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.
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.
The role of serum fatty acid binding proteins (FABPs) in arachidonic acid (AA) uptake by murine peritoneal macrophages has been studied. The kinetics of ^sup 3^Harachidonic acid uptake by the cells ...was investigated over a wide range of AA concentration (10^sup -10^-10^sup -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^sup-10^-10^sup -6^M in the presence of serum FABPs and 10^sup -10^-10^sup -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.PUBLICATION ABSTRACT
The influence of 10
−10–10
−6 M morphine on the release of
3Harachidonic acid and its metabolites (
3HAAM) from prelabeled resident peritoneal murine macrophages was investigated. Morphine enhanced
...3HAAM release from A23187- and LPS-stimulated macrophages, as well as the basal release of
3HAAM. Dose-response curves showed a maximum at 10
−8 M morphine. Naloxone had no effect on morphine enhancement of
3HAAM release. These results are in agreement with the hypothesis that
3HAAM may be involved in the effects of morphine.
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 PGE
2, PGF
2a and thromboxane B
2, spleen macrophages produce PGE
2 and PGF
2a, 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 PGE
2 concentrations (10
−14–10
−7 M). The suppressive effect of PGE
2 decreased when its concentration was lowered and disappeared at 10
−9 M PGE
2 (this concentration corresponded to the basal level of non-stimulated macrophage synthesis of PGE
2). Further lowering of the concentration became essential for the proliferation process once again, and at picomolar concentrations PGE
2 caused a suppressive effect comparable with that for 10
−8 M PGE
2. We also found that PGE
2 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) PGE
2 concentrations. This phenomenon of PGE
2 biphasic control of lymphocyte proliferation may play an important role in cellular homeostasis, in particular in immune cell function regulation.
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 Ca2+-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 TxB2and PGE2. 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 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 3HAA 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 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 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 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.
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