Accumulation of Hydrogen Peroxide in Chloroplasts of SO2-Fumigated Spinach Leaves

Illuminated chloroplasts isolated from SO₂-fumigated spinachleaves accumulated more H₂O₂ than those from non-fumigated ones.This H₂O₂ formation was dependent on light and was inhibitedby DCMU. It also was depressed by cytochrome c and superoxidedismutase (EC 1.15.1.1 [EC] ). The addition of sulfite to rupturedchloroplasts isolated from non-fumigated leaves caused an H₂O₂accumulation that accompanied O₂ uptake. Spinach leaves losttheir catalase (EC 1.11.1.6 [EC] ), ascorbate peroxidase and glutathionereductase (EC 1.6.4.2 [EC] ) activities at the beginning of SO₂ fumigation,when H₂O₂ was accumulated. These results suggest that the accumulationof H₂O₂ in SO₂-fumigated spinach leaves is caused by the increasein O₂^–production, the precursor for H₂O₂, with a sulfite-mediatedchain reaction at the reducing site of photosystem I, and byinactivation of the H₂O₂ scavenging system. (Received October 7, 1981; Accepted June 16, 1982)     

Further Evidence for Inactivation of Fructose-1,6-bisphosphatase at the Beginning of SO2 Fumigation. Increase in Fructose-1,6-bisphosphate and Decrease in Fructose-6-phosphate in SO2-Fumigated Spinach Leaves

The substrate level of the photosynthetic reductive pentosephosphate cycle in spinach leaves during SO₂ fumigation wassurveyed. At the beginning of SO₂ fumigation, fructose-1,6-bisphosphateincreased and fructose-6-phosphate decreased, while ribulose-1,5-bisphosphateremained unchanged and 3-phosphoglyceric acid rapidly decreased.These results suggested that the inhibition of photosynthesisin spinach leaves with SO₂ might be due to inactivation of fructose-1,6-bisphosphatase. (Received May 26, 1982; Accepted September 27, 1982)     

Monodehydroascorbate Reductase in Spinach Chloroplasts and Its Participation in Regeneration of Ascorbate for Scavenging Hydrogen Peroxide

The primary reaction product of chloroplast ascorbate peroxidaseactivity was shown to be monodehydroascorbate radical (MDA).MDA reductase (EC 1.6.5.4 [EC] ) was localized in spinach chloroplaststroma. The MDA reductase activity of spinach chloroplasts,using NAD(P)H as electron donor, could account for the regenerationof ascorbate from MDA produced by ascorbate peroxidase activity.In the absence of MDA reductase, MDA disproportionated to ascorbate(AsA) and dehydroascorbate (DHA). The DHA was reduced to AsAby DHA reductase (EC 1.8.5.1 [EC] ) in chloroplasts. Both NADH andNADPH served as the electron donor of partially purified MDAreductase from spinach leaves. (Received September 24, 1983; Accepted January 23, 1984)     

Presence of Organelle-Associated Serine Enzymes in Spinach Leaves

Particulate fractions prepared from spinach leaves by differentialcentrifugation were analyzed for proteins capable of bindingdi-isopropyl phosphorofluoridate (DFP); [3H]-labeled DFP andSDS-polyacrylamide gel electrophoresis were used. The chloroplast-richfraction contained one kind of DFP-binding protein, whose bindingwas inhibited by phenylmethylsulfonyl fluoride (PMSF). The mitochondrion-richfraction contained another DFP-binding protein, whose bindingwas sensitive to PMSF and L-1-tosylamide-2-phenylethyl chloromethylketone (TPCK). The microsome-rich fraction contained three PMSF-sensitiveDFP-binding proteins; one was sensitive to both Np-tosyl-L-lysinechloromethyl ketone (TLCK) and TPCK, one was sensitive to TLCKand one to TPCK. These DFP-binding proteins are believed tobe serine proteases. (Received April 30, 1983; Accepted October 17, 1983)     

Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and Root Nodules of Medicago sativa

Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 have been analyzed for the presence of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC 1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol of nodules possess CN⁻-insensitive SOD activities. Studies of SOD inactivation with H₂O₂ indicate that, very likely, a Mn-SOD is present in the bacteroids, and suggest that the cytosol contain both Mn-SOD and Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By contrast, the nodule cytosol exhibits an elevated peroxidase activity as compared with the foliar tissue; this activity was completely inhibited by 50 to 100 micromolar KCN. The significantly lower contents of H₂O₂ and malondialdehyde (a product of lipid peroxidation) in nodules with respect to those in leaves reveal that the above-mentioned bacteroid and cytosol enzymes act in an efficient and combined manner to preserve integrity of nodule cell membranes and to keep leghemoglobin active.     

Inactivation of Ascorbate Peroxidase in Spinach Chloroplasts on Dark Addition of Hydrogen Peroxide: Its Protection by Ascorbate

Dark addition of hydrogen peroxide to intact spinach chloroplastsresulted in the inactivation of ascorbate peroxidase accompaniedby a decrease in ascorbate contents. This was also the casein reconstituted chloroplasts containing ascorbate, NADP⁺, NAD⁺and ferredoxin. The addition of hydrogen peroxide during light,however, showed little effect on ascorbate contents and ascorbateperoxidase activity in either the intact or reconstituted chloroplasts.In contrast to ascorbate peroxidase, the enzymes participatingin the regeneration of ascorbate in chloroplasts (monodehydroascorbatereductase, dehydroascorbate reductase and glutathione reductase)were not affected by the dark addition of hydrogen peroxide.Ascorbate contents increased again by illumination of the chloroplastsafter the dark addition of hydrogen peroxide. These resultsshow that the inactivation of the hydrogen peroxide scavengingsystem on dark addition of hydrogen peroxide [Anderson et al.(1983) Biochim. Biophys. Acta 724: 69, Asada and Badger (1984)Plant & Cell Physiol. 25: 1169] is caused by the loss ofascorbate peroxidase activity. Ascorbate peroxidase activitywas rapidly lost in ascorbate-depleted medium, and protectedby its electron donors, ascorbate, isoascorbate, guaiacol andpyrogallol, but not by GSH, NAD(P)H and ferredoxin. (Received June 14, 1984; Accepted August 15, 1984)     

Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts

Intact spinach chloroplasts scavenge hydrogen peroxide witha peroxidase that uses a photoreductant as the electron donor,but the activity of ruptured chloroplasts is very low [Nakanoand Asada (1980) Plant & Cell Physiol. 21 : 1295]. Rupturedspinach chloroplasts recovered their ability to photoreducehydrogen peroxide with the concomitant evolution of oxygen afterthe addition of glutathione and dehydroascorbate (DHA). In rupturedchloroplasts, DHA was photoreduced to ascorbate and oxygen wasevolved in the process in the presence of glutathione. DHA reductase(EC 1.8.5.1 [EC] ) and a peroxidase whose electron donor is specificto L-ascorbate are localized in chloroplast stroma. These observationsconfirm that the electron donor for the scavenging of hydrogenperoxide in chloroplasts is L-ascorbate and that the L-ascorbateis regenerated from DHA by the system: photosystem IferredoxinNADPglutathione.A preliminary characterization of the chloroplast peroxidaseis given. (Received April 16, 1981; Accepted June 3, 1981)     

Inactivation of Ascorbate Peroxidase by Thiols Requires Hydrogen Peroxide

The hydrogen peroxide-dependent oxidation of ascorbate by ascorbateperoxidase from tea leaves was inhibited by thiols, such asdithiothreitol, glutathione, mercaptoethanol and cysteine. Thesethiols themselves did not inactivate the enzyme. However, theyinactivated the enzyme when hydrogen peroxide was produced bythe metal-catalyzed oxidation of thiols or when exogenous hydrogenperoxide was added. Thiols were oxidized by ascorbate peroxidaseand hydrogen peroxide to thiyl radicals, as detected by theESR spectra of the thiyl radical-5,5'-dimethyll- pyrroline-N-oxidieadducts. Inactivation of ascorbate peroxidase by thiols andhydrogen peroxide is caused by the interaction of the enzymewith the thiyl radicals produced at its reaction center. (Received September 10, 1991; Accepted December 9, 1991)     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

植物叶片中过氧化氢含量测定方法的改进

Ti（Ⅳ）-H₂O₂比色法因背景物质干扰而测得的植物叶片内H₂O₂含量偏高，5％三氯乙酸抽提，活性炭脱色，Ti（Ⅳ）-4-(2-吡啶偶氮)间苯二酚(PAR)比色法测得的H₂O₂含量偏低.萃取法有效地脱去丙酮提液中的色素，且H₂O₂的回收率在95％以上.用过氧化氢酶(CAT）处理作空白对照，利用H₂O₂与Ti（Ⅳ）-PAR的显色反应，建立了一种简便、快速、准确的植物叶片内的H₂O₂含量测定方法，H₂O₂的最低检测浓度为0.25 μmol·L^－1.用该方法测得多种植物叶片中H₂O₂的含量在0.1～0.8 μmol·g^－1.     

Drought Influences the Activity of Enzymes of the Chloroplast Hydrogen Peroxide Scavenging System

Smirnoff, N. and Colomb?, S. V. 1988. Drought influences theactivity of enzymes of the chloroplast hydrogen peroxide scavengingsystem.—J. exp. Bot. 39: 1097–1108. The effect of drought on the activity of ascorbate peroxidase(AP), glutathione reductase (GR) and monodehydroascorbate reductase(MDAR) in leaves of barley (Hordeum vulgare) and tef {Eragrostistef) was studied. These enzymes are components of the chloroplasthydrogen peroxide scavenging system. Severe leaf water deficit(<–30 M Pa) resulted in increased activity (leaf dryweight basis) of GR and MDAR in barley and of AP and MDAR intef. The specific activity of all the scavenging enzymes wasgreater in droughted plants. The activities (dry weight basis)of two ‘control’ enzymes, malate dehydrogenase (barley)and phosphoenolpyruvate carboxylase (tef), not directly involvedin the scavenging system, were not affected by drought. These data suggest that drought may cause an increase in thecapacity of the hydrogen peroxide scavenging system and thatit may, therefore, increase the rate of hydrogen peroxide formationin chloroplasts. Key words: Water stress, hydrogen peroxide, enzyme activity     

Inactivation of Lipoxygenase by Hydrogen Peroxide,Cysteine and Some Other Reagents

The polarographic data show that H₂O₂ is not formed during the course of the coupled oxidation of antioxidants by lipoxygenase from defatted soybean meal. A lower concentration of H₂O₂ or autoxidizing cysteine has been found to induce an irreversible inactivation of the enzyme. Inactivation activity of cysteine is reduced either by the addition of catalase or under anaerobic condition. These facts are indicative of the oxidative function of autoxidizing cysteine for the enzyme. The inactivation by cysteine and H₂O₂ respectively is in additive and is impeded by the addition of competitive inhibitors such as linolelaidic and conjugated linoleic acids, indicating a possible reaction with a certain amino acid residue involved in the enzymic catalysis. The experimental evidences obtained with H₂O₂ and some other modifying reagents have been integrated to furnish a basis of later identification of the residue that is exerting the specific catalytic function.     

Starch Synthesizing Enzymes in Chloroplasts of Developing Leaves of Spinach (Spinacea oleracea L.)

In experiments in which RuDP carboxylase activity was used asa marker for the integrity of isolated chloroplasts, more than90% of the starch synthase activity and more than 80% of theADP-glucose pyrophosphorylase activity of spinach leaves wasfound to be located in chloroplasts. Less than 2% of the UDP-glucosepyrophosphorylase was associated with chloroplasts. The activityof starch synthase per chloroplast remained almost constantduring plastid replication in developing leaves on whole plantsand in leaf discs cultured for 7 d on agar under different lightconditions. The ADP-glucose pyrophosphorylase activity of chloroplastsincreased during leaf development and was much lower in dark-growntissues. The results suggest that the synthesis of starch iscontrolled by the synthesis of ADP-glucose pyrophosphorylaseas well as by the previously known control of activity by metabolitessuch as 3-phosphoglyceric acid and inorganic phosphate.     

Influence of Ethephon and 2,5-Norbornadiene on Antioxidative Enzymes and Proline Content in Salt-Stressed Spinach Leaves

The effects of ethephon, an ethylene generating compound, and 2,5-norbornadiene (NBD), an inhibitor of ethylene action, on peroxidase (POD; EC 1.11.1.7), catalase (CAT; EC 1.11.1.6), polyphenol oxidase (PPO; EC 1.14.18.1) activities and proline content in salt-stressed spinach leaves were investigated. POD and PPO activities were increased by NaCl + ethephon + NBD combination and reduced by NBD. Also, ethephon increased the CAT activity while ethephon + NBD reduced CAT activity. NaCl + ethephon increased proline content. The antagonistic effect of ethephon and NBD was seen on POD and PPO activity and proline accumulation, but was not on CAT activity.     

Oxidation of NAD(P)H in a Reconstituted Spinach Chloroplast Preparation Using Ascorbate and Hydrogen Peroxide

The conversion of fructose-1,6-bisphosphate to glycerate-3-phosphate (PGA) was studied in a reconstituted spinach (Spinacia oleracea L.) chloroplast preparation to determine whether a chloroplast-localized NAB(P)H-oxidizing system (Kow, Smyth, Gibbs 1982 Plant Physiol 69: 72-76 with substrates of ascorbate, NAD(P)H, and H₂O₂ could serve as a coupling enzyme in the recycling of NAD(P)H. The rate of PGA formation was monitored as an indicator of NAD(P) generation. With NAD as a cofactor, ascorbate enhanced PGA formation, and an additional increase resulted upon addition of glucose-glucose oxidase, a H₂O₂-generating enzyme. This increase in PGA formation due to H₂O₂ was eliminated by the addition of catalase. With NADP and ferredoxin as cofactors, the recycling of NADP apparently was catalyzed both by ferredoxin-NADP reductase coupled to O₂ and by the NAD(P)H-oxidizing system.     

衰老叶片和叶绿体中H_2O_2的累积与膜脂过氧化的关系

在自然衰老和ABA处理的叶片和叶绿体中活性氧H_2O_2均比对照明显增高。外加H_2O_2刺激水稻叶绿体膜脂过氧化作用。叶绿体的丙二醛含量随H_2O_2浓度、光照时间、光照强度及叶绿体完整性而变化。AsA、GSH、SOD、甘露醇和过氧化氢酶对外源H_2O_2引起的膜脂过氧化有缓解作用,Fe~(2+)有刺激作用。而H_2O_2对叶绿体过氧化损伤主要是转化为OH之故。     

Hydrogen Peroxide in Human Blood

Blood and plasma of humans and rats were analyzed for hydrogen peroxide. The samples were analyzed after deproteinization with trichloroacetic acid, immediately after they were withdrawn from human volunteers or rats. A radio-isotopic technique based on peroxide-dependent decarboxylation of 1-¹⁴C-alpha-ketoacids and consequent liberation of ¹⁴CO₂ was used. The results demonstrate the presence ofmicromolar levels of H₂O₂, both, in the plasma as well as in the whole blood. The values in the whole blood were substantially greater than the plasma. This was true for rats as well as humans. The presence of such significant quantities of H₂O₂ in the blood have been demonstrated for the first time. The investigation, therefore, opens a newer avenue of research on diseases purported to be related to the generation of oxygen radicals in vivo.     

菠菜叶片中硝态氮代谢库的测定（简报）

随着培养时间的延长 ,菠菜组织中还原产生的亚硝态氮量呈波动型上升 ;以 pH 7.5的磷酸缓冲液为培养介质的亚硝态氮累积量显著高于以KCl和CaCl2 混合溶液 ( pH 6.65 )为介质的 ;培养前不通氮气 ,培养后期亚硝态氮的生成量明显高于通氮气的。亚硝态氮生成第一峰值出现的时间随硝态氮含量增高而后延 ,峰值却随硝态氮含量的增高而升高。用 pH 7.5的 0 .0 5mol·L-1磷酸缓冲液且不通氮气对组织进行培养 ,亚硝态氮生成的第一个峰值代表硝态氮代谢库的大小。