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The kinetics of 3H-labeled arachidonic acid (AA, 10(-10)-10(-5) M) incorporation into murine peritoneal macrophages was investigated. During the incorporation of AA into the cells, the steady state was reached at 10 h. The level of incorporation consisted of 48-50% for nanomolar concentrations and 28-30% for micromolar concentrations of AA. Exogenous AA in micromolar but not nanomolar concentrations stimulated [3H]AA release from intracellular stores of pre-labeled cells. A mathematical model fitting the behavior of the experimental system is proposed. The difference in the level of uptake of AA in nanomolar and micromolar concentrations is explained by the activation of AA release from intracellular stores at high concentrations of exogenous AA.

Prostaglandin H synthase was isolated in the form of microsomes from sheep vesicular glands and immobilized on silica gel. This system of prostaglandin synthesis was activated by calcium ions and stabilized by adrenaline. Microsomes immobilized in the presence of adrenaline and calcium ions were stable upon storage at 4 degrees C. After two months, their activity was 80% of the initial activity. Immobilized microsomes were able to catalyze several cycles of prostaglandin E2 synthesis with no substantial loss of activity: after eight utilization cycles, they retained 66% of their initial enzymic activity.

This chapter describes biosensors for characterization and correction of thrombosis and hemostasis. A large group of cardio-vascular pathologies are related to the platelets aggregation. This process in the blood system is much defined by the synthesis of two antagonistic prostanoids: thromboxane (T X A 2 ) and prostacyclines (PGI). The precursors of prostanoids are polyunsaturated C-20 fatty acids. It is showed that in myocardial infarction, the blood plasma is far more enriched with arachidonic (C-20:4) acid compared to (C-20:3) and (C-20:5) acids. The level and ratio of C-20 polyunsaturated fatty acids in human organism are much dependent on the diet. A proper diet can notably correct the platelet aggregation processes. In view of this, it proves important to solve the problems of: (1) quantifying C-20 polyunsaturated fatty acids in human blood plasma; (2) quantifying C-20 polyunsaturated fatty acids in foods; (3) discriminating between C-20:3, C-20:4, and C-20:5 acids. To this end, two biosensors have been devised that enable to determine the range of C-20 polyansaturated fatty acids and to quantify selectively C-20:3 acid. These biosensors are based on application of prostaglandin-Hsynthetase from vesicular glands of animals as well as of vegetal lipooxygenase.

The properties of prostaglandin H synthase (PGHS) as a protein and as a rate-limiting enzyme of physiologically active prostanoid synthesis have been considered. Fast and dramatic changes in the protein structure were shown to accompany inactivation of PGHS in the course of the reaction. The conclusions derived from the study of the steady-state kinetics of the enzyme action are discussed, and a minimal kinetic scheme is proposed. The mechanism of substrate-induced inactivation of PGHS is suggested as a basis for the regulation at the prostanoid level. The mechanism is hypothesized to be a new regulatory mechanism of enzyme activity in biological systems.

The authors examined the ratios of blood free polyunsaturated fatty acids (PUFA), such as 20:3n6, 20:4n6, 20:5n3, and 22:6n3, which are substrates and inhibitors of synthesis of thromboxane A2 and prostacyclins that regulate both normal blood fluidity, and platelet adhesion and primary thrombogenesis. The object of the study was plasma from healthy subjects and 4 groups of patients with cardiovascular diseases: 1) large myocardial infarction; 2) resting and exercise-induced angina pectoris; 3) large myocardial infarction; and 4) recurrent myocardial infarction. The levels of plasma free PUFA were measured by gas chromatography. Assessment of the PUFA ratios indicated that the risk for thrombogenesis increased in large and recurrent myocardial infarctions as compared to small myocardial infarction and angina pectoris both by reducing the relative levels of 20:3n6 and, in particular, 20:5n3, substrates of synthesis of only thrombolytics and vasodilators and by more greatly inhibiting the synthesis of prostacyclins than thromboxane with elevated 22:6n3 levels.

It was shown that the ability of sheep and horse haptoglobins differing in their immunological properties to inhibit PGH synthetase is about the same. It was found that haptoglobin inhibits the PGH synthetase-catalyzed enzymatic reaction, the inhibiting effect being non-competitive with respect to the electron donor, adrenaline. The degree of PGH synthetase inhibition by haptoglobin depends on the glycoprotein concentration, incubation time and enzyme activity.

Benzofurocaine similarly to ortophen (voltaren) causes dose-dependent suppression of acute inflammatory reactions induced in rats by carragheenin, serotonin and histamine, protects animals from absolutely lethal dose of cellulose sulphate. When administered in a dose of 0.79 X 10(-3) M, benzofurocaine exerts no effect on the activity of prostaglandin synthetase.

1. α-Chymotrypsin (CT), enclosed in positively and negatively charged polyelectroiyte gels based on methacrylate, with a preservation of high catalytic activity, was produced. 2. The pH profile of the enzymatic activity of CT enclosed in gel is shifted in absolute magnitude by approximately 1 pH unit, while the sign of the shift agrees with the theory. 3. The incorporation of CT into a negatively charged gel leads to a sharp (600-fold) increase in its stability with respect to monomolecular thermal inactivation. The stabilization is due to the influence of the polymer matrix on the protein globule of the enzyme.

Prostaglandin H synthetase solubilized in octane with the aid of hydrated reversed micelles of Aerosol OT (AOT) exhibits a catalytic activity dependent on hydration of the surface-active substance and its concentration. The maximum rate of the reaction is attained at the H2O/AOT molar ratio equal to 20 and amounts to a value close to the one observed in an aqueous solution. The inactivation rate of the enzyme in the course of the reaction does not depend on the water content in the system and is described by a Kin value commensurable with the Kin in an aqueous solution.

The kinetic regularities of the prostaglandin endoperoxide synthetase in human platelets microsomes were studied. It was shown that at low equilibrium of the reaction the reaction product yield linearly depends on the enzyme concentration. The value of the inactivation rate constant is not dependent on the enzyme concentration, is not changed within the pH range of 6-9 and is markedly increased with a rise in hemin concentration. The kinetics of thermoinactivation of the enzyme at 4 degrees and 32 degrees was studied. It was concluded that inactivation occurs via intermediate enzyme-substrate complexes. The enzyme is also inactivated in the presence of hemin and oxygen. The latter process is eliminated in the presence of an electron donor (epinephrine) in a medium.

Partially purified preparations of prostaglandin endoperoxide-synthetase (PGH-synthetase) and thromboxane synthetase (PGH-convertase) were obtained from human platelets by ion exchange chromatography. The kinetics of prostaglandin H2 enzymatic conversion was studied in the presence of thromboxane synthetase from human platelets. It was found that prostaglandin H2 conversion into both thromboxane A2 and malonic dialdehyde is a catalytic process, i. e., its rate increases as the protein concentration rises. The linear dependence of the enzymatic reaction velocity on substrate concentration at a prostaglandin H2 concentrations below 10-15 microM was demonstrated. PGH-synthetase and PGH-convertase from human platelets exhibit similar enzymatic activity dependence on pH and temperature, PGH-convertase being more thermostable.

A kinetic scheme of the prostacyclin-thromboxane system has been evolved on the basis of the authors experimental data and the results described elsewhere. The kinetic behavior of the model has been analysed with the aid of computer technology by varying the following parameters: phospholipase activities, free arachidonic acid exchange rates between platelets and endothelium, PGH-synthetase biosynthesis rates, velocities of arachidonic acid pathways other than the cyclooxygenase ones. It has been demonstrated that the biological system is capable of sustaining prostacyclin and thromboxane concentrations at steady fixed levels within a wide range of kinetic parameters.

The dynamic replies of the multienzyme system of blood prostanoid synthesis to the introduction of an irreversible inhibitor of prostaglandin H synthetase (PGH synthetase) have been analysed by using kinetic modelling. The alterations of arachidonic acid and PGH synthetase concentrations in platelets and endothelium and the concentrations of thromboxane and prostacyclin have been demonstrated. Particularities of kinetic behaviour of the system probably providing the therapeutic effect of non-steroidal anti-inflammatory drugs have been shown. Namely, the kinetic wave of free arachidonic acid and prostacyclin concentration with respect to thromboxane concentration appears after introduction of the drugs.

The quantitative study of the processes that accompany nonenzymatic degradation of prostaglandin H2 has been carried out. The thiobarbituric acid test which shows the content of malonic dialdehyde in the reaction mixture has been used to study kinetics of the degradation. The apparent rate constants of this process have been pH-independent over the pH-range 5,5-9,5, and the calculated conversion halftime changes from 5,8 to 3,6 min at these pH values. Thromboxane synthetase from human platelets has been chosen to demonstrate the possibility of application of thiobarbituric acid test for determination of the activity of prostaglandin endoperoxide convertase. It has been shown that the apparent rate constant of the reaction in the presence of the enzyme is the linear function of its concentration.

A photocolorimetric method has been employed for kinetic investigation of aggregation of human platelets under the effects of arachidonic acid (AA) and the products of its metabolism in the platelets. The platelet aggregation was induced by AA and prostaglandin (PG)H2, the precursors of thromboxane (Tx). The initial rate of aggregation depended on AA and PGH2, concentrations. The nature of the influence of selective inhibitors of PGH-synthetase (indomethacin) and PGH-convertase (imidazole) on the rate of aggregation is suggestive of the rate-limiting type of enzymatic reactions of AA conversion into TxA2. Addition to the platelet suspension of solubilized PGH-convertase accelerates the PGH2-induced aggregation. Analysis of platelet aggregation processes induced by AA and PGH2 demonstrated that the enzymatic synthesis of TxA2 is one of the rate-limiting steps within the framework of molecular modifications responsible for the platelet aggregate formation.

A kinetic model has been suggested for the thromboxane synthesis polyenzyme system, taking into account the inactivation of a limiting enzyme, prostaglandin H-synthetase, in the course of the reaction. The model also includes the effect of phospholipase, adenylate cyclase, as well as the "outflows" of the components from the system, and basic regulatory effects in the system. A mathematical description of the model is given, and numerical solutions of the system of equations obtained for a wide variety of parameters. An analysis of the kinetic responses of the polyenzyme system shows that: 1) the system is highly conserved with respect to thromboxane concentration changes; 2) two cardinally different modes of the system are registered; they correspond to two insignificantly differing thromboxane steady-state levels; 3) transition of the system from one mode to the other is controlled by the phospholipase activity level which is sensitive to a change in the concentration of the regulator (cyclo-AMP or Ca ions) in the system. It is presumed that the two thromboxane steady-state levels are of physiological significance.

Enzymatic preparations of prostaglandin synthetase were immobilized by various methods, i.e., covalent binding to the carrier, incorporation into the matrix of polyacrylamide gel and adsorption on ion-exchangers. The most representative results were obtained with the use of the absorption technique. In this case, the enzyme activity either decreased very insignificantly or remained unaffected. The stability of immobilized enzyme preparations was studied. Some kinetic characteristics of immobilized prostaglandin synthetase were obtained.

The effect of protohemin IX and its modified analogs (monomethyl ester, dimethyl ester, as well as monoamides with Val-Phe-OCH3 or Leu-His-OCH3) has been examined on the activity of prostaglandin endoperoxide synthetase from sheep vesicular glands (PGH-synthetase, EC 1.14.99.1, isolated as apoenzyme). For holoenzymes having the above compounds as prosthetic groups, the dissociation constants, relative activities and the apparent inactivation constants in the course of the reaction have been determined. The effect of Tween 20 on the indicated parameters for holoenzymes with protohemin IX and its mono- and dimethyl esters has been studied. Modification of one of the two carboxylic groups of protohemin IX markedly increases the dissociation constant for the respective holoenzyme and virtually does not affect catalytic activity. Modification of both carboxylic groups of protohemin IX hinder the binding with the apoenzyme and strongly reduces the catalytic activity of the holoenzyme.

A study was made of the character and kinetics of interaction of acetylsalicylic acid and indomethacin with endoperoxide prostaglandin synthetase (PGH-synthetase) of sheep vesicular glands and human platelets. Enzymatic activity of PGH-synthetase was determined polarographically with the aid of Clark's electrodes. Acetylsalicylic acid was found to inhibit PGH-synthetase of sheep vesicular glands and human platelets at concentrations of the order of 1 x 10(-6) and 1 x 10(-4) M, whereas indomethacin at concentrations of 1 x 10(-6) and 1 x 10(-7) M, respectively. Acetylsalicylic acid inhibited PGH-synthetase from sheep vesicular glands and that from human platelets at an equal rate. Indomethacin inhibited the enzyme from sheep vesicular glands to a higher degree. Indomethacin reversibly interacted with PGH-synthetase. Meanwhile acetylsalicylic acid inhibited this enzyme irreversibly.

The adsorption of inorganic pyrophosphatase from baker's yeast by alkylated polysaccharides involves both electrostatic and hydrophobic interactions. The enzyme is eluted by NaCl solutions of different ionic strengths depending on the adsorbent hydrophobicity. The degree of purification on different adsorbents varies from 2- to 80-fold.

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 comparison of peptide maps of sheep foetus haptoglobin (Hp) phenotypes revealed a significant difference in 6 peptides. The map of multicomponent Hp2 shows the presence of a group of neutral peptides, which was absent in the case of a single-component Hp1. This difference is probably due to the presence of the alpha-subunit in Hp2.

A combined effect on the irreversible inhibitor aspirin and fast reversible inhibitors ibuprofen, naproxen and sodium salicylate on prostaglandin-H-synthetase was studied on microsomal fractions from ram vesicular glands. The fast reversible inhibitors were shown to bind to prostaglandin-H-synthetase at the same site as aspirin and thereby to protect the enzyme against irreversible inactivation by aspirin.

The influence of some reagents modifying NH2-, SH-groups or imidazole moiety, on the prostaglandin endoperoxide synthetase activity was studied. Acetaldehyde, pyridoxal phosphate, dithiobis (nitrobenzoic) acid and iodoacetamide were found not to affect the enzyme activity. The activity was abolished as a result of the interaction with p-chloromercuribenzoic acid and diethyl pyrocarbonate. The hemin completely protected the apo-enzyme against the inactivation with diethyl pyrocarbonate. The assumption about the presence of imidazole moiety in the active site of the enzyme was made.

The authors studied the pattern and mechanism of ibuprophen and naproxen interaction with endoperoxideprostaglandin synthetase (PGH synthetase) of sheep vesicular glands. The enzymatic activity of PGH synthetase was determined polarographically with the aid of a Clark electrode. Ibuprophen and naproxen were found to inhibit completely PGH synthetase at concentrations of the order of 1 X 10(-5) M. As regards the mechanism of action both the drugs are competitive inhibitors of this enzyme with reference to arachidonic acid and incompetitive inhibitors with reference to adrenaline, an electron donor.

The effect of vinyl groups of protohemin IX on its cofactor properties with respect to prostaglandin H synthetase has been studied. It was shown that substitution of ethyl groups or a hydrogen for vinyl groups affects neither binding of the prosthetic group to the apoenzyme nor catalytic properties of holo-prostaglandin H synthetase. Replacement of vinyl groups with bulkier substituents (hydroxyethyl or acetyl groups) decreases holoenzyme stability and catalytic activity. By comparison of the cofactor properties of protoporphyrin and hematoporphyrin macrocycles with different central ions (Fe3+, Mn2+, 2H+ in the case of protoporphyrin, and Fe3+, Mg2+, Cd2+ and Cu2+ in the case of hematoporphyrin), the presence of Fe3+ ions was shown to be mandatory for prostaglandin H synthetase activity. It was demonstrated that the cofactor structure modifications do not affect the holo-prostaglandin H synthetase inactivation rate constant in a reaction.