Hydroperoxide Substrate Research Articles

Crystallization and preliminary X-ray analysis of the catalytic core of the alkylhydroperoxide reductase component AhpF from Escherichia coli.

Alkylhydroperoxide reductases (AhpR, E.C. 1.6.4.x) are essential for the oxygen tolerance of aerobic organisms, converting otherwise toxic hydroperoxides of lipids or nucleic acids to their corresponding alcohols. The AhpF component (521 amino-acid residues, 56.2 kDa) belongs to the family of pyridine nucleotide-disulfide oxidoreductases and channels electrons from NAD(P)H via a series of disulfides towards the AhpC component, which finally reduces the hydro-peroxide substrates. Crystals of the proteolytically truncated AhpF component (residues Asn208-Ala521) of the alkyl hydroperoxide reductase from Escherichia coli were grown under oxidizing conditions. The crystals belong to space group P3(2)21, with unit-cell parameters a = 60.4, c = 171.8 A. X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation. A molecular-replacement solution was found using the structure of thioredoxin reductase from Arabidopsis thaliana as a search model.

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.

Dirofilaria immitis:Molecular Cloning and Expression of a cDNA Encoding a Selenium-Independent Secreted Glutathione Peroxidase

A cDNA clone, Di29, encoding a homolog of glutathione peroxidase, was isolated from aDirofilaria immitisadult female cDNA expression library by a combination of polymerase chain reaction amplification with primers designed from theBrugia pahangiglutathione peroxidase gene sequence and hybridization screening ofD. immitiscDNA libraries. The Di29 nucleotide and deduced amino acid sequences were very similar to those described for lymphatic filariae and predicted a secreted form of glutathione peroxidase with a cysteine residue substituted for selenocysteine in the active site. The cDNA clone was expressed inEscherichia coliandSpodoptera frugiperdaSf9 insect cells, and the resulting recombinant proteins were purified for antibody production and assessment of enzymatic properties, respectively. An antiserum generated against theE. coli-expressed protein detected a protein of 29 kDa inD. immitisvia immunoblotting. This protein is expressed in adult worms (both sexes) and fourth stage larvae generated via 6 days ofin vitroculture, but was undetectable in microfilariae, and third stage larvae obtained either directly from mosquitoes or following 2 days of culture. The Di29-encoded recombinant protein was secreted from Sf9 insect cells and displayed low-level glutathione peroxidase activity against a range of hydroperoxide substrates, including hydrogen peroxide.

Interleukin-1-induced nuclear factor kappa B activation is inhibited by overexpression of phospholipid hydroperoxide glutathione peroxidase in a human endothelial cell line.

Oxygen radicals are commonly accepted mediators in the tumour necrosis factor-mediated nuclear factor kappa B (NF kappa B) signalling cascade, but evidence for their role during interleukin-1 (IL-1) signalling is lacking. To test the involvement of hydroperoxides we investigated whether IL-1-induced NF kappa B activation could be influenced by glutathione peroxidases (GPx). These enzymes remove hydroperoxides with various specificities for the hydroperoxide substrate. By overexpressing phospholipid hydroperoxide glutathione peroxidase (PHGPx), which characteristically reacts with lipophilic hydroperoxides, the roles of H2O2 and lipid hydroperoxides were assessed. A human umbilical endothelial cell line, ECV 304, was stably transfected with the genes for both PHGPx and selenophosphate synthetase (selD), which provides selenophosphate for selenoprotein biosynthesis. When grown in selenium-deficient culture medium, the double-transfected clone (ECVPHGPx+SelD+) expressed 5-fold higher (P<0.005) PHGPx activity (measured by phosphatidylcholine hydroperoxide removal) than controls. The rate of H2O2 removal was also significantly (P<0.01) higher in this clone. When grown with high levels of extracellular selenium (up to 100 nM selenite), PHGPx activity and H2O2 removal were enhanced substantially in control cells and transfected cells. Under these conditions, PHGPx activity was 1.7-fold (P<0.005) higher in ECVPHGPx+SelD+, but H2O2 removal was the same as in controls. IL-1-induced NF kappa B activation was inhibited by selenium supplementation in control cells. In ECVPHGPx+SelD+ under conditions of selenium restriction, IL-1 induced NF kappa B activation only to a similar extent as under conditions of selenium supplementation in controls, and activation was abolished with 50 nM sodium selenite. These results show that overexpressed PHGPx is sufficient to inhibit NF kappa B activation, and suggests that NF kappa B activation by IL-1 is mediated by a preferential substrate of PHGPx, such as a fatty acid hydroperoxide, rather than by H2O2, the preferred substrate of the more abundant cytosolic GPx.

Identification of a naturally occurring peroxidase-lipoxygenase fusion protein.

A distant relative of catalase that is specialized for metabolism of a fatty acid hydroperoxide was identified. This heme peroxidase occurs in coral as part of a fusion protein, the other component of which is a lipoxygenase that forms the hydroperoxide substrate. The end product is an unstable epoxide (an allene oxide) that is a potential precursor of prostaglandin-like molecules. These results extend the known chemistry of catalase-like proteins and reveal a distinct type of enzymatic construct involved in the metabolism of polyunsaturated fatty acids.

Prostaglandin H synthase-catalyzed oxidation of all-trans- and 13-cis-retinoic acid to carbon-centered and peroxyl radical intermediates.

Due to the importance of all-trans-retinoic acid (RA) in the treatment of various dermatological conditions and the wide distribution of prostaglandin H synthase (PGHS) in tissues, we have further examined the mechanisms involved in the hydroperoxide-dependent cooxidation of RA and its isomer, 13-cis-retinoic acid ((13Z)-RA), by PGHS. Hydroperoxide-dependent, PGHS-catalyzed oxidation of RA and (13Z)-RA was shown to form free radical adducts, using electron spin resonance (ESR) spin trapping techniques and 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-OOH-18:2) as hydroperoxide substrates. Utilization of the spin trap alpha-phenyl-N-tert-butylnitrone (PBN) resulted in the detection of (13Z)-RA-PBN and RA-PBN adducts whose spectra were characterized by hyperfine coupling constants of aH = 4.16/aN = 15.69 and aH = 3.01/aN =15.92, respectively. Identical experiments under anaerobic conditions were carried out using the spin trap 2-methyl-2-nitrosopropane (NtB) which yielded nitroxide adducts whose spectra were characterized by a triplet of doublets with values of aH = 3.49/aN = 15.84 for the (13Z)-RA adduct and aH = 3.49/aN = 15.88 for the RA adduct. These results are indicative of secondary carbon-centered radical formation. We also used (+)-benzo[a]pyrene 7(S),8(S)-dihydrodiol ((+)-BP-7,8-diol) as a peroxyl radical probe. The results demonstrated the formation of (+)-BP-7,8-diol-derived tetrols, with the trans-anti tetrol representing the major oxidation product in systems undergoing PPHP-dependent, PGHS-catalyzed oxidation of (13Z)-RA or RA. These results are consistent with the formation of peroxyl radicals in these systems. In all experiments, the (13Z)-RA isomer appeared to be a better substrate for the enzyme compared to the all-trans isomer. Collectively these results provide further evidence to support the previously proposed mechanism for retinoid oxidation by PGHS involving the intermediacy of C4 carbon-centered radicals which subsequently react with dioxygen, yielding retinoid-derived peroxyl radicals.

Heterologous Expression and Enzymatic Properties of a Selenium-independent Glutathione Peroxidase from the Parasitic Nematode Brugia pahangi

A full-length cDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera frugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). The recombinant protein was glycosylated and secreted from the cells in tetrameric form. The purified protein showed glutathione peroxidase activity with a range of organic hydroperoxides, including L-alpha-phosphatidylcholine hydroperoxide, but no significant activity against hydrogen peroxide. Glutathione was the only thiol tested that served as a substrate for the enzyme, which showed no activity with the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). No glutathione-conjugating activity was detected against a range of electrophilic compounds that are common substrates for glutathione S-transferases. The apparent (pseudo)m for glutathione was determined as 4.9 mM at a fixed concentration of linolenic acid hydroperoxide (3 microM). The enzyme showed low affinity for hydroperoxide substrates (apparent Km for linolenic acid hydroperoxide and L-alpha-phosphatidylcholine hydroperoxide of 3.8 and 9.7 mM, respectively at a fixed glutathione concentration of 3 mM).

Reactivity of Plasma Glutathione Peroxidase with Hydroperoxide Substrates and Glutathione

We studied enzyme kinetics parameters of plasma glutathione peroxidase (GSHPx-P) and the major cellular enzyme, GSHPx-1, for the substrates, H 2O 2, linoleic acid hydroperoxide (LinOOH), and glutathione (GSH). The major objectives were to determine whether the relatively slow GSHPx-P enzyme had a lower reactivity with hydroperoxides or with GSH and to identify favored hydroperoxide substrates. The rate constants describing the reactivity of human GSHPx-P and human GSHPx-1 with LinOOH and H 2O 2 are in the same range; GSHPx-P is more reactive with LinOOH and GSHPx-1 is more reactive with H 2O 2. GSHPx-P also has a low level of reducing activity toward cholesterol 7α-OOH and no detectable activity with the 5α-OOH isomer in contrast to phospholipid hydroperoxide glutathione peroxidase (PHGPx) which readily reduced both isomers. GSHPx-P catalytic activity toward phospholipid hydroperoxides is demonstrable in the absence of detergents, enhanced at low concentrations by deoxycholate, and strongly inhibited by Triton X-100 and incorporation into liposomes. These properties are the opposite of PHGPx. These results suggest that GSHPx-P largely lacks the membrane interfacial properties of PHGPx. GSHPx-P exhibits a smaller GSH rate constant than GSHPx-1. This property partially explains the slower turnover of GSHPx-P with several hydroperoxide substrates; the low reactivity with GSH is not consistent with efficient GSHPx function in the bulk plasma volume. GSHPx-P kinetic properties suggest that it would function best as a free fatty acid hydroperoxidase in GSH-rich microenviroments. Minimally, the secretion of reduced enzyme would permit it to scavenge free fatty acid hydroperoxides.

Formation of epoxyalcohols by a purified allene oxide synthase. Implications for the mechanism of allene oxide synthesis.

The allene oxide synthase (hydroperoxide dehydrase) of flaxseed is a cytochrome P450 that exhibits an exceptionally high catalytic turnover (> or = 1000/s) for hydroperoxy substrates. In a previous study, using a crude extract of flaxseed, we detected a secondary activity that could offer an insight into the mechanism of the enzymatic transformation of hydroperoxides. We observed that the substrate 8R-hydroxy-15S-hydroperoxyeicosa-5,9,11,13,17-pentaenoic acid is converted not only to allene oxide, but also to epoxyalcohol derivatives (Brash, A. R., Baertschi, S. W., and Harris, T. M. (1990) J. Biol. Chem. 265, 6705-6712). The transformation of hydroperoxides to epoxyalcohols has been investigated extensively in other systems, and heterolytic or homolytic cleavage of the hydroperoxide is associated with characteristic rearrangements and stereochemistry of the epoxyalcohol products. Using the purified enzyme, we established that the epoxyalcohols are products of the allene oxide synthase. Their structures were determined by UV, gas chromatography-mass spectrometry, and NMR. The major epoxyalcohol is 8R,13R-dihydroxy-14R,15S-epoxyeicosa-5Z,9E ,11Z,17Z-tetraenoic acid, a trans-epoxide with an alpha-hydroxyl in the relative threo configuration. Two minor products are the corresponding 11E isomer and a cis-epoxide identified as 8R,13-dihydroxy-14S,15S-epoxyeicosa-5Z,9E,11E,++ +17Z-tetraenoic acid. Gas chromatography-mass spectrometry analysis of a reaction with [18O2]hydroperoxide substrate indicated complete retention of the hydroperoxy oxygens in the epoxyalcohol products. Mechanistic precedents support a homolytic hydroperoxide cleavage as the initial step in the synthesis of these epoxyalcohols. We suggest that the same process initiates allene oxide synthesis, a conclusion that is also most compatible with the known chemistry of cytochromes P450.

A Laccase Electrode for Organic-Phase Enzymatic Assays

The biocatalytic activity of catalase in non-aqueous environments is exploited for developing an organic-phase amperometric biosensor. The catalase is immobilized within the Eastman-AQ film on the glassy carbon surface. Various experimental variables influencing the response of the t-butyl hydroperoxide substrate are explored for optimum performance.

Purification and Characterization of a Glutathione Peroxidase from the Aloe vera Plant

Extracts from the parenchymous leaf-gel of the Aloe vera plant (Aloe barbadensis Miller) were shown to contain glutathione peroxidase (GSHPx) activity. The activity was purified to homogeneity by ion exchange and gel filtration (FPLC) chromatography in the presence of 0.5 mM glutathione. The native enzyme has an apparent molecular weight of 62 kD as determined by gel filtration. In the presence of sodium dodecylsulfate (SDS), the molecular weight was estimated to be about 16 kD as determined by polyacrylamide-gel electrophoresis (SDS-PAGE). The native enzyme is proposed to be constituted of four identical subunits; it also contains one atom of selenium per subunit, as found with most glutathione peroxidases from animal sources. The Km values were determined to be 3.2 mM for glutathione and 0.26 mM for the hydroperoxide substrate, cumene hydroperoxide. The enzyme is competitively inhibited by N, S, bis-FMOC glutathione (Ki = 0.32 mM), a potent inhibitor of glyoxalase II. Inhibitors of glyoxalase I (e.g. S-octylglutathione) have no effect on the peroxidase activity.

Purified prostaglandin synthase activates aromatic amines to derivatives that are mutagenic to Salmonella typhimurium

Prostaglandin H synthase (PHS) is widely distributed in mammalian tissues and has the ability to oxidize a variety of mutagens and carcinogens. It may therefore play a key role in the metabolic activation of xenobiotics. The present study documents that highly purified PHS can be used in conjunction with 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP), a relatively stable and non-mutagenic hydroperoxide substrate, for the metabolic activation of aromatic amines to mutagenic derivatives that can be detected in short-term Salmonella typhimurium mutagenesis assays. The PHS-based activation system alone was not mutagenic for these tester strains, nor were the test compounds significantly toxic for the bacteria over the concentration range tested. When used in conjunction with Salmonella strains TA98 and TA100 in a modified Ames assay, this system should prove useful for screening of a wide range of compounds for metabolic activaton by this mammalian peroxidase. The potential broad utility of this purified PHS-dependent metabolic activation system was investigated by evaluating the activation of 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5- f]quinoline (MeIQ), which are representative of a group of mutagenic and carcinogenic heterocyclic arylamines to which humans are exposed via their diet. Both IQ and MeIQ were activated by PHS to potent mutagens and confirm the utility of the PPHP/PHS system for the activation of premutagens. Whereas the extent of activation of aromatic amines by S9-based systems is significantly greater than for the PHS activation system described herein, PHS may play a significant role in target tissues in which it is present at significantly greater levels than P450 isoenzymes. Moreover, it is likely that the substrate specificity of PHS differs sufficiently from that of P450 isoenzymes so that PHS may activate some compounds that are not efficiently activated by mixed-function oxidase based systems.

A kinetic stidy of the catalysed oxidation of 1',3-dimethylferrocene ethylamine by cytochrome c peroxidase

The kinetics of the catalysed oxidation of 1',3-dimethylferrocene ethylamine by cytochrome c peroxidase was studied in the presence of two hydroperoxide substrates using cyclic voltammetry. The second order rate constants for the reduction of oxidised CcP were determined as 3.57 × 10 6 dm 3 mol −1 s −1 in the presence of hydrogen peroxide and 5.00 × 10 5 dm 3 mol −1 s −1 in the presence of m-chloroperoxybenzoic acid. Hydrogen peroxide therefore proved to be a more efficient electron acceptor than m-chloroperoxybenzoic acid. The relative rates of reaction of the two substrates were found to be similar to those for the immobilised enzyme. In addition we have also presented some preliminary results for the coupling of a range of other ferrocene derivatives with CcP.

A spectrophotometric assay for hydroperoxide lyase

AbstractSpectrophotometric assays for hydroperoxide lyase have traditionally measured the loss in absorption at 234 nm due to the disruption of conjugated diene in the fatty acid hydroperoxide. However, that assay does not distinguish between hydroperoxide lyase and hydroperoxide dehydrase activities, both of which cause a loss of conjugation in the substrate hydroperoxide. A new assay has been developed which is specific for hydroperoxide lyase. It is based on the ability of hydroperoxide lyase products, aldehydes and ω‐oxoacids, to serve as substrates for yeast alcohol dehydrogenase. Thus, the new hydroperoxide lyase assay is a coupled enzyme assay, which is conducted spectrophotometrically at 340 nm and measures the oxidation of nicotinamide adenenine dinucleotide, reduced form (NADH). In addition to its specificity for hydroperoxide lyase, the coupled assay allows higher concentrations of both fatty acid hydroperoxide substrate and crude enzyme extracts than the assay conducted at 234 nm.

Biochemical effects of combined gases of nitrogen dioxide and ozone. IV. Changes of lipid peroxidation and antioxidative protective systems in rat lungs upon life span exposure

Lipid peroxide production, antioxidant contents and activities of antioxidative protective enzymes were examined in lungs of rats exposed to clean air (control group), 0.05 ppm O 3, 0.05 ppm O 3 + 0.04 ppm NO 2 and 0.05 ppm O 3 + 0.4 ppm NO 2 for 22 months. The results were compared with our previous data in rats exposed to 0.04 ppm NO 2, 0.4 ppm NO 2 and 4 ppm NO 2 for their life span (Sagai et al., Toxicol. Appl. Pharmacol., 73, (1984) 444–456). TBA values used as an index of lipid peroxidation in the lungs were increased maximally at 9 months, but were decreased below control values in animals exposed for 18 and 22 months. Nonprotein sulfhydryl (NPSH) contents were increased maximally at 9 months, and after 18 and 22 months were decreased significantly below values. Vitamin E (VE) contents showed a similar trend. On the other hand, enzyme activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR), glutathione peroxidase measured by using cumene hydroperoxide (cum · OOH) substrate (GPx-cum · OOH), glutathione peroxidase measured by using H 2O 2 as a substrate (GPx-H 2O 2), glutathione S-transferase (GSH-Tase) and superoxide dismutase (SOD) did not show any significant changes during this experiment. The results show that lipid peroxidation in lungs was increased synergistically by a combination of NO 2 and O 3 at ambient levels, and that the time of maximum lipid peroxide production was shorter than with NO 2 alone. The protective ability against lipid peroxides was higher with increased lipid peroxide levels, but the inducibility was not maintained through a life span exposure to the combined gases. Additionally, two small adenomas were observed in 2 out of 18 rats in the 0.05 ppm O 3 + 0.04 ppm NO 2 group and a large adenoma was observed in 1 out of 18 animals in the 0.05 ppm + 0.4 ppm NO 2 group exposed for 22 months.

Glutathione transferase in the free-living nematode Panagrellus redivivus

The free-living nematode Panagrellus redivivus was found to have relatively high cytosolic glutathione transferase activity. Chromatofocusing indicated that at least four GSH transferase forms were present in the nematode cytosol. An endogenous molecular factor interfered with the binding of the cytosolic GSH transferases to a glutathione affinity matrix and binding only occurred after a partial purification step. The major resolved GSH transferase form B was a basic enzyme that showed no biochemical homology to the three mammalian multigene GSH transferase families and appeared to interact with plant phenols, possible natural substrates. A minor GSH transferase form AT showed a biochemical relationship to the mammalian α family including catalytic activity with a model lipid hydroperoxide substrate.

Evidence for the presence of glutathione peroxidase activity toward an organic hydroperoxide in larvae of the cabbage looper moth, Trichoplusia ni

Using enzyme preparations of mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, we have detected glutathione peroxidase activity against a model organic hydroperoxide substrate, cumene hydroperoxide. Specific activity, pmol NADPH oxidized/mg protein/min at 25°C and pH 7.0 was 54.3 for 600 g (10 min) and 57.3 for 850 g (15 min) supernatant. Cysteamine and cyanide which inhibit the peroxidase activity of glutathione transferase, significantly inhibited the enzyme activity, while azide, an inhibitor of catalase and peroxidase had no effect. Specific activities associated with the nuclear, microsomal and cytosolic fractions were 74.0, 34.6 and 13.2, respectively. No activity was detected in the mitochondrial fraction. In the absence of a Se-dependent glutathione peroxidase in T. ni, the GSH-dependent peroxidase activity appears to conform to the peroxidase activity analogous to the mammalian non-Se glutathione transferase.

Modification of microsomally mediated oxygen uptake by alterations in dietary lipids

Rats were fed a basal diet with 5% or 20% soy oil that was either fresh, oxidized or partially hydrogenated, both with and without the cytochrome P-450 inducers, phenobaritone and β-naphthoflavone. Changes in the qualitative character of the oil altered the hepatic cytochrome P-450 mediated rate of oxygen uptake by the liver microsomes and linoleic hydroperoxide substrates; the quality and quantity of the oil affected the non-enzymatic uptake of oxygen mediated by ascorbate. These changes in oxygen transfer rates were attributed to both changes in lipid composition of the endoplasmic reticulum and changes in the constituent forms of P-450. The implications of these effects in oxidative cell injury are discussed in light of the recently reported reaction that describes a very rapid uptake of oxygen in a system which utilizes molecular oxygen, cytochrome P-450 and hydroperoxide as the only reactants.

Singlet oxygen production by bleomycin. A comparison with heme-containing compounds.

Fe(III)-bleomycin catalyzes the decomposition of 13-hydroperoxylinoleic acid and of 15-hydroperoxyarachidonic acid to produce small quantities of singlet oxygen. No singlet oxygen is produced when hydrogen peroxide, ethyl hydroperoxide, cumene hydroperoxide, and t-butyl hydroperoxide are used as substrates. The heme-containing catalysts, methemoglobin and hematin, have identical hydroperoxide substrate requirements for singlet oxygen production. The hydroperoxide requirements for singlet oxygen production correlate with those reported by Dix et al. (Dix, T.A., Fontana, R., Panthani, A., and Marnett, L.J. (1985) J. Biol. Chem. 260, 5358-5365) for the production of peroxyl radicals in the hematin-catalyzed decomposition of hydroperoxides. The bimolecular reaction of peroxyl radicals is a plausible reaction mechanism for the singlet oxygen production in the systems studied.

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, interface substrates.