Membrane Protein Thiol Oxidation Research Articles

Erythrocyte glutathione depletion impairs resistance to haemolysis in women consuming alcohol.

Alcohol abuse is known to cause an array of ethanol induced abnormalities in men but very few reports are available on the effect of alcohol in women. None of them discuss the effect of ethanol consumption on erythrocyte membrane. In the present study, erythrocytes in women who consume alcohol showed significant decrease in their ability to resist haemolysis with HPLC studies. Erythrocyte membrane indicates decreased phospholipid (p<0.05) levels, which increased the cholesterol/phospholipid ratio significantly (p<0.01) in women who consume alcohol. This can decrease the fluidity of membrane, which appears to be related to the effect of ethanol on erythrocyte membrane. Also the protection against exogenous and endogenous peroxides in the erythrocytes of alcoholic women is considerably affected due to decreased (p<0.05) activity of catalase, glucose-6-phosphate dehydrogenase, protein–SH group and glutathione (GSH). Enhanced free radical generation induced oxidation of oxyHb to metHb in alcoholics. Increased methemoglobin leads to significant reduction in membrane GSH, which may cause protein thiol oxidation. Thus peroxidative damage to membrane lipids and oxidation of membrane protein thiols potentially harmful to membrane fluidity and flexibility is responsible for decreased resistance to haemolysis as demonstrated in women who consume alcohol.

Vimang ( Mangifera indica L. extract) induces permeability transition in isolated mitochondria, closely reproducing the effect of mangiferin, Vimang's main component

Mitochondrial permeability transition (MPT) is a Ca 2+-dependent, cyclosporin A (CsA)-sensitive, non-selective inner membrane permeabilization process. It is often associated with apoptotic cell death, and is induced by a wide range of agents or conditions, usually involving reactive oxygen species (ROS). In this study, we demonstrated that Mangifera indica L. extract (Vimang), in the presence of 20 μM Ca 2+, induces MPT in isolated rat liver mitochondria, assessed as CsA-sensitive mitochondrial swelling, closely reproducing the same effect of mangiferin, the main component of the extract, as well as MPT-linked processes like oxidation of membrane protein thiols, mitochondrial membrane potential dissipation and Ca 2+ release from organelles. The flavonoid catechin, the second main component of Vimang, also induces MPT, although to a lesser extent; the minor, but still representative Vimang extract components, gallic and benzoic acids, show respectively, low and high MPT inducing abilities. Nevertheless, following exposure to H 2O 2/horseradish peroxidase, the visible spectra of these compounds does not present the same changes previously reported for mangiferin. It is concluded that Vimang-induced MPT closely reproduces mangiferin effects, and proposed that this xanthone is the main agent responsible for the extract's MPT inducing ability, by the action on mitochondrial membrane protein thiols of products arising as a consequence of the mangiferin's antioxidant activity. While this effect would oppose the beneficial effect of Vimang's antioxidant activity, it could nevertheless benefit cells exposed to over-production of ROS as occurring in cancer cells, in which triggering of MPT-mediated apoptosis may represent an important defense mechanism to their host.

Mitochondrial permeability transition induced by chemically generated singlet oxygen.

Pure singlet molecular oxygen (1O2) generated by thermal decomposition of the 3,3'-(1,4-naphthylidene) dipropionate endoperoxide (NDPO2), inhibited respiration of isolated rat liver mitochondria supported by NADH-linked substrates or succinate, but not by N,N,N,N-tetramehyl-p-phenylene-diamine (TMPD)/ascorbate. Under the latter conditions, mitochondria treated with 2.7 mM NDPO2 exhibited a decrease in transmembrane potential (deltapsi) in manner dependent on NDPO2 exposure time. This process was sensitive to the mitochondrial permeability transition inhibitors EGTA, dithiothreitol, ADP, and cyclosporin A. The presence of deuterium oxide (D2O), that increases 1O2 lifetime, significantly enhanced NDPO2-promoted mitochondrial pereabilization. In addition, NDPO2-induced mitochondrial permeabilization was accompanied by DTT or ADP-sensitive membrane protein thiol oxidation. Taken together, these results provide evidence that mitochondrial permeability transition induced by chemically generated singlet oxygen is mediated by the oxidation of membrane protein thiols.

Curcumin induces the mitochondrial permeability transition pore mediated by membrane protein thiol oxidation

Curcumin is a natural compound showing antiproliferative properties. Recent studies suggest that these properties might be due to its ability to induce apoptosis in tumor cells. As mitochondria play a pivotal role in the induction of the apoptotic process, we analyzed the effect of curcumin on mitochondrial function. Curcumin induced an increase in rat liver mitochondrial membrane permeability, resulting in swelling, loss of membrane potential and inhibition of ATP synthesis. These effects were mediated by the opening of the permeability transition pore. Curcumin pore induction involved the oxidation of membrane thiol functions and required the presence of low Ca 2+ concentrations. These data suggest that mitochondria might be a target by which curcumin induces apoptosis of tumor cells.

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1–6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4–6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (14C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (∼ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.

The participation of pyridine nucleotides redox state and reactive oxygen in the fatty acid-induced permeability transition in rat liver mitochondria

The ability of low concentrations (5–15 μM) of long-chain fatty acids to open the permeability transition pore (PTP) in Ca 2+-loaded mitochondria has been ascribed to their protonophoric effect mediated by mitochondrial anion carriers, as well as to a direct interaction with the pore assembly [M.R. Wieckowski and L. Wojtczak, FEBS Lett. 423 (1998) 339–342]. Here, we have compared the PTP opening ability of arachidonic acid (AA) with that of carbonyl cyanide- p-trifluoromethoxyphenylhydrazone (FCCP) at concentrations that cause similar quantitative dissipation of the membrane potential (Δ Ψ) in Ca 2+-loaded rat liver mitochondria respiring on succinate. The initial protonophoric effects of AA and FCCP were only slightly modified by carboxyatractyloside and were followed by PTP opening, as indicated by a second phase of Δ Ψ disruption sensitive to EGTA, ADP, dithiothreitol and cyclosporin A. This second phase of Δ Ψ dissipation could also be prevented by rotenone or NAD(P)H-linked substrates which decrease the pyridine nucleotide (PN) oxidation that follows the stimulation of oxygen consumption induced by AA or FCCP. These results suggest that, under the experimental conditions used here, the PTP opening induced by AA or FCCP was a consequence of PN oxidation. Exogenous catalase also inhibited both AA- and FCCP-induced PTP opening. These results indicate that a condition of oxidative stress associated with the oxidized state of PN underlies membrane protein thiol oxidation and PTP opening.

Inhibition of mitochondrial permeability transition by low pH is associated with less extensive membrane protein thiol oxidation.

Ca2+ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H2O2 production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca2+ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues.

Ca2+-Stimulated Mitochondrial Reactive Oxygen Species Generation and Permeability Transition Are Inhibited by Dibucaine or Mg2+

Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca2+andt-butyl hydroperoxide or inorganic phosphate, but not 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or phenylarsine oxide, are inhibited by the local anesthetic dibucaine. Dibucaine promotes an inhibition of the Ca2+-induced increase in mitochondrial H2O2generation measured by the oxidation of scopoletin in the presence of horseradish peroxidase. This decrease in mitochondrial H2O2generation may be attributed to the reduction of Ca2+binding to the membrane induced by dibucaine, as assessed by measuring45Ca2+binding to the mitochondrial membrane. Mg2+also inhibited Ca2+binding to the mitochondrial membrane, mitochondrial swelling, membrane protein thiol oxidation, and H2O2generation induced by Ca2+. Together, these results demonstrate that the mechanism by which dibucaine and Mg2+inhibit mitochondrial permeability transition is related to the decrease in reactive oxygen species generation induced by Ca2+-promoted alterations of inner mitochondrial membrane properties.

3,5,3′-Triiodothyronine Induces Mitochondrial Permeability Transition Mediated by Reactive Oxygen Species and Membrane Protein Thiol Oxidation

Ca2+-loaded rat liver mitochondria treated with 3,5,3′-triiodothyronine (T3) undergo nonspecific inner membrane permeabilization, as evidenced by mitochondrial swelling, a decrease in membrane potential (ΔΨ), and an increase in the rate of oxygen uptake. T3analogues thyroxine (T4), 3′,5′-diiodothyronine (T2), and 3,5′,3′-triiodothyronine (reverse T3), in decreasing order of potency, resulted in a similar but less extensive effect. Permeabilization induced by T3is dependent on Ca2+(1 μM) and T3(0.5–25 μM) concentrations and is inhibited by cyclosporin A, a known inhibitor of mitochondrial permeability transition. Catalase or dithiothreitol also prevents membrane permeabilization, suggesting the participation of membrane protein thiol group oxidationinduced by reactive oxygen species. The determination of the mitochondrial membrane protein thiol group content after treatment with Ca2+and T3shows a significant decrease, due to thiol oxidation. When mitochondria are incubated in the presence of inorganic phosphate and the protonophore carbonyl cyanidep-trifluoromethoxyphenylhydrazone, mitochondrial swelling still occurs after treatment with T3and high Ca2+concentrations, suggesting that mitochondrial permeabilization is not dependent on T3-induced ΔΨ or matrix pH alterations. Under these experimental conditions, when no oxygen is present in the incubation medium, no permeabilization occurs, suggesting that the permeabilization is dependent on mitochondrial-generated reactive oxygen species. Confirming this hypothesis, superoxide generation in a suspension of submitochondrial particles is increased when T3is present. Our results lead to the conclusion that T3induces a situation of oxidative stress in isolated liver mitochondria, with Ca2+-mediated membrane protein thiol oxidation and nonspecific inner membrane permeabilization.

The Thiol-specific Antioxidant Enzyme Prevents Mitochondrial Permeability Transition: EVIDENCE FOR THE PARTICIPATION OF REACTIVE OXYGEN SPECIES IN THIS MECHANISM

Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca2+ and inorganic phosphate are inhibited in a dose-dependent manner either by catalase, the thiol-specific antioxidant enzyme (TSA), a protein recently demonstrated to present thiol peroxidase activity, or ebselen, a selenium-containing heterocycle which also possesses thiol peroxidase activity. This inhibition of mitochondrial permeability transition is due to the removal of mitochondrial-generated H2O2 which can easily diffuse to the extramitochondrial space. Whereas ebselen required the presence of reduced glutathione as a reductant to grant its protective effect, TSA was fully reduced by mitochondrial components. Decrease in the oxygen concentration of the reaction medium also inhibits mitochondrial permeabilization and membrane protein thiol oxidation, in a concentration-dependent manner. The results presented in this report confirm that mitochondrial permeability transition induced by Ca2+ and inorganic phosphate is reactive oxygen species-dependent. The possible importance of TSA as an intracellular antioxidant, avoiding the onset of mitochondrial permeability transition, is discussed in the text.

4,6-Dinitro- o-cresol uncouples oxidative phosphorylation and induces membrane permeability transition in rat liver mitochondria

The effect of the herbicide 4,6-dinitro- o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioenergetics and inner membrane permeability of isolated rat liver mitochondria was studied. We observed that DNOC (10–50 μM) acts as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respiration in the presence or absence of ADP and a decrease in transmembrane potential. The protonophoric activity of DNOC was evidenced by the induction of mitochondrial swelling in hyposmotic K +-acetate medium, in the presence of valinomycin. At higher concentrations ( > 50 μM), DNOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be observed. The addition of uncoupling concentrations of DNOC to Ca 2+-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Cyclosporin A, which inhibits mitochondrial permeability transition, prevented DNOC-induced mitochondrial swelling in the presence of Ca 2+, which was accompanied by a decrease in mitochondrial membrane protein thiol content, owing to protein thiol oxidation. Catalase partially inhibits mitochondrial swelling and protein thiol oxidation, indicating the participation of mitochondrial-generated reactive oxygen species in this process. It is concluded that DNOC is a potent protonophore acting as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria by dissipating the proton electrochemical gradient. Treatment of Ca 2+-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transition, associated with membrane protein thiol oxidation by reactive oxygen species.

Opening of the mitochondrial permeability transition pore by uncoupling or inorganic phosphate in the presence of Ca 2+ is dependent on mitochondrial-generated reactive oxygen species

In this study, we show that mitochondrial membrane permeability transition in Ca 2+-loaded mitochondria treated with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or inorganic phosphate (P i) is preceded by enhanced production of H 2O 2. This production is inhibited either by ethylene glycol-bis( b-aminoethyl ether) N, N, N′, N′-tetraacetic acid (EGTA) or Mg 2+, but not by cyclosporin A. Permeability transition is prevented either by EGTA, catalase or dithiothreitol, suggesting the involvement of Ca 2+, H 2O 2 and oxidation of membrane protein thiols in this mechanism. When mitochondria are incubated under anaerobiosis, no permeabilization or H 2O 2 production occurs. Based on these results we conclude that mitochondrial permeability transition induced by P i or FCCP-uncoupling is dependent on mitochondrial-generated reactive oxygen species.

Ca(2+)-induced mitochondrial membrane permeabilization: role of coenzyme Q redox state.

Rotenone-poisoned rat liver mitochondria energized by succinate addition, after a 5-min period of preincubation in presence of 10 microM Ca2+, produce H2O2 at much faster rates, undergo extensive swelling, and are not able to retain the membrane potential and accumulated Ca2+. Similar results were obtained when a suspension of rat liver mitochondria preincubated in anaerobic medium for 5 min was reoxygenated. The addition of either ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ruthenium red, catalase, or dithiothreitol, just before succinate or O2 addition, prevented mitochondrial swelling, indicating the involvement of Ca2+, reactive oxygen species, and oxidation of membrane protein thiols in this process of membrane permeabilization. Inhibition of mitochondrial swelling by cyclosporin A suggests that the membrane alterations observed under these experimental conditions are related to opening of the permeability transition pore. The presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which prevents Ca2+ cycling across the membrane, did not inhibit mitochondrial swelling when Ca2+ influx into the mitochondrial matrix was driven by a high Ca2+ gradient. When rotenone plus antimycin A-poisoned mitochondria were energized by N,N,N',N'-tetramethyl-p-phenylenediamine, which reduces respiratory chain complex IV, mitochondrial swelling did not occur, unless succinate, which reduces coenzyme Q, was also added. It is concluded that reduced coenzyme Q is the electron source for oxygen radical production during Ca(2+)-stimulated oxidative damage of mitochondria.

Oxidant Damage to Erythrocyte Membrane in Glucose-6-Phosphate Dehydrogenase Deficiency: Correlation With In Vivo Reduced Glutathione Concentration and Membrane Protein Oxidation

AbstractChronic nonspherocytic hemolytic anemia has been observed in a recently described glucose-6-phosphate dehydrogenase (G6PD) variant, G6PDWayne. The mechanical properties of these erythrocytes and other G6PD variants were examined. The deformability of G6PD-deficient erythrocytes was normal, as determined by osmotic scan ektacytometry. and was not significantly affected by hemolytic crisis. In the common varieties of G6PD deficiency, the mechanical stability of the red blood cell (RBC) membrane was greater than normal, but G6PDWayna membranes were abnormally susceptible to shear-induced fragmentation. There was no evidence for a concurrent genetic defect in spectrin, because self-association constants and tryptic digests were normal. The fragility of G6PDWayne membranes appeared to be a consequence of oxidative damage to membrane thiol groups associated with a low glutathione (GSH) level in these RBCs. Associations among GSH level, thiol oxidation, and membrane instability were also found when a larger group of G6PD-deficient RBCs were examined. In normal erythrocytes, 1-chloro-2, 4-dinitro-benzene was used to reduce GSH levels by 50%. Membrane thiol oxidation and membrane fragility both increased when these cells were kept at 4°C for 3 to 5 days. Our findings suggest that chronic depletion of GSH leads to the destabilization of membrane skeleton through oxidation of membrane protein thiols.

Oxidant damage to erythrocyte membrane in glucose-6-phosphate dehydrogenase deficiency: correlation with in vivo reduced glutathione concentration and membrane protein oxidation

Chronic nonspherocytic hemolytic anemia has been observed in a recently described glucose-6-phosphate dehydrogenase (G6PD) variant, G6PDWayne. The mechanical properties of these erythrocytes and other G6PD variants were examined. The deformability of G6PD-deficient erythrocytes was normal, as determined by osmotic scan ektacytometry, and was not significantly affected by hemolytic crisis. In the common varieties of G6PD deficiency, the mechanical stability of the red blood cell (RBC) membrane was greater than normal, but G6PDWayne membranes were abnormally susceptible to shear-induced fragmentation. There was no evidence for a concurrent genetic defect in spectrin, because self- association constants and tryptic digests were normal. The fragility of G6PDWayne membranes appeared to be a consequence of oxidative damage to membrane thiol groups associated with a low glutathione (GSH) level in these RBCs. Associations among GSH level, thiol oxidation, and membrane instability were also found when a larger group of G6PD-deficient RBCs were examined. In normal erythrocytes, 1-chloro-2,4-dinitrobenzene was used to reduce GSH levels by 50%. Membrane thiol oxidation and membrane fragility both increased when these cells were kept at 4 degrees C for 3 to 5 days. Our findings suggest that chronic depletion of GSH leads to the destabilization of membrane skeleton through oxidation of membrane protein thiols.

The effect of α-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membranelipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between α-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with α-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, α-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.

Vesiculation of Sickle Erythrocytes During Thermal Stress

Abstract Since it is not known why sickle RBCs tend to undergo microvesiculation, we have investigated their susceptibility to thermal stress. While normal RBCs start to vesiculate at 49.0 +/- 0 degrees C (n = 14), sickle RBCs begin to vesiculate at 47.9 +/- 0.5 degrees C, with a range of 46.5 to 48.5 degrees C (n = 14). This abnormality is reproduced by treating normal RBCs with phenazine methosulfate (PMS), which stimulates the excess intracellular generation of superoxide characteristic of sickle RBCs. For PMS-treated RBCs, there is a strong correlation between membrane protein thiol oxidation and vesiculation temperature (r = .977, P less than .001). The abnormal vesiculation temperature of both unmanipulated sickle RBCs and PMS-treated RBCs is significantly improved by treatment of the RBCs with dithiothreitol. The most dense sickle RBCs are most prone to vesiculation during thermal stress, and they are the subpopulation having the greatest amount of thiol oxidation. We conclude that the tendency of sickle RBCs to vesiculate during thermal stress is further evidence for functional abnormality of the RBC cytoskeleton due to thiol oxidation.

Vesiculation of sickle erythrocytes during thermal stress

Since it is not known why sickle RBCs tend to undergo microvesiculation, we have investigated their susceptibility to thermal stress. While normal RBCs start to vesiculate at 49.0 +/- 0 degrees C (n = 14), sickle RBCs begin to vesiculate at 47.9 +/- 0.5 degrees C, with a range of 46.5 to 48.5 degrees C (n = 14). This abnormality is reproduced by treating normal RBCs with phenazine methosulfate (PMS), which stimulates the excess intracellular generation of superoxide characteristic of sickle RBCs. For PMS-treated RBCs, there is a strong correlation between membrane protein thiol oxidation and vesiculation temperature (r = .977, P less than .001). The abnormal vesiculation temperature of both unmanipulated sickle RBCs and PMS-treated RBCs is significantly improved by treatment of the RBCs with dithiothreitol. The most dense sickle RBCs are most prone to vesiculation during thermal stress, and they are the subpopulation having the greatest amount of thiol oxidation. We conclude that the tendency of sickle RBCs to vesiculate during thermal stress is further evidence for functional abnormality of the RBC cytoskeleton due to thiol oxidation.

Inhibition of erythrocyte Ca 2+-ATPase by activated oxygen through thiol- and lipid-dependent mechanisms

We have studied erythrocyte Ca 2+-ATPase as a model target for elucidating effects of activated oxygen on the erythrocyte membrane. Either intracellular or extracellular generation of activated oxygen causes parallel decrements in Ca 2+-ATPase activity and cytoplasmic GSH, oxidation of membrane protein thiols, and lipid peroxidation. Subsequent incubation with either dithiothreitol or glucose allows only partial recovery of Ca 2+-ATPase, indicating both reversible and irreversible components which are modeled herein using diamide and t-butyl hydroperoxide. The reversible component reflects thiol oxidation, and its recovery depends upon GSH restoration. The irreversible component is largely due to lipid peroxidation, which appears to act through mechanisms involving neither malondialdehyde nor secondary thiol oxidation. However, some portion of the irreversible component could also reflect oxidation of thiols which are inaccessible for reduction by GSH, since we demonstrate existence of different classes of thiols relevant to Ca 2+-ATPase activity. Activated oxygen has an exaggerated effect on Ca 2+-ATPase of GSH-depleted cells. Sickle erythrocytes treated with dithiothreitol show a heterogeneous response of Ca 2+-ATPase activity. These findings are potentially relevant to oxidant-induced hemolysis. They also may be pertinent to oxidative alteration of functional or structural membrane components in general, since many components share with Ca 2+-ATPase both free thiols and close proximity to unsaturated lipid.