p-Bromophenacyl bromide prevents cumene hydroperoxide-induced mitochondrial permeability transition by inhibiting pyridine nucleotide oxidation

Mitochondrial permeability transition is commonly characterized as a Ca2+ -dependent non-specific increase in inner membrane permeability that results in swelling of mitochondria and their de-energization. In the present study, the effect of different inhibitors of phospholipase A2--p-bromophenacyl bromide, dibucaine, and aristolochic acid--on hydroperoxide-induced permeability transitions in rat liver mitochondria was tested. p-Bromophenacyl bromide completely prevented the hydroperoxide-induced mitochondrial permeability transition while the effects of dibucaine or aristolochic acid were negligible. Organic hydroperoxides added to mitochondria undergo reduction to corresponding alcohols by mitochondrial glutathione peroxidase. This reduction occurs at the expense of GSH which, in turn, can be reduced by glutathione reductase via oxidation of mitochondrial pyridine nucleotides. The latter is considered a prerequisite step for mitochondrial permeability transition. Among all the inhibitors tested, only p-bromophenacyl bromide completely prevented hydroperoxide-induced oxidation of mitochondrial pyridine nucleotides. Interestingly, p-bromophenacyl bromide had no affect on mitochondrial glutathione peroxidase, but reacted with mitochondrial glutathione that prevented pyridine nucleotides from being oxidized. Our data suggest that p-bromophenacyl bromide prevents hydroperoxide-induced deterioration of mitochondria via interaction with glutathione rather than through inhibition of phospholipase A2.     

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.     

Hydroperoxide-induced loss of pyridine nucleotides and release of calcium from rat liver mitochondria

It has been previously reported (L?tscher, H. R., Winterhalter, K. H., Carafoli, E., and Richter, C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4340-4344) that in Ca2+-loaded mitochondria hydroperoxides induce a release of Ca2+ from mitochondria and an irreversible oxidation of mitochondrial pyridine nucleotides. Here we show that in the presence of Ca2+ oxidized mitochondrial pyridine nucleotides are hydrolyzed inside mitochondria and that nicotinamide is released from mitochondria. The extent of the hydrolysis of NAD(P)+ is dependent on the amount of both hydroperoxide and Ca2+. The hydrolysis is reversible in the presence of added nicotinamide. The release of Ca2+ from mitochondria is electroneutral, and is directly or indirectly dependent on oxidized mitochondrial pyridine nucleotides. By contrast, the uptake of Ca2+ most probably does not require the present of reduced pyridine nucleotides. Control experiments show that even under the most drastic conditions employed in this study (100 nmol of Ca2+ and 85 nmol of t-butylhydroperoxide/mg of protein) mitochondria retain a considerable degree of functional integrity.     

Rat lung glutathione release: response to oxidative stress and selenium deficiency

We performed experiments to characterize the glutathione-dependent metabolism occurring during tert-butyl hydroperoxide infusion in isolated perfused rat lungs and to examine the effect of selenium deficiency on this metabolism. Selenium deficiency resulted in decreased lung glutathione peroxidase activity but normal glutathione reductase activity and glutathione content. Infusion of the hydroperoxide into control lungs caused a proportional increase in tissue glutathione disulfide (GSSG) concentration and release of GSSG into the perfusate up to an infusion rate of 250 nmol of tert-butyl hydroperoxide X min-1 X 100 g body wt-1. Infusion rates greater than this resulted in continued rise of tissue GSSG concentrations but GSSG release into the perfusate plateaued. Infusion of tert-butyl hydroperoxide into selenium-deficient rat lungs resulted in much lower concentrations of tissue GSSG and GSSG release into the perfusate; however, release in the selenium-deficient rat lung was also found to be saturable at infusion rates of 450 nmol of tert-butyl hydroperoxide X min-1 X 100 g of body wt-1. Selenium deficiency in the rat decreases the rate of reduction of infused tert-butyl hydroperoxide by glutathione and may predispose the lung to free radical damage.     

Relation between glutathione redox changes and biliary excretion of taurocholate in perfused rat liver

The influence of the intracellular glutathione status on bile acid excretion was studied in the perfused rat liver. Perturbation of the thiol redox state by short term additions of diamide (100 microM) or hydrogen peroxide (250 microM) or t-butyl hydroperoxide (250 microM) led to a reversible inhibition of biliary taurocholate release without affecting hepatic uptake; inhibition amounted to 45% for diamide and 90% for the hydroperoxides. Concomitantly, the bile acid accumulated intracellularly. Bile flow increased from 1.3 to 2.0 microliters X min-1 X g liver-1 upon infusion of taurocholate (10 microM); the latter value was suppressed to 1.2 microliters X min-1 X g liver-1 by the addition of t-butyl hydroperoxide (250 microM). Similarly, the hepatic disposition of another bile constituent, bilirubin, was suppressed by 70% upon addition of hydrogen peroxide. While the addition of hydrogen peroxide inhibited also the endogenous release of bile acids almost completely, endogenous bile flow was much less affected, decreasing from 1.3 to 1.0 microliters X min-1 X g liver-1. Measurement of [14C]erythritol clearance showed bile/perfusate ratios of about unity both in the absence and presence of hydrogen peroxide, suggesting canalicular origin of the bile under both conditions. In livers from Se-deficient rats low in Se-GSH peroxidase (less than 5% of controls), hydrogen peroxide inhibited taurocholate transport substantially less, providing evidence for the involvement of glutathione in mediating the inhibition observed in normal livers. The percentage inhibition of taurocholate release and intracellular glutathione disulfide (GSSG) content were closely correlated. The addition of t-butyl hydroperoxide caused a several-fold increase of biliary GSSG release, whereas biliary GSH release was even decreased. The results establish a role of glutathione in canalicular taurocholate disposition.     

Regulation of cyclosporin A sensitive mitochondrial permeability transition by the redox state of pyridine nucleotides

The mechanisms involved in the induction of cyclosporine A sensitive mitochondrial swelling by oxidative stress were investigated in isolated guinea pig liver mitochondria. The aim of our study was to investigate, if swelling is inevitably associated with the oxidation of pyridine nucleotides, and if the oxidized pyridine nucleotides have to be hydrolysed for the induction of mitochondrial swelling. Quantitative measurement of oxidized pyridine nucleotides was performed with HPLC. Mitochondrial swelling was recorded by monitoring the decrease in light scattering of the mitochondrial suspension. Reduction and oxidation of pyridine nucleotides were followed by monitoring the changes of the autofluorescence signal of reduced pyridine nucleotides. Qualitative measurement of mitochondrial membrane potential was performed with the fluorescence indicator rhodamine 123. Neither t-butyl hydroperoxide nor the dissipation of the mitochondrial inner membrane potential with FCCP (carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone) induced the opening of the membrane permeability transition pore, unless an extensive oxidation of mitochondrial pyridine nucleotides took place. Mitochondrial swelling induced by our experimental conditions was always sensitive to cyclosporine A and accompanied by a cyclosporine A sensitive release of inner mitochondrial pyridine nucleotides without pyridine nucleotide hydrolysis. Not the cycling of calcium across the mitochondrial inner membrane but the accumulation of calcium inside the mitochondria was a prerequisite for mitochondrial swelling. The mitochondrial membrane permeability transition is neither caused nor accompanied by the hydrolysis of mitochondrial pyridine nucleotides.     

The relative effectiveness of human plasma glutathione peroxidase as a catalyst for the reduction of hydroperoxides by glutathione

To reveal clues to the function of human plasma glutathione peroxidase (GPx), we investigated its catalytic effectiveness with a variety of hydroperoxides. Comparisons of hydroperoxides as substrates for plasma GPx based on the ratio ofV _max /K _m were blocked by the limited solubility of the organic hydroperoxides, which prevented kinetic saturation of the enzyme at the chosen glutathione concentration. Therefore, we compared the hydroperoxides by the fold increase in the apparent first-order rate constants of their reactions with glutathione owing to catalysis by plasma GPx. The reductions of aromatic and small hydrophobic hydroperoxides (cumene hydroperoxide,t-amyl hydroperoxide,t-butyl hydroperoxide, paramenthane hydroperoxide) were better catalyzed by plasma GPx than were reductions of the more “physiological” substrates (linoleic acid hydroperoxide, hydrogen peroxide, peroxidized plasma lipids, and oxidized cholesterol).     

The role of selenium peroxidases in the protection against oxidative damage of membranes

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.     

Phospholipase C-dependent hydrolysis of phosphatidylcholine hydroperoxides to diacylglycerol hydroperoxides and its reduction by phospholipid hydroperoxide glutathione peroxidase

We have shown that 1,2-diacylglycerol hydroperoxides activate protein kinase C (PKC) as efficiently as does phorbol ester [Takekoshi S, Kambayashi Y, Nagata H, Takagi T, Yamamoto Y, Watanabe K. Activation of protein kinase C by oxidized diacylglycerol. Biochem Biophys Res Commun 1995; 217: 654-660]. 1,2-Diacylglycerol hydroperoxides also stimulate human neutrophils to release superoxide whereas their hydroxides do not [Yamamoto Y, Kambayashi Y, Ito T, Watanabe K, Nakano M. 1,2-Diacylglycerol hydroperoxides induce the generation and release of superoxide anion from human polymorphonuclear leukocytes. FEBS Lett 1997; 412: 461-464]. One of the proposed mechanisms for the formation of 1,2-diacylglycerol hydroperoxides is the hydrolysis of phosphatidylcholine hydroperoxides by phospholipase C (PLC). To confirm this hypothesis, we incubated 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) liposomes containing PLPC hydroperoxides (PLPC-OOH) with Bacillus cereus PLC and found 1-palmitoyl-2-linoleoylglycerol (PLG) and its hydroperoxide (PLG-OOH) were produced. PLC hydrolyzed the two substrates without preference, as the yields of PLG and PLG-OOH were the same even though cholesterol was incorporated into liposomes to increase bilayer integrity. Phospholipid hydroperoxide glutathione peroxidase (PHGPX) reduced PLG-OOH to its hydroxide in the presence of glutathione while the conventional cytosolic glutathione peroxidase did not. These data suggest that PLC hydrolyzes oxidized biomembranes to give 1,2-diacylglycerol hydroperoxides for PKC stimulation but PHGPX may prevent neutrophil stimulation by reducing 1,2-diacylglycerol hydroperoxides to their hydroxides.     

Effect of ebselen on Ca2+ transport in mitochondria

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclosporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.     

The role of hydroperoxides as calcium release agents in rat brain mitochondria

Hydroperoxides have previously been shown to induce Ca2+ release from intact rat liver mitochondria via a specific release pathway. Here it is reported that, in rat brain mitochondria, a hydroperoxide-induced Ca2+ release is also operative but is of minor importance. Hydroperoxide stimulates Ca2+ release in the presence of ruthenium red about twofold at a Ca2+ load of 40 nmol/mg mitochondrial protein. After addition of hydroperoxide, Ca2+ release from brain mitochondria can still be evoked by Na+. In the presence of succinate and rotenone, hydroperoxide induces only a very limited oxidation of pyridine nucleotides, most probably due to the low level of glutathione peroxidase (EC 1.11.1.9) and glutathione reductase (EC 1.6.4.2) found in brain mitochondria. Similar to liver mitochondria, a NADase (EC 3.2.2.5) activity is found in brain mitochondria. Its localization and sensitivity toward ADP and ATP, however, is different from that of the liver mitochondrial enzyme.     

Lipid peroxidation during ischemia depends on ischemia time in warm ischemia and reperfusion of rat liver

Prolonged hepatic warm ischemia has been incriminated in oxidative stress after reperfusion. However, the magnitude of oxidative stress during ischemia has been controversial. The aims of the present study were to elucidate whether lipid peroxidation progressed during ischemia and to clarify whether oxidative stress during ischemia aggravated the oxidative damage after reperfusion. Rats were subjected to 30 to 120 min of 70% warm ischemia alone or followed by reperfusion for 60 min. Lipid peroxidation (LPO) was evaluated by amounts of phosphatidylcholine hydroperoxide (PC-OOH) and phosphatidylethanolamine hydroperoxide (PE-OOH) as primary LPO products. Total amounts of malondialdehyde and 4-hydroxy-2-nonenal (MDA + 4-HNE), degraded from hydroperoxides, were also determined. PC-OOH and PE-OOH significantly increased at 60 and 120 min ischemia with concomitant increase of oxidized glutathione. These hydroperoxides did not increase at 60 min reperfusion after 60 min ischemia, whereas they did increase at 60 min reperfusion after 120 min ischemia with deactivation of phospholipid hydroperoxide glutathione peroxidase and superoxide dismutase. The amount of MDA + 4-HNE exhibited similar changes, but the velocity of production dropped with ischemic time longer than 60 min. In conclusion, oxidative stress progressed during ischemia and triggered the oxidative injury after reperfusion. Secondary LPO products are less sensitive, especially during ischemia, which may cause possible underestimation and discrepancy.     

Phospholipase C-dependent hydrolysis of phosphatidylcholine hydroperoxides to diacylglycerol hydroperoxides and its reduction by phospholipid hydroperoxide glutathione peroxidase

Abstract

We have shown that 1,2-diacylglycerol hydroperoxides activate protein kinase C (PKC) as efficiently as does phorbol ester [Takekoshi S, Kambayashi Y, Nagata H, Takagi T, Yamamoto Y, Watanabe K. Activation of protein kinase C by oxidized diacylglycerol. Biochem Biophys Res Commun 1995; 217: 654-660]. 1,2-Diacylglycerol hydroperoxides also stimulate human neutrophils to release superoxide whereas their hydroxides do not [Yamamoto Y, Kambayashi Y, Ito T, Watanabe K, Nakano M. 1,2-Diacylglycerol hydroperoxides induce the generation and release of superoxide anion from human polymorphonuclear leukocytes. FEBS Lett 1997; 412: 461-464]. One of the proposed mechanisms for the formation of 1,2-diacylglycerol hydroperoxides is the hydrolysis of phosphatidylcholine hydroperoxides by phospholipase C (PLC). To confirm this hypothesis, we incubated 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) liposomes containing PLPC hydroperoxides (PLPC-OOH) with Bacillus cereus PLC and found 1-palmitoyl-2-linoleoylglycerol (PLG) and its hydroperoxide (PLG-OOH) were produced. PLC hydrolyzed the two substrates without preference, as the yields of PLG and PLG-OOH were the same even though cholesterol was incorporated into liposomes to increase bilayer integrity. Phospholipid hydroperoxide glutathione peroxidase (PHGPX) reduced PLG-OOH to its hydroxide in the presence of glutathione while the conventional cytosolic glutathione peroxidase did not. These data suggest that PLC hydrolyzes oxidized biomembranes to give 1,2-diacylglycerol hydroperoxides for PKC stimulation but PHGPX may prevent neutrophil stimulation by reducing 1,2-diacylglycerol hydroperoxides to their hydroxides.     

Purification and characterization of a novel monomeric glutathione peroxidase from rat liver

A novel glutathione peroxidase, which is active toward hydroperoxides of phospholipid in the presence of a detergent, has been purified to homogeneity from a rat liver postmicrosomal supernatant fraction by ammonium sulfate fractionation and three different column chromatographies. From a DE52 column, glutathione peroxidase active toward phosphatidylcholine dilinoleoyl hydroperoxides was eluted in one major and two minor peaks. The enzyme in the major peak was found to be separated from the "classic" glutathione peroxidase and glutathione S-transferases and further purified by Sephacryl S-200 and Mono Q column chromatographies. The purified enzyme was found to be homogeneous on polyacrylamide gel electrophoresis under nondenaturing conditions as well as that in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 22,000, and that by gel filtration was comparable, indicating that the enzyme protein is a single polypeptide. The purified enzyme was found to catalyze the reduction of phosphatidylcholine dilinoleoyl hydroperoxides to the corresponding hydroxy derivatives. The isoelectric point of the enzyme was found at pH 6.2, and the optimum pH for the enzyme activity was 8.0. The enzyme was active toward cumene hydroperoxide, H2O2, and 1-monolinolein hydroperoxides in the absence of a detergent. The enzyme activity toward phospholipid hydroperoxides was minute in the absence of a detergent but was remarkably enhanced by the addition of a detergent. From these results, the presently purified enzyme is obviously different from the classic glutathione peroxidase and also from phospholipid hydroperoxide glutathione peroxidase purified from pig heart (Ursini, F., Maiorino, M., and Gregolin, C. (1985) Biochim. Biophys. Acta 839, 62-70), though considerably similar to the latter.     

Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione peroxidase

We have recently characterized the major hydroperoxide-reducing enzyme of human plasma as a glutathione peroxidase (Maddipati, K. R., Gasparski, C., and Marnett, L. J. (1987) Arch. Biochem. Biophys. 254, 9-17). We now report the purification and kinetic characterization of this enzyme. The purification steps involved ammonium sulfate precipitation, hydrophobic interaction chromatography on phenyl-Sepharose, anion exchange chromatography, and gel filtration. The purified peroxidase has a specific activity of 26-29 mumol/min/mg with hydrogen peroxide as substrate. The human plasma glutathione peroxidase is a tetramer of identical subunits of 21.5 kDa molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is different from human erythrocyte glutathione peroxidase. The plasma peroxidase is a selenoprotein containing one selenium per subunit. Unlike several other glutathione peroxidases this enzyme exhibits saturation kinetics with respect to glutathione (Km for glutathione = 4.3 mM). The peroxidase exhibits high affinity for hydroperoxides with Km values ranging from 2.3 microM for 13-hydroperoxy-9,11-octadecadienoic acid to 13.3 microM for hydrogen peroxide at saturating glutathione concentration. These kinetic parameters are suggestive of the potential of human plasma glutathione peroxidase as an important regulator of plasma hydroperoxide levels.     

New Plant Growth Retardants

Phosphatidylcholine hydroperoxide was assayed with phospholipase A₂ and glutathione peroxidase, based on fluorometry with N(9-acridinyl)maleimide. The hydroperoxide was poorly reduced by glutathione peroxidase, and was converted by phospholipase A₂ into reactable forms of glutathione peroxidase. A linear relationship was found between hydroperoxides assayed by the enzymatic and chemical methods in the range from 0.05 to 5.0 nmol with 0.5 to 1.5 mg of the sample. The hydroperoxides of fatty acids, triacylglycerol and phosphatidylcholine were assayed in their mixtures and in commercial lecithins.     

Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity

Automated assays for catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase are presented. The assay for catalase is based on the peroxidatic activity of the enzyme. The glutathione peroxidase and reductase assays measure the consumption of NADPH following the reduction of t-butyl hydroperoxide and oxidized glutathione, respectively. The assay for superoxide dismutase is based on the reduction of cytochrome c. All assays utilize the Cobas FARA clinical automated analyzer and provide considerable time savings over the manual assays.     

Oxygen toxicity in the perfused rat liver and lung under hyperbaric conditions.

1. In the lung and liver of tocopherol-deficient rats, the activities of glutathione peroxidase and glucose 6-phosphate dehydrogenase were increased substantially, suggesting an important role for both enzymes in protecting the organ against the deleterious effects of lipid peroxides. 2. Facilitation of the glutathione peroxidase reaction by infusing t-butyl hydroperoxide caused the oxidation of nicotinamide nucleotides and glutathione, resulting in a concomitant increase in the rate of release of oxidized glutathione into the perfusate. Thus the rate of production of lipid peroxide and H2O2 in the perfused organ could be compared by simultaneous measurement of the rate of glutathione release and the turnover number of the catalase reaction. 3. On hyperbaric oxygenation at 4 X 10(5)Pa, H2O2 production, estimated from the turnover of the catalase reaction, was increased slightly in the liver, and glutathione release was increased slightly, in both lung and liver. 4. Tocopherol deficiency caused a marked increase in lipid-peroxide formation as indicated by a corresponding increase in glutathione release under hyperbaric oxygenation, with a further enhancement when the tocopherol-deficient rats were also starved. 5. The study demonstrates that the primary response to hyperbaric oxygenation is an elevation of the rate of lipid peroxidation rather than of the rate of formation of H2O2 or superoxide.     

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase

The reduction of membrane-bound hydroperoxides is a major factor acting against lipid peroxidation in living systems. This paper presents the characterization of the previously described 'peroxidation-inhibiting protein' as a 'phospholipid hydroperoxide glutathione peroxidase'. The enzyme is a monomer of 23 kDa (SDS-polyacrylamide gel electrophoresis). It contains one gatom Se/22 000 g protein. Se is in the selenol form, as indicated by the inactivation experiments in the presence of iodoacetate under reducing conditions. The glutathione peroxidase activity is essentially the same on different phospholipids enzymatically hydroperoxidized by the use of soybean lipoxidase (EC 1.13.11.12) in the presence of deoxycholate. The kinetic data are compatible with a tert-uni ping-pong mechanism, as in the case of the 'classical' glutathione peroxidase (EC 1.11.1.9). The second-order rate constants (K1) for the reaction of the enzyme with the hydroperoxide substrates indicate that, while H2O2 is reduced faster by the glutathione peroxidase, linoleic acid hydroperoxide is reduced faster by the present enzyme. Moreover, the phospholipid hydroperoxides are reduced only by the latter. The dramatic stimulation exerted by Triton X-100 on the reduction of the phospholipid hydroperoxides suggests that this enzyme has an 'interfacial' character. The similarity of amino acid composition, Se content and kinetic mechanism, relative to the difference in substrate specificity, indicates that the two enzymes 'classical' glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are in some way related. The latter is apparently specialized for lipophylic, interfacial substrates.