Cooperative binding of substrates to transketolase from Saccharomyces cerevisiae.

Catalytic activity of two active sites of transketolase and their affinity towards the substrates (xylulose-5-phosphate and ribose-5-phosphate) has been studied in the presence of Ca2+ and Mg2+. In the presence of Ca2+, the active sites exhibit negative cooperativity in binding both xylulose-5-phosphate (donor substrate) and ribose-5-phosphate (acceptor substrate) and positive cooperativity in the catalytic transformation of the substrates. In the presence of Mg2+, nonequivalence of the active sites is not observed.

The changes in the energy metabolism of human muscle induced by training.

The biochemical effects of training programmes have been studied with a kinetic model of central metabolism, using enzyme activities and metabolite concentrations measured at rest and after 30 s maximum-intensity exercise, collected before and after long and short periods of training, which differed only by the duration of the rest intervals. After short periods of training the glycolytic flux at rest was three times higher than it had been before training, whereas during exercise the flux and energy consumption remained the same as before training. Long periods of training had less effect on the glycolytic flux at rest, but increased it in response to exercise, increasing the contribution of oxidative phosphorylation.

[Elaboration and introduction of an algorithm of rendering the anesthetic-and-resuscitation care to a big number of wounded person in terrorist attacks]. / Razrabotka i vnedrenie algoritma okazaniia anesteziologo-reanimatsionnoi pomoshchi ranenym pri terroristicheskikh aktakh.

Medical records of 320 persons, who were injured in terrorist attacks in 1999-2003, were analyzed. The main errors in rendering the anesthetic-and-resuscitation care were pointed out. An algorithm of actions is elaborated for the anesthesiologist working at the reception, surgical and intensive care departments of a hospital. Owing to the mentioned algorithm, the lethality rate of wounded persons dropped from 18 to 10%.

Acceptor substrate inhibits transketolase competitively with respect to donor substrate.

Two substrates of the transketolase reaction are known to bind with the enzyme according to a ping-pong mechanism [1]. It is shown in this work that high concentrations of ribose-5-phosphate (acceptor substrate) compete with xylulose-5-phosphate (donor substrate), suppressing the transketolase activity (Ki = 3.8 mM). However, interacting with the donor-substrate binding site on the protein molecule, the acceptor substrate, unlike the donor substrate, does not cause any change in the active site of the enzyme. The data are interesting in terms of studying the regulatory mechanism of the transketolase activity and the structure of the enzyme-substrate complex.

Kinetic investigation of cooperativity in coenzyme binding by transketolase active sites.

The two-step mechanism of coenzyme (thiamine diphosphate, ThDP) binding with two initially identical active sites of apotransketolase has been examined with a kinetic model. Cooperativity between sites in the primary ThDP binding and in the following conformational transition has been analyzed. The only reliable difference between sites is shown to be the tenfold difference in the backward rate constants of the conformational transition; this means that the cooperative interaction between sites takes place only after termination of both steps of ThDP binding in both sites.

A model of mitochondrial Ca(2+)-induced Ca2+ release simulating the Ca2+ oscillations and spikes generated by mitochondria.

Recent evidence underlines a key role of mitochondrial Ca2+ fluxes in cell Ca2+ signalling. We present here a kinetic model simulating the Ca2+ fluxes generated by mitochondria during mitochondrial Ca(2+)-induced Ca2+ release (mCICR) resulting from the operation of the permeability transition pore (PTP). Our model connects the Ca2+ fluxes through the ruthenium redsensitive Ca2+ uniporter, the respiration-dependent and passive H+ fluxes, the rate of oxygen consumption, the movements of weak acids across the mitochondrial membrane, the electrical transmembrane potential (delta psi), and operation of the PTP. We find that two factors are crucial to account for the various mCICR profiles that can be observed experimentally: (i) the dependence of PTP opening and closure on matrix pH (pHi), and (ii) the relative inhibition of the respiratory rate consecutive to PTP opening. The resulting model can simulate irreversible Ca2+ efflux from mitochondria, as well as the genesis of damped or sustained Ca2+ oscillations, and of single Ca2+ spikes. The model also simulates the main features of mCICR, i.e. the threshold-dependence of mCICR triggering, and the all-or-nothing nature of mCICR operation. Our model should appear useful to further mathematically address the consequences of mCICR on the spatiotemporal organisation of Ca2+ signals, as a 'plug-in' module for the existing models of cell Ca2+ signalling.

Kinetic mechanism of active site non-equivalence in transketolase.

The two-step mechanism of coenzyme (TDP) binding to apotransketolase has been examined by kinetic modeling, and the rate and equilibrium constants for each binding step for two active sites have been determined. The dissociation constants for the primary fast binding step and the forward rate constants for the secondary slow binding step have been shown to be similar for two active sites. The backward rate constants for the secondary binding step are different for two active sites, providing the kinetic mechanism of their non-equivalence in TDP binding.

Determination of constants of substrate primary binding with baker's yeast transketolase by kinetic modelling.

A kinetic model of bisubstrate reaction catalyzed by baker's yeast transketolase is proposed. The model considers individual stages of substrates reversible primary binding. The model corresponds to the observed kinetics of product accumulation within a wide range of initial substrate concentrations. Kinetic parameters for the best simulation of the experimental data are defined. The equilibrium constants of the primary binding of both the initial and produced ketose and also the initial aldose were unequivocally determined by varying the initial substrate concentrations. The dissociation constants of the primary enzyme-substrate complex for the initial ketose (xylulose 5-phosphate) and the reaction product (sedoheptulose 7-phosphate) were found to differ by more than by two orders of magnitude. The result is discussed in the context of the hypothesis of flip-flop functioning of the transketolase active sites.

[Analysis and interaction of myosin active centers during a mechanochemical cycle]. / Analiz vzaimodeistviia aktivnykh tsentrov miozina v protsesse mekhanokhimicheskogo tsikla.

A model of myosin complex with two active sites is considered. Each of the sites is supposed to pass through four states during a cycle of ATP hydrolysis. The model describes the distribution of the complex between states and the ATP hydrolysis rate as dependent on ATP concentration and reaction constants. Some of the constants were determined experimentally [6], but these values reproduce experimental curves [5] only with the assumption of cooperativity. If the first site is bind to actin, the rate of binding for the second one is shown to increase tenfold. If one of the sites is bind to actin by a "pulling" bridge, and the second site is in "rigor" state, then the ATP binding for the second site is about ten times faster. The transition into the rigor state proceeds much faster if both sites form pulling bridges. The rate of binding to actin is the same for one and for two sites.

Activity oscillations predicted for pyruvate dehydrogenase complexes.

A kinetic model for the pyruvate dehydrogenase complex is analyzed. The model takes into account intermediate channeling through the lipoyl network attached to the complex core, as well as inter-related regulatory effects of protein X acetylation and enzyme phosphorylation. The model predicts undamped oscillations of enzyme activity.

Unusual kinetic behavior predicted for alpha-keto acid dehydrogenase complexes.

A novel regulation type, which may be observed as an unusual kinetic 'cooperativity', is predicted for the alpha-keto acid dehydrogenase complexes. The inter-relationship of this regulation with the well-known regulatory effect of enzyme phosphorylation is discussed.

[Mathematical model of oscillations in mammalian erythrocytes. The role of calmodulin-dependent Ca-activated Ca-pump]. / Matematicheskaia model' kolebanii v éritrotsitakh mlekopitaiushchikh.Rol' kal'modulinzavisimogo Ca-aktiviruemogo Ca-nasosa.

A kinetic model of ionophore-induced oscillation of ion fluxes in mammalian erythrocytes is proposed. The model is based on the regulation by Ca2(+)-ions and calmodulin of (Ca2(+) + Mg2+)-dependent ATPase of erythrocytes. The results obtained are in a good agreement with the experimental data available.

Graph-theoretic approach to metabolic pathways.

A graph-theoretic approach is shown to be applicable within the framework of the metabolic control analysis. Kinetic differential equations linearized near a steady state are presented as kinetic graphs (schemes), their structure being correlated with kinetic properties of corresponding metabolic networks. The global properties may be expressed in terms of the local properties for steady states of metabolic systems. Instability, bistability, and concentrational oscillations are shown to be induced by specific graph fragments. The approach is illustrated by an example of systems showing the oscillatory kinetic behaviour.

[A study of the effect of radiation on megakaryocytopoiesis using a mathematical model]. / Issledovanie deistviia radiatsii na megakariotsitopoéz s pomoshch'iu matematicheskoi modeli.

Mathematical modelling used in analysing the postirradiation changes in megakaryocytopoiesis permitted to determine the level of radiation-induced injury in each experiment conducted and to show that megakaryocytopoiesis regulation followed the same mechanism after irradiation as it does normally and after the effect of hydroxyurea and anti-thrombocyte serum. The analysis has demonstrated that after the stem cell death induced by ionizing radiation, the regeneration can be provided by the committed cells, and the level of regeneration is determined by the maturity of precursors.

[Megakaryocytopoiesis studied in a mathematical model. 2. Regulation of cell differentiation following damage to part of the proliferating bone marrow pool by hydroxyurea]. / Izuchenie megakariotsitopoéza s pomoshch'iu matematicheskoi modeli. 2. Reguliatsiia differentsirovki kletok posle porazheniia chasti proliferiruiushchego pula kostnogo mozga oksimochevinoi.

Analysis of the earlier obtained data on the effect of hydroxyurea on megakaryocytopoiesis by a simulation model has shown that differentiation of proliferating precursors of the megakaryocytes depends on their amount in bone marrow; the time of their involvement in the proliferating pool increases if the number of proliferating cells decreases. Within 24 h after the treatment with hydroxyurea (900 mg/kg), the transit time for the mouse megakaryocytes is reduced by about 14 h due to acceleration of the latest developmental stages. A hypothesis is put forward which accounts for correlation between the duration of proliferative period and the volume and maturation time of megakaryocytes.

[Megakaryocytopoiesis studied in a mathematical model. 3. Regulation of cell differentiation in immune thrombocytopenia]. / Izuchenie megakariotsitopoéza s pomoshch'iu matematicheskoi modeli. 3. Reguliatsiia differentsirovki kletok pri immunnoi trombotsitopenii.

Simulation experiments have shown that just after antiplatelet serum of rabbit blood is introduced into mice the transit time of early proliferating precursors of the megakaryocytes is reduced by about 17 h, the transit time for megakaryoblasts increases, and within 24 h after the treatment the transit time for mature megakaryocytes is reduced b about 17 h. The data obtained confirmed our earlier hypothesis on correlation between the duration of proliferative period and the mean size of megakaryocytes.

[Megakaryocytopoiesis studied in a mathematical model. 4. Regulation of cell flow in a transit population]. / Izuchenie megakariotsitopoéza s pomoshch'iu matematicheskoi modeli. 4. Reguliatsiia potoka kletok v tranzitnoi populiatsii.

The role of cells capable of formation of megakaryocytic colonies (CFUm) in regulation of thrombocytopoiesis was studied using a simulation model of megakaryocytopoiesis. The CFUm were shown to be the least differentiated cells involved in regulation of megakaryocytopoiesis in mice upon immune thrombocytopenia and after intravenous injection of hydroxyurea. A correlation was found between the CFUm population productivity and the number of cells in a transit population of the megakaryocytic line which makes it possible to reproduce experimental kinetics in the model. The duration of cell development from CFUm to megakaryocytes is about 3 days.