[Trigger factor dependent refolding of bacterial luciferases in Escherichia coli cells: kinetics, efficiency and effect of the bichaperone system, DnaKJE-ClpB].

Here were determined the basic parameters of the Tigger Factor (TF) -dependent refolding of thermal inactivated bacterial luciferases. The TF-dependent refolding is less efficient and requires more time than DnaKJE-dependent refolding. The increase in the intracellular concentration of TF leads to an apparent decrease in the level of the thermal inactivated bacterial luciferase refolding. For thermolabile luciferases, the level of TF-dependent refolding is significantly higher, than for thermostable luciferases: 30-40%--for the thermolabile Aliivibrio fischeri and Photobacterium leiognathi luciferases, and 10 and 0.5% for the thermostable Vibrio harveyi and Photorhabdus luminescens luciferases, respectively. The negative effect of the ClpB protein on the TF-dependent refolding was shown: in Escherichia coli clpB::kan TF-dependent refolding is more efficient than in the E. coli clpB+.

[A kinetic model of beta-galactosidase of Escherichia coli].

On the basis of previously published experimental data, the catalytic cycle of Escherichia coli galactosidase has been reconstructed. The enzyme plays a key role in the transcription regulation of lac operon. Using the proposed scheme, we have built a kinetic model that takes into account the reactions of hydrolysis, isomerization as well as galactosylation. Using the experimental data and optimization algorithms, all parameters of the model have been estimated. On the basis of the model, the features of beta-galactosidase functioning have been predicted in a wide range of conditions.

[A kinetic model of functioning and regulation of Escherichia coli isocitrate dehydrogenase].

A Rapid Equilibrium Random Bi Ter mechanism of formation of two dead-end complexes was proposed to describe the experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH). A kinetic model for the enzyme functioning was constructed, which assumes that it is regulated through reversible phosphorylation by its kinase/phosphatase, which in turn is regulated by IDH substrates and central metabolites such as pyruvate (Pyr), 3-phosphoglycerate (3-PG), and AMP. It was shown using the model that increasing the concentration of these effectors results in an increase of the active part of IDH, thus leading to an increase in the Krebs cycle flux. We predict that the ratio of the phosphorylated and free forms of IDH (IDHP/IDH) is more sensitive to AMP, NADPH, and isocitrate concentrations than to Pyr and 3-PG. The model allows a realistic prediction of changes in the IDHP/IDH ratio, which would occur under changes of biosynthetic and energetic loading of the E. coli cell.

[Kinetic modeling of energy metabolism and generation of active forms of oxygen in hepatocyte mitochondria]. / Kineticheskoe modelirovaniie énergeticheskogo metabolizma i generatsii aktivnyhkh form kisloroda v mitokhondriiakh gepatotsita.

Direct nonenzymatic oxidation of semiquinone by oxygen is one of the main sources of superoxide radicals (O2.-) in mitochondria. By using all the known data on hepatocyte mitochondria, we have revealed the correlation between the rate of superoxide generation by the bc1 complex and the transmembrane potential (delta psi). If the main electrogenic stage of the Q cycle is suggested to be the electron transfer between the cytochrome b hemes, then the rate of superoxide generation sharply increases when delta psi grows from 150 mV to 180 mV. However, this interrelation is ambiguous. Indeed, the increase of the generation rate with the growth of the potential can occur faster when succinate dehydrogenase is inhibited by malonate than when external ADP is exhausted. When the potential is changed by adding phosphate or potassium (K+), the rate of O2.- production remains constant, although the comparison of the rate values at the same delta psi reveals the effect of phosphate or potassium. It turned out that the rate of O2.- generation is a function of delta mu H rather than any of its components. Phosphate and K+ have practically no influence on delta mu H, since the change in delta psi is compensated by delta pH. The rate of superoxide generation by the bc1 complex is a multiple function of the electron-transfer activity of enzymes, the processes determining the membrane potential (e.g., loading), and of the oxygen concentration. The kinetic model proposed in this work may serve a tool to understand how the superoxide production is regulated.

[Effect of Ca2+ ions on the activity of pyruvate dehydrogenase complex in ascites Ehrlich carcinoma cells]. / Vliianie ionov Ca2+ na aktivnost' piruvatdegidrogenaznogo kompleksa v kletkakh astsitnoi kartsinomy Erlikha.

The effect of [Ca2+] at saturating concentrations of other substrates on the activity of pyruvate dehydrogenase complex (PDC) from Ehrlich ascite carcinoma cells was studied. The effect is biphasic (bell-shaped) and maximum of PDC activity is at 600 nM [Ca2+]. The experimental curve is in agreement with theoretical plot constructed by analysis of PDC kinetic model. The kinetic model considers the data on the structural and functional relationships between PDC components including pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2), dihydrolipoyl dehydrogenase (E3), and protein X. This model can be used for analysis of experimental activation by low Ca2+ concentrations and inhibition by increasing Ca2+ concentrations and can predict changes in the system caused by changes in the substrate concentrations.