[Production of timolol containing calcium-phosphate nanoparticles and evaluation of their effect on intraocular pressure in experiment].

Methodology for production of calcium-phosphate nanoparticles is developed and its efficacy as a drug carrier system is estimated by example of timolol. Conditions for production of particles with optimal size and resistance are determined, methodology of loading of particles with timolol is developed. Physical parameters of particles (form, size, relief), kinetics of saturation with drug and its release are studied. Packaging of timolol into calcium phosphate nanoparticles was showed to enhance and prolong its hypotensive effect in experiment on healthy rabbits.

[Design of the composition of baculovirus agents].

The ability of 30 compounds to protect infection bacteria and baculoviruses from the damaging effect of ultraviolet (UV) irradiation was investigated. For this B. thuringiensis var. israelensis spores and gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus were mixed with different components and exposed to UV irradiation at 0.25 WI cm2 for 60 min. Then spore viability and viral pathogenicity were studied in third instar gypsy moth larvae. The composition comprising sodium alginate, albumin, and ascorbic acid ensured the most effective protection of the viruses and bacteria. These components were shown to provide protection from exposure to UV irradiation even at a low concentration. Their incorporation into biopesticides will assist in enhancing the efficiency of their use.

[The properties of the Pseudomonas aeruginosa bacteriophage phiPMG1].

The properties of the isolated Pseudomonas aeruginosa bacteriophage phiPMG1 include the lytic infection cycle, and the formation of a broad halo (semi-transparent zone) around the plaques. We consider phiPMG1 as a potential member of therapeutic cocktails of live phages, and as a source of peptidoglycan and lipopolysaccharide degrading enzymes. Partial sequencing of phiPMG1 genome has revealed high similarity with known temperate P. aeruginosa phage D3. An open reading frame encoding lytic transglycosilase was identified in the genome. This enzyme PMG MUR was obtained in recombinant form, and its activity and substrate specificity has been studied.

[Development of Streptococcus pyogenes cells inactivation for turbidimetric determination of bacteriolytic activity of phage-associated enzyme].

AIM: Development a method of treatment of Streptococcus pyogenes bacteria that does not disrupt the integrity of surface structure of cell and provides optimal reproducibility of enzyme preparation activity test results. MATERIALS AND METHODS: Museum cultures of S. pyogenes M29 and S. pyogenes T1 were used, as well as standard strain S.pyogenes T5 (ATCC) and 3 phage-associated lysine PlyC preparations (enzybiotics): 2 isolated from phage C1, third--recombinant enzyme obtained by cloning phage C1 DNA. 3 methods of S. pyogenes cells treatment were used: inactivation by chloroform, antibiotics and heating. RESULTS: Treatment of S. pyogenes cells by rifampicin and gentamicin allows simultaneous turbidimetric determination of enzyme preparations activity and streptococci lysis effectiveness with a good reproducibility of test results. Comparison of kinetic curves of streptococci lysis killed by heating with curves of live culture lysis showed that heat treatment of cells results in a decrease oflysis depth and a reduction of enzyme activity. Pattern and effectiveness of lysis of cells incubated with chloroform approached curve of live streptococci lysis, however this method did not exclude lysis of part of cells and required presence of equipment for work with chemical substances. CONCLUSION. S. pyogenes test culture inactivation method by 2-step treatment of culture with antibiotics that does not disrupt the integrity of surface structure of cells and provides optimal reproducibility of enzyme preparation activity test results was developed.

[Bacteriophage enzymes for the prevention and treatment of bacterial infections: stability and stabilization of the Streptococcus pyogenes cell lysing enzyme].

The effect of various compounds on the activity and stability of a phage-associated enzyme lysing cells of streptococci of groups A and C (PlyC) was investigated. Substantial inhibition of the enzyme activity was revealed at an increased ionic strength (in the presence of NaCl) and upon the addition of carbohydrates (mono-, di-, and polysaccharides), i.e., agents stabilizing many enzymes. It was established that the enzyme activity was substantially reduced in the presence of positively charged polyelectrolytes and surfactants, whereas incubation with micelle-forming substances and negatively charged polyelectrolytes led to PlyC activation and stabilization. It was shown that, in the mycelial polyelectrolyte composition M16, the enzyme retained its activity for 2 months; while in a buffer solution under the same conditions (pH 6.3, room temperature), it practically completely lost its activity in 2 days. Characteristics of the enzyme thermal inactivation were found, in particular, its semiinactivation time at various temperatures; these allowed us to estimate its behavior at any temperature and to recommend conditions for its storage and use.

[Effect of chemical modification of lipase on the regulation of its lipolytic activity in reversed micelles].

Hydrophilized and hydrophobized forms of the lipase from Mucor miehei were obtained by its chemical modification with cellobiose and N-hydroxysuccinimidyl palmitate with a modification degree of 4 in both cases. A comparative analysis of the regulation of the catalytic activities of the native and modified lipases was carried out in the system of reversed micelles of OT aerosol (AOT) in isooctane. The level of catalytic activity of all the lipase preparations in the micellar medium was found to be higher than that in aqueous solution. The chemical modification of lipase did not result in a change in the regulation of the oligomeric composition of the enzyme controlled by the degree of micelle hydration omega0 (micelle size). The kcat dependences on omega0 for each lipase preparation exhibit two maxima, corresponding to the functioning of lipase monomers and tetramers. The changes in the hydrophilic-lipophilic balance of the lipase surface significantly affect the character of the regulation of enzyme activity due to changes in the surfactant concentration (the number of micelles). The lipase hydrophobization results in a decrease in the enzyme activation effect with an increase in the AOT concentration in comparison with the native lipase. The lipase hydrophilization dramatically decreases the activity of lipase tetramer when the AOT concentration is increased. The catalytic activity of the monomer of hydrophilized lipase is practically independent of the AOT concentration. Kinetic data indicate a mixed type of activation of both oligomeric forms of the native and the hydrophobized lipase by AOT molecules and the noncompetitive type of the activation and AOT inhibition of the monomer and the tetramer of the hydrophilized lipase, respectively. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

[The lipase/lipoxygenase bienzyme system in AOT reversed micelles in octane]. / Bifermentnaia sistema lipaza/lipoksigenaza v obrashchennykh mitsellakh AOT v oktane.

In this work it is shown that the bienzyme lipase/lipoxygenase system can function in reversed micelles of bis(2-ethyl)hexyl sulfosuccinate (AOT) in octane. As a lipase substrate, a fish fat preparation (fat of sea mammals) with a high content of polyunsaturated fatty acids was used. It was demonstrated that the bienzyme reaction proceeded in a stationary mode and had a rate-limiting step catalyzed by lipase. Under optimal conditions, the efficacy of functioning of the bienzyme system was by an order of magnitude higher than that in water. The lipase/lipoxygenase bienzyme system can be used as a new method of spectrophotometric determination of lipase activity. The English version of the paper.

[Synthesis of alkyl glycosides, catalyzed by beta-glycosidases in a reversed micelle system]. / Sintez alkilglikozidov kataliziruemyi beta-glikozidazami v sisteme obrashchennykh mitsell.

A basic possibility of enzymic synthesis of alkyl glycosides in a system of the Aerosol-OT (AOT) reverse micelles was studied. Octyl beta-D-galactopyranoside and octyl beta-D-glucopyranoside were synthesized from the corresponding sugars (lactose or glucose) and octyl alcohol under catalysis with glycolytic enzymes, beta-galactosidase and beta-glucosidase, respectively. The transglycosylation/hydrolysis ratio was shifted toward transglycosylation by using octyl alcohol, one of the substrates, as an organic solvent. The alkyl glycosides were thus obtained in one step from a hydrophilic mono- or disaccharide and a hydrophobic aliphatic alcohol. The direction of the reaction was shown to depend on the pH of aqueous solution immobilized in nerves micelles. The maximum yields were 45% and 40% for octyl galactoside and octyl glucoside, respectively; they markedly exceeded the yields of enzymic syntheses in a two-phase system reported previously.

[Regulation of the supramolecular structure and catalytic activity of D-glyceraldehyde-3-phosphate dehydrogenase in systems of reversed Aerosol OT micelles in octane]. / Reguliatsiia nadmolekuliarnoi struktury i kataliticheskoi aktivnosti D-glitseral'degid-3-fosfatdegidrogenazy v sistemakh obrashchennykh mitsell aérozolia OT v oktane.

Reversible formation of various oligomeric forms of rabbit skeletal muscle D-glyceraldehyde-3-phosphate dehydrogenase (GAPD) in reversed micelles of Aerosol OT in octane at variable degrees of hydration (size) of the micelles, has been demonstrated. All the oligomeric forms of the enzyme possess a catalytic activity in the reversed micelle system. Data from the sedimentation analysis provide evidence for the formation of a monomer, a dimer and a tetramer of GAPD. The peculiarities of the structure of oligomeric forms of the enzyme revealed in fluorescence studied are discussed.

[Penicillin acylase from Escherichia coli: catalytically active subunits]. / Penitsillinatsilaza iz Escherichia coli: kataliticheski aktivnye sub''edinitsy.

Gel filtration under denaturing conditions was used to isolate the alpha- and beta-subunits of penicillin acylase (PA). Refolded subunits were obtained through removing urea by dialysis. Both renatured subunits were catalytically active during hydrolysis of phenylacetic acid p-nitroanilide; this activity decreased after addition of a serine-specific inhibitor--phenylmethanesulfonyl fluoride. The subunits were also active in reversed micelles of Aerosol OT (AOT) in octane, the optimum hydration degree being 11.9 and 17.5 for the light (alpha) and heavy (beta) subunits, respectively. The positions of the maxima were consistent with both theoretically calculated optimum hydration degrees and the earlier reported profile of enzymatic activity for native PA in reversed micelles.

[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.

[Artificial oligomerization of enzymes--a new way of regulating their catalytic activity in reversed micellar systems]. / Iskusstvennaia oligomerizatsiia fermentov--novyi put' k regululiatsii ikh kataliticheskoi aktivnosti v sistemakh obrashchennykh mitsell.

Comparative studies were carried out in the catalytic activity regulation of native alpha-chymotrypsin and its artificially produced hexameric form as an example of non-dissociating oligomeric enzyme (covalently cross-linked by means of succinimidyl-3-(2-pyridylthiopropionate] in the Aerosol OT reversed micelles in octane. Native (monomeric) alpha-chymotrypsin exhibits maximal catalytic activity in the reversed micelles at the hydration degree w0 = 10, when the radius of the micelle inner cavity is equal to the radius of the alpha-chymotrypsin globule. For the alpha-chymotrypsin hexamer, optimum is observed at w0 = 45, with the inner micellar cavity radius (r = 68 A) being approximately equal to the radius of the sphere surrounding the octahedral combination of the six monomeric alpha-chymotrypsin molecules (r = 61 A). Thus, construction of the corresponding oligomeric structures is made easy, with the optimal catalytic activity in a preset range of the hydration degrees.

[Alkaline phosphatase in reverse micelles of surfactants in organic solvent]. / Shchelochnaia fosfataza v obrashchennykh mitsellakh PAV v organicheskom rastvoritele.

A dimeric enzyme (alkaline phosphatase from calf intestinal mucosa) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. The dependence of the enzyme's activity on the hydration degree (on the size of micelles) is a curve with two optima corresponding to the hydration degrees [H2O]/[AOT] = 17 and 25; when the inner cavity radii of reversed micelles are equal to the size of the enzyme's monomer (Mr = 70 000) and of the dimer (Mr = 140 000). Ultracentrifugation experiments showed that a reversible dissociation of the enzyme into subunits takes place as a result of the change of the hydration degree; the first and second maxima corresponding to the functioning of the monomeric and dimeric forms of the enzyme, respectively.

[Comparative study of the regulation of catalytic activity of soluble and membrane forms of enzymes in reverse micellar systems. Gamma-glutamyltransferase and aminopeptidase]. / Sravnitel'noe izuchenie reguliatsii kataliticheskoi aktivnosti rastvorimykh i membrannykh form fermentov v sistemakh obrashchennykh mitsell. Gamma-glutamiltransferaza i aminopeptidaza.

The regulations of functioning of water soluble and membrane forms of enzymes in the systems of reversed micelles of surfactants in organic solvents are compared. By an examples of gamma-glutamyltransferase (in AOT reversed micelles in octane) and amino-peptidase (in Brij 96 reversed micelles in cyclohexane) the principal difference in the catalytic activity regulation of water soluble and membrane forms is demonstrated. The catalytic activity of the membrane form depends largely on the surfactant concentration at the constant hydration degree, whereas the activity of the water soluble form is constant under these conditions. The catalytic activity dependence on the surfactant concentration is regarded as a "test for the enzyme's membrane activity".

[Interconnection between activity and conformational mobility of alpha-chymotrypsin in reverse micelle systems]. / Vzaimosviaz' aktivnosti i konformatsionnoi podvizhnosti alpha- khimotripsina v sistemakh obrashchennykh mitsell.

A comparative study of the catalytic activity of alpha-chymotrypsin and the spin label rotation frequency in the alpha-chymotrypsin active center of reverse micellar systems solvated by H2O-organic mixtures was carried out. It was found that the decrease in the label rotation frequency resulting from the substitution of water in the micellar inner cavity by glycerol, 2.3-butanediol and dimethylsulfoxide (up to 95%) caused a marked increase (20-fold in the case of 2.3-butanediol) of the enzyme catalytic activity. The experimental results are quantitatively interpreted in terms of a simple kinetic scheme postulating the existence of the enzyme in two interconvertible forms differing in the conformational (intramolecular) mobility, i.e., the resting one predominantly existing in aqueous solution, and the tense one whose proportion rises with an increase in the concentration of the water-miscible organic solvent in the reverse micellar system. The value of kcat (2.4 s-1) for the tense form of the enzyme exceeded by more than 25 times the catalytic activity of chymotrypsin in aqueous solution (0.09 s-1) for the resting form.

[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.

[Relaxation phenomena in systems of protein-containing reverse micelles of surfactants in organic solvents]. / Relaksatsionnye iavleniia v sistemakh beloksoderzhashchikh obrashchennykh mitsell poverkhnostno-aktivnykh veshchestv v organicheskikh rastvoriteliakh.

The research was aimed to establish the equilibrium processes in protein-containing systems of AOT reverse micelles in octane. As chromophore label for tracing the kinetics of the process, the acid-base indicator, p-nitrophenol, was used. The establishing of the equilibrium in the reverse micelle system notably decelerated in the presence of a solubilized protein (native and stearoylated alpha-chymotrypsin). During the establishing of the equilibrium, the solubilized enzyme can be irreversibly inactivated. The level of the residual activity of the enzyme in the equilibrium system depended on the procedure of micellar system preparation. The methods have been offered to set up the equilibrium in the reverse micelle system without inactivation of the solubilized enzyme.