Induction of heat resistance of wheat coleoptiles by salicylic and succinic acids: Connection of the effect with the generation and neutralization of reactive oxygen species

The influence of salicylic (SaA) and succinic (SuA) acids on the generation of reactive oxygen species (ROS) and the heat resistance of wheat (Triticum aestivum L.) coleoptiles has been studied. The treatment of coleoptiles with 10 μM SaA or SuA results in the accumulation of hydrogen peroxide and enhanced formation of a superoxide anion radical. This effect was partially suppressed by both α-naphthol (the NADPH oxidase inhibitor) and salicylhydroxamic acid (peroxidase inhibitor). SaA and SuA cause an increase in the activity of antioxidant enzymes, such as superoxide dismutase, catalase, and soluble peroxidase, and improve the heat resistance of coleoptiles. Antioxidant ionol and inhibitors of the NADPH oxidase and peroxidase significantly reduce the positive influence of SaA and SuA on the heat resistance of wheat coleoptiles. ROS are considered to be intermediates for heat resistance induction in coleoptiles, treated with SaA and SuA; enhanced ROS generation can be caused by an increased activity of the NADPH oxidase and peroxidase.     

The role of reactive oxygen species and calcium ions in the implementation of the stress-protective effect of brassinosteroids on plant cells

The effect of the brassinosteroids (BS) 24-epibrassinolide and 24-epicastasterone on the thermoresistance of wheat coleoptiles (Triticum aestivum L.) and their generation of the superoxide anion radical and antioxidant enzymes activity were investigated. The treatment of coleoptiles with 10 nM solutions of BS caused a transient increase in O ₂ ^⊙? generation and a subsequent increase in the activity of superoxide dismutase and catalase and an improvement in heat resistance. Pretreatment of coleoptiles with the NADPH oxidase inhibitor imidazole leveled the increase in production of the superoxide anion radical and prevented an increase in the activity of antioxidant enzymes and the development of cell thermostability. The investigated effects of BS were also depressed by the pretreatment of coleoptile segments with extracellular calcium chelator EGTA and inhibitor of ADP-ribosyl cyclase nicotinamide. A conclusion was made about the participation of calcium ions and reactive oxygen species generated by the action of NADPH oxidase in the implementation of the stress-protective effect of the BS in the cells of wheat coleoptiles.     

Elevated temperature effects on the oxidant/antioxidant balance in submerged batch cultures of the filamentous fungus Aspergillus niger B1-D

In the present study the relationship between oxidative stress and elevated culture temperature was examined in an industrially relevant fungal culture, Aspergillus niger B1-D. For the first time, both the intracellular levels of the main stressor species (superoxide radical [O(2) (.-)]) and activities of cellular defensive enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxide [GPx]) were quantified at varying temperature (25, 30, 35, 40 degrees C) to more fully characterize culture response in different growth phases. Elevated culture temperature led to increased O(2) (.-) levels in various culture phases. In the exponential phase this was due to an enhanced generation of O(2) (.-), whereas in stationary phase a decreased dismutation rate may also have contributed. CAT activities generally increased with culture temperature, whereas GPx activity changed little as temperature rose, indicating that GPx played only a minor role in destroying H(2)O(2) in this A. niger. The combination of elevated temperature (35 degrees C) and increased O(2) supply (50% enrichment) led to decreased levels of O(2) (.-) compared to the cultivation at 35 degrees C gassed with air, probably due to enhanced activity of the alternative fungal respiratory pathway. Our findings indicate that while elevated cultivation temperature does clearly induce oxidative stress events, mechanistically, it does so by a rather more complex route than previous studies indicate. Elevated temperature caused a marked disparity in the activities of SOD and CAT, very distinct from the integrated increase in activity of these enzymes in response to oxidative stress.     

Salicylic and jasmonic acids in regulation of the proantioxidant state in wheat leaves infected by Septoria nodorum Berk

Influence of mediators of the signal systems of salicylic (SA) and jasmonic (JA) acids and their mixture on reactive oxygen species' (ROS) (superoxide radical O2*- and H2O2) generation and activity of oxidoreductases (oxalate oxidase, peroxidase and catalase) in leaves of wheat Triticum aestivum L. infected by Septoria leaf blotch pathogen Septoria nodorum Berk. has been studied. Presowing treatment of seeds by SA and JA decreased the development rate of fungus on wheat leaves. SA provided earlier inductive effect on production of O2*- and H2O2 compared with JA. The protective effect of the salicylic and jasmonic acids against Septoria leaf blotch pathogen was caused by activation of oxalate oxidase, induction of anion and cation peroxidases, and decrease of catalase activity. Ability of compounds to stimulate ROS in the plant tissues can be used as criteria for evaluation of immune-modulating activity of new substances for protection of the plants.     

Effect of sodium nitroprusside on heat resistance of wheat coleoptiles: Dependence on the formation and scavenging of reactive oxygen species

The effect of nitric oxide donor sodium nitroprusside (SNP) on resistance of coleoptiles of 4-day-old etiolated seedlings of wheat (Triticum aestivum L., cv. Elegiya) to damaging heating (10 min at 43°C) and possible dependence of this effect on changes in the activities of enzymes producing and scavenging reactive oxygen species (ROS) were studied. Treatment of coleoptiles with 500 μM SNP considerably boosted generation of superoxide anion radical therein. This effect was substantially suppressed by blocker of calcium channels (lanthanum chloride), calmodulin antagonist (chlorpromazine), and inhibitor of NADPH-oxidase (imidazole) but not by peroxidase inhibitor (salicylhydroxamic acid). NO donor activated antioxidant enzymes (superoxide dismutase, catalase, and soluble peroxidase) and elevated heat resistance of wheat coleoptiles. NO scavenger methylene blue, antioxidant agent ionol, calcium antagonists, and NADPH-oxidase inhibitor imidazole substantially reduced the elevation of heat resistance of wheat coleoptiles induced by NO donor. It was concluded that SNP-induced heat resistance of coleoptiles depended on calcium and ROS, whose production is probably boosted by activation of NADPH-oxidase.     

Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth

Hydroxyl radicals (*OH), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for *OH generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize ( Zea mays L.) coleoptiles and sunflower ( Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause *OH generation by POD in vitro. The effects of NADH on wall loosening, growth, and *OH production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of *OH in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the *OH-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the *OH-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O2*-) (and H2O2 utilized by POD for) *OH production in the cell wall.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.     

Apoplastic superoxide production and peroxidase activity by intact and excised axenically grown seedling roots of sunflower

Excised and cold-preincubated sunflower seedling roots were compared with intact non-preincubated roots to test the effect of the injury stress and cold preincubation on the oxidative burst measured as apoplastic superoxide (O (2) (.-) ) generation and exocellular peroxidase (ECPOX) activity. Preincubated excised or intact roots released into the medium apoplastic proteins with peroxidase activity. Intact and excised roots responded to methyl jasmonate by an immediate oxidative burst that could not be induced by salicylic acid; both phytohormones also induced a slight and slow O (2) (.-) generation and ECPOX activity on excised roots, when added to the cold preincubation medium. The results with cyanide, azide, SHAM (ECPOX inhibitors) and diphenylene iodonium (inhibitor of trans-plasma membrane NAD(P)H-oxidases (NOX)-respiratory burst oxidase homologue in plants (RBOH), the trans-plasmamembrane nicotinamide adenine dinucleotide phosphate oxidase) are consistent with the hypothesis that different systems may be the origin of O (2) (.-) in intact and excised roots; ECPOX was an important component of them in both, together with NOX-RBOH in intact roots, but in excised roots the last one was replaced by an oxidase sensitive to the same inhibitors as the alternative mitochondrial oxidase. According to our hypothesis, these results could be explained if the electron flux would be deviated to different interconnected plasma membrane-redox systems, with different terminal oxidases, activated by different effectors or stresses.     

Participation of Nitric Oxide in 24-Epibrassinolide-Induced Heat Resistance of Wheat Coleoptiles: Functional Interactions of Nitric Oxide with Reactive Oxygen Species and Ca Ions

Nitric oxide (NO) effects on heat resistance of wheat (Triticum aestivum L.) coleoptiles induced by 24-epibrassinolide (24-EB) have been investigated. Coleoptiles’ survival after damaging heating (43°С, 10 min) increased when they were treated preliminarily with 5–200 nM of 24-EB. After 24-EB treatment, transient amplification of nitric oxide (NO) and also ROS (superoxide anion-radical (O ₂ ^?? ) and hydrogen peroxide) generation by coleoptiles was noted. Coleoptiles pretreatment with inhibitors of nitrate reductase and an enzyme similar to animal NO-synthase partially removed the increase of NO content caused by the action of 24-EB. Amplification of superoxide anion-radical generation caused by 24-EB was depressed under the influence of imidazole (NADPH-oxidase inhibitor). Calcium antagonists (EGTA and neomycin) largely neutralized the 24-EB-induced increase in generation of both O ₂ ^?? and NO. The increase in NO content in coleoptile tissues caused by 24-EB was almost completely leveled by antioxidants and partly by imidazole. 24-EB-induced enhancement of the superoxide anion-radical generation was partially suppressed by the action of NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and the inhibitors of nitrate reductase and an enzyme similar to animal NO-synthase. Positive 24-EB effect on the heat resistance of wheat coleoptiles was leveled by PTIO, inhibitors of enzymes that generate NO, antioxidants, an inhibitor of NADPH-oxidase imidazole, and calcium antagonists. A conclusion was made on the role of NO in brassinosteroid signal transduction inducing heat resistance development of coleoptiles and on the functional interaction between NO, ROS, and calcium ions as the signal mediators.     

Effect of jasmonic acid on the pro-/antioxidant system of wheat coleoptiles as related to hyperthermia tolerance

The effects of treatment with jasmonic acid (JA) of wheat (Triticum aestivum L, cv. Elegia) coleoptiles on the generation of superoxide anion-radical (O ₂ ^·? ), the activity of extracellular peroxidase, enzymatic and non-enzymatic components of the antioxidant system were studied. During the first hour after the start of coleoptile treatment with 1 μM JA, the generation of O ₂ ^·? was enhanced and the extracellular peroxidase was activated. During following 23 h, these effects were gradually reduced. JA-enhanced O ₂ ^·? generation was partially suppressed by coleoptile treatment with the inhibitor of peroxidase salicylhydroxamic acid, the inhibitor of NADPH-oxidase imidazol, and also the calcium chelator EGTA. Under the influence of JA treatment, antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and soluble guaiacol peroxidase) in wheat coleoptiles were activated. Treatment with JA improved coleoptile tolerance to damaging heating (10 min at 43°C); it favored the maintenance of the pools of enzymatic and non-enzymatic antioxidants. The inhibitors of NADPH-oxidase and peroxidase, and also calcium chelator reduced a positive JA influence on coleoptile thermotolerance. The role of changes in the pro-/antioxidant balance in plant tissues for the realization of stress-defensive JA effects is discussed.     

The activity of antioxidant enzymes in Arabidopsis thaliana exposed to colchicine and H2O2

Studies on the possible interference of colchicine and H2O2 with the activity of some antioxidant enzymes were carried out on Arabidopsis thaliana v. Columbia grown in Murashige and Skooge nutrient medium. Measurements of superoxide dismutase (SOD), guaiacol peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT) activities were conducted spectrophotometrically. In the presence of colchicine, SOD activity increased, while CAT, APX and POX activities decreased. Inhibitory H2O2 effects on the activity of the enzymes were found. Colchicine pre-treatment resulted in an increase in CAT activity and a further increase in SOD activity in plants treated with H2O2.     

Characteristics of Eliciting Effects of Furostanol Glycosides on Cultured Yam Cells

To unravel mechanisms of elicitor action of furostanol glycosides (FGs), the formation of superoxide anion after the addition of FGs to a suspension culture of yam (Dioscorea deltoidea Wall. ex Griseb) cells was studied. The substantial increase in superoxide level, evaluated by nitroblue tetrazolium (NBT) reduction to formazan, was found at the exponential phase of cell growth. The involvement of NADPH oxidase in the superoxide generation was revealed by means of inhibitory analysis. Diphenyliodonium chloride (DPI), the inhibitor of NADPH oxidase, compromised the action of FGs. Meanwhile, the elimination of apoplastic peroxidase did not affect the accumulation of formazan, which suggests the involvement of NADPH oxidase but not peroxidase in the superoxide generation. In addition to NBT-test, the superoxide formation was judged by changes in activity of superoxide dismutase (SOD). Exogenous FGs activated the enzyme due to the increased production of superoxide anion. In this case, DPI decreased SOD activity that conforms to the NADPH oxidase involvement in the superoxide generation. The analysis of antioxidant activity of FGs by inhibition of radicals of 2,2-diphenyl-1-picrylhydrazyl showed that FGs are weak reductants in comparison with ascorbic acid. The results of the work allow for the suggestion that, supposing a weak reducing capacity of FGs, the special feature of their exogenous action on cultured yam cells is the increase in the level of superoxide anion radical mainly produced by NADPH oxidase.     

Cell surface peroxidase--generator of superoxide anion in wheat root cells under wound stress

Development of wound stress in excised wheat roots is known to be accompanied with an increase in reactive oxygen species (ROS) production, fall of membrane potential, release of K+ from cells, alkalization of extracellular solution, changes in respiration and metabolism of structural lipids. Dynamics of superoxide release correlates with changes in other physiological parameters, indicating the cross-reaction of these processes. Activity of peroxidase in extracellular solution after a 1 h incubation and removal of roots was shown to be stimulated by the range of organic acids, detergents, metals, and to be inhibited by cyanide. Superoxide production was sensitive to the addition of Mn2+ and H2O2. Increase in superoxide production correlates with the enhancement of peroxidase activity at the application of organic acids and detergents. The results obtained indicate that cell surface peroxidase is one of the main generators of superoxide in wounded wheat root cells. Different ways of stimulation of the ROS producing activity in root cells is supposed. By controlling superoxide and hydrogen peroxide formation, the cell surface peroxidase can control the adaptation processes in stressed plant cells.     

6-苄（基）腺嘌呤和抗坏血酸对渗透胁迫时杨树光合作用光抑制的影响

研究了6-BA和A5A对渗透胁迫时杨树幼苗叶片光合作用光抑制和活性氧代谢的影响．结果表明,渗透胁迫时杨树叶片净光合速率(Pn)和表观量子效率(AQY)降低,光合作用光抑制加剧,超氧化物歧化酶(SOD)活性升高,抗坏血酸过氧化物酶(APX)活性降低,O2产生加快,H2O2和膜脂过氧化产物丙二醛(MDA)含量升高．6-BA和A5A预处理使胁迫时叶片SOD和APx活性升高。O2生成减少。H2O22和MDA含量降低,同时缓解了光合作用的光抑制．相关分析表明,杨树叶片活性氧水平和MDA含量与Pn和AQY呈负相关．胁迫时杨树叶片活性氧的积累与光合作用光抑制有一定关系,6-BA和A5A对光抑制的缓解作用与其对活性氧清除系统的促进作用有关。     

Generation of Activated Oxygen and Change of Cell Defense Enzyme Activity in Leaves of Korean Pine Seedling Under Low Temperature

Changes of activated oxygen O2 and H2O2, malondialdehyde (MDA) content and activity of enzymes involving cell defense in leaves of Korean pine (Pinus koraiensis Sieb. et. Zucc) seedling under different time of low temperature stress were studied. With the increase of stress time,the rate of O2 generation and H2O 2 content increased to a certain degree and then decreased. The increase of MDA content fluctuated as well as its decrease after the low-temperature stress was removed. The activity of cell defense enzymes, viz. superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascrobate peroxidase (ASP) were gradually decreased. However, after cold pre-treatment of the seedlings all these indexes showed marked changes and different ways of adaptivity to cold stress; thus enhanced the cold tolerance of the plant.     

Early production of activated oxygen species in root apoplast of wheat following copper excess

Wheat seedlings (Triticum durum Desf.) were incubated in a solution containing 100 microM CuSO(4) for increasing time ranging from 1 min to 6h. Copper rapidly accumulated into the roots, and its amount increased significantly until 360 min. During the experiment, copper did not cause any lipid peroxidation and K(+) leakage. Up to 60 min of copper treatment the superoxide (O2(*-)) production in root apoplast decreased concomitantly with increase in superoxide dismutase (SOD) activity. In contrast, after 60 min of incubation, SOD decreased and this facilitated an increase in O2(*-) production. In the presence of the SOD inhibitor diethyldithiocarbamic acid, O2(*-) production was more than two times higher and showed a biphasic increase. Very high SOD activity in the apoplast, due to the presence of three different isozymes, one Mn-SOD and two CuZn-SODs, dismutated the radical giving rise, at least in part, to an increase in hydrogen peroxide. The highest value of H(2)O(2) was detected at 15 min, when peroxidase (POD) activity reached the lowest value. Root apoplast showed the presence of at least five different isoforms of PODs, whose pattern did not change during the entire treatment.     

The Participation of calcium ions and reactive oxygen species in the induction of antioxidant enzymes and heat resistance in plant cells by hydrogen sulfide donor

The effect of hydrogen sulfide (H₂S) donor sodium hydrosulfide (NaHS) on the heat resistance of wheat (Triticum aestivum L.) coleoptile cells, the formation of reactive oxygen species (ROS), and the activity of the antioxidant enzymes in them was investigated. The treatment of coleoptiles with 100 µM NaHS caused transient enhancement of the generation of the superoxide anion radical (O₂ ^?) and an increased hydrogen peroxide content. The activities of antioxidant enzymes—superoxide dismutase, catalase, and guaiacol peroxidase— and coleoptile resistance to damaging heat was later found to have increased. The biochemical and physiological effects of the hydrogen sulfide donor described above were inhibited by the treatment of wheat coleoptiles with the hydrogen peroxide scavenger dimethylthiourea, the NADPH oxidase inhibitor imidazole, the extracellular calcium chelator EGTA, and the phosphatidylinositol-specific phospholipase C inhibitor neomycin. A conclusion was made on the role of ROS generation, which is dependent on the activity of NADPH oxidase and calcium homeostasis, in the transduction of the H₂S signal, which induces antioxidant enzymes and the development of plant cell heat resistance.     

Effects of Exogenous Spermidine on Antioxidant System Responses of Typha latifolia L. Under Cd^2＋ Stress

The effects of foliar spraying with spermidine (Spd), ranging in concentration from 0.25 to 0.50 mmol/L, on the antioxidant system under Cd^2 stress (range 0.1- 0.2 mmol/L Cd^2 ) in Typha latifolia L. grown hydroponically were investigated in order to offer a referenced evidence for an understanding of the mechanism by which polyamines (PAs) relieve the damage to plants by heavy metal and improve the phytoremediation efficiency of heavy metal-contaminated water. The results showed that Cd^2 stress induced oxidative injury, as evidenced by an increase in the generation of superoxide anion (O2), as well as the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in both leaves and caudices. With the exception of superoxide dismutase (SOD) activity in the leaves, an increase in the activities of catalase (CAT), guaiacol peroxidase (GPX), and glutathione reductase (GR) was observed in both leaves and caudices, SOD activity was increased in caudices, and ascorbate peroxidase (APX) activity was increased in leaves following Cd^2 treatment. The reduced glutathione (GSH) content in both leaves and caudices and the reductive ascorbate content in leaves was obviously increased, which were prompted by the application of exogenous Spd. Spraying with Spd increased the activity of GR and APX in both leaves and caudices, whereas the activity of SOD, CAT, and GPX was increased only in caudices following spraying with Spd. The generation of O2 and the H2O2 and MDA content in both leaves and caudices decreased after spraying with Spd. The decrease in MDA was more obvious following the application of 0.25 than 0.50 mmol/L Spd. It is supposed that exogenous Spd elevated the tolerance of T. latifolia under Cd^2 stress primarily by increasing GR activity and the GSH level.     

二氧化硫对小麦的氧化胁迫及其某些信号分子的调节

通过在密闭的培养箱中一次性通入不同体积浓度的SO₂气体,研究了小麦幼苗超氧自由基O2-含量和3种抗氧化酶活性的变化,探讨了信号分子水杨酸、乙烯和过氧化氢对SO₂氧化胁迫的调节作用.结果表明,当通入10和40 μl·L^-1 SO₂时,小麦叶片中O2-含量递增,过氧化物酶(POD)和过氧化氢酶(CAT)活性增强,但超氧化物歧化酶(SOD)活性降低.当SO₂浓度达到50 μl·L^-1时,POD和CAT活性也开始降低,此时叶片尖端出现坏死,叶片绿色部位滋生大量真菌.用1 mmol·L^-1水杨酸(SA)(pH6.5)浸泡小麦干种子6 h,或用10 mmol·L^-1 H₂O₂浸泡幼苗,O2-含量低于对照植株,而3种酶的活性高于对照植株,均可有效地减轻SO₂的氧化胁迫.在SO₂熏蒸下,乙烯显著抑制3种酶的活力,提高O2-的形成速率.SA与乙烯同时使用时,SA几乎完全消除了乙烯的负面作用.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.