A novel approach to distinguish between enzyme mechanisms: quasi-steady-state kinetic analysis of the prostaglandin H synthase peroxidase reaction.

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.

Experimental and theoretical investigation of arachidonic acid uptake in macrophages.

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.

Kinetics of prostanoid synthesis by macrophages is regulated by arachidonic acid sources.

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.

[Prostaglandin-H-synthase: stability and activity during immobilization on silica gel].

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.

Arachidonic acid regulation of prostanoid synthesis in macrophages.

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.

Lack of direct connection between arachidonic acid release and prostanoid synthesis upon differentiation of U937 cell.

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 TxB2 and PGE2.

Prostaglandin E2 biphasic control of lymphocyte proliferation: inhibition by picomolar concentrations.

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.

Prostaglandin H synthase of mouse macrophages: inhibiting and activating action of ibuprofen.

The effect of low (10(-10)-10(-14 M) ibuprofen concentrations on the release of labeled arachidonic acid metabolites by mouse peritoneal macrophages containing a constitutive isoform of prostaglandin H synthase was investigated. It was found that during the activation the cells metabolized AA through the cyclooxygenase pathway, synthesizing PGE2 (110 +/- 10 ng per 10(6) cells), PGF(2 alpha) (120 +/- 15 ng per 10(6) cells), and TxB2 (48 +/- 5 ng per 10(6) cells). Incubation of the macrophages with 10(-12) M ibuprofen leads to a sharp increase of PGf2 and PGF(2 alpha) synthesis (315 +/- 84 and 320 +/- 20 ng per 10(6) cells, respectively).

Ratios of substrates and inhibitors of prostaglandin synthesis in blood plasma of patients with heart ischemia.

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).

Ultralow concentrations of ibuprofen activate cell prostaglandin synthesis.

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.

Low concentrations of nonsteroidal anti-inflammatory drugs affect cell functions.

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.

[Prostaglandin H synthase. Chemical modification of histidine residues in various forms of the enzyme by diethylpyrocarbonate].

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).

Prostaglandin H synthase as a limiting enzyme of prostaglandin synthesis: substrate-induced inactivation as a new kind of enzyme activity regulation.

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.

Direct influence of morphine on the release of arachidonic acid and its metabolites.

The influence of 10(-10)-10(-6) M morphine on the release of [3H]arachidonic acid and its metabolites ([3H]AAM) from prelabeled resident peritoneal murine macrophages was investigated. Morphine enhanced [3H]AAM release from A23187- and LPS-stimulated macrophages, as well as the basal release of [3H]AAM. Dose-response curves showed a maximum at 10(-8) M morphine.

Prostaglandin H synthase. Inactivation of the enzyme in the course of catalysis is accompanied by fast and dramatic changes in protein structure.

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.

[Identification of haptoglobin as an endogenous inhibitor of prostaglandin H synthetase in the cytosol of sheep vesicular gland cells].

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.

[Changes in the level of polyunsaturated fatty acids, substrates and inhibitors of thromboxane and prostacyclin synthesis, in the blood of patients with cardiovascular diseases].

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.

Identification of haptoglobin as an endogenous inhibitor of prostaglandin H synthase in the cytosol fraction of primary cells from sheep vesicular glands.

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.

[The effect of haptoglobin on prostaglandin H synthase from ram vesicular glands].

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.

[The effect of benzofurocaine on acute inflammation in rats and on the activity of prostaglandin synthetase in vitro].

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.

[Prostaglandin-like inhibitors of prostaglandin-H-synthase].

The effect of none prostaglandin-like cyclopentanone derivatives on the prostaglandin H synthase activity was studied. Seven substances proved to be inhibitors of the enzyme, some of the being similar to the well-known nonsteroid antiinflammatory drugs with respect to their inhibitory activity.

[2-component inhibition of prostaglandin H synthetase by nonsteroidal anti-inflammatory preparations].

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.

[Isolation and properties of prostaglandin H synthetase immobilized in insoluble polyelectrolyte complexes].

Immobilization of prostaglandin-H-synthetase (EC 1.14.99.

[Kinetic model of a multienzyme system of blood prostanoid synthesis. II. Dynamic responses to pharmacological agents--inhibitors of prostaglandin H synthetases].

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.

[Kinetic model of a multienzyme system of blood prostanoid synthesis. I. Mechanism of stabilization of thromboxane and prostacyclin levels].

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.