Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

The reaction of antimycin with a cytochrome b preparation active in reconstitution of the respiratory chain

1. Succinate-cytochrome c reductase activity was reconstituted by incubating a mixture of succinate dehydrogenase, cytochrome c₁, ubiquinone-10, phospholipid and a preparation of cytochrome b, made by the method of .

2. Preparations of cytochrome b active in reconstitution contained 5–28% native cytochrome b, as adjudged by reducibility with succinate in the reconstituted preparation and by lack of reaction with CO. Preparations of cytochrome b containing no native cytochrome b according to this criterion were inactive in reconstitution.

3. With a fixed amount of cytochrome b, the activity of the reconstituted preparation increased with increasing amounts of cytochrome c₁ until a ratio of about 2b (total): 1c₁ (allowing for the cytochrome c₁ present in the cytochrome b preparation) was reached.

4. The amount of antimycin necessary for maximal inhibition of the reconstituted enzyme is a function of the amount of the cytochrome b and is independent of the amount of cytochrome c₁. It is equal to about one half the amount of native cytochrome b.

5. Preparations of intact or reconstituted succinate-cytochrome c reductase or of cytochrome b completely quench the fluorescence of added antimycin, until an amount of antimycin equal to onehalf the amount of native cytochrome b present was added. Antimycin added in excess of this amount fluoresces with normal intensity. The quenching is only partial in the presence of Na₂S₂O₄. Denatured cytochrome b does not quench the fluorescence.

6. Since preparations of cytochrome b active in reconstitution contained cytochrome c₁ in an amount exceeding one half the amount of native cytochrome b present in the preparation, there is no evidence that native cytochrome b has been resolved from cytochrome c₁. The stimulatory action of cytochrome c₁ may be due to the restoration of a damaged membrane conformation.

7. Based on the assumption that the bc₁ segment of the respiratory chain contains 2b:1c₁:1 antimycin-binding sites, the specific quenching of antimycin fluorescence by binding to cytochrome b enables an accurate determination of the absorbance coefficients of cytochromes b and c₁. These are 25.6 and 20.1 mM⁻¹×cm⁻¹ for the wavelength pairs 563–577 nm and 553–539 nm, respectively, in the difference spectrum reduced minus oxidized.     

Autocatalytic peroxidation of ferrocytochrome c

1. Ferricytochrome c acts as a catalyst in the peroxidation of ferrocytochrome c thereby giving rise to an autocatalytic reaction.

2. The rate of the peroxidation reaction is proportional to the concentration of H₂O₂ and ferricytochrome c but is independent of the concentration of ferrocytochrome c in the concentration ranges studied.

3. Integration of the rate equation, d[c³⁺]/dt = k[c³⁺][H₂O₂], gives a theoretical expression which fits the experimental time courses for the ferrocytochrome c peroxidation reaction.

4. No direct spectral evidence was found for the formation of a catalytically active ferricytochrome c-H₂O₂ derivative. Kinetic evidence is presented, however, which indicates the existence of such an intermediate.

5. Ferricytochrome c was more susceptible than ferrocytochrome c to an apparent degradation reaction caused by excess H₂O₂, thus supporting the idea that the cytochrome c heme iron is more accessible in the oxidized form.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

The deleterious effects of H₂O₂ on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H₂O₂ resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H₂O₂ on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H₂O₂ on respiratory complexes was almost completely recovered by β-mercaptoethanol treatment. H₂O₂ treatment resulted in full resistance to Q_O site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Q_O) of complex III is the target for H₂O₂. H₂O₂ did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H₂O₂ addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.     

Myxothiazol, a new inhibitor of the cytochrome b-c1 segment of the respiratory chain

Myxothiazol inhibited oxygen consumption of beef heart mitochondria in the presence and absence of 2,4-dinitrophenol, as well as NADH oxidation by submitochondrial particles. The doses required for 50% inhibition were 0.58 mol myxothiazol/mol cytochrome b for oxygen consumption of beef heart mitochondria, and 0.45 mol/mol cytochrome b for NADH oxidation by submitochondrial particles. Difference spectra with beef heart mitochondria and with cell suspensions of Saccharomyces cerevisiae revealed that myxothiazol blocked the electron transport within the cytochrome b-c₁ segment of the respiratory chain. Myxothiazol induced a spectral change in cytochrome b which was different from and independent of the shift induced by antimycin. Myxothiazol did not give the extra reduction of cytochrome b typical for antimycin. Studies on the effect of mixtures of myxothiazol and antimycin on the inhibition of NADH oxidation indicated that the binding sites of the two inhibitors are not identical.     

Further studies on the NADH oxidase of the cytoplasmic membrane of Mycobacterium tuberculosis

The cytoplasmic membrane of the H₃₇Ra strain of Mycobacterium tuberculosis has been isolated free of cell wall.

These membrane preparations contain very small quantities of cytochromes c, b and cytochrome oxidase. The cytochrome c is not extracted by any method attempted. The cytochrome b is reducible only by dithionite and is believed not to be involved in the direct transfer of electrons during the oxidation of NADH by these preparations. The NADH oxidase activity of the membrane is inhibited by high concentrations of cyanide and also by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide (HQNO). The cytochrome oxidase of the membrane contains both cytochromes a and a₃ and is present in low concentrations relative to cytochrome c. The cytochrome a₃ component was identified by characteristic complexes with both CO and cyanide and shows a γ-band absorption maximum at a slightly lower wavelength than the cytochrome oxidase of mammalian mitochondria (442 nm vs. 445 nm). The functional activity of the cytochrome oxidase is indicated by the inhibition of reoxidation of reduced cytochromes c and a in the presence of cyanide.     

Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode

Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant (k_s) of 17.6 s⁻¹ and the charge-transfer coefficient (a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2.0 × 10⁻⁵ to 2.4 × 10⁻⁴ mol l⁻¹. The detection limit (3σ) for determination of H₂O₂ has been found to be 1.46 × 10⁻⁵ mol l⁻¹.     

Low-temperature spectral properties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata

Highly purified mitochondrial preparations from the trypanosomatid hemoflagellate, Crithidia fasciculata (A.T.C.C. No.11745), were examined by low-temperature difference spectroscopy. The cytochrome a+a₃ maximum of hypotonically-treated mitochondria reduced with succinate, was shifted from 605 nm at room temperature to 601 nm at 77 °K. The Soret maximum, found at 445 nm at 23 °C, was split at 77 °K into two approximately equally absorbing species with maxima at 438 and 444 nm. A prominent shoulder observed at 590 nm with hypotonically-treated mitochondria was not present in spectra of isotonic controls.

The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent.

Unlike other soluble c-type cytochromes, the maximum of cytochrome c₅₅₅ was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane.

Using cytochrome c₅₅₅-depleted mitochondria, a cytochrome c₁ pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c₅₅₅-depleted preparations exhibited an maximum at 553 nm at room temperature.

The reduced rate of succinate oxidation by cytochrome c₅₅₅-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c₁, even in the presence of antimycin, indicated that cytochrome c₅₅₅-mediated electron transfer between cytochromes c₁ and a+a₃ in a manner analagous to that of cytochrome c in mammalian mitochondria.     

Energy transduction in photosynthetic bacteria VI. Respiratory sites of energy conservation in membranes from dark-grown cells of Rhodopseudomonas capsulata

Membranes prepared from Rhodopseudomonas capsulata grown heterotrophically in the dark perform phosphorylation linked to oxidation of NADH and succinate, with P/2e⁻ ratios of about 0.5 and 0.15, respectively. The localization of the sites of energy conservation was investigated by observing the respiration-induced quenching of the fluorescence of atebrine.

Energization of the membrane can be demonstrated when NADH is oxidized by O₂, ferricyanide or Q₁, when succinate is oxidized by O₂ or by oxidized diaminodurene, and during the oxidation of reduced diaminodurene.

Antimycin A completely inhibits energization between succinate and O₂ or succinate and diaminodurene; however, it only inhibits partially NADH or succinate oxidases and energization between NADH and O₂. KCN inhibits NADH oxidase in a biphasic way: the first level of inhibition is observed at concentrations which block the oxidation of exogenous cytochrome c or of diaminodurene and energization between succinate or ascorbate-diaminodurene and O₂. The second level corresponds to the inhibition of the antimycin-insensitive oxidase.

The results are interpreted as evidence of the presence in these bacteria of a respiratory chain branching after the dehydrogenase system, one arm of the chain being sensitive to antimycin A and low concentrations of KCN and capable of energy conservation, the other being represented by a completely uncoupled system.     

Studies on yeast mitochondria: Some properties of mitochondria isolated from Saccharomyces Carlsbergensis grown in normal glucose medium

1. Mitochondria isolated from Saccharomyces Carlsbergensis are found to have three phosphorylation sites in the respiratory chain for the oxidation of NADH and NAD⁺-linked substrates and two for succinate oxidation. Freshly isolated mitochondria exist in an inhibited state with no respiratory control, but on ageing for 2–3 h a good coupled state is obtained. -Ketogultarate and -glycerophosphate are poorly oxidized in these mitochondria.

2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low K_m. However, one-third of the added NADH is not available for oxidation probably due to some form of compartmentation. Studies of both oxygen uptake and the redox changes of cytochrome b show complete oxidation of two-third of the added NADH.

3. Difference spectra of yeast mitochondria at liquid-nitrogen temperatures show all the characteristic peaks of cytochromes a (600 nm), b (558, 525 and 428 nm), c₁ (552 nm) and c (545 and 516 nm).

4. The reduction of cytochrome b by dicumarol in antimycin A inhibited mitochondria provides evidence for an energy conservation site on the substrate side of cytochrome b.

5. In the absence of added ADP, the oxidation of malate and pyruvate occurs in the yeast mitochondria in a new respiratory state (State X) where the oxygen uptake occurs at State 4 rate but the redox level of the flavins, cytochrome b and c are similar to State 3. State X respiration is believed to be due to depletion of the high energy intermediate C I caused by the substrate anions accumulation.

6. The responses of yeast mitochondria to Ca²⁺ are qualitatively similar to those in rat liver mitochondria, particularly with respect to respiratory stimulation, membrane alkalinization and its accumulation in the mitochondria with succinate as the substrate in the presence and absence of acetate.     

Extramitochondrial release of hydrogen peroxide from insect and mouse liver mitochondria using the respiratory inhibitors phosphine, myxothiazol, and antimycin and spectral analysis of inhibited cytochromes

The fumigant insecticide phosphine (PH3) is known to inhibit cytochrome c oxidase in vitro. Inhibition of the respiratory chain at this site has been shown to stimulate the generation of superoxide radicals (O2-), which dismutate to form hydrogen peroxide (H2O2). This study was performed in order to investigate the production of H2O2 by mitochondria isolated from granary weevil (Sitophilus granarius) and mouse liver on exposure to PH3. Other respiratory inhibitors, antimycin, myxothiazol, and rotenone were used with insect mitochondria. Hydrogen peroxide was measured spectrophotometrically using yeast cytochrome c peroxidase as an indicator. Insect and mouse liver mitochondria, utilizing endogenous substrate, both produced H2O2 after inhibition by PH3. Insect organelles released threefold more H2O2 than did mouse organelles, when exposed to PH3. Production of H2O2 by PH3-treated insect mitochondria was increased significantly on addition of the substrate alpha-glycerophosphate. Succinate did not enhance H2O2 production, however, indicating that the H2O2 did not result from the autoxidation of ubiquinone. NAD(+)-linked substrates, malate and pyruvate also had no effect on H2O2 production, suggesting that NADH-dehydrogenase was not the source of H2O2. Data obtained using antimycin and myxothiazol, both of which stimulated the release of H2O2 from insect mitochondria, lead to the conclusion that glycerophosphate dehydrogenase is a source of H2O2. The effect of combining PH3, antimycin, and myxothiazol on cytochrome spectra in insect mitochondria was also recorded. It was observed that PH3 reduces cytochrome c oxidase but none of the other cytochromes in the electron transport chain. There was no movement of electrons to cytochrome b when insect mitochondria are inhibited with PH3. The spectral data show that the inhibitors interact with the respiratory chain in a way that would allow the production of H2O2 from the sites proposed previously.     

Reducibility of cytochromes b in aerobic beef-heart mitochondria treated with antimycin

Under anaerobic conditions cytochrome b in beef-heart mitochondria is partially reduced in the presence of NADH, whereas other cytochromes are completely reduced. Addition of antimycin together with oxygen under these conditions causes an immediate reduction of cytochromes b-558, b-562 and b-566 and oxidation of cytochrome c. During the subsequent transient aerobic steady state cytochromes b-558 and b-566 are rapidly re-oxidized without changes in redox state of cytochrome c, but cytochrome b-562 remains reduced. When oxygen is consumed by the leak through or around the antimycin-inhibition site, cytochrome b-562 becomes oxidized with concomitant reduction of cytochrome c.

The cytochromes b in lyophilized beef-heart mitochondria are more readily accessible to electrons from NADH, and in the presence of antimycin and NADH a complete and stable reduction is obtained under both aerobic and anaerobic conditions. Gradual addition of rotenone under these conditions causes re-oxidation of cytochromes b in which oxidation of cytochromes b-558 and b-566 precedes that of cytochrome b-562.

It is concluded that (1) the effect of antimycin in the presence of oxygen involves all three cytochromes b, (2) the reducibility of the cytochromes b in the aerobic steady state of antimycin-treated mitochondria is dependent upon the potential of the substrate redox couple registered on the cytochromes, and (3) the midpoint potential of cytochrome b-562 in the presence of antimycin is higher than that of cytochrome b-558 or b-566.     

Purification of cytochrome c peroxidase for monitoring H2O2 production.

One of the most precise methods of determining hydrogen peroxide (H₂O₂) formation by biological systems is based on measuring the rate of enzyme-substrate complex formation between H₂O₂ and cytochrome c peroxidase (CCP). The main problem with this method is that CCP is not commercially available and has to be prepared in the laboratory. We have modified some currently available methods for purifying a highly active preparation of CCP in about 4 d. It includes a batch extraction of protein using DEAE-sepharose followed by concentration either by lyophilization or by passing the extract through a small DEAE-sepharose column instead of by ultrafiltration. The concentrated preparation is passed through a Sephadex G-75 column and the final CCP crystallized against water. The final preparations had a purity index (PI, ratio of absorbance at 408 nm/280 nm, equivalent to heme/protein ratio) above 1.2. These changes make the overall procedure very simple, preserving enzyme activity and spectral properties. In addition, we point out that special care has to be taken to eliminate cytochrome c from crude CCP extracts. Cytochrome c not only introduces an artifact when determining PI, but is also may act as a hydrogen donor for CCP when monitoring H₂O₂ formation, thus decreasing the sensitivity of this method.     

The b-type cytochromes of bovine heart mitochondria: Absorption spectra, enzymatic properties, and distribution in the electron transfer complexes

1. 1. Three b-type cytochromes (b_557.5, b₅₆₀, and b_562.5), plus a chromophore with an absorption peak at 558 nm at 77 °K, have been found to be associated with the electron transport system of bovine heart mitochondria. The reduced minus oxidized spectra of these components at 77 °K, as well as that of cytochrome c₁, have been recorded with a wavelength accuracy of ± 0.1 nm and presented to the nearest 0.5 nm. All the major and β absorption peaks of cytochromes b_557.5, b₅₆₀, b_562.5, c₁ and c have been shown by fourth derivative analysis to be present in the dithionite-reduced minus oxidized spectra of mitochondria and submitochondrial particles.

2. 2. The distribution of the above components has been studied in the four electron transfer complexes of the respiratory chain. Cytochromes b₅₆₀, b_562.5 and c₁, as well as chromophore-558, were found to fractionate into Complex III (reduced ubiquinone-cytochrome c reductase), whereas cytochrome b_557.5 was found in Complex II (succinate-ubiquinone reductase).

3. 3. Cytochrome b₅₆₀ was readily reduced by NADH or succinate, but b_562.5 was not reduced by substrates unless the preparation was treated with antimycin A. In antimycin-treated preparations pre-reduction of c₁ with ascorbate inhibited the subsequent reduction of b_562.5 by substrates. These results indicate that b₅₆₀ and b_562.5 correspond, respectively, to b_K and b_T previously described by Chance et al.¹⁴ (1970, Proc. Natl. Acad. Sci. U.S. 66, 1175–1182).

4. 4. Similar to b₅₆₀, chromophore-558 can be reduced by substrates in the absence or presence of antimycin A. However, in antimycin-treated preparations, pre-reduction of c₁ inhibits its subsequent reduction by substrates. This property is similar to that of b_562.5.

5. 5. Cytochrome b_557.5, which occurs in Complex II, appears to have a low mid-point potential. It can be reduced with dithionite and oxidized by fumarate or ubiquinone. CO treatment of dithionite-reduced b_557.5 neither modified the spectrum of this cytochrome nor diminished the extent of b_557.5 reoxidation by fumarate.

6. 6. Antimycin A treatment does not appear to alter the spectra of the above cytochromes. However, small amounts (< 4%) of ethanol or methanol, which are usually added to particles as solvent for antimycin A, have a pronounced effect on the peaks of cytochrome c₁. The spectrum of cytochrome c₁ at 77 °K as modified by 3% (v/v) ethanol is shown.

Phenotypic analysis of the ccp1Delta and ccp1Delta-ccp1W191F mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H₂O₂ to H₂O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H₂O₂. Transformation of ccp1Δ with ccp1^W191F, which encodes the CCP^W191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H₂O₂ of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1^W191F exhibited wild-type tolerance to H₂O₂, which exceeded that of ccp1Δ. Challenge with H₂O₂ caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H₂O₂ exposure in ccp1Δ than in ccp1Δ-ccp1^W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1^W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.     

Hydrogen peroxide is necessary for abscisic acid-induced senescence of rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced rice leaf senescence is investigated. ABA treatment resulted in H₂O₂ production in rice leaves, which preceded the occurrence of leaf senescence. Dimethylthiourea, a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced senescence, ABA-increased malondialdehyde (MDA) content, ABA-increased antioxidative enzyme activities (superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase), and ABA-decreased antioxidant contents (ascorbic acid and reduced glutathione) in rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, and KCN and NaN₃, inhibitors of peroxidase, prevented ABA-induced H₂O₂ production, suggesting NADPH oxidase and peroxidase are H₂O₂-generating enzymes in ABA-treated rice leaves. DPI, IMD, KCN, and NaN₃ also inhibited ABA-promoted senescence, ABA-increased MDA contents, ABA-increased antioxidative enzyme activities, and ABA-decreased antioxidants in rice leaves. These results suggest that H₂O₂ is involved in ABA-induced senescence of rice leaves.     

Inhibition of respiration and destruction of cytochrome a3 by light in mitochondria and cytochrome oxidase from beef heart

Irradiation of beef-heart mitochondria and of cytochrome oxidase purified from beef-heart mitochondria with blue light inhibited electron transport from substrate (succinate for the mitochondria and reduced cytochrome c for the cytochrome oxidase) to O₂. The irradiation treatment also destroyed cytochrome a₃ as assayed by the absorption band for the reduced cyanide-cytochrome a₃ complex at 587 nm in the low-temperature absorption spectrum. Irradiation under anaerobic conditions was not inhibitory. Cytochrome a₃ was protected against photodestruction if cyanide was present during the irradiation.     

Hydroxylamine potentiates the effect of low dose hydrogen peroxide in glioma cells independent of p53

We had earlier shown that higher concentration of hydrogen peroxide (H₂O₂) induced p53-dependent apoptosis in glioma cell line with wild type p53 but had minimal effect on cells with mutated p53. Here we show a potentiating effect of hydroxylamine (HA), an inhibitor of catalase, on a nontoxic dose of H₂O₂ in glioma cells. HA sensitized both p53 wild type and mutated glioma cells to 0.25 mM H₂O₂. Potentiating effect of HA was independent of p53. Higher levels of reactive oxygen species (ROS) generation were observed in cells treated with HA+H₂O₂ as compared to cells treated with each component alone in both the cell lines. Dimethyl sulfoxide (DMSO) protected cells. Cytosolic cytochrome c and activated caspase 3 were detected at 4 h. The results suggest that higher levels of intracellular ROS, generated by HA+H₂O₂ act as a molecular switch in activating a rapidly acting p53-independent mitochondrial apoptotic pathway.     

Properties and function of the respiratory chain of freshwater mussel spermatozoa in relation to motility

1. The spermatozoa of the freshwater mussel (Hyriopsis schlegelii) contain cytochromes aa₃, b and c, flavoproteins and nicotinamide nucleotides in molar ratios of 1.0:0.9:1.8:1.8:8.7. Cytochrome c₁ is not detectable even at liquid-N₂ temperature, but a c₁-like cytochrome with an -band at 550 mμ is found at liquid-N₂ temperature in a cell preparation from which cytochrome c is completely removed.

2. The near-ultraviolet difference spectrum of whole cells reveals an absorption peak at 315 mμ with a shoulder around 350 mμ.

3. Both the endogenous respiration and motility of spermatozoa are completely blocked by 0.2 mM CN⁻ and by 0.2 μM antimycin A. 2,4-Dinitrophenol and pentachlorophenol completely inhibit motility at the maximal stimulation of respiration. Rotenone strongly inhibits NADH oxidase of spermatozoa, although it has no effect on the respiration of whole cells.

4. It is concluded that the motility of mussel spermatozoa is tightly coupled to respiration, and the respiratory chain phosphorylating process is the only energy-supplying system for motility.