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The zeta-crystallin (ZCr) gene P1 of Arabidopsis thaliana, known to confer tolerance toward the oxidizing drug 1,1'-azobis(N, N-dimethylformamide) (diamide) to yeast [Babiychuk, E., Kushnir, S., Belles-Boix, E., Van Montagu, M.Inzé, D. (1995) J. Biol. Chem. 270, 26224], was expressed in Escherichia coli to characterize biochemical properties of the P1-zeta-crystallin (P1-ZCr). Recombinant P1-ZCr, a noncovalent dimer, showed NADPH:quinone oxidoreductase activity with specificity to quinones similar to that of guinea-pig ZCr. P1-ZCr also catalyzed the divalent reduction of diamide to 1,2-bis(N,N-dimethylcarbamoyl)hydrazine, with a kcat comparable with that for quinones. Two other azodicarbonyl compounds also served as substrates of P1-ZCr. Guinea-pig ZCr, however, did not catalyze the azodicarbonyl reduction. Hence, plant ZCr is distinct from mammalian ZCr, and can be referred to as NADPH:azodicarbonyl/quinone reductase. The quinone-reducing reaction was accompanied by radical chain reactions to produce superoxide radicals, while the azodicarbonyl-reducing reaction was not. Specificity to NADPH, as judged by kcat/Km, was1000-fold higher than that to NADH both for quinones and diamide. N-Ethylmaleimide and p-chloromercuribenzoic acid inhibited both quinone-reducing and diamide-reducing activities. Both NADPH and NADP+ suppressed the inhibition, but NADH did not, suggesting that sulfhydryl groups reside in the binding site for the phosphate group on the adenosine moiety of NADPH. The diamide-reducing activity of P1-ZCr accounts for the tolerance of P1-overexpressing yeast to diamide. Other possible physiological functions of P1-ZCr in plants are discussed.

The effect of benzyl alcohol on the transverse mobility and repartition of phospholipids in the human erythrocyte membrane was investigated using electron spin resonance and morphological modification of red blood cells. Transmembrane internalization rates and equilibrium distribution in red blood cells of short-chain spin-labeled phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were strongly modified by treatment with 10-70 mM benzyl alcohol. A dual effect was observed: (a) at 4 degrees C and 37 degrees C there was an N-ethylmaleimide-sensitive, long lasting and fully reversible increase in the spin-labeled phosphatidylserine and phosphatidylethanolamine internalization rate; (b) at 37 degrees C, an enhancement of N-ethylmaleimide-insensitive fluxes of all the labeled phospholipids through the membrane occurred. Both effects were dose-dependent. Erythrocytes submitted to benzyl alcohol incubation also showed dose-dependent shape changes: an immediate one from discocytes to echinocytes, followed by a slower N-ethylmaleimide- and ATP-dependent change to stomatocytes. Moreover, benzyl alcohol treatment was shown to lead to enhanced hydrolysis of intracellular ATP. All the effects of benzyl alcohol can be described as an accumulation of labeled phosphatidylethanolamine (and labeled phosphatidylcholine at 37 degrees C) in the inner leaflet. This can be interpreted as a perturbation of the erythrocyte membrane, leading to an energy-consuming specific increase in aminophospholipid translocase activity, in addition to a slow and passive bidirectional flux of all phospholipids at 37 degrees C.

Gluconeogenesis in rat kidney cortex slices was reversibly inhibited by diazenedicarboxylic acid bis(N,N-dimethylamide). The rate of formation of glucose from pyruvate, malate, succinate and D-fructose decreased at least 3-fold in the presence of this inhibitor, commonly called diamide. The extent of inhibition of gluconeogenesis from pyruvate was dependent on the concentration of diamide and closely correlated with the degree of inhibition of protein kinase. The inhibition of gluconeogenesis and protein kinase were completely reversed after removal of the inhibitor. The effects of diamide on particulate enzymes which may be involved at rate-limiting steps in transport processes which support gluconeogenesis in renal tissue was examined. Several enzymes present in kidney brush border and microsomal membrane preparations were inhibited by diamide. Na+, K+-dependent ATPase and glucose-6-phosphatase were inhibited, but Mg2+-dependent ATPase, alkaline phos-phatase and y-glutamyl transpeptidase, which are also present in these same subcellular fractions, were not inhibited by diamide. Based on the results obtained in these studies it is proposed that diamide may inhibit gluconeogenesis at rate-limiting steps catalyzed by enzymes which are regulated by protein kinase. These enzymes, in turn, appear to play a role in the transfer of substrates across the plasma membrane. The mechanism of inhibition of protein kinase by diamide was also investigated. Protein kinase present in a wide variety of rat tissues as well as phosphorylase kinase and pyruvate dehydrogenase kinase was inhibited by diamide. Kinetic studies showed that the inhibition of protein kinase by diamide was non-competitive with respect to Mg2+, ATP, protein substrates and cyclic AMP. Sedimentation studies in sucrose-density gradients containing histone and cyclic AMP showed that diamide inhibited both the intact and dissociated forms of protein kinase. These results support earlier observations which showed that diamide did not bind at the active site of protein kinase, but instead formed inactive complexes by binding to hydrophobic areas on the surface of the enzyme.

We recently demonstrated activation of 5-lipoxygenase activity in human polymorphonuclear leukocytes (PMN) on preincubation of the cells with glutathione-depleting agents, namely 1-chloro-2,4-dinitrobenzene (Dnp-C1) and azodicarboxylic acid bis[dimethylamide] (diamide). In this paper we show that Dnp-C1, but not diamide, impairs the reduction of added organic peroxides in whole PMN. Also, since co-incubation of fatty acid hydroperoxides with arachidonate caused activation of 5-lipoxygenase, we propose that Dnp-C1 increases the peroxide level in PMN which is required for the onset of lipoxygenase activity. This could be substantiated in PMN homogenates by a glutathione-dependent depression of arachidonate 5-lipoxygenation. At higher arachidonate concentrations and in the presence of Ca2+ the glutathione effect was not observed but additional glutathione peroxidase also blocked this maximally stimulated 5-lipoxygenase. Together with other experiments, it became obvious that the formation of leukotrienes, but also of 15-lipoxygenase products, requires a sharply defined threshold level of fatty acid hydroperoxides which are generated by the lipoxygenases and counteracted by glutathione-dependent peroxidase(s). Dnp-C1 influences this equilibrium by removing glutathione and thereby inhibiting glutathione-dependent peroxidase activity. From our data we conclude that it is the physiological function of the peroxidase activity in PMN to determine an efficiently regulated threshold level of hydroperoxide products, below which no activation of 5-lipoxygenase or 15-lipoxygenase can occur.

The influence of the glutathione status of human polymorphonuclear leukocytes (PMN) on 5-lipoxygenase activity was studied by treating cells with increasing concentrations of 1-chloro-2,4-dinitrobenzene (Dnp-Cl) or azodicarboxylic acid bis(dimethylamide) (Diamide). Subsequent incubation with arachidonate resulted in an up to tenfold-stimulated formation of 5-hydroxyeicosatetraenoic acid, leukotriene B4, leukotriene B4 isomers and omega-hydroxyleukotriene B4. Higher concentrations of the GSH reagents were inhibitory. At maximal stimulation by Dnp-Cl, 5-hydroperoxyeicosatetraenoic acid started to be built up at the expense of 5-HETE at glutathione levels which were diminished by about 50% compared to resting cells. No increase in cytosolic Ca2+ could be measured under these conditions by the fura-2 method. In PMN homogenates Dnp-Cl and Diamide were without effect and even caused inhibition when 5-lipoxygenase was stimulated by Ca2+ and ATP. 15-Lipoxygenase was either unchanged in the case of Diamide, or even increased after pretreatment with Dnp-Cl. The results allow us to conclude that 5-lipoxygenase activity in intact PMN is regulated not only by Ca2+ but in a complex manner also by the glutathione redox status. Conditions of oxidative stress increase the activity which may reflect the in vivo situation under phagocytosis and oxidative burst.

The intracellular thiol-oxidising diazenes, diazenedicarboxylic acid bis-N,N-dimethylamide and diazenedicarboxylic acid bis-N'-ethylpiperazinide, have been used in the study of red cells. A difference in the consequences of diazene oxidant treatment between the human red cell and rat red cells has been found in respect to the quantity of oxidant needed for glutathione (GSH) oxidation, to the fate of GSH, and to the reactivity of hemoglobin. In the first place, significantly more oxidant is needed for GSH oxidation in the rat red cell than in the human cell. Secondly, in the human cell, all of the GSH is converted to glutathione disulfide (GSSG), from which GSH is regenerated. In the rat cell, GSH disappears without being converted to GSSG, and GSH is not regenerated. Thirdly, a decrease in rat hemoglobin thiol groups, but no change in human hemoglobin, is found. Sterically unhindered thiol groups in the rat hemoglobin are thought to react with the usual adduct intermediate in GSH oxidation by diazene (formed from RCON = NCOR + GSH leads to RCON(SG)NHCOR) to produce mixed disulfides, from which GSH is not easily regenerated. The results support the idea that reduction of mixed disulfides of GSH and protein is not carried out directly by GSSG reductase but necessitates thiol transferase and GSH. The thiol-oxidising diazenes may be of use in mapping of exposed, reactive thiol groups in proteins.

Thin-gel isoelectric focusing (IEF) is a simple and sensitive method of quantifying S-thiolation of individual proteins (protein mixed-disulfide formation). IEF of rat liver cytosol identified one major protein (pI 7.0) which underwent S-thiolation with glutathione disulfide to produce two acidic bands with pIs 6.4 and 6.1. The S-thiolated forms of the protein were purified by preparative isoelectric focusing. An apparent molecular mass of 30 kDa was determined by SDS/polyacrylamide gel electrophoresis. The 30-kDa protein amounted to 7 +/- 2% of the total cytosolic protein on IEF. The most abundant soluble protein of freshly isolated hepatocytes, with an identical isoelectric point to the liver 30-kDa protein, was modified in a similar manner in response to oxidative stress induced by model compounds. Addition of 50 microM tert-butyl hydroperoxide, 50 microM diamide [1,1-azobis(N,N'-dimethylformamide)] or 20 microM menadione (2-methyl-1,4-naphthoquinone) initiated the S-thiolation within less than 2 min in the hepatocytes. These compounds, at the concentrations employed, did not result in cell death. Menadione produced slowly progressive S-thiolation of the protein, while tert-butyl hydroperoxide or diamide produced rapid S-thiolation that decreased quickly after 2 min.

1 The inhibitory effect of t-butyl hydroperoxide on O2 uptake by perfused rat liver and by isolated hepatocytes was investigated with isolated mitochondria. 2 O2 uptake by mitochondria oxidizing the ketoacids, 2-oxoglutarate and pyruvate, was substantially decreased upon addition of t-butyl hydroperoxide, that of isocitrate was only slightly decreased, and that of succinate and of 3-hydroxybutyrate was practically unchanged. The reduction product of the hydroperoxide, t-butyl alcohol, showed no effect. The inhibitory effect of the hydroperoxide was reversed upon addition of dithioerythritol, a thiol reductant. The inhibitory effect of the hydroperoxide was mimicked by the penetrant disulfide, cystamine, and by the thiol-oxidizing agent, diamide. 3 Mitochondrial extracts from rats fed a selenium-deficient diet were shown to have virtually no measurable GSH peroxidase activity. The hydroperoxide had almost no inhibitory effect on 2-oxoglutarate-dependent O2 uptake in mitochondria from selenium-deficient rats. 4 These observations demonstrate effects of GSH peroxidase activity in the mitochondrial matrix on the pattern of substrate oxidations, possibly with the ketoacid oxidases, dependent on coenzyme A and lipoamide, as main target sites. In view of the known steady-state formation of mitochondrial O2− and H2O2, a connection between the resulting oxidation of mitochondrial GSH and NADPH and the regulation of mitochondrial substrate oxidations is proposed.