Thiol-oxidizing Agent Diamide Research Articles

Erythrocyte (Ca+2 + Mg+2)-ATPase activity: increased sensitivity to oxidative stress in glucose-6-phosphate dehydrogenase deficiency.

The effect of the thiol-oxidizing agent diamide on erythrocyte (Ca+2 + Mg+2)-ATPase activity was measured in normal and glucose-6-phosphate-dehydrogenase-deficient (G6PD-) cells. Although the enzyme activity before the oxidative stress was similar in both groups, diamide induced a markedly greater inhibition in the enzyme activity in the G6PD- cells than in the normal controls. These data indicate dependence of erythrocyte (Ca+2 + Mg+2)-ATPase, in part, on the redox status of the cell. The increased vulnerability of (Ca+2 + Mg+2)-ATPase to oxidative stress in G6PD- may be of pathophysiological relevance to their premature destruction in oxidant-induced hemolysis.

Plasmodium falciparum: Thiol status and growth in normal and glucose-6-phosphate dehydrogenase deficient human erythrocytes

The relationship of the thiol status of the human erythrocyte to the in vitro growth of Plasmodium falciparum in normal and in glucose-6-phosphate dehydrogenase (G6PD)-deficient red cells was investigated. Pretreatment with the thiol-oxidizing agent diamide led to inhibition of growth of P. falciparum in G6PD-deficient cells, but did not affect parasite growth in normal cells. Diamidetreated normal erythrocytes quickly regenerated intracellular glutathione (GSH) and regained normal membrane thiol status, whereas G6PD-deficient cells did not. Parasite invasion and intracellular development were affected under conditions in which intracellular GSH was oxidized to glutathione disulfide and membrane intrachain and interchain disulfides were produced. An altered thiol status in the G6PD-deficient erythrocytes could underlie the selective advantage of G6PD deficiency in the presence of malaria.

Role of glutathione in the regulation of inorganic pyrophosphatase activity in Streptococcus faecalis.

When the thiol-oxidizing agent diamide was added to a batch culture of Streptococcus faecalis during exponential growth, the specific activity of inorganic pyrophosphatase (EC 3.6.1.1) decreased to the level observed in the stationary phase. The effect of diamide was completely reversed by reduced glutathione. Furthermore, the ratio of reduced to oxidized glutathione and the specific activity of inorganic pyrophosphatase changed in a very similar fashion during batch culture. These findings, together with our earlier results, suggest that the activity of inorganic pyrophosphatase in S. faecalis is regulated by glutathione via the ratio of reduced to oxidized glutathione.

Diamide, Temperature and spontaneous transmitter release at the neuromuscular junction: Stimulation of exocytosis by a direct effect on membrane fusion?

The thiol-oxidizing agent diamide markedly increases m.e.p.p. frequency at the frog neuromuscular junction, even at low [Ca 2+] 0 and also when the mitochondria are uncoupled with DNP. The effect is reversed by dithioerythritol and is very temperature-sensitive, with a marked transition at 16°C; m.e.p.p. frequency is raised 2- to 5-fold at 13–15°C and 55- to 60-fold at 17–20°C. Diamide increases the frequency of large amplitude m.e.p.p.s, the effect being explicable as the fusion of two or more vesicles. It is concluded that (a) diamide does not act at the Ca 2+ channels of the plasma membrane, nor at the mitochondria. It affects the release system directly via an alteration of membrane protein—SH groups; (b) the eventual decline in m.e.p.p. frequency after DNP treatment is because of the exhaustion of mitochondrial Ca 2+ rather than a depletion of quanta; (c) the major effect of temperature is on the release mechanism, perhaps via a phase-change in the phospholipoproteins of the plasmalemma or vesicles, rather than an elevation of [Ca 2+] i; (d) either diamide or temperature above 16°C make Ca 2+ more effective in promoting vesicle-plasmalemma fusion.

Divalent cation dependent ATPase activities of red blood cell membranes: influence of the oxidation of membrane thiol groups close to each other.

An Mg2+-dependent low ATPase activity can be detected in erythrocyte "white membranes," in addition to that of the well known (Ca2+ + Mg2+)-ATPase. The thiol oxidizing agent diamide affects both activities. The oxidation of neighboring thiols seems to leave the mechanism of the (Ca2+ + Mg2+)-ATPase amplification system evoked by Ca2+ largely unaffected. The perturbation caused by diamide in the membranes seems to affect primarily a step of the ATP hydrolysis mechanism that is common to both ATPase activities. The effectiveness of diamide seems to be the same when either Ca2+ and Mg2+, or Mg2+ alone are present during the reagent action. Reduction of disulfide bonds by DTE after diamide treatment restores the (Ca2+ + Mg2+)-ATPase activity but is unable to take the Mg2+-ATPase activity back to the original level. The hypothesis is discussed that the redox state of one (or more than one) couple of --SH close to each other and possibly connected to the active site, may be an important factor in optimizing the efficiency of Ca action on the (Ca2+ + Mg2+)-ATPase.

The effect of oxidant stress on diamide-treated human granulocytes

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xanthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.

The thiol-oxidizing agent diamide increases transmitter release by decreasing calcium requirements for neuromuscular transmission in the frog

Diamide, which in concentrations of10 −5 M and higher oxidizes glutathione intracellularly, produces a dose-related increase in the frequency of miniaure end-plate potentials (MEPPs). With high enough doses, quantal release is blocked, apparently through exhaustion. The early phase of MEPP frequency increase is accompanied by an increase in EPP amplitude that may reach more than 10-fold and is therefore not produced by depolarization of axon terminals. Subsequently, EPP amplitude is reduced and falls to zero, associated with failure of invasion of the nerve action into the terminals while the MEPP frequency remains elevated. Both facilitation and PTP follow the time course of change in EPP amplitude. The increase in MEPP frequency with diamide does not require external Ca 2+ but raising external Ca 2+ increases the MEPP rate in the presence of diamide. External Ca 2+ is necessary for EPP appearance and also potentiates the diamide effects. Conversely diamide reduces the requirements for Ca 2+ in releasing ACh. Diamide substitutes for external Ca 2+ in K + evoked MEPP release and in the absence of external Ca 2+, diamide-evoked MEPP release is increased by raising external Mg 2+ levels. The action of diamide may be dependent on the actual release of Ca 2+ from intracellular stores or it may work through mimicking some of the actions of Ca 2+. The action of diamide bears close resemblance to the effects of prolonged stimulation of the motor axon at 10 Hz.

Glutathione: V. The effects of the thiol-oxidizing agent diamide on initiation and translation in rabbit reticulocytes

1. 1. Addition of the thiol-oxidizing agent, diamide ((CH 3) 2NCON=NCON(CH 3) 2), to rabbit reticulocytes, which are actively synthesizing protein, halts instantaneously the incorporation of labeled amino acid into soluble protein along with the oxidation of glutathione (GSH) to the disulfide (GSSG). 2. 2. Both translation and initiation are affected. 3. 3. Processes involved in translation (and release) recover after a short lag, following regeneration of 40–60 % of the original GSH. 4. 4. Initiation is more sensitive than translation to treatment with diamide, recovers after regeneration of 70–80 % of the original GSH, but after long periods of zero GSH, only incompletely. 5. 5. The rapidity of the response of the protein synthesizing system to diamide treatment suggests that thiol groups are intimately and directly linked to various stages in protein synthesis. 6. 6. The remarkable reversibility observed in protein synthesis after diamide treatment offers a new probe into the details of the complex process.