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.     

Protein and lipid oxidation of banked human erythrocytes: role of glutathione

In banked human erythrocytes (RBCs), biochemical and functional changes are accompanied with vesiculation and reduced in vivo survival. We hypothesized that some of these changes might have resulted from oxidative modification of membrane lipids, proteins, or both as a result of atrophy of the antioxidant defense system(s). In banked RBCs, we observed a time-dependent increase in protein clustering, especially band 3; carbonyl modification of band 4.1; and malondialdehyde, a lipid peroxidation product. Examination of the antioxidative defense system showed a time-dependent decline in glutathione (GSH) concentration and glutathione-peroxidase (GSH-PX) activity, with a concomitant increase in extracellular GSH, cysteine, and homocysteine, and unchanged catalase activity. When subjected to acute oxidant stress by exposure to ferric/ascorbic acid or tert-butylhydroperoxide (tert-BHT), catalase activity showed a steeper decline compared with GSH-PX. The results demonstrate that GSH and GSH-PX appear to provide the primary antioxidant defense in stored RBCs, and their decline, concurrent with an increase in oxidative modifications of membrane lipids and proteins, may destabilize the membrane skeleton, thereby compromising RBC survival.     

Amelioration of glucose induced hemolysis of human erythrocytes by vitamin E

Cells under aerobic condition are always threatened with the insult of reactive oxygen species, which are efficiently taken care of by the highly powerful antioxidant systems of the cell. The erythrocytes (RBCs) are constantly exposed to oxygen and oxidative stress but their metabolic activity is capable of reversing the injury under normal conditions. In vitro hemolysis of RBCs induced by 5, 10 and 20 mM glucose was used as a model to study the free radical induced damage of biological membranes in hyperglycemic conditions and the protection rendered by vitamin E on the same. RBCs are susceptible to oxidative damage, peroxidation of the membrane lipids, release of hemoglobin (hemolysis) and alteration in activity of antioxidant enzymes catalase and superoxide dismutase. The glucose induced oxidative stress and the protective effect of vitamin E on cellular membrane of human RBCs manifested as inhibition of membrane peroxidation and protein oxidation and restoration of activities of superoxide dismutase and catalase, was investigated.Thiobarbituric acid reactive substances are generated from decomposition of lipid peroxides and their determination gives a reliable estimate of the amount of lipid peroxides present in the membrane. Vitamin E at 18 μg/ml (normal serum level) strongly enhanced the RBC resistance to oxidative lysis leading to only 50–55% hemolysis in 24 h, whereas RBCs treated with 10 and 20 mM glucose without vitamin E leads to 70–80% hemolysis in 24 h. Levels of enzymic antioxidants catalase, superoxide dismutase and nonenzymic antioxidants glutathione showed restoration to normal levels in presence of vitamin E. The study shows that vitamin E can protect the erythrocyte membrane exposed to hyperglycemic conditions and so a superior antioxidant status of a diabetic patient may be helpful in retarding the progressive tissue damage seen in chronic diabetic patients.     

Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings.

Leaves of maize (Zea mays L.) seedlings were supplied with different concentrations of abscisic acid (ABA). Its effects on the levels of superoxide radical (O(2)(-)), hydrogen peroxide (H(2)O(2)) and the content of catalytic Fe, the activities of several antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), the contents of several non-enzymatic antioxidants such as ascorbate (ASC), reduced glutathione (GSH), alpha-tocopherol (alpha-TOC) and carotenoid (CAR), and the degrees of the oxidative damage to the membrane lipids and proteins were examined. Treatment with 10 and 100 microM ABA significantly increased the levels of O(2)(-) and H(2)O(2), followed by an increase in activities of SOD, CAT, APX and GR, and the contents of ASC, GSH, alpha-TOC and CAR in a dose- and time-dependent pattern in leaves of maize seedlings. An oxidative damage expressed as lipid peroxidation, protein oxidation, and plasma membrane leakage did not occur except for a slight increase with 100 microM ABA treatment for 24 h. Treatment with 1,000 microM ABA led to a more abundant generation of O(2)(-) and H(2)O(2) and a significant increase in the content of catalytic Fe, which is critical for H(2)O(2)-dependent hydroxyl radical production. The activities of these antioxidative enzymes and the contents of alpha-TOC and CAR were still maintained at a higher level, but no longer further enhanced when compared with the treatment of 100 microM ABA. The contents of ASC and GSH had no changes in leaves treated with 1,000 microM ABA. These results indicate that treatment with low concentrations of ABA (10 to 100 microM) induced an antioxidative defence response against oxidative damage, but a high concentration of ABA (1,000 microM) induced an excessive generation of AOS and led to an oxidative damage in plant cells.     

Phosphatidylserine transport in cell life and death

Phosphatidylserine (PS) is a negatively charged glycerophospholipid found mainly in the plasma membrane (PM) and in the late secretory/endocytic compartments, where it regulates cellular activity and can mediate apoptosis. Export of PS from the endoplasmic reticulum, its site of synthesis, to other compartments, and its transbilayer asymmetry must therefore be precisely regulated. We review recent findings on nonvesicular transport of PS by lipid transfer proteins (LTPs) at membrane contact sites, on PS flip-flop between membrane leaflets by flippases and scramblases, and on PS nanoclustering at the PM. We also discuss emerging data on cooperation between scramblases and LTPs, how perturbation of PS distribution can lead to disease, and the specific role of PS in viral infection.     

Effects of piperine on antioxidant pathways in tissues from normal and streptozotocin-induced diabetic rats

Using diabetes mellitus as a model of oxidative damage, this study investigated whether subacute treatment (10 mg/kg/day, intraperitoneally for 14 days) with the compound piperine would protect against diabetes-induced oxidative stress in 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione (GSH and GSSG, respectively) content, and activities of the free-radical detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. Piperine treatment of normal rats enhanced hepatic GSSG concentration by 100% and decreased renal GSH concentration by 35% and renal glutathione reductase activity by 25% when compared to normal controls. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Treatment with piperine reversed the diabetic effects on GSSG concentration in brain, on renal glutathione peroxidase and superoxide dismutase activities, and on cardiac glutathione reductase activity and lipid peroxidation. Piperine treatment did not reverse the effects of diabetes on hepatic GSH concentrations, lipid peroxidation, or glutathione peroxidase or catalase activities; on renal superoxide dismutase activity; or on cardiac glutathione peroxidase or catalase activities. These data indicate that subacute treatment with piperine for 14 days is only partially effective as an antioxidant therapy in diabetes.     

Concentration dependent antioxidant/pro-oxidant activity of curcumin studies from AAPH induced hemolysis of RBCs

The antioxidant properties of curcumin have been studied by evaluating its ability to protect RBCs from AAPH (2,2'-azobis (2-amidinopropane) hydrochloride) induced oxidative damage. RBCs are susceptible to oxidative damage, resulting in peroxidation of the membrane lipids, release of hemoglobin (hemolysis), release of intracellular K(+) ions and depletion of glutathione (GSH). In this paper, lipid peroxidation, hemolysis and K(+) ion loss in RBCs were assessed respectively by formation of thiobarbituric acid reactive substances (TBARS), absorbance of hemoglobin at 532nm and flame photometry. The treatment of RBCs with curcumin showed concentration dependant decrease in level of TBARS and hemolysis. The IC(50) values for inhibition of lipid peroxidation and hemolysis were estimated to be 23.2+/-2.5 and 43+/-5microM respectively. However in contrast to the above mentioned effects, curcumin in similar concentration range, did not prevent release of intracellular K(+) ions during the process of hemolysis, rather curcumin induced its release even in the absence of hemolysis. The ability of curcumin to prevent oxidation of intracellular GSH due to hemolysis showed mixed results. At low concentrations of curcumin (<10microM) it prevented GSH depletion and at higher concentrations, the GSH levels decreased gradually. Curcumin scavenges the peroxyl radical generated from AAPH. Based on these results, it is concluded that curcumin exhibits both antioxidant/pro-oxidant activity, in a concentration dependent manner.     

Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii

The effect of salinity on the antioxidative system of root mitochondria and peroxisomes of a cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) was studied. Salt stress induced oxidative stress in Lem mitochondria, as indicated by the increased levels of lipid peroxidation and H(2)O(2). These changes were associated with decreased activities of superoxide dismutase (SOD) and guaiacol peroxidases (POD) and contents of ascorbate (ASC) and glutathione (GSH). By contrast, in mitochondria of salt-treated Lpa plants both H(2)O(2) and lipid peroxidation levels decreased while the levels of ASC and GSH and activities of SOD, several isoforms of ascorbate peroxidase (APX), and POD increased. Similarly to mitochondria, peroxisomes isolated from roots of salt-treated Lpa plants exhibited also decreased levels of lipid peroxidation and H(2)O(2) and increased SOD, ascorbate peroxidase (APX), and catalase (CAT) activities. In spite of the fact that salt stress decreased activities of antioxidant enzymes in Lem peroxisome, oxidative stress was not evident in these organelles.     

Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs

Oxidant stress, in vivo or in vitro, isknown to induce oxidative changes in human red blood cells (RBCs). Ourobjective was to examine the effect of augmenting RBC glutathione(GSH) synthesis on 1) degenerative protein loss and2) RBC chemokine- and free radical-scavenging functions inthe oxidatively stressed human RBCs by using banked RBCs as a model.Packed RBCs were stored up to 84 days at 1-6°C in Adsol or inthe experimental additive solution (Adsol fortified with glutamine,glycine, and N-acetyl-L-cysteine). Supplementingthe conventional additive with GSH precursor amino acids improved RBCGSH synthesis and maintenance. The rise in RBC -glutamylcysteineligase activity was directly proportional to the GSH content andinversely proportional to extracellular homocysteine concentration,methemoglobin formation, and losses of the RBC proteins band 3, band4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffyantigen (P < 0.01). Reduced loss of Duffy antigencorrelated well with a decrease in chemokine RANTES (regulated uponactivation, normal T-cell expressed, and secreted) concentration. Weconclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSHsynthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, inwhich RBCs are subjected to chronic/acute oxidant stresses.

     

UV-B and cadmium induced changes in pigments,photosynthetic electron transport activity,antioxidant levels and antioxidative enzyme activities of <Emphasis Type="Italic">Riccia</Emphasis> sp.

UV-B and cadmium, alone and together, induced changes in photosynthetic pigment levels, photosynthetic electron transport activity, enzymatic and non-enzymatic (low molecular weight) antioxidants, level of hydrogen peroxide and lipid peroxidation in Riccia sp. were evaluated. Chlorophyll content was found to decrease with the rising concentration of cadmium and UV-B exposure alone and its level further declined when both the stresses were applied together. In contrast to this, carotenoids exhibited varied response, as it showed enhancement with UV-B (15, 30 and 45 min exposure) and low concentration of Cd (1 and 10 μM) treatment alone and in combination. Both the stresses caused strong inhibitory effect on PS II activity (H₂O → p-BQ), while PS I activity (DCPIP/ASC → MV) appeared to be less sensitive. Total peroxide content increased with simultaneous increase in lipid peroxidation. The level of non-enzymatic antioxidant ascorbate and enzymatic antioxidants superoxide dismutase and peroxidase activity were found to increase with simultaneous decrease in catalase activity following UV-B and Cd treatments. These results indicate that 45 min of UV-B exposure and 10, 100 and 1000 μM cadmium alone and together, strongly arrested electron flow through PS II which caused accelerated generation of reactive oxygen species (H₂O₂) and excess accumulation of H₂O₂ due to significant inhibition of catalase activity, led to the oxidative damage in Riccia sp.     

Prothrombinase acceleration by oxidatively damaged phospholipids

The optimally efficient production of thrombin by the prothrombinase complex relies on suitable positioning of its component factors and substrate on phosphatidylserine-containing lipid membranes. The presence of oxidatively damaged phospholipids in a membrane disrupts the normal architecture of a lipid bilayer and might therefore be expected to interfere with prothrombinase activity. To investigate this possibility, we prepared phosphatidylserine-containing lipid vesicles containing oxidized arachidonoyl lipids, and we examined their ability to accelerate thrombin production by prothrombinase. Oxidized arachidonoyl chains caused dose-dependent increases in prothrombinase activity up to 6-fold greater than control values. These increases were completely attenuated by the presence of alpha-tocopherol, gamma-tocopherol, or ascorbate. Over the course of a 300-min oxidation, the ability of arachidonoyl lipids to accelerate prothrombinase peaked at 60 min and then declined to base-line levels. These results suggest that instead of being impeded by oxidative membrane damage, prothrombinase activity is enhanced by one or more products of nonenzymatic lipid oxidation.     

UV-C对紫杉针叶叶绿体膜脂过氧化及PSⅡ电子传递活性的影响

在实验室条件下,用12W·m^-2剂量的紫外线C(UV-C,254nm)辐射紫杉针叶离体叶绿体．结果表明。随辐射时间的延长,活性氧清除系统中类胡萝卜素(Car)、谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)活性有不同程度的下降;脂质过氧化产物丙二醛(MDA)含量和膜相对透性有不同程度的增加;光系统Ⅱ(PSⅡ)电子传递活性显著下降,这种下降与光合活性光(PAR)强度呈反比;叶绿素对UV-C辐射不敏感．根据以上结果推测,UV-C辐射诱导叶绿体膜脂过氧化是导致PSⅡ电子传递活性下降的原因之一．     

Oxidative modification of rat liver 5'-nucleotidase: the mechanisms for protection and re-activation

The effect of oxidative stress catalysed by transition metals appears to have a critical relevance for the structure and function not only of membrane lipids but also of integral membrane proteins in a complex lipid-protein assembling, and membrane-dependent function. The integral membrane enzyme 5'-nucleotidase is susceptible to Fe((2+))-ion catalysed oxidative modification, and the extent of enzyme inhibition is in inverse relationship (r = -0.820) with lipid peroxidation (MDA) level. This work is also a comparative study about possible effectiveness of different Fe-ion chelators (deferoxamine, Na-citrate, Na-salicylate, ammonium oxalate and EDTA), antioxidants (GSH, GSH/GSH-Px system, Cu, Zn-SOD and mannitol) and metal cations (Mg(2+) and Mn(2+)) to protect or restore Fe(2+)-ion induced 5'-nucleotidase inhibition and to suppress Fe(2+)-ion enhanced lipid peroxidation. Among the examined chelators it was only deferoxamine and Na-citrate that exerted a fully protective and reactivating ability; among the antioxidants it was only GSH; among the metal cations it was only Mn(2+). The ability to protect or restore 5'-nucleotidase activity and to diminish chain-induced lipid peroxidation is explicable in terms of: metal-binding ability, capacity of taking iron away from a biological molecule, or ability of transferring the damage to itself. After a short incubation period, the iron associated with enzyme or lipid hydroperoxides could be in a labile coordinative linkage, still able to interact with possible ligands or metal cations.     

Oxidative Stress-Induced Membrane Shedding from RBCs is Ca Flux-Mediated and Affects Membrane Lipid Composition

Phosphatidylserine (PS), which is normally localized in the cytoplasmic leaflet of the membrane, undergoes externalization during aging or trauma of red blood cells (RBCs). A fraction of this PS is shed into the extracellular milieu. Both PS externalization and shedding are modulated by the oxidative state of the cells. In the present study we investigated the effect of calcium (Ca) flux on oxidative stress-induced membrane distribution of PS and its shedding and on the membrane composition and functions. Normal human RBCs were treated with the oxidant t-butyl hydroperoxide, and thalassemic RBCs, which are under oxidative stress, were treated with the antioxidant vitamin C or N-acetylcystein. The intracellular Ca content was modulated by the Ca ionophore A23187 and by varying the Ca concentration in the medium. Ca flux was measured by Fluo-3, PS externalization and shedding were measured by quantitative flow cytometry and membrane composition was measured by ¹H-NMR analysis of the cholesterol and phospholipids. The results indicated that increasing the inward Ca flux induced PS externalization and shedding, which in turn increased the membrane cholesterol/phospholipid ratio and thereby increased the RBC osmotic resistance. In addition, these processes modulated the susceptibility of RBCs to undergo phagocytosis by macrophages; while PS externalization increased phagocytosis, the shed PS prevented it. These results indicate that PS redistribution and shedding from RBCs, which are mediated by increased calcium, have profound effects on the membrane composition and properties and, thus, may control the fate of RBCs under physiological and pathological conditions.     

Impact of iron toxicity on oxidative metabolism in young Eugenia uniflora L. plants

In excess, iron can induce the production and accumulation of reactive oxygen species (ROS), causing oxidative stress. The objective of this work was to evaluate the impact of toxic concentrations of iron (Fe) on the antioxidative metabolism of young Eugenia uniflora plants. Forty-five-day-old plants grown in Hoagland nutrient solution, pH 5.0, were treated with three Fe concentrations, in the form of FeEDTA, during three periods of time. At the end of the treatment, the plants were harvested and relative growth rate, iron content, lipid peroxidation and enzymes and metabolites of the antioxidative metabolism were determined. Iron-treated plants showed higher iron contents, reduced relative growth rates and iron toxicity symptoms in both leaves and roots. There was an increase in lipid peroxidation with increasing Fe, only in the leaves. The enzymatic activities of superoxide dismutase (SOD) and glutathione reductase (GR) increased with increasing Fe concentration and treatment exposure time. The activities of catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APX) also increased with increasing Fe concentration but decreased with increasing treatment exposure time. Glutathione peroxidase activity (GPX) decreased with increasing Fe concentration and exposure time. The ascorbate (AA) and reduced glutathione (GSH) contents and the AA/DHA and GSH/GSSG ratios, in general, increased with increasing Fe concentration and treatment exposure time. The results indicate that under toxic levels of Fe, young E. uniflora plants suffer increased oxidative stress, which is ameliorated through changes in the activities of antioxidative enzymes and in the contents of the antioxidants AA and GSH.     

Oxidative stress in the hippocampus after pilocarpine-induced status epilepticus in Wistar rats

The role of oxidative stress in pilocarpine-induced status epilepticus was investigated by measuring lipid peroxidation level, nitrite content, GSH concentration, and superoxide dismutase and catalase activities in the hippocampus of Wistar rats. The control group was subcutaneously injected with 0.9% saline. The experimental group received pilocarpine (400 mg.kg(-1), subcutaneous). Both groups were killed 24 h after treatment. After the induction of status epilepticus, there were significant increases (77% and 51%, respectively) in lipid peroxidation and nitrite concentration, but a 55% decrease in GSH content. Catalase activity was augmented 88%, but superoxide dismutase activity remained unaltered. These results show evidence of neuronal damage in the hippocampus due to a decrease in GSH concentration and an increase in lipid peroxidation and nitrite content. GSH and catalase activity are involved in mechanisms responsible for eliminating oxygen free radicals during the establishment of status epilepticus in the hippocampus. In contrast, no correlations between superoxide dismutase and catalase activities were observed. Our results suggest that GSH and catalase activity play an antioxidant role in the hippocampus during status epilepticus.     

Trp2063 and Trp2064 in the factor Va C2 domain are required for high-affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex

Interactions between factor Va and membrane phosphatidylserine (PS) regulate activity of the prothrombinase complex. Two solvent-exposed hydrophobic residues located in the C2 domain, Trp(2063) and Trp(2064), have been proposed to contribute to factor Va membrane interactions by insertion into the hydrophobic membrane bilayer. However, the prothrombinase activity of rHFVa W(2063, 2064)A was found to be significantly impaired only at low concentrations of PS (5 mol %). In this study, we find that 10-fold higher concentrations of mutant factor Va are required for half-maximal prothrombinase activity on membranes containing 25% PS. The ability of the mutant factor Va to interact with factor Xa on a membrane was also impaired since 4-fold higher concentrations of factor Xa were required for half-maximal prothrombinase activity. The interaction of factor Va with 25% PS membranes was also characterized using fluorescence energy transfer and surface plasmon resonance. We found that the affinity of mutant factor Va for membranes containing 25% PS was reduced at least 400-fold with a K(d) > 10(-7) M. The binding of mutant factor Va to 25% PS membranes was markedly enhanced in the presence of factor Xa, indicating stabilization of the factor Va-factor Xa-membrane complex. Our findings indicate that Trp(2063) and Trp(2064) play a critical role in the high-affinity binding of factor Va to PS membranes. It remains to be determined whether occupancy of this PS binding site in factor Va is also required for high-affinity binding to factor Xa.     

Influence of epithelial lining fluid lipids on NO(2)-induced membrane oxidation and nitration

Within the pulmonary epithelial lining layer (ELF), antioxidants such as ascorbic acid (AH(2)) and glutathione (GSH) react with inhaled nitrogen dioxide ((*)NO(2)) to produce reactive oxygen species (ROS) that induce cellular oxidation. Because the ELF contains unsaturated fatty acids (UFA), which potentially react with (*)NO(2) and/or the antioxidant-derived ROS, we studied the influence of aqueous phase model UFA [egg phosphatidylcholine (EggPC) liposomes] on exposure-induced oxidation and nitration of membranes. Our lung surface model used gas phase (*)NO(2) exposures of immobilized red cell membranes (RCM) overlaid with defined aqueous phases. Acetyl cholinesterase (AChE) activity, TBARS, and 3-nitrotyrosine (3-NT) were used to assess protein and lipid oxidation and RCM nitration, respectively. During (*)NO(2) exposure, AH(2) and GSH induced AChE loss and TBARS, which were unchanged with buffer only. Exposures of EggPC generated extensive TBARS but not AChE loss; addition of AH(2)/GSH to EggPC resulted in smaller AChE declines and fewer TBARS. 3-NT formation occurred with or without EggPC, low concentration antioxidants, SOD, catalase, or DTPA, but was inhibitable by desferrioxamine or high antioxidant concentrations. The data suggest that reaction/diffusion limitations govern (*)NO(2) distribution, that (*)NO(2) per se directly nitrates tyrosine residues within hydrophobic regions, and that the induction of secondary oxidative processes is dependent on nonlinear relationships among (*)NO(2) flux rates, antioxidant concentrations, and diffusivity of secondary reactive species.     

Efficacy of soluble phospholipids in the prothrombinase reaction

The prothrombinase complex is comprised of an enzyme, factor Xa, and a cofactor, factor Va, that each bind peripherally to membranes containing phosphatidylserine (PS) and activate the substrate, prothrombin. The mechanism by which the membrane contributes to enhanced catalytic efficacy of prothrombinase is not precisely known but is generally attributed to some aspect of enzyme and substrate assembly on the multisite surface of the membrane. A recent proposal has suggested a radically different role in which individual phospholipid molecules, either in the membrane or as single soluble molecules, act by an entirely allosteric mechanism that does not involve the multisite feature of the membrane [Zhai, X., Srivastava, A., Drummond, D. C., Daleke, D., and Lentz, B. R. (2002) Biochemistry 41, 5675-5684]. Our study measured prothrombinse activity in the presence of phospholipids such as short-chain phosphatidylserine and lysophosphatidylserine (lyso-PS). Both enhanced prothrombinase activity, and the increase was consistent with the requirement for extended bilayer structure. Even then, prothrombinase activity was low when compared with activity on bilayer membranes of mixed PS and phosphatidylcholine (PC). Lyso-PS approached the activity of PS/PC membranes only when it was mixed with PC bilayers. The results suggest that the two-dimensional membrane bilayer surface is necessary for the support of full prothrombinase activity.     

A Chinese Hamster Ovary Cell Mutant Resistant to Phosphatidylserine Is Defective in Transbilayer Movement of Cell Surface Phosphatidylserine

A mammalian plasma membrane protein(s) which catalyzes ATP-dependent transbilayer movement (flip-flop) of phosphatidylserine (PS) has been suggested to be involved in the formation and maintenance of membrane lipid asymmetry. Flip-flop of PS in the cell surface of nucleated cells was first described by O. C. Martin and R. E. Pagano (1987,J. Biol. Chem.262, 5890–5898). It has been suggested that flip-flop is involved in the internalization of exogenous PS in cultured cells. In the present study we report that incubation with an excess amount of PS is cytotoxic to Chinese hamster ovary (CHO) cells, while the same amount of phosphatidylcholine gives no effect. This effect allowed us to obtain PS-resistant cells among mutagenized CHO cells. Endocytosis-independent internalization of exogenous fluorescent PS analog was defective in 40% of the PS-resistant mutants. One of the mutants, PSR (phosphatidylserine resistant) 406 was further characterized. Unlike wild-type CHO cells, this mutant did not transport fluorescent PS significantly at 15°C. Fluorescent PS was not metabolized at 15°C in either wild-type or mutant cells. These results suggest that transbilayer movement of cell surface PS is defective in PS-resistant cells.