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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.

The role of serum fatty acid binding proteins (FABPs) in arachidonic acid (AA) uptake by murine peritoneal macrophages has been studied. The kinetics of [3H]arachidonic acid uptake by the cells was investigated over a wide range of AA concentration (10−10–10−5 M). It was shown that these putative fatty acid transporters dramatically change the uptake processes. In the presence of FABPs, the time-course curves of AA uptake exhibited two distinct periods: one with a rapid AA uptake during the first hour with an equilibrium in 1–2.5 h and another with an equilibrium reached in 20 h, whereas in the absence of FABPs the uptake curves were smooth without kinks and with the equilibrium reached in 10 h. In addition, it was shown that the amount of incorporated AA was linearly dependent on the concentration of AA over the range of 10−10–10−6 M in the presence of serum FABPs and 10−10–10−7 M in their absence. We assume that the changes in the character of AA uptake by macrophages in the presence of FABP soccur due to the interaction of FABPs with the cell plasma membrane.

The dependence of prostanoid synthesis on the nature of free arachidonic acid (AA) appearance was investigated in mouse peritoneal macrophages. AA delivery from intracellular sources to the constitutive prostaglandin (PG)H synthase was provided by action of calcium-ionophore A23187; and from extracellular sources by AA addition to the culture medium. It was found that the kinetics of prostanoid synthesis dramatically depends on the sources of AA. Free AA concentration used for prostanoid synthesis is either a constant or a variable value depending upon the sources. The kinetics of cellular prostanoid synthesis can be regulated by the following processes: (a) the irreversible inactivation of PGH-synthase in the course of the reaction (kin), (b) prostanoid metabolism (kr), and (c) incorporation of exogenous AA into cellular membranes (ka). From our experiments and mathematical calculation these parameters were found to be kin = 0.20 +/- 0.02 min-1, kr = 0.17 +/- 0.03 min-1 in the case of stimulation with A23187, and kin = 0.0156 min-1, kr = 0. 0134 min-1, ka = 0.0025 min-1 in the case of exogenous AA addition. The studies of prostanoid biosynthesis by macrophage microsomes led to independent determination of kin = 0.20 +/- 0.02 min-1. This value perfectly fits the kinetics of the prostanoid cell synthesis under endogenous AA supply but shows a 10-fold decrease in the case of exogenous AA supply. Our study on the kinetics of prostanoid synthesis by mouse peritoneal macrophages clearly demonstrate that AA is able to regulate cellular prostanoid synthesis in the presence of constitutive PGH-synthase only. A regulation mechanism based on the co-operation of the constitutive PGH-synthase isoform and the availability of free AA is proposed and could be confirmed by mathematical modelling.

To check the proposed hypothesis that the relative content of individual polyunsaturated fatty acids (PUFAs)--substrates and inhibitors of prostanoid synthesis--in plasma can be regarded as a quantitative risk factor of blood clotting, a test was conducted on free fatty acids content in blood plasma of healthy people (group 0) and patients with heart ischemia before (group 1) and after (group 2) they were treated for a month with a food additive called "Eiconol," enriched with PUFA omega 3. Different proportions of PUFAs have been calculated in all cases, accounting for the contribution of each acid to the process of primary clotting. Comparison of PUFA rations among the three groups showed significant differences of means between groups 0 and 1 and also group 1 and 2 for 6 out of 7 proposed coefficients, which disappeared after "Eiconol" treatment (comparison of groups 0 and 2). The results led to the conclusion that out of the proposed PUFA proportions, the coefficients describing the relative content of arachidonic acid in blood plasma may be the most informative for diagnosis and treatment efficiently in evaluation of heart and vascular diseases.

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 H2O2. 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 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.

The dynamics of prostaglandin (PG) E2 synthesis by mouse peritoneal macrophages during the delivery of the basic substrate, arachidonic acid (AA), from different sources to the enzyme system of the cells was investigated. The dynamics of PGE2 synthesis in these cells was studied both after addition of exogenous AA and after stimulating the liberation of AA from intracellular pools with the calcium ionophore A23187. The kinetics of PGE2 synthesis when AA was supplied from intracellular and extracellular sources were absolutely different. PGE2 metabolism and the inactivation of the key enzyme of PG synthesis (PGH-synthase) during the reaction may be the regulating factors in the kinetics of PGE2 synthesis in the cells. For the different sources of AA in the cells, the rate constants of PGE2 consumption (k2) and PGH-synthase inactivation in the course of the reaction (kin) were calculated. The experimentally determined value of the apparent rate constant kin was identical to the theoretically calculated kin value for the case when AA was provided from an intracellular source. An observed deceleration in the PGE2 synthesis kinetics from exogenous AA is characterized by a 10-fold drop in the apparent kin and k2 values. The possibility of prostanoid synthesis regulation at the level of the traditional, constitutive isoenzyme PGH-synthase-1 is discussed.

It was shown that cytosol of primary sheep vesicular gland cells inhibits peroxidase activity of prostaglandin H synthase (PGHS). The degree of the enzyme inactivation depends on cytosol concentration. It was established that cytosol contains glycoprotein haptoglobin that is one of the cytosol basic components responsible for its property to inhibit PGHS. Haptoglobin is supposed to participate in endogenous regulation of PGHS activity in sheep vesicular glands.

The changes in AA incorporation and release as well as prostanoid synthesis upon differentiation of human premonocytic cell line, U937, induced by three functionally diverse agents—phorbol ester (TPA), dimethyl sulfoxide (DMSO), and retinoic acid (RA) have been investigated. The rate of AA incorporation into the cells remained unchanged whereas a 3- to 6-fold increase in AA release upon stimulation with Ca 2+ -ionophore A23187 as compared to undifferentiated cells was observed. While undifferentiated cells were incapable to metabolise AA via the cyclooxygenase pathway all three types of differentiated U937 cells produced TxB 2 and PGE 2 . Only TPA-differentiated cells responded with a 6-fold increase of prostanoid synthesis after A23187 stimulation, whereas in DMSO-differentiated cells prostanoid synthesis was slightly stimulated by A23187 and in RA-differentiated cells it was not stimulated at all. Thus, agonist-induced prostanoid synthesis in differentiated cells is dependent on the nature of differentiating agent and does not correlate with AA liberation.

The interest in the prostaglandin (PG) synthesis by animal cells today grows steadily because of the difficulties in obtaining them by any other way. Murine peritoneal macrophages can under certain conditions synthesize large amounts of PGs. The effect of well-known nonsteroidal anti-inflammatory drug ibuprofen on PG synthesis by the cells using a high-performance liquid chromatography (HPLC) method with fluorescence detection of 4-bromomethyl-7-methoxy-coumarin (BrMMC) derivatives was studied. In our case, the main metabolites were PGE2 and PGF2a. The PG synthesis activation effect was shown by ibuprofen concentrations in the 10(-10)-10(-14) M range with the maximum effect of 10(-12)M. In this case, the ibuprofen effect was comparable in value with the effect of well-known cell PG synthesis activator--calcium ionophore A23187. Although the exact mechanism of such an effect is not clear at the moment, at low concentration, ibuprofen itself is able to activate PG synthesis in murine peritoneal macrophages.

The effect of 10 −14 − 10 −4 M ibuprofen and aspirin both on arachidonic acid metabolism in peritoneal murine macrophages and on the concanavalin A-induced proliferation of murine splenocytes were investigated. It was shown that 10 −7 − 10 −4 M ibuprofen inhibits the arachidonic acid metabolism. On the other hand, 10 −12 −10 −11 M ibuprofen causes pronounced activation of arachidonic acid metabolism. The low concentration (10 −14 − 10 −10 M) effects also take place when non-steroidal anti-inflammatory drugs influence other functions of the immune system: that is, they activate the splenocyte mitogen-induced proliferative response. These results are in accord with our suggestion that the low concentration effects of these drugs do not depend upon cell types and may have an important physiological significance.

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.

Prostaglandin H synthase (PGHS) as apo- and holoenzyme and the enzyme inactivated during the conversion of arachidonic acid into prostaglandin H2 has been modified by diethyl pyrocarbonate (DEPC). DEPC (40 mol/l mol protein) rapidly, but quantitatively differently interacted with the three forms of the enzyme (pH 6.0, 25 degrees C). The exhausted reaction with DEPC corresponded to modification of seven histidine residues in apo-PGHS and four residues in holo-PGHS. All of the 18 histidine residues were available for modification in the enzyme inactivated during the catalysis. The modification of apo-PGHS was accompanied by a concerted loss of the combined cyclooxygenase plus peroxidase and peroxidase activities. The velocities of the tryptic cleavage of the three forms of the enzyme into the 33 and 38 kDa polypeptides were essentially different, but the modification of each enzyme form did not affect the velocity of its cleavage. Two of the three histidine residues essential for the interaction with the heme within the 38 kDa fragment might be His-309 and His-388. Based on the comparison of availability for the reaction with DEPC of all the 18 histidine residues in the enzyme molecule inactivated by the interaction with arachidonic acid and on the abnormally high velocity of the tryptic cleavage of this form of PGHS, a hypothesis has been put forward about the fast and dramatic changes in the protein structure in the course of catalysis.

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.

It was shown that sheep vesicular gland cytosol inhibits the peroxidase activity of prostaglandin H synthetase (PGHS). The degree of enzyme inactivation depends on cytosol concentration and incubation time. It was found that cytosol contains a glycoprotein, haptoglobin, which is one of the cytosolic basic components responsible for its ability to inhibit PGHS. Haptoglobin is supposed to participate in endogenous regulation of the PGHS activity in sheep vesicular glands.

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.

Physiological concentrations of arachidonic acid in deproteinized serum can be determined amperometrically in the presence of prostaglandin endoperoxide synthetase and potassium hexacyanoferrate(II). A determination takes 1.5–2 min, by measuring the current at a glassy carbon electrode at 0.0 V vs. SCE.

A kinetic scheme of the prostacyclin-thromboxane system has been evolved on the basis of our own experimental material 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, prostaglandin H (PGH) synthetase biosynthesis rates, velocities of arachidonic acid pathways other than 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.

A new method is proposed for assaying transketolase activity. The method is based on determining erythrulose, the product of the transketolase reaction, by its optical activity.

Different methods have been developed for the determination of the amount of protein immobilized on solid supports. The most frequently used procedure is quantitative amino acid analysis after acid hydrolysis of the protein-carrier conjugate. The application of less time-consuming spectrophotometric methods to the measurement of the amount of protein bound to agarose beads was delineated by Koelsch et al. (1). In this report a modified procedure for the direct spectrophotometric protein determination with the intact gel beads is described. It is based on the measurement of optical density of the suspension of immobilized protein in polyethylene glycol. Due to the high sensitivity and reproducibility of the method, it is applicable to the measurement of small amounts of Sepharose-bound protein.

Thiamine pyrophosphate (TPP), when bound with transketolase (TK) induces some changes in the absorption of the enzyme and coenzyme which can be registered by difference spectrophotometry. The binding of a donor substrate to the binary complex give rise to changes in the absorption region of the TPP thiazolium ring and in the charge transfer spectrum. With low concentrations of hydroxypyruvate, the kinetics of these changes may be revealed. The possibility is discussed of the charge transfer complex (CTC) being involved in the catalytic reaction.

Prostaglandins (PGs) have an important physiological role in the modulation of various cell immune functions. The main sources of PGs during immune responses are monocyte cells. We report here the ability of non-stimulated macrophages to synthesize prostanoids and show that peritoneal mouse macrophages synthesize PGE2, PGF2a and thromboxane B2, spleen macrophages produce PGE2 and PGF2a, and in a fresh medium this synthesis reaches a constant basal level in a few hours. We studied the kinetics of Con A-induced proliferation of murine splenocytes under the influence of a wide range of PGE2 concentrations (10(-14)-10(-7) M). The suppressive effect of PGE2 decreased when its concentration was lowered and disappeared at 10(-9) M PGE2 (this concentration corresponded to the basal level of non-stimulated macrophage synthesis of PGE2). Further lowering of the concentration became essential for the proliferation process once again, and at picomolar concentrations PGE2 caused a suppressive effect comparable with that for 10(-8) M PGE2. We also found that PGE2 significantly inhibited cell proliferation when it was added 1 h before the addition of mitogen, as compared with simultaneous mitogen addition. The effect was obtained for both low (10(-12) M) and high (10(-8) M) PGE2 concentrations. This phenomenon of PGE2 biphasic control of lymphocyte proliferation may play an important role in cellular homeostasis, in particular in immune cell function regulation.

Prostaglandin H synthase (PGHS) as apo-PGHS, holo-PGHS, and holo-PGHS, inactivated in the course of catalysis was studied using chemical modification with diethyl pyrocarbonate (DEPC). The exhausted reaction with DEPC corresponded to the modification of 7 histidine residues in apo-PGHS and 4 in holo-PGHS. All 18 histidine residues became accessible for modification with DEPC in the enzyme, inactivated in the course of catalysis. The velocities of tryptic cleavage of all the three forms into two fragments were fairly different but independent of modification. Based on the results we hypothesize fast and dramatic changes in the protein structure in the course of the substrate conversion.