Erythrocyte and plasma glutathione levels in patients with chronic renal insufficiency

Reduced glutathione (GSH) levels were investigated in the erythrocytes and plasma of nondialyzed patients with varying degrees of renal insufficiency and also of patients on regular hemodialysis treatment. GSH levels were from 19 to 70% higher in the erythrocytes of examined patients as compared to their corresponding age-matched controls. A correlation was found between the degree of renal insufficiency and the erythrocyte GSH level. No variations in plasma GSH levels which could be related to the degree of renal deterioration were observed. A routine hemodialysis did not significantly affect erythrocyte and plasma GSH levels. No significant differences in GSH levels between anemic and nonanemic uremic patients were observed.     

Photooxidation of cell membranes in the presence of hematoporphyrin derivative: reactivity of phospholipid and cholesterol hydroperoxides with glutathione peroxidase

The susceptibility of photodynamically-generated lipid hydroperoxides to reductive inactivation by glutathione peroxidase (GPX) has been investigated, using hematoporphyrin derivative as a photosensitizing agent and the human erythrocyte ghost as a target membrane. Photoperoxidized ghosts were reactive in a glutathione peroxidase/reductase (GPX/GRD)-coupled assay only after phospholipid hydrolysis by phospholipase A2 (PLA2). However, enzymatically determined lipid hydroperoxide values were consistently approx. 40% lower than iodometrically determined values throughout the course of photooxidation. Moreover, when irradiated ghosts were analyzed iodometrically during PLA2/GSH/GPX treatment, a residual 30-40% of non-reactive lipid hydroperoxide was observed. The possibility that cholesterol product(s) account for the non-reactive lipid hydroperoxide was examined by tracking cholesterol hydroperoxides in [14C]cholesterol-labeled ghosts. The sum of cholesterol hydroperoxides and GPX/GRD-detectable lipid hydroperoxides was found to agree closely with iodometrically determined lipid hydroperoxide throughout the course of irradiation. Thin-layer chromatography of total lipid extracts indicated that cholesterol hydroperoxide was unaffected by PLA2/GSH/GPX treatment, whereas most of the phospholipid peroxides were completely hydrolyzed and the released fatty acid peroxides were reduced to alcohols. It appears, therefore, that the GPX-resistant lipid hydroperoxides in photooxidized ghosts were derived primarily from cholesterol. Ascorbate plus Fe3+ produced a burst of free-radical lipid peroxidation in photooxidized, PLA2-treated ghosts. As expected for fatty acid hydroperoxide inactivation, the lipid peroxidation was inhibited by GSH/GPX, but only partially so, suggesting that cholesterol hydroperoxide-derived radicals play a major role in the reaction.     

The yeast glutaredoxins are active as glutathione peroxidases

The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, which are active as glutathione-dependent oxidoreductases. Our studies show that changes in the levels of glutaredoxins affect the resistance of yeast cells to oxidative stress induced by hydroperoxides. Elevating the gene dosage of GRX1 or GRX2 increases resistance to hydroperoxides including hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide. The glutaredoxin-mediated resistance to hydroperoxides is dependent on the presence of an intact glutathione system, but does not require the activity of phospholipid hydroperoxide glutathione peroxidases (GPX1-3). Rather, the mechanism appears to be mediated via glutathione conjugation and removal from the cell because it is absent in strains lacking glutathione-S-transferases (GTT1, GTT2) or the GS-X pump (YCF1). We show that the yeast glutaredoxins can directly reduce hydroperoxides in a catalytic manner, using reducing power provided by NADPH, GSH, and glutathione reductase. With cumene hydroperoxide, high pressure liquid chromatography analysis confirmed the formation of the corresponding cumyl alcohol. We propose a model in which the glutathione peroxidase activity of glutaredoxins converts hydroperoxides to their corresponding alcohols; these can then be conjugated to GSH by glutathione-S-transferases and transported into the vacuole by Ycf1.     

NMR studies of exchange between intra- and extracellular glutathione in human erythrocytes

Glutathione is the main source of intracellular antioxidant protection in the human erythrocyte and its redox status has frequently been used as a measure of oxidative stress. Extracellular glutathione has been shown to enhance intracellular reduced glutathione levels in some cell types. However, there are conflicting reports in the literature and it remains unclear as to whether erythrocytes can utilise extracellular glutathione to enhance the intracellular free glutathione pool. We have resolved this issue using a 13C-NMR approach. The novel use of L-gamma-glutamyl-L-cysteinyl-[2-13C]glycine allowed the intra- and extracellular glutathione pools to be distinguished unequivocally, enabling the direct and non-invasive observation over time of the glutathione redox status in both compartments. The intracellular glutathione redox status was measured using 1H spin-echo NMR, while 13C[1H-decoupled] NMR experiments were used to measure the extracellular status. Extracellular glutathione was not oxidised in the incubations, and did not affect the intracellular glutathione redox status. Extracellular glutathione also did not affect erythrocyte glucose metabolism, as measured from the lactate-to-pyruvate ratio. The results reported here refute the previously attractive hypothesis that, in glucose-starved erythrocytes, extracellular GSH can increase intracellular GSH concentrations by releasing bound glutathione from mixed disulfides with membrane proteins.     

Reaction of ozone with sulfhydryls of human erythrocytes

Oxidation of GSH by ozone yielded 60% GSSG. Exposure of human erythrocytes to ozone caused oxidation of intracellular GSH. Between 4 and 6% of the administered ozone caused GSH oxidation. No more than 30% of the GSH oxidized by ozone could be accounted for by GSSG in the erythrocyte. The GSSG formed in the erythrocyte was rapidly reduced and the pentose phosphate pathway was stimulated. When GSH and unsealed erythrocyte ghosts were simultaneously exposed to ozone, 6–11% of the oxidized GSH could be accounted for as mixed disulfide of protein and GSH. When GSH and cytoplasmic proteins from the erythrocyte were simultaneously exposed to ozone, 5–7% of the oxidized GSH could be accounted for as mixed disulfide. Ozone generated membrane protein disulfide crosslinks when erythrocyte ghosts, but not intact erythrocytes, were exposed. Ozone had no effect on glucose uptake and did not change oxyhemoglobin content of the erythrocytes.     

Plant catechols prevent lipid peroxidation in human plasma and erythrocytes

The antioxidant activity of several plant catechol derivatives was tested in buffer, plasma, and human erythrocytes. In buffer, chlorogenic acid (CGA), caffeic acid (CA), and dihydrocaffeic acid (DCA) reduced ferric iron equally well in the ferric reducing antioxidant power (FRAP) assay. Low concentrations of the polyphenols enhanced the ability of plasma to reduce ferric iron by about 10%. In plasma, lipid hydroperoxide and F₂-isoprostane formation induced by a water-soluble free radical initiator were reduced by CGA at concentrations as low as 20 M. During incubation at 37°C, human erythrocytes took up DCA, but not CGA, and intracellular DCA enhanced the ability of erythrocytes to reduce extracellular ferricyanide. When intact erythrocytes were exposed to oxidant stress generated by liposomes containing small amounts of lipid hydroperoxides, extracellular CGA at a concentration of 5 M decreased both lipid peroxidation in the liposomes, and spared -tocopherol in erythrocyte membranes. These results suggest that the catechol structure of these compounds convey the antioxidant effect in plasma and in erythrocytes.     

Hydroperoxide-metabolizing systems in rat liver.

1. Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions. 2. Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3--4 mumol X min-1 X gram liver-1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per mumol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity. 3. GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 mumol X min-1 X gram liver-1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole. 4. A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed. 5. Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation. 6. Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts. 7. A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.     

The role of human glutathione S-transferases hGSTA1-1 and hGSTA2-2 in protection against oxidative stress.

In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human alpha-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver. We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli. Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide. In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates. For example, the catalytic efficiency (kcat/Km) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s-1 mM-1, respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s-1 mM-1, respectively. Immunotitration studies with human liver extracts showed that the antibodies against human alpha-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively. GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates. These results show that the alpha-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver. Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetraenoic acid, an important intermediate in the 5-lipoxygenase pathway.     

Anomeric specificity of glycolysis in a non glucokinase-containing cell

In rat erythrocyte homogenates, the phosphorylation of D-glucose measured at 30 degrees C over a wide range of glucose concentrations (50 microM to 20 mM) yielded in a double reciprocal plot a single straight line with a Km close to 0.06 mM and a maximal velocity close to 47 nmol/60 min per mg hemoglobin. At 8 degrees C, the rate of glucose phosphorylation was 60% higher in the presence of beta-D-glucose than alpha-D-glucose. Yet, in intact erythrocytes incubated at 8 degrees C in the presence of beta-D-glucose (4 or 7 mM), the glucose-induced increment in lactic acid output represented no more than 39 to 74% of that found in the presence of alpha-D-glucose. Thus, a greater rate of glycolysis in the presence of alpha-D-glucose was observed in a cell devoid of glucokinase and containing a hexokinase with preference for beta-D-glucose. These findings indicate that the anomeric specificity of glycolysis in intact cells cannot be predicted and does not necessarily depend on the anomeric preference of glucose-phosphorylating enzyme(s).     

Beneficial effect of succinic acid monoethyl ester on erythrocyte membrane bound enzymes and antioxidant status in streptozotocin-nicotinamide induced type 2 diabetes

Succinic acid monoethyl ester (EMS) was recently proposed as an insulinotropic agent for the treatment of non-insulin dependent diabetes mellitus. In the present study the effect of EMS and metformin on erythrocyte membrane bound enzymes and antioxidants activity in plasma and erythrocytes of streptozotocin-nicotinamide induced type 2 diabeteic model was investigated. Succinic acid monoethyl ester was administered intraperitonially for 30 days to control and diabetic rats. The effect of EMS on glucose, insulin, hemoglobin, glycosylated hemoglobin, TBARS, hydroperoxide, superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (Gpx), glutathione-S-transferase (GST), vitamins C and E, reduced glutathione (GSH) and membrane bound enzymes were studied. The effect of EMS was compared with metformin, a reference drug. The levels of glucose, glycosylated hemoglobin, TBARS, hyderoperoxide, and vitamin E were increased significantly whereas the level of insulin and hemoglobin, as well as antioxidants (SOD, CAT, Gpx, GST, vitamin C and GSH) membrane bound total ATPase, Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase were decreased significantly in streptozotocin-nicotinamide diabetic rats. Administration of EMS to diabetic rats showed a decrease in the levels of glucose, glycosylated hemoglobin, lipid peroxidation markers and vitamin E. In addition the levels of insulin, hemoglobin, enzymic antioxidants, vitamin C, and GSH and the activities of membrane bound enzymes also were increased in EMS and metformin treated diabetic rats. The present study indicates that the EMS possesses a significant beneficial effect on erythrocyte membrane bound enzymes and antioxidants defense system in addition to its antidiabetic effect.     

Glutathione transferases: a possible role in the detoxication and repair of DNA and lipid hydroperoxides

Two types of GSH peroxidase occur in the cell both of which detoxify fatty acid hydroperoxides, thymine hydroperoxide and DNA hydroperoxides. One is a Se-dependent enzyme which also detoxifies H2O2. The other contains members of the GSH transferase supergene family. These non-selenium dependent GSH peroxidases do not detoxify H2O2 and have substrate specificities varying markedly with the isoenzyme. Of particular interest is GSH transferase 5*-5* an enzyme extracted from the nucleus with urea which has a relatively high activity towards DNA hydroperoxide. The possible role of these enzymes in the detoxication of lipid and DNA hydroperoxides is discussed and it is pointed out that they may be important participants in mechanism for the repair of free-radical damage.     

The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31P NMR study.

1. The polymorphic phase behaviour of aqueous dispersions of phosphatidylethanolamines isolated from human erythrocytes, hen egg yolk and Escherichia coli have been investigated employing 31P NMR techniques. All species exhibit well defined, reversible bilayer to hexagonal (H11) phase transitions as the temperature is increased. The temperatures at which these transition take place (10, 25--30 and 55--60 degrees C for erythrocyte, egg yolk and E. coli phosphatidylethanolamine, respectively) are sensitive to the fatty acid composition, occurring at a temperature up to 10 degrees C above the high temperature end of the hydrocarbon phase transition as detected by differential scanning calorimetry. In some cases the bilayer to hexagonal (H11) transitions may also be detected employing calorimetric techniques. 2. The addition of equimolar concentrations of cholesterol to these naturally occurring phosphatidylethanolamines does not dramatically affect the bilayer-hexagonal (H11) transition temperature, producing changes of up to 10 degrees C. 3. 18 : 1t/18 : 1t phosphatidylethanolamine undergoes the bilayer to hexagonal (H11) phase transition as the temperature is increased through the interval 50--55 degrees C. Alternatively, hydrated 12 : 0/12 : 0 phosphatidylethanolamine remains in the bilayer phase at temperatures up to 90 degrees C (50 degrees C above the hydrocarbon phase transition temperature). 4. The presence of 100 mM NaCl or 10 mM CaCl2 in aqueous dispersions of egg yolk phosphatidylethanolamine does not alter the temperature-dependent polymorphic phase behaviour significantly. However, at 40 degrees C, increasing the p2H above 8.0 results in progressive inhibition of the hexagonal (H11) phase and the appearance of a phase possibly of cubic structure at p2H 9.0. At p2H 10.0 the bilayer phase is preferred. 5. It is suggested that in biomembranes containing phosphatidylethanolamine as a majority species (such as that of E. coli) the fatty acid composition may primarily reflect the need to maintain bilayer structure. Alternatively, it is pointed out that in mammalian membranes such as that of the erythrocyte, phosphatidylethanolamine tends to destabilize bilayer structure. The resulting possibility that transitory non-bilayer lipid configurations may occur may be directly related to many important properties of biological membranes.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

Mechanisms of ascorbic acid recycling in human erythrocytes.

Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of D-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM D-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by D-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75-90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of D-glucose metabolism in the pentose cycle, but that in the absence of D-glucose DHA reduction can also utilize NADH.     

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides in solution

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides has been performed, as a model for the reactions of lipid hydroperoxides with vitamin E radical in biological systems. The rates of reaction of hydroperoxides (n-butyl hydroperoxide 1, sec-butyl hydroperoxide 2, and tert-butyl hydroperoxide 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl 4) in benzene solution have been determined spectrophotometrically. The second-order rate constants, k-1, obtained are 1.34 x 10(-1) M-1s-1 for 1, 2.42 x 10(-1) M-1s-1 for 2, and 3.65 x 10(-1) M-1s-1 for 3 at 25.0 degrees C. The result indicates that the rate constants increase as the total electron donating capacity of the alkyl substituents at alpha-carbon atom of hydroperoxides increases. The above rates, k-1, are about seven order of magnitude lower than those, k1, for the reaction of vitamin E with peroxyl radical.     

Phospholipid hydroperoxides are substrates for non-selenium glutathione peroxidase.

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl- or sn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM(-1) min(-1)) were k(1) = 1-3 x 10(5) and k(2) = 4-11 x 10(4). In the presence of 0.36 mM GSH, apparent K(m) was 120-130 microM and apparent V(max) was 1.5-1.6 micromol/min/mg of protein. Assays with H(2)O(2) and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7-8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K(i) 4 miroM. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.     

Rat liver cytosolic glutathione peroxidase: reactivity with linoleic acid hydroperoxide and cumene hydroperoxide.

The reactivity of rat liver glutathione (GSH) peroxidase with two hydroperoxides was determined using integrated rate equations. The bimolecular rate constant for the reaction of GSH peroxidase with linoleic acid hydroperoxide is approximately four times the rate constant with cumene hydroperoxide. The reactivity toward reduced glutathione is not altered by different hydroperoxides. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for lipid hydroperoxide in rat liver is approximated at 9.5 × 10^?5 min.     

Anomeric preference of glucose phosphorylation and glycolysis in human erythrocytes

Lactate output from the alpha and beta anomers of glucose was measured in intact human erythrocytes at 37 degrees C; and glucose anomer phosphorylation, in human erythrocyte homogenates. The rates of both glucose metabolism (lactate output) and phosphorylation were higher in the presence of beta-D-glucose as distinct from alpha-D-glucose at three glucose concentrations used (2, 5, and 10 mM). Thus, the v beta/v alpha ratios of metabolism and phosphorylation of glucose at 2 mM were 1.24 and 1.22, respectively. The results indicate that the beta preference of hexokinase, a rate-limiting enzyme in glycolysis, is reflected in beta-preferential glycolysis.     

Evidence for a role of taurine in the in vitro oxidative toxicity of neutrophils toward erythrocytes

Production of hydrogen peroxide and secretion of myeloperoxidase by stimulated neutrophils resulted in myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl), the reaction of HOCl with taurine to yield taurine monochloramine (TauNHCl), and accumulation of TauNHCl in the extracellular medium. When erythrocytes were present, the yield of TauNHCl was lower as the result of uptake of TauNHCl into erythrocytes. The zwitterion taurine was not taken up, but the anion TauNHCl and other anionic oxidants including taurine dichloramine (TauNCl2) and L-alanine chloramines were transported into erythrocytes by the anion-transport system. Oxidation of intracellular components such as glutathione (GSH) by taurine chloramines resulted in reduction of the chloramines and trapping of taurine within erythrocytes. At high oxidant:erythrocyte ratios, TauNHCl also oxidized hemoglobin (Hb) and depleted ATP, but caused little lysis. TauNCl2 was much more effective as a lytic agent. At low oxidant:erythrocyte ratios, the chloramines caused net loss of GSH when no glucose was provided, but Hb was not oxidized and GSH content returned to normal when glucose was added. Therefore, anionic chloramines may mediate oxidative toxicity when the neutrophil:erythrocyte ratio is high. Under more physiologic conditions, chlorination of taurine by neutrophils and the uptake and reduction of TauNHCl by erythrocytes prevents accumulation of oxidants and may protect blood cells, plasma components, and tissues against oxidative toxicity.     

Attachment of Sendai virus particles to cell membranes: dissociation of adsorbed virus particles with dithiothreitol.

Sendai virus particles bind to human erythrocytes at 4 degrees C and fuse with them at 37 degrees C. The present work describes a new method by which adsorbed virus particles can be removed from human erythrocytes, allowing quantitative determination of the number of virus particles which can bind and fuse with human erythrocyte membranes. Through the use of 125I-labeled Sendai virus particles, it is shown that incubation with 50 mM dithiothreitol removed about 90 to 95% of adsorbed virus particles. Fused virus particles were resistant to treatment with dithiothreitol. Negligible amounts of 125I-labeled Sendai virus particles were removed by treatment with dithiothreitol after incubation of virus-cell complexes at 37 degrees C. Trypsinized virus particles were able to attach to, but not fuse with, human erythrocytes even after prolonged incubation at 37 degrees C. Treatment with dithiothreitol removed as much as 80 to 85% of trypsinized virus particles incubated with human erythrocytes at 37 degrees C. A quantitative determination revealed that about 1,000 to 1,200 and 600 to 800 Sendai virus particles can bind to or fuse with human erythrocytes, respectively.