[Cultured cell systems for revelation of new drugs antiatherosclerotic effects and their mechanisms investigation].

Cultured cell models have been developed to study the cholesterol efflux from the arterial wall as an integral indicator of reverse cholesterol transport. The models and the results of in vivo and ex vivo experiments can be used to propose new antiatherosclerotic drugs and elucidate their mood of action.

[The effect of glycolipids [correction of glycoproteins] on the oligomeric structure and catalytic activity of alpha-L-fucosidase from human kidneys]. / Vliianie glikolipidov [correction of glikoproteidov] na oligomernuiu strukturu i katatlicheskuiu aktivnost' alpha-L-fukozidazy pochek cheloveka.

alpha-L-Fucosidase (EC 3.2.1.51) has been isolated from human kidney and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose and fucosylamino-Sepharose. The catalytic activity and oligomeric structure of the enzyme were studied in a reversed micelle system of aerosol OT in octane. Depending on the degree of hydration (a parameter determining the geometrical sizes of the inner aqueous cavity of micelles), fucosidase is present within micelles as 53 kDa monomers, 110 kDa dimers, 230 kDa tetramers and 480 kDa octamers. Association of the monomers into tetra- or octamers causes a 3-4 fold increase in the specific catalytic activity of alpha-L-fucosidase. At pH and ionic strength values corresponding to intralysosomal ones alpha-L-fucosidase is isolated from tissues exclusively in a tetrameric form. After treatment with sodium cholate and subsequent dialysis this tetramer irreversibly dissociates into monomers; this reaction is accompanied by 2-3-fold decreases in the specific catalytic activity of alpha-L-fucosidase. The enzyme tetrameric structure and specific catalytic activity may be reconstituted in a reversed micelle system in the presence of glycolipids-di- and trihexosylceramides, GM1-ganglioside and a mixture of bovine brain gangliosides.

[Human glycosidase in the AOT reversed micelle system: features of properties and kinetic regularities of catalysis]. / Glikozidazy cheloveka v sisteme obrashchennykh mitsell PAV: osobennosti svoistv i kineticheskie zakonomernosti kataliza.

Kinetic of hydrolysis, by lysosomal glycosidases, of their synthetic substrates were studied in systems of the Aerosol OT (AOT) reversed micelles in octane. Catalytic activity of all the tested enzymes, viz., GM1-galactosidase, beta-hexosaminidases A and B, neuraminidase, and galactocerebrosidase, in reversed micelles proved to be the same as or higher than in the water buffer. In the reversed micelles an effective inhibition of the enzymatic reactions by the resulting carbohydrates was however observed. The dependence of the enzymes' activity on the hydration degree was represented by curves with one or several maxima, corresponding to various oligomeric forms of the enzymes. Dependencies of effective Km on the hydration degree in reversed micelles are similar dependencies of the enzyme activities on the same parameter, which can be explained by corresponding changes of local substrate concentration near the enzyme active site. Dependencies of the enzymatic activity on the surfactant's concentration in the reversed micellar system were also studied. Catalytic activity of the soluble lysosomal glycosidases was found to be unaffected by the micelles concentration. Activity of the membrane lysosomal glycosidase, galactocerebrosidase, strongly increased when the surfactant's concentration decreased. Under optimal conditions the activity of galactocerebrosidase in reversed micelles was 10-fold as compared with its activity in the water buffer.

[Regulation of the catalytic activity and supermolecular structure of penicillin acylase from Escherichia coli in an aerosol OT reversed micelle system in octane]. / Reguliatsiia kataliticheskoi aktivnosti i nadmolekuliarnoi struktury penitsillinatsilazy iz Escherichia coli v sistem obrashchennykh mitsell aérozolia OT v oktane.

The properties of penicillin acylase from E. coli solubilized by hydrated reversed micelles of Aerozol OT (AOT) in octane were studied. The catalytic activity dependence on the hydration degree, a parameter which determines the size of the micelle inner cavity, represents a curve with three optima, each corresponding to the enzyme functioning either in a dimer form (omega 0 = 23) or in the form of separate subunits--heavy, beta, and light, alpha, at omega 0 = 20 and 14, respectively. Reversible dissociation of the enzyme was confirmed by ultracentrifugation followed by electrophoresis. Preparative isolation of penicillin acylase subunits, their catalytic activity being retained, was shown to be possible.

[Hexosaminidase A-hexosaminidase B complex from human kidneys]. / Kompleks geksozaminidazy A i geksozaminidazy B iz pochek cheloveka.

Isolation and purification on human kidney hexosaminidases A and B (EC 3.2.1.30) were carried out. The regulation of the supramolecular organization and catalytic activity of the hexosaminidases in the AOT reversed micellar system modelling the enzyme microenvironment in the lysosomes, were investigated. It was found that under these conditions hexosaminidases A and B associate to form a 280-300 kDa dimeric complex. At pH 4.75 hexosaminidases A and B can be isolated from kidney tissue only within the composition of the complex.

[Regulation of supermolecular organization and catalytic activity of the lysosomal complex GM1-galactosidase and neuraminidase in vitro]. / Reguliatsiia nadmolekuliarnoi organizatsii i kataliticheskoi aktivnosti lizosomnogo kompleksa GM!-galaktozidazy i neiraminidazy in vitro.

The regulation of the catalytic activity and supramolecular organization of human kidney Gm1-galactosidase and neuraminidase was investigated in the reversed micellar systems of Aerosol OT in octane. It was found that in the reversed micellar systems the Gm1-galactosidase can exist in the monomeric, tetrameric or octameric forms depending on the H2O/surfactant ratio in the system which determines the micelle size. The association of Gm1-galactosidase monomers into octameric structure characteristic of Gm1-galactosidase in the lysosomes results in a two fold increase of the specific catalytic activity of the enzyme. 32 kDa "protective" protein--the component of Gm1-galactosidase--neuraminidase native complex was found to improve significantly this association.

[A new approach to titrating active enzyme centers upon the use of surface-active micellar substances in organic solvents]. / Novyi podkhod k titrovaniiu aktivnykh tsentrov fermentov pri ispol'zovanii mitsell poverkhnostno-aktivnykh veshchestv v organicheskikh rastvoriteliakh.

Micellar solutions of surfactant in organic solvents with rubber additions are proposed for determination of active enzyme concentration. A kinetic theory of enzymatic reactions in reversed micellar systems is developed, suggesting the intermicellar transport of the substrate to be the limiting step in viscous medium. Under these conditions, it is shown that fraction of the product formed after quick transformation of the substrate located in the enzyme-containing micelles depends upon active enzyme concentration and aggregation number of surfactant molecules. The proposed approach is used for the active-site titration of trypsin and cellobiase and for the determination of the aggregation number of Aerosol OT (AOT) molecules in the ternary system AOT/water/cyclohexane.