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The kinetic mechanism of the interaction of nonsteroidal anti-inflammatory drugs (NSAIDs) with their main pharmacological target, prostaglandin H synthase (PGHS), has not yet been established. We showed that inhibition of PGHS-1 from sheep vesicular glands by naproxen (a representative of NSAIDs) demonstrates a non-competitive character with respect to arachidonic acid and cannot be described within a framework of the commonly used kinetic schemes. However, it can be described by taking into account the negative cooperativity of naproxen binding to the cyclooxygenase active sites of the PGHS-1 homodimer (the first naproxen molecule forms a more stable complex (K 1 = 0.1 µM) with the enzyme than the second naproxen molecule (K 2 = 9.2 µM)). An apparent non-competitive interaction of PGHS-1 with naproxen is due to slow dissociation of the enzyme-inhibitor complexes. The same experimental data could also be described using commonly accepted kinetic schemes, assuming that naproxen interacts was a mixture of two enzyme species with the inhibition constants K α = 0.05 µM and K β = 18.3 µM. Theoretical analysis and numerical calculations show that the phenomenon of kinetic convergence of these two models has a general nature: when K 2 >> K 1 , the kinetic patterns (for transient kinetics and equilibrium state) generated by the cooperative model could be described by a scheme assuming the presence of two enzyme forms with the inhibition constants K α = K 1 /2, K β = 2·K 2 . When K 2 << K 1 , the cooperative model can be presented as a scheme with two inhibitor molecules simultaneously binding to the enzyme with the observed inhibition constant K (K = K 1 ·K 2 ). The assumption on the heterogeneity of the enzyme preparation in relation to its affinity to the inhibitor can be used instead of the assumption on the negative cooperativity of the enzyme-inhibitor interactions for convenient and easy practical description of such phenomena in enzymology, biotechnology, pharmacology, and other fields of science.

Reaction mixture for PGHS (prostaglandin-H-synthase) is a two-phase system including micellar hydrophobic phase and hydrophilic aqueous phase. Reagents added to the mixture are distributed between phases, thus concentrations of reagents dissolved in phases can differ significantly from their overall contents. Using dynamic light scattering we found that the hydrophobic phase produced by tween-20 consists of micelles, which radius (4-5nm) does not depend on either tween-20 overall content (0.1%-1% v/v) or arachidonic acid (AA) addition (10-1000μM) or PGHS addition (1μM). Tween-20 overall content changing from 0.1% to 2% v/v dramatically affected COX kinetic, but accounting AA distribution between phases allowed us to estimate "true" parameters, independent of the tween-20 overall content and the concentration of another substrate: KM(Ox) equals 9.8μM O2 in the aqueous phase or 0.0074bar in the gaseous phase, KM(AA) equals 5400μM AA in the phase of tween-20 micelles and 5400/PμM AA in the aqueous phase (P is the distribution ratio for the AA between the aqueous phase and the hydrophobic phase (P≫1000)). This approach allowed to evaluate PS, the distribution ratio for the AA between the hydrophobic phase and the PGHS active center (PS ~310). This coefficient indicates the AA selectivity toward the cyclooxygenase active center., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Quantitative evaluation of the accuracy of the rapid equilibrium assumption in the steady-state enzyme kinetics was obtained for an arbitrary mechanism of an enzyme-catalyzed reaction. This evaluation depends only on the structure and properties of the equilibrium segment, but doesn't depend on the structure and properties of the rest (stationary part) of the kinetic scheme. The smaller the values of the edges leaving equilibrium segment in relation to values of the edges within the equilibrium segment, the higher the accuracy of determination of intermediate concentrations and reaction velocity in a case of the rapid equilibrium assumption.

With the use of a graph theory new relations for steady-state enzyme kinetics are derived and strictly proved for the arbitrary mechanism of an enzyme-catalysed reaction containing a reversible segment. Using these relations, a general principle for rapid equilibrium assumption is formulated and proved: the reversible bound segment can be considered as an equilibrium segment only when the values of the base trees that are not proper to this segment can be neglected (within a prescribed accuracy) in relation to the values of the base trees that belong to this segment. In contrast with the foreign base trees the base trees that are proper to the segment have the following properties: the tree that is directed to the base within this segment does not contain the edges leaving this segment; and the tree that is directed to the base outside the segment contains only one edge leaving this segment. Equilibrium variations are assessed for steady-state intermediates concentrations of the equilibrium segment, numerical expressions are obtained for the accuracy of determination of the intermediates concentrations as well as for the accuracy of determination of the rate of enzyme-catalysed reaction in case of using rapid equilibrium assumption.

The possible application of the quasi-equilibrium assumption for an arbitrary mechanism of enzymatic reaction is considered. It is shown at what ratios of kinetic constants a segment consisting of two, three, and four intermediates may be considered as an equilibrium one. Expressions for evaluation of accuracy of distribution of intermediate concentrations inside the equilibrium segment and accuracy of determination of intermediate concentrations inside and outside the equilibrium segment as a function of the ratio of kinetic constants are derived. A method for determination of the limitations on the ratio of rate constants for an equilibrium segment of arbitrary structure is suggested.

For the cyclooxygenase reaction of prostaglandin-H-synthase isolated from ram vesicular glands, dependences of the initial reaction rate, the maximal yield of the product, and the rate constant of enzyme inactivation in the course of reaction on oxygen concentration were studied in the absence and in the presence of electron donor in the reaction medium. It is shown that in the absence of electron donor the cyclooxygenase reaction is strictly governed by Michaelis-Menten kinetics over a wide range of oxygen concentrations (5-800 µM). In the presence of electron donor in the reaction medium it was found that cyclooxygenase reaction is inhibited by an excess of dissolved oxygen: the maximal values of the initial reaction rate and yield of the product are attained at oxygen concentration 50 µM, and its increase to 500 µM causes twofold decrease in the initial rate and maximal yield. The rate constant of enzyme inactivation in the course of reaction increases on increase in oxygen concentration both in the presence and in the absence of electron donor.

Application of the quasi-equilibrium assumption for the steady-state kinetics of bisubstrate irreversible enzymatic reactions in the case of ordered binding of substrates (Bi-Bi ordered mechanism) is considered. The necessary and sufficient conditions for application of the quasi-equilibrium assumption have been found and accuracy of this assumption has been numerically evaluated. The limitations on application of the quasi-equilibrium assumption have been shown and errors of its application have been analyzed. It is shown that possible discrimination of substrate binding order using asymmetrical expressions grounded on the quasi-equilibrium assumption is inconsistent because such asymmetrical expressions arise from incorrect application of the quasi-equilibrium assumption. Moreover, it has been proved in the general case that mechanisms generating such substrate-asymmetrical expressions for the steady-state rate of enzymatic reaction do not exist. The error source when using graphical interpretation for discrimination of mechanisms of bisubstrate enzymatic reactions has been determined. The strategy to avoid such errors is pointed out.

Steady-state kinetics of compulsory-ordered single-substrate irreversible and reversible enzyme reactions with two, three, and arbitrary number of intermediates were observed. Necessary and sufficient conditions for application of the quasi-equilibrium assumption and restrictions of this assumption were found in cases of two and three intermediates in the equilibrium segment. For all cases, accuracy of the quasi-equilibrium assumption was evaluated.

To study the interrelation between the spectral and structural properties of fluorescent proteins, structures of mutants of monomeric red fluorescent protein mRFP1 with all possible point mutations of Glu66 (except replacement by Pro) were simulated by molecular dynamics. A global search for correlations between geometrical structure parameters and some spectral characteristics (absorption maximum wavelength, integral extinction coefficient at the absorption maximum, excitation maximum wavelength, emission maximum wavelength, and quantum yield) was performed for the chromophore and its 6 A environment in mRFP1, Q66A, Q66L, Q66S, Q66C, Q66H, and Q66N. The correlation coefficients (0.81-0.87) were maximal for torsion angles in phenolic and imidazolidine rings as well as for torsion angles in the regions of connection between these rings and chromophore attachment to beta-barrel. The data can be used to predict the spectral properties of fluorescent proteins based on their structures and to reveal promising positions for directed mutagenesis.

Prostaglandin-H-synthase is a bifunctional enzyme catalyzing conversion of arachidonic acid into prostaglandin H2 as a result of cyclooxygenase and peroxidase reactions. The dependence of the rate of the cyclooxygenase reaction on oxygen concentration in the absence and in the presence of electron donor was determined. A two-dimensional kinetic scheme accounting for independent proceeding and mutual influence of the cyclooxygenase and peroxidase reactions and also for hierarchy of the rates of these reactions was used as a model. In the context of this model, it was shown that there are irreversible stages in the mechanism of the cyclooxygenase reaction between points of substrate donation (between donation of arachidonic acid and the first oxygen molecule and also between donation of two oxygen molecules).

A steady-state approximation of the generalized two-dimensional model of a bifunctional enzyme catalyzing independent proceeding of two one-pathway reactions is considered in a case of mutual influence of the active sites. Coexistence of fast and slow catalytic cycles in the reaction mechanism is analyzed. Conditions when the hierarchy of fast and slow catalytic cycles allows simplification of a two-dimensional model and its reduction to the one-dimensional cyclic schemes were determined. Kinetic equations describing these simplified schemes are presented.

Kinetic models of inactivation of cyclooxygenase and peroxidase activities of prostaglandin-H-synthase (PGHS) during cyclooxygenase and peroxidase reactions catalyzed by the enzyme and also on preincubation with H2O2 have been developed; these models account for data obtained by the authors as well as data from the literature. Being rather simple, these models simultaneously describe the processes of cyclooxygenase and peroxidase inactivation of PGHS, using the minimal set of experimental parameters.

Prostaglandin-H-synthase (PGHS) is a bifunctional enzyme catalyzing cyclooxygenase and peroxidase reactions and undergoing irreversible inactivation during catalysis. A new method for kinetic studies of both PGHS activities in the course of cyclooxygenase as well as peroxidase reactions and also preincubation with hydroperoxides is suggested. It is shown that peroxidase activity is retained after complete cyclooxygenase inactivation and cyclooxygenase activity is retained after complete peroxidase inactivation. Two-stage cyclooxygenase inactivation occurs on preincubation of PGHS with hydrogen peroxide. Studies on inactivation under various conditions indicate that chemical mechanisms of cyclooxygenase and peroxidase inactivation are different. The data allow development of kinetic models.

Prostaglandin-H-synthase (PGHS, EC 1.14.99.1) catalyzes the first committed step in biosynthesis of all prostaglandins, thromboxanes, and prostacyclins by converting arachidonic acid to prostaglandin H(2) (PGH(2)). PGHS exhibits two enzymatic activities: cyclooxygenase activity converting arachidonic acid to prostaglandin G(2) (PGG(2)) and peroxidase activity reducing the hydroperoxide PGG(2) to the corresponding alcohol, PGH(2). Despite the many investigations of the kinetics of PGHS, many features such as the absence of competition and mutual activation between the cyclooxygenase and peroxidase activities cannot be explained in terms of existing schemes. In this work we have studied the influence of different electron donors (N,N,N ,N -tetramethyl-p-phenylenediamine, L-epinephrine, 2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid), potassium ferrocyanide) on the PGHS activities. The proposed scheme describes independent but interconnected cyclooxygenase and peroxidase activities of PGHS. It also explains the experimental data obtained in the present work and known from the literature.

Topology of the neutral form of the DsRed fluorescent protein chromophore as a residue of [(4-cis)-2-[(1-cis)-4-amino-4-oxobutanimidoyl]-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1H-imidazol-1-yl]acetic acid was calculated with OPLS-AA force field. Use of this topology and molecular dynamics simulation allows calculating the parameters of proteins that contain such residue in their polypeptide chains. The chromophore parameters were obtained by ab initio (RHF/6-31G**) quantum chemical calculations applying density functional theory (B3LYP). Using this chromophore, we have calculated the molecular dynamics trajectory of tetrameric fluorescent protein DsRed in solution at 300 K (4 nsec). Correctness of the chromophore parametrization was revealed by comparison of quantitative characteristics of the chromophore structure obtained from the molecular dynamic simulations of DsRed protein with the quantitative characteristics of the chromophore based on the crystallographic X-ray data of fluorescent protein DsRed (PDB ID: 1ZGO, 1G7K, and 1GGX), and also with the quantitative characteristics of the chromophore obtained by quantum chemical calculations. Inclusion of the neutral form of DsRed protein chromophore topology into the OPLS-AA force field yielded the extended force field OPLS-AA/DsRed. This force field can be used for molecular dynamics calculations of proteins containing the DsRed chromophore. The parameter set presented in this study can be applied for similar extension in any other force fields.

A method of analysis for steady-state kinetic data has been developed that allows relationships between key partial reactions in the catalytic cycle of a functioning enzyme to be determined. The novel approach is based on a concept of scalar and vector 'kinetic connectivities' between enzyme intermediates in an arbitrary enzyme mechanism. The criterion for the agreement between experimental data and a proposed kinetic model is formulated as the kinetic connectivity of intermediate forms of the enzyme. This concept has advantages over conventional approaches and is better able to describe the complex kinetic behaviour of prostaglandin H synthase (PGHS) when catalysing the oxidation of adrenaline by H(2)O(2). To interpret the experimental data for PGHS, a generalized model for multi-substrate enzyme reactions was developed with provision for irreversible enzyme inactivation. This model showed that two enzyme intermediates must undergo inactivation during the catalytic cycle. These forms are proposed to be PGHS compound I and a compound I-adrenaline complex.

The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state of the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.

The red fluorescent protein, DsRed, recently cloned from coral Discosoma sp. has one of the longest fluorescence waves and one of the most complex absorbance spectra among the family of fluorescent proteins. In this work we found that with time DsRed fluorescence decreases under mildly acidic conditions (pH 4.0-4.8) in a pH-dependent manner, and this fluorescence inactivation could be partially recovered by subsequent re-alkalization. The DsRed absorbance and circular dichroism spectra under these conditions revealed that the fluorescence changes were caused by denaturation followed by partial renaturation of the protein. Further, analytical ultracentrifugation determined that native DsRed formed a tight tetramer under various native conditions. Quantitative analysis of the data showed that several distinct states of protein exist during the fluorescence inactivation and recovery, and the inactivation of fluorescence can be caused by protonation of a single ionogenic group in each monomer of DsRed tetramer.

The steady-state approximation of the generalized model of a bifunctional multisubstrate enzyme is considered. Cases when the reactions catalyzed by the bifunctional enzyme are independent of one another and when one reaction influences the other are considered. Shunting of the kinetic mechanisms and activation of one reaction by the second reaction catalyzed by the bifunctional enzyme are analyzed. The kinetic equations are derived and qualitatively analyzed. It is shown in all cases discussed that the functional relationships between the kinetic parameters observed and the concentrations of the substrates participating in the reactions can be unambiguously defined on the basis of the concept of relationship of the intermediate enzyme forms in the enzymatic process.

General schemes of unbranched multisubstrate enzyme reactions associated with enzyme inactivation during catalysis are analyzed. Equations of integral kinetics at constant substrate concentrations and at depletion in one of the substrate are presented. Experimental dose-response curve characterized by additivity of some enzyme intermediates (absorption spectra, fluorescence, EPR, etc.) are theoretically analyzed. Also rapid equilibrium at certain stages of enzyme reaction is considered. The interrelationship of enzyme intermediates is a criterion of the kinetic behavior of enzyme reaction mechanism. New coordinates are suggested for the analysis of the integral kinetics of self-inactivating enzymes. Results of the analysis are used for interpretation of the data on arachidonic cascade enzymes.

A number of substituted pyridylisoxazoles and their 4,5-dihydro analogs were synthesized by 1,3-dipolar cycloaddition of substituted nitrile oxides to either alkenes or alkynes. The synthesized compounds inhibit arachidonic acid-induced aggregation of human thrombocytes at concentrations of 10(-6) to 10(-3) M. Due to low toxicity, these compounds can be regarded as potential antithrombosis medicines.

The phenomenon of supercooperativity in platelet aggregation is manifested by the occurrence of clear-cut thresholds in dose-response relationships; in such cases the Hill coefficient has unusually high values. Approximation, by the Hill equation, of the relationship of the rate of arachidonate-induced platelet aggregation to the concentrations of either the inducer or inhibitors such as substituted pyridyl isoxazoles (synthesized by us), indomethacin, and pinane thromboxane A2, demonstrated that the Hill coefficients ranged from 30 to 100. 3-(3-Pyridyl)-5-phenylisoxazole, which exhibited maximal anti-aggregatory activity among the synthesized compounds, inhibited neither cyclooxygenase nor thromboxane synthase. The compounds affected the signal transduction pathway at/or posterior to the stage of thromboxane A2 reception.

Incubation of human platelet-rich plasma (PRP) or washed platelets with merthiolate (MT; sodium ethylmercurithiosalicylate; an inhibitor of lysophosphatide: arachidonoyl transferase) leads to irreversible platelet aggregation which is parallelled by an increase in thromboxane A2 synthesis. MT-induced aggregation is preceded by a pronounced lag-period (0.5-10 min). Duration of the latter is inversely related to the concentration of MT ([MT]). Platelet responses to MT are similar to those triggered by arachidonate (AA) in that the relationships of the aggregation rates both to [MT] and [AA] are threshold and exhibit characteristic super-high values of the apparent Hill coefficients (h > 30). A typical MT-induced response can be subdivided in two sequential phases: i) cyclooxygenase-independent slow aggregation, and ii) indomethacin-abrogated rapid aggregation. MT-induced responses are blocked by PGE1 or ajoene (which inhibits binding of fibrinogen to its cell surface receptor, GPIIb/IIIa). The obtained data are interpreted both quantitatively and qualitatively in terms of a model assuming the existence of: i) a relationship between the rate of MT-inhibitable AA incorporation into phospholipids and the concentration of intracellular free AA, [AA]i; ii) a certain threshold value of [AA]i essential for triggering the second phase of the aggregation.

Concentration-response relationships of human platelet aggregation rates were analyzed for a variety of agonists and inhibitors. Their approximation by the Hill equation showed that the values of the Hill coefficient (h) were agonist-dependent and increased as follows: hADP = hL-EPINEPHRINE = hPAF = hPGH2 = hU46619 less than hPMA less than hA23187 less than hMERTHIOLATE = hARACHIDONATE. The results were interpreted in terms of a model assuming varying degrees of cooperativity for each step of signal transduction involved in platelet aggregation. Super-high values of h (greater than 30) obtained with arachidonate and merthiolate, as well as in the case of inhibition of an arachidonate-induced response by indomethacin and PTA2, suggested that at least one region of signal transduction pathway leading to aggregation exhibited supercooperative properties.

Incubation of human platelets (in the form of platelet rich plasma or washed platelet suspension) with sodium merthiolate (ethyl mercuric salicylate inhibiting the arachidonic acid incorporation into phospholipids) induces their irreversible aggregation, which is accompanied by TxB2 synthesis. The merthiolate-induced aggregation has a lag-period of 0.5-10 min, whose magnitude is inversely correlated with the merthiolate concentration. The concentration dependencies of the rate of the merthiolate-induced and arachidonate-induced aggregation are threshold ones; the Hill coefficients are more than 30. The merthiolate-induced aggregation occurs in two phases: a slow phase which is independent of the arachidonic acid cyclooxygenase metabolism and a fast phase which is fully blocked by indomethacin. This aggregation is inhibited by PGE1 and ajoene (an inhibitor of the fibrinogen interaction with the fibrinogen receptor, GPIIb/IIIa). Quantitative and qualitative analyses of the experimental data were performed, using a model which took account of: (a) increase in the concentration of free endogenous arachidonic acid resulting from the inhibition by merthiolate of the arachidonic acid re-incorporation into phospholipids, and (b) existence of a threshold intracellular arachidonic acid concentration needed for the irreversible aggregation of platelets.

The author proposes a functional model of the cell as a set of the simplest functional blocks each of which consists of a cellular signal receptor, a system of signal transmission in the chain of secondary intermediaries and an element whose numerical value characterizes the cellular response. The steady-state approximation of the dependence of the cellular response on the concentration of an activator and inhibitors was analyzed quantitatively and qualitatively. Mathematic criteria for the existence were defined. Analysis was made of the properties of the maximal signal enhancement mode, of the signal enhancement lack mode, and of the intermediate and bifurcation modes. Equations were derived, connecting the experimentally observed parameters of the concentration dependencies with the parameters of the simplest functional blocks.

A study was made of a model of platelet aggregation in shear flow taking into account the kinetics of intercellular fibrinogen bond formation limited by the turning time of the doublet of collided platelets. The energy curve for two platelets was calculated. One fibrinogen bond was sufficient to form a doublet stable in the flow. The equation for the rate of single platelet disappearance with regard to the kinetics of intercellular fibrinogen bond formation, the stochastic character of bond distribution on the contacts of collided platelets, hydrodynamically controlled time of their interaction was derived. Approximation of the obtained dependencies for the platelet aggregation rate by Hill's equation was suggested and its parameters were determined. A qualitative criterion for kinetic behavior of the system of aggregating platelets was introduced.

A platelet aggregation model in shear flow taking into account the kinetics of intercellular fibrinogen bond formation limited by aggregated platelets rotation time was considered. For this consideration the average duration of platelets interaction in flow with shear rate value G is shown to be pi/4G. One fibrinogen bond is sufficient to form a solid aggregate between two platelets. The equation for single platelets disappearance rate concerned with intercellular fibrinogen bond formation, stochastic character of bond distribution in collided platelets and hydrodynamically controlled interaction time was obtained. The Hill's approximation for the obtained aggregation rate dependences was suggested and appropriate constants were determined. The qualitative criterion of platelets aggregating systems behavior was introduced.

The mechanism of inactivation of the electron-transport chain of chloroplasts within the framework of a previously described kinetic model based on two steps, i.e. activation of the electron-transport chain and its irreversible inactivation, was studied. It was found that activation results from a decrease in the transmembrane gradient of pH on the photosynthetic membrane during inactivation. The kinetics of irreversible inactivation of different parts of the electron-transport chain of chloroplasts were studied. The water decompositon system was shown to be the most labile part of the chain (at a point lying before the carrier accepting the electrons from 1,5-diphenylcarbazide). The inactivation kinetics of the electron-transport chain of chloroplasts under different environmental conditions were studied. The stabilizing effects of definite concentrations of hydrogens ions, glycerol and bovine serum albumin were demonstrated. The mechanisms of these effects are discussed. A 200-fold stabilization of the chloroplasts with respect to their irreversible inactivation due to optimization of the medium was achieved.

The influence of external factors, viz. reaction mixture temperature, presence of low- and high-molecular (tween-20) organic compounds, urea and the solution high ionic strength on kinetic properties of PGH synthetase has been studied. Some factors (high ionic strength, addition of organic compounds) were found to slow down irreversible inactivation of PGH synthetase in the course of the reaction. Sensitivity of the enzyme to the change of the solution ionic strength depends on environmental factors, rising on the microsomal enzyme solubilization with tween-20 and decreasing on further enhancement of the tween-20 concentration.

A method has been developed that makes it possible to obtain [5,6-3H2]PGE1 with a yield of 35% and a molar radioactivity of 1.7-1.8 TBq/mmol. The binding of [5,6-3H2]PGE1 to native platelets proved to be specific, saturating and reversible. It is characterized by low values (approximately 10(-9) M) of dissociation constants for high-affinity sites, correlates with the inhibition of ADP-induced aggregation of platelets and can be considered as receptor binding. Specific binding of 10 +/- 2 molecules of PGE1 with one platelet was found to cause 50% inhibition of the ADP-induced aggregation.

The effects of glutaric aldehyde on pea leave chloroplasts and their inactivation kinetics were studied. Optimization of the chloroplasts fixation by glutaric aldehyde resulted in a 5-fold increase of stability of the chloroplasts. Immobilization of the chloroplasts in agar-agar gels was performed; the ability of chloroplasts for photooxidation of H2O was thereby retained. Immobilization did not actually affect the stability of chloroplasts. The inactivation kinetics of fixed and immobilized chloroplasts are in good agreement with the previously described model for inactivation of native chloroplasts.

A generalized scheme of ordered multisubstrate enzymatic reactions under steady-state conditions is described. The equations for the reaction rate and product yield as well as the observed rate constant for the enzyme inactivation as functions of concentrations of substrates, reaction products, reversible and irreversible inhibitors were derived and analyzed. It was demonstrated that the kinetic behaviour of such systems can adequately be described in terms of interrelationship of intermediate forms of the enzyme.

Kinetic parameters of enzymatic and non-enzymatic transformations of [3H]prostaglandin H2 (PGH2) were determined; the maximum yield of [3H]PGD2 being obtained at the keobs/koobs ratio equal to 10. The two-stage enzymatic synthesis of [3H]PGD2 with high molar radioactivity (3.15 TBq/mmol) from [3H]arachidonic acid carried out. Its identity in properties to the natural PGD2 was shown in experiments on the inhibition of ADP-induced aggregation of thrombocytes and on enzymatic oxidation with 15-hydroxyprostaglandin dehydrogenase.