in public life (agriculture, health, security). It is timely that cholinergic research focuses on elucidation of non-synaptic ChEs, and on analyzing non-neuronal cholinergic systems (NNCS) in general.