Alevtina T. Mevkh

Immobilized dimers of D-glyceraldehyde-3-phosphate dehydrogenase

Immobilization of enzymes has proved useful for elucidating the problem of intersubunit interactions in the oligomeric proteins. Avaluable approach to the study of matrix-bound protein subunits was developed by Chan and applied to investigation of a number of enzymes [l-3] . Recently we have bound tetrameric glyceraldehyde-3-phosphate dehydrogenase to Sepharose, the catalytic activity of the enzyme being retained to a considerable degree [4 ] . In the present communication we describe the method for dissociating the matrixbound tetramer into enzymatically active dimers and characterize some of the properties of the immobilized dimers.

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. 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 the charge transfer complex in the transketolase catalyzed reaction

Abstract 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.

A new method of determination of transketolase activity by asymmetric synthesis reaction.

Abstract 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.

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. Naloxone had no effect on morphine enhancement of [3H]AAM release. These results are in agreement with the hypothesis that [3H]AAM may be involved in the effects of morphine.

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. Haptoglobin is supposed to participate in endogenous regulation of PGHS activity in sheep vesicular glands.

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. 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.

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. 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.

Ultralow concentrations of ibuprofen activate cell prostaglandin synthesis.

Abstracthe 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 con ditions 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 10-10-10-14M range with the maximum effect at the 10-12M. In this case, the ibupro fen effect was comparable in value with the effect of the 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 activiate PG synthesis in murine peritoneal macrophages.

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. 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.