[SPR-biosensor assay for analysis of small compounds interaction with human cytochrome P450 51A1 (CYP51A1)].

The SPR assay for human cytochrome P450 51A1's (CYP51A1) ligand screening was developed. Assay has been validated with known azole inhibitors of cytochrome P450s. The studied azoles selectively interacted with human cytochrome P450 51A1, which showed the highest affinity towards ketoconazole. The efficiency of the SPR assay was showed with 19 steroid and triterpene compounds, which were not investigated as potential ligands of CYP51A1.

[Thermodynamic analysis of dimerization inhibitors binding to HIV protease monomers].

Here, we describe the analysis of kinetic and thermodynamic parameters for binding of peptide and nonpeptide dimerization inhibitors to immobilized HIV protease (HIVp) monomers by using surface plasmon resonance. Molecular interactions were investigated at different inhibitors concentrations (0-80 microM) and temperatures (15-35 degrees C). The kinetic, equilibrium and thermodynamic parameters have been determined. It was found that both inhibitors were characterized by similar interaction parameters. The complex formation is entropically driven process for both inhibitors. The entropic term(-TdeltaS) had the value about -20 kcal/mol while the enthalpic term (deltaH) had the positive value about 14 kcal/mol and counteracted the complex formation.

[Specificity of molecular recognition in oligomerization of bacterial L-asparaginases].

Bacterial L-asparaginases, which are widely used in the antitumor therapy, act only as homotetramers, because their active sites are located at the interface between the subunits of the enzyme. Since salt bridges substantially stabilize L-asparaginase tetramers, we have supposed that oligomerization of bacterial L-asparaginase is a high-avidity process. This assumption was proved by bioinformatic and biosensoric methods. It was shown, that a stable tetrameric complex can be formed only by the subunits of the same L-asparaginase. Using two mutants of L-asparaginase Helicobacter pylori it was shown that specificity of molecular recognition is significantly reduced even by single point mutation at the interface of high-homologous closely-related subunits.

[Biosensor analysis of interaction of potential dimerization inhibitors with HIV-1 protease].

Currently inhibitors of protein-protein interactions are considered as perspective prototypes of new generation of drugs. The most attractive targets for such inhibitors are the oligomeric enzymes which active sites are formed by amino acid residues from different subunits. The classic example of such enzyme is HIV protease (HIVp), active only in the homodimeric form. We have developed a new approach for experimental screening of HIVp dimerization inhibitors. It is based on the original biosensoric test-system for differential analysis of interaction of tested substances with HIVp dimers and monomers. Using this test-system we have analyzed the most perspective candidate substances predicted by method of virtual screening, and some derivatives of glycyrrhizin, triterpenic and steroid glycosides. As a result we found one compound, which mainly interacts with HIVp monomers and inhibits in vitro activity of this enzyme with IC50 of about 10(-6) M.

[Oligomerization of L-asparaginase from Erwinia carotovora].

Bacterial L-asparaginases catalyzing hydrolysis of L-asparagine up to L-aspartate and ammonia, are used in medical practice for treatment of acute lymphoblastic leukemia. The long-term therapy with these preparations is accompanied by a number of side effects, which are attributed to glutaminase activity of L-asparaginase. Substrate specificity and activity of L-asparaginases are directly associated with the process of enzyme oligomerization. It is active only in the tetrameric form as the active sites are located in contact areas between monomers. The present work is devoted to homology modeling of spatial structure of L-asparaginase from Erwinia carotovora, the comparative molecular-graphic analysis of subunits interfaces, as well as development of experimental approach for enzyme oligomerization study. L-asparaginase was immobilized on a CM5 chip surface of optical biosensor Biacore 3000 based on the surface plasmon resonance technology. The dissociation process of enzyme tetrameric complexes up to monomers and subsequent oligomerization process have been registered.

[Interaction investigation of trypsin inhibitor from sea anemone Radianthus macrodactylus with proteases].

The interaction of inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated. The following enzymes were tested: serine proteases (trypsin, alpha-chymotrypsin, plasmin, thrombin, kallikrein), cysteine protease (papain) and aspartic protease (pepsin). Inhibitor VJ interacted only with trypsin and 6-chymotrypsin. Kinetic and thermodynamics parameters of intermolecular complexes formation were determined: KD = 7,38 x 10(-8) M and 9,93 x 10(-7) M for pairs InhVJ/trypsin and InhVJ/alpha-chymotrypsin, respectively.

[Optical biosensor study of interaction between trypsin and trypsin inhibitor].

The formation of complexes between trypsin (T) and soybean tripsin inhibitor (STI) was analyzed, using a two-channel IAsys+ optical biosensor. The temperature dependence of complex formation (with its major parameters--the association rate constants (k(on)), the dissociation rate constants (k(off)) and the equilibrium constants (Kp)) was determined. The equilibrium constants obtained well correlate with those obtained by other methods. The association rate constant for the binding of T/STI complex increased with temperature, while the dissociation rate constant practically remained unchanged. The association and dissociation rate constants activation parameters were calculated from their concentration dependence. Based on the temperature dependence the activation energy, enthalpy and entropy of complex formation were calculated. It was shown that the entropy component plays a key role in T/STI interaction.

Capacitive and ionic currents in BLM from phosphatidic acid in Ca2+-induced phase transition.

The development of electric current with time in a bilayer lipid membrane (BLM) formed from dipalmitoylphosphatidic acid on introducing Ca2+ ions into the medium was studied at constant temperature and pH. The phase transition in the Ca2+-induced BLM is accompanied by the initial capacitive current followed by the occurrence of single ionic channels. The amount of transported charges in the capacitive current is 5 C/ microF. The conductivity of the single ionic channels ranges from 50 to 100 pSm.

[Ca2+ ions induce successive capacity and ion currents in BLM from phosphatidic acid as a result of phase transition]. / Iony Ca2+ indutsiruiut posledovatel'noe poiavlenie emkostnogo i ionnogo tokov v BLM iz fosfatidnoi kisloty v rezul'tate fazovogo perekhoda.

Time dependence of Ca2+-induced electric current in BLM formed from DPPA was studied at constant temperature and pH. The phase transition in BLM is accompanied by capacity current and following appearance of single ionic channels. It was shown that transferred charge was 5 nC/F, conductivity of single ionic channels--500-100 pSm.

[Cholesterol ester increases the permeability of erythrocyte membrane]. / Efir kholesterina uvelichivaet pronitsaemost' membran éritrotsitov.

The effect of cholesteryl palmitate on erythrocyte membrane permeability for K+ and hemoglobin was studied. Cholesterol ether was incorporated into the erythrocyte membrane by liposomes containing cholesteryl palmitate and lecithin or by dispersion of cholesteryl palmitate. It was shown that cholesteryl palmitate considerably increases permeability of the erythrocyte membrane for K+ and hemoglobin. The leakage of K+ and hemoglobin from red blood cells is not accompanied by cell destruction.

[Cholesterol esters increase the permeability of lecithin bilayer membranes]. / Efiry kholesterina uvelichivaiut pronitsaemost' letsitinovykh bisloinykh membran.

Conductivity of lecithin bilayers containing cholesterol and its esters was studied. It was shown that cholesterol causes a considerable decrease of conductivity, the original conductivity of bilayers being (5.8 +/- 0.9) . 10(-8) Ohm-1 cm-2. Maximal effect was observed when concentration of cholesterol was about 30 mol%. Further increase of cholesterol content does not change the conductivity of bilayers. It was found that cholesterol esters in contrast to cholesterol cause the increase of ion permeability of bilayers. The same effect was also observed at high membrane cholesterol content. The obtained results were discussed in relation to cell membrane destructions at atherosclerosis.

[Conductivity of bilayer lipid membranes of phosphatidic acid at phase transitions, induced by temperature and pH]. / Provodimost' bisloinykh lipidnykh membran iz fosfatidnoi kisloty pri fazovom perekhode, indutsiruemom temperaturoi i pH.

Conductivity of bilayer lipid membranes formed from synthetic phosphatic acid and its thioanalogs was studied. Conductivity of bilayer lipid membranes was shown to change spasmodically in the phase transition region. The temperature of conductivity jump decreased with pH shift from acid to alkaline region. The amplitude of conductivity jump attained 1.5-2 orders. The jump can be also obtained at constant temperature by changing the medium pH. The observed conductivity jumps are suggested to determine the phenomena of thermo- and chemoreception in biological membranes.

[Initiation of anion-selective ionic channels in bilayer membranes at lipid phase transitions]. / Vozniknovenie anion-selektivnykh ionnykh kanalov v bisloinykh membranakh pri fazovykh perekhodakh lipidov.

A study was carried out to electric parameters of single ionic channels initiated at phase transition of bromidmetilate 1,2-distearoyl-rac-glycero-3-(O-beta-dimethylaminoethyl)-methylphosphonate, whose molecules under conditions given below are possibly charged. It has been shown that changes of transmembrane current appear at phase transition temperature. Comparison between ionic selectivity of channels initiated at Tph.t in the membranes of DSL and its phosphate analog suggests that the channel walls initiated at phospholipid phase transitions are covered with polar groups of molecules.