E. V. Rozengart

The Chelating Ability of the Anticoccidial Drug 1,3-bis(p-chlorobenzilideneamino)guanidine: the Complexes with Ca2+ and La3+

Among the few drugs for treatment and prevention of protozoal diseases manufactured in Russia, Chimkoktsid (1,3bis ( p -chlorobenzilideneamino)guanidinium, Cl-BAG), a highly effective anticoccidial preparation against animal coccidioses and toxoplasmosis, occupies a special place [1]. This preparation is also of interest for medicine as a potential drug that may be used for human toxoplasmosis treatment [1]. However, the molecular mechanism of Cl-BAG action remains obscure. Earlier, it was shown by the method of molecular mechanics that folded conformations of Cl-BAG are the most preferable due to attraction between the benzene rings [2]:

Catalytic Properties of Cholinesterases from the Brain and Blood Serum of Mink (Mustela Vison Bris.)

The family of cholinesterases (CEs), which displays prominent species and tissue specificity, is one of the most intensely studied groups of enzymes [1, 2]. However, the list of kinetically characterized cholinesterases of animals at different stages of evolution is not very long [3]. There are even fewer examples of detailed characterization of CEs from different tissues of the same animal. It is believed that mammalian nervous tissue and erythrocytes usually contain acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7), whereas blood serum contains butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). These two types of cholinesterases significantly differ in their substrate and inhibitor specificity [1, 2]. To broaden the notion on the properties of these cholinesterases, in this study we, for the first time, performed the substrate and inhibitor analysis of cholinesterase from the brain and blood serum of the American mink ( Mustela vison Bris.), a predator which is only now being domesticated and which retains most behavioral characteristics of its wild relatives [4, 5].

On the noncyclic mechanism of cholinesterase-mediated catalysis.

The Brestkin and Rozengart scheme [1], which illustrates the mechanism of cholinesterase-mediated catalysis and has been commonly accepted for the last several decades, is reproduced in the major monographs on cholinesterases [2–5]. The rest of this scheme is formation of the cholinesterase active site in the presence and with participation of the substrate, which entails formation of Michaelis’ cyclic enzyme– substrate complex:

Chromogenic Indophenyl Esters: Hydrophobic Substrates of Cholinesterases of Various Origin

Indophenyl acetate (IPA) and a number of its halogenated derivatives that yield intensely colored indophenolate ions as a result of their hydrolysis were previously studies as cholinesterase substrates [1–4]. These experiments resulted in the design of a highly sensitive automated procedure of determination of the cholinesterase enzymatic activity [5]. The second peak of interest to IPA was accounted for by unusual properties of acetylcholinesterase modified by alkyl aziridinium derivatives at the active site: along with an almost complete loss of the ability to hydrolyze choline substrates, the rate of IPA hydrolysis considerably increased [6, 7]. Based on this finding, it was assumed that the sorption sites on the catalytic surface of acetylcholinesterase in hydrophilic choline substrates and hydrophobic IPA are different [7]. In all previous studies, indophenyl esters were used only in the studies on erythrocyte acetylcholinesterase and serum butyrylcholinesterase. Thus, a comparative enzymological problem arises: it is interesting to know how other enzymes belonging to the large cholinesterase family catalyze hydrophobic indophenol substrates. In this work, cholinesterases from the optical ganglia of mature Pacific squid Todarodes pacificus (the northwestern region of the Pacific Ocean) and Comandor squid Berryteuthis magister from different parts of its range, including Pribilof–Alaskan (center) and Olyutorsk–Navarin (north) regions of the Bering Sea and the area near the central Kuril Islands (south), were used. Supernatants obtained by centrifugation of aqueous homogenates of the optical ganglion (3 mg/ml) at 800 g for 15 min served as the enzyme source. Hemolymph of the Pacific gastropod Neptunea eulimata caught in the Peter the Great Bay of the Sea of Japan was collected by breaking the shell and cutting the vessel; the hemolymph was subsequently frozen at –18 ° C. We also used purified preparations of human erythrocytic acetylcholinesterase (EC 3.1.1.7) and horse serum butyrylcholinesterase (EC 3.1.1.8) with specific activities of 1.2 and 9.6 U, respectively. Purified enzymes were obtained from the Perm Research Institute of Vaccines and Sera (Russia). Indophenol substrates were synthesized as described in [4, 8] (Table 1), and iodides of acetylcholine (AC) and acetylthiocholine (ATC) were obtained from Chemapol. Enzymatic hydrolysis of chromogenic substrates in a 0.05 M phosphate buffer, pH 8.5, was monitored by differential photometry [4]. To stimulate color development in the experiments with IPA ( p K a of indophenol is 8.1 [4]), 0.07 M phosphate buffer (pH 11.2) was added to the reaction mixture after termination of the reaction, so that pH of the mixture was 9.5 [4]. Hydrolysis of AC and ATC was studied by means of long-term potentiometric titration [5, 9] and colorimetrically [10], respectively. Kinetic parameters of the enzymatic reaction were determined by the analytical method [11].

The cholinesterase activity of the brain of northern Pacific flatfish of different species and genera of the family Pleuronectidae: substrate specificity.

With respect to abundance and biomass, as well as the species diversity, flatfish takes up one of the leading places in the benthic ichthyofauna of the shelf of Far Eastern seas (in the northern part of the Pacific Ocean, family Pleuronectidae is represented by forty-five flounder species and four halibut species) [1]. All flatfish species do not perform significant horizontal migrations: their movings are limited both by the seabottom characteristics (deep trenches, canyons, and protruding capes separating different parts of the shelf from one another and seasonal changes in the depth of their habitats within certain local areas to which the population groups are confined. Therefore, one of the topical problems of the Far Eastern fisheries is the determination of the characteristics of the population structure of food flatfish species. It was of interest to accomplish the well-studied biological characteristics of flatfish (size, weight, etc.) [1–4] with the evolutionary-biochemical data on comparative enzymology of cholinesterases, a large group of enzymes that play a key role in the cholinergic system of the nerve-impulse transmission in animals [5]. In addition, specific properties of cholinesterases are used in the enzymological method of identification of hydrobionts species, which was proposed by us earlier [6, 7].

The Substrate Specificity of the Cholinesterase Activity of the Brain of the Sea Pacific Herring (Cluippea pallasi Val) from Different Populations

The pacific herring (( Cluippea pallasi Val.) is classified with the numerous fluctuating fish species widely spread in the Sea of Japan, Sea of Okhotsk, and Bering Sea of the northern water area of the Pacific Ocean. This species is represented by three ecological forms (sea, littoral, and lake–lagoon) and thirty-four populations, eight of which consist of the sea form. Sea herring spend all life in saline sea or oceanic water. They are confined to the littoral shelf area and frequently perform long-term foraging, wintering, and spawning migrations within this water area [1]. On the northwestern Pacific region, sea herring is represented by several populations, which differ in biological, ecological, and morphological characteristics, as well as in the abundance and location of reproduction areas. Different populations are often mixed during the growing period and wintering. The Okhotsk population, which in summer growing period occupies the whole western half of the Sea of Okhotsk (including the northeastern coast of Sakhalin) [2, 3], is of great economic importance. Among other, less abundant populations, a special place is occupied by the herring of the Anadyr population, which inhabits Anadyr Bay of the bering Sea [4], and the herring of the Der Kastri population, whose life cycle is spent in the northern part of the Tatar Strait, along the continental and Sakhalin coasts.

Brain Cholinesterase Activity of Pacific Salmonids (Family Salmonidae): Substrate–Inhibitor Specificity

Modern studies of the evolution of salmonids (family Salmonidae) take into consideration various aspects of fish morphology, genetics, molecular biology, etc. [1, 2]. On the other hand, determination of the population structure of commercially important species of Pacific salmonids is one of the most important problems facing the Far East salmon fishery, because salmonids are of paramount importance for national economy (annual catches in some years reach 200000 t [1]). Pacific salmons of the genus Oncorhynchus belong to the subfamily Oncorhynchinidae. According to current views, this group includes six widely spread species: the cherry salmon, silver salmon, quinnat, dog salmon, blue-back salmon, and humpback salmon. The results of analysis of the phenotypical similarity between these species suggest that this group of Pacific salmonids is subdivided into two subgroups: cherry salmon–silver salmon–quinnat and dog salmon–blue-back salmon–humpback salmon [1]. It was of considerable interest to supplement these biological characteristics with the evolutional biochemical data on the comparative enzymology of cholinesterases (CEs), large group of enzymes in which both molecular protein and catalytic properties have been studied in detail [3, 4]. In addition, CE was used in the enzymological method of identification of species of hydrobionts suggested in our earlier works [5, 6]. The following representatives of the genera Oncorhynchus and Salmo of the family Salmonidae were studied: humpback salmon Oncorhynchus gorbuscha (Walbaum, 1792), dog salmon Oncorhynchus keta (Walbaum, 1792), silver salmon Oncorhynchus kisutch (Walbaum, 1792), blue-back salmon Oncorhynchus nerka (Walbaum, 1792), quinnat Oncorhynchus tschawytscha (Walbaum, 1792), and Atlantic salmon Salmo salar. Fish brain CE was isolated by centrifugation ( 800 g , 15 min) of an aqueous homogenate (3 mg/ml) of brain tissue of both male and female salmons caught in the northwest area of the Pacific Ocean. The enzymatic activity of CE was measured colorimetrically by the method of Ellman [7] at 25°ë and pH 7.5. The kinetic parameters of enzymatic hydrolysis, namely, the maximum reaction rate ( V ), Michaelis constant ( K M ), and their ratio V / K M ) were determined as described in [8]. Iodides of acetylthiocholine (ATC), propyonylthiocholine (PTC), and butyrylthiocholine (BTC) (Chemapol) were used as substrates. The following inhibitors were tested:

The Relationship between the Anticholinesterase Effect of Organophosphorous Inhibitors and the Extent of Shielding (Accessibility) of the Phosphorus Atom

It is known that irreversible inhibition of cholinesterases by organophosphorous inhibitors (OPIs) is based on phosphorylation of the serine hydroxyl in the enzyme active site by the mechanism of nucleophilic substitution [1–3]. The efficiency of this process apparently depends not only on the strength of the ester bond broken, but also on the steric accessibility of the phosphorus atom for a nucleophilic group [4]. Using the method of molecular mechanics, we developed the procedure of determination of the extent of steric accessibility of the phosphorus atom [4, 5] and explained the inability of some cyclic organophosphorous inhibitors to irreversibly inhibit acetylcholinesterase [4, 6]. In this study, we calculated stable conformations of 42 organophosphorous inhibitors by the method of molecular mechanics, estimated the phosphorus atom accessibility, and performed comparative correlation analysis with the kinetic parameters of inhibition of erythrocytic acetylcholinesterase and butyrylcholinesterase.

Conformational Aspects of the Interaction of Various Cholinesterases with Polymethylene-bis(trimethylammonium) Derivatives

dides, (CH 3 ) 3 –(CH 2 ) n – (CH 3 ) 3 (I · n ), have been known for a long time as cholinergically active compounds that block the nicotine choline receptor [1] and reversibly inhibit cholinesterases [2–4]. However, the assumptions on the mechanism of their action are restricted to the hypothesis on a polyfunctional interaction with the active surface [2, 5]. Moreover, these compounds are widely used to measure the distances between the anionogenic groups of the active surface of biomolecules [1]. At first glance, the completely extended conformation with the trans position of all C − C bonds is the most advantageous in terms of the electrostatic repulsion energy of the ammonium groups. However, due to conformational lability of the polymethylene chain, the solution should also contain less advantageous forms; i.e., the series I · n represents a mixture of conformers, with the probability of existence of each conformer being determined by the conformation energy. Accurate data of the conformational possibilities of these compounds may be obtained using the method of molecular mechanics [6, 7]. In addition, the differences in the cholinesterase specificity with respect to these reversible inhibitors in various animal species are known [3, 4]; however, these differences were not studied in connection with the conformational characteristics of these compounds. In view of this, it was interesting to study the conformation–function relationship for the series of derivatives I · n (namely, I · 4 –I · 10 ) during their interaction with cholinesterases of various origin. We used purified preparations of human erythrocyte acetylcholinesterase (EC 3.1.1.7) and horse blood serum butyrylcholinesterase (EC 3.1.1.8) with specific activities of 1.2 and 9.6 units, respectively (Perm’ N + N + Research Institute of Vaccines and Sera). Cholinesterases from aqueous solutions of the frog ( Rana temporaria ) brain and the Pacific squid ( Todarodes pacificus ) visual ganglion (protein concentrations of 45 and 3 mg/ml, respectively) were also studied [4]. The catalytic activity of the enzymes was determined colorimetrically at 25°ë and pH 7.5 using acetylthiocholine iodide (Chemapol) as a substrate [8]. We used the following polymethylenebis (trimethylammonium) diiodides, [CH 3 ) 3 N(CH 2 ) n N(CH 3 ) 3 ] 2+ · 2I – · ( I · n ), as cholinesterase inhibitors: n = 4 (I · 4), n = 5 (I · 5), n = 6 (I · 6), n = 7 (I · 7), n = 8 (I · 8), n = 9 ( I · 9 ), and n = 10 ( I · 10 ) [4]. The efficiency of reversible inhibitors was

Guanidinium derivatives as reversible inhibitors of cholinesterases of various origins.

It is known that 1,3bis ( p -chlorobenzylideneamino)guanidinium (compound I) occupies a special place among the biologically active guanidinium derivatives because of its high effectivity against animal coccidioses and toxoplasmosis [1]. However, the molecular mechanism of its effect remains obscure. The molecular mechanics study showed [2] that, in compound I, folded conformations are the most preferable due to the attraction between benzene rings. In this case, the most remote chlorine atoms can closely approach each other (at a distance of 3.7 Å), which implies the possibility of formation of chelate complexes with Ca 2+ . This assumption was confirmed by the results of comparative spectrophotometrical studies based on the second derivative of differential UV spectra [2]. In addition, when studying the mechanism of the biological effect of compound I and some its derivatives, we found that these compounds have an anticholinesterase effect [3, 4]. To test this hypothesis, the anticholinesterase activities of guanidinium and its amino derivatives were studied in more detail. On the one hand, these compounds simulate the hydrophilic moiety of compound I. On the other hand, when protonated, these compounds have different extents of charge delocalization and are of interest as reversible inhibitors of cholinesterases of various origins [5, 6].