Inhibition of Photosynthetic Energy Conversion by Cupric Ion : EVIDENCE FOR Cu-COUPLING FACTOR 1 INTERACTION

This study describes a specific Cu²⁺ and light-dependent inhibition of spinach (Spinacia oleracea L.) chloroplast reactions involving coupling factor 1 function. A primary effect is an inhibition of photophosphorylation induced by illumination of Class II chloroplasts with micromolar Cu²⁺ and pyocyanine in the absence of ADP, Mg²⁺, and HPO₄²⁻. The inhibition, which is dependent on free Cu²⁺ as indicated by protection by ethylene diamine tetraacetic acid and dithiothreitol, requires illumination (electron flow) for establishment of the specific inhibition to be noted. Protection is also afforded by uncouplers and some partial protection is provided by micromolar concentrations of ADP and ATP. The data strongly suggest that Cu²⁺ causes an O₂-independent oxidation of sulfhydryl groups on coupling factor 1, which are essential to catalytic function. This conclusion is supported by the reduction of energy-dependent ³H-N-ethylmaleimide labeling of the γ subunit of coupling factor 1 by the Cu²⁺-light pretreatment.     

Reversible sporicidal action of cuprous ions

At 10 mM, Cu⁺ was highly protective against killing of spores of Bacillus megaterium ATCC 19213 by H₂O₂, while at higher concentrations, from 15–100 mM, killing was augmented. In contrast, Cu²⁺, Fe²⁺, Fe³⁺, Co²⁺ or Co³⁺ ions acted only protectively. Cu⁺ itself was sporicidal in the absence of H₂O₂ or ascorbate, and its sporicidal action did not depend on generation of highly reactive oxygen species. It appeared that killing involved either inhibition of germination or copper toxicity to germinated cells in that Cu⁺-inactivated spores did not germinate readily but chemical decoating of the cells prior to plating on a solid medium resulted in reversal of the sporicidal effect. Received 12 July 1996/ Accepted in revised form 03 November 1996     

Capturing a Reactive State of Amyloid Aggregates: NMR-BASED CHARACTERIZATION OF COPPER-BOUND ALZHEIMER DISEASE AMYLOID β-FIBRILS IN A REDOX CYCLE*

The interaction of redox-active copper ions with misfolded amyloid β (Aβ) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer disease. Despite intensive studies, it is still not conclusive how the interaction of Cu⁺/Cu²⁺ with Aβ aggregates leads to ROS production even at the in vitro level. In this study, we examined the interaction between Cu⁺/Cu²⁺ and Aβ fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of ∼60 μm hydrogen peroxide (H₂O₂) from a solution of 20 μm Cu²⁺ ions in complex with Aβ(1–40) in fibrils ([Cu²⁺]/[Aβ] = 0.4) within 2 h of incubation after addition of biological reducing agent ascorbate at the physiological concentration (∼1 mm). Furthermore, SSNMR ¹H T₁ measurements demonstrated that during ROS production the conversion of paramagnetic Cu²⁺ into diamagnetic Cu⁺ occurs while the reactive Cu⁺ ions remain bound to the amyloid fibrils. The results also suggest that O₂ is required for rapid recycling of Cu⁺ bound to Aβ back to Cu²⁺, which allows for continuous production of H₂O₂. Both ¹³C and ¹⁵N SSNMR results show that Cu⁺ coordinates to Aβ(1–40) fibrils primarily through the side chain N_δ of both His-13 and His-14, suggesting major rearrangements from the Cu²⁺ coordination via N_ϵ in the redox cycle. ¹³C SSNMR chemical shift analysis suggests that the overall Aβ conformations are largely unaffected by Cu⁺ binding. These results present crucial site-specific evidence of how the full-length Aβ in amyloid fibrils offers catalytic Cu⁺ centers.     

The mechanism of methanol decomposition by CuO. A theoretical study based on the reaction force and reaction electronic flux analysis

A theoretical study of methanol decomposition using a model representing the initial step of the reaction CH ₃ OH + CuO → CH ₂ O + H ₂ O + Cu is presented. Theoretical calculations using B3LYP/6-31 G along with Lanl2DZ pseudopotentials on metallic centers were performed and the results discussed within the framework of the reaction force analysis. It has been found that the reaction takes place following a stepwise mechanism: first, copper reduction (Cu ⁺² → Cu ⁺) accompanies the oxygen transposition and then a second reduction takes place (Cu ⁺ → Cu ⁰) together with a proton transfer that produce formaldehyde and release a water molecule.     

Inhibition by cupric ions of the hydration of CO2 catalyzed by carbonic anhydrase II

The inhibition by cupric ions of the hydration of CO₂ catalyzed by carbonic anhydrase II is interesting because of the results of Tuet al. obtained at chemical equilibrium, indicating that Cu²⁺ inhibits specifically a proton transfer in the catalytic pathway. We have measured this inhibition at steady state, using stopped-flow methods. The inhibition by Cu²⁺ of the hydration of CO₂ catalyzed by carbonic anhydrase II had aK _I near 1×10^–6 M atpH 7.0 and gave inhibition that is noncompetitive atpH 6.0 and mixed, but close to uncompetitive, atpH 6.8. ThepH dependence of this binding is consistent with a binding site for Cu²⁺ on the enzyme with apK _a near 7. The binding interaction between Cu²⁺ and the fluorescent inhibitor 5-dimethylaminonaphthalene-l-sulfonamide on carbonic anhydrase II was noncompetitive, indicating that the binding site for Cu²⁺ is distinct from the coordination sphere of zinc in which the actual interconversion of CO₂ and HCO ₃ ^– and the binding of sulfonamides takes place.     

Interrelation of active oxygen species,membrane damage and altered calcium functions

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine —xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O₂ ^– in the presence of CaCl₂ enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tertbutyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca²⁺ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.     

Reaction of nitric oxide with the oxidized di-heme and heme–copper oxygen-reducing centers of terminal oxidases: Different reaction pathways and end-products

Inhibition of terminal oxidases by nitric oxide (NO) has been extensively investigated as it plays a role in regulation of cellular respiration and pathophysiology. Cytochrome bd is a tri-heme (b₅₅₈, b₅₉₅, d) bacterial oxidase containing no copper that couples electron transfer from quinol to O₂ (to produce H₂O) with generation of a transmembrane protonmotive force. In this work, we investigated by stopped-flow absorption spectroscopy the reaction of NO with Escherichia coli cytochrome bd in the fully oxidized (O) state. We show that under anaerobic conditions, the O state of the enzyme binds NO at heme d with second-order rate constant k_on = 1.5 ± 0.2 × 10² M⁻¹ s⁻¹, yielding a nitrosyl adduct (d³⁺–NO or d²⁺–NO⁺) with characteristic optical features (an absorption increase at 639 nm and a red shift of the Soret band). The reaction mechanism is remarkably different from that of O cytochrome c oxidase in which the heme–copper binuclear center reacts with NO approximately three orders of magnitude faster, forming nitrite. The data allow us to conclude that in the reaction of NO with terminal oxidases in the O state, Cu_B is indispensable for rapid oxidation of NO into nitrite.     

Pulse radiolytically determined superoxide dismutase mimicking activity of copper-putrescine-pyridine,a diSchiff base coordinated copper complex

In order to exclude possible interferences in the many indirect superoxide dismutase (SOD) activity measurements using the copper-putrescine-pyridine complex (Cu-PuPy) a pulse radiolytic study on this diSchiff base copper complex has been devised and sucessfully performed. The reaction kinetics and rate constants of pulse radiolytically generated superoxide in the presence of Cu-PuPy reveal pseudo first-order characteristics. The rate constant (k ₂ = 6 ± 1 × 10⁸ m ^–1 s ^–1) is comparable to that of an Fe-SOD and is approximately a factor of 3 lower than that of bovine Cu₂Zn₂-SOD. The superoxide dismutating activity of Cu-PuPy shows a more pronounced temperature dependence compared with that of Cu₂Zn₂-SOD. Arrhenius analyses yielded activation energies of 7.8 ±0.3 and 4.6 ± 0.2 kcal mol^–1 for Cu-PuPy and Cu₂Zn₂-SOD, respectively. The rate constant of the reaction of superoxide and Cu-PuPy is highest at pH 5.0. The possible application of Cu-PuPy for new therapeutic strategies on all types of inflammatory diseases appears to be promising.     

Isolation and characterization of anthocyanin 5-O-glucosyltransferase from flowers of Dahlia variabilis

In the cyanic flowers ofDahlia variabilis (Asteraceae), an enzyme was demonstrated which catalyzes a glucosyl group transfer from UDP-glucose to the 5 position of anthocyanidin 3-O-glucoside and 3-O-malonylglucoside. The anthocyanin 5-O-glucosyltransferase (5GT) was purified 88-fold at 8 percnt; yield by (NH₄)₂SO₄ precipitation followed by successive chromatography on DEAE-cellulose, Sephacryl S-200 and Mono P. 5GT exhibited a pH optimum at 8.0 and a pI of 4. 2. Its apparent molecular weight calculated from Sephacryl S-200 was 53 kDa. Its activity was stimulated by 2-ME and DTE but strongly inhibited by PCMB and NEM. It was slightly activated by Mg²⁺ and Ca²⁺ but strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, Fe³⁺ and Al³⁺. No effect of EDTA was observed. The apparent Km values for cyanidin 3-O-glucoside, cyanidin 3-O-(6′′-O-malonyl)glucoside and UDP-glucose were 120 μmol/L, 75 μmol/L and 250 μmol/L, respectively. Pelargonidin 3-O-glucoside and malonylglucoside were also considerable substrates, but low relative activity was observed for delphinidin 3-O-glucoside which has yet not been found inDahlia flowers.Dahlia 5GT showed substrate specificities different from those reported forSilene, Petunia, Matthiola andPerilla. Neither ADP-glucose nor UDP-galactose could serve as glycosyl donor.     

Influence of chelators and iron ions on the production and degradation of H2O2 by β-amyloid–copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Thiosulfate stimulates growth and alleviates silver and copper toxicity in tomato root cultures

The influence of supplemented thiosulfate (S₂O₃ ²⁻) as well as a complex of either Ag⁺ or Cu²⁺ with S₂O₃ ²⁻ in the culture medium on proliferating root cultures of tomato (Solanum lycopersicum) was investigated. The presence of 10–300 μM sodium thiosulfate (Na₂S₂O₃) in half-strength Murashige and Skoog (MS) basal salt medium promoted root elongation and proliferation of lateral roots. Growth was enhanced by 1–2 μM AgNO₃, but was completely arrested at 5 μM AgNO₃; moreover, growth inhibition was elicited by dissolved silver (Ag⁺) and by silver in silver precipitate particles. Root elongation was also inhibited by 50 μM CuSO₄ supplemented to the basal medium. Roots subjected to either AgNO₃ or CuSO₄ growth inhibiting treatments were unable to recover following transfer to medium lacking either Ag⁺ or Cu²⁺. When the basal medium was supplemented with either silver or copper in the form of silver thiosulfate complex or copper thiosulfate complex, root cultures continued to elongate and proliferate, thus either completely alleviating or diminishing the inhibitory effects of Ag⁺ and Cu²⁺, respectively. It was concluded that tomato roots sensed and responded to S₂O₃ ²⁻, hence root proliferation could be promoted by adding Na₂S₂O₃ to the medium. Moreover, a complex of Ag⁺ with S₂O₃ ²⁻ detoxified dissolved Ag⁺ and prevented the generation of toxic silver particle precipitates. Consequently, silver thiosulfate was superior to AgNO₃ in enhancing root culture. Finally, a complex of Cu²⁺ with S₂O₃ ²⁻ ligand reduced toxicity of Cu²⁺ to root cultures of tomato.     

The effect of copper on frog skin. The role of sulphydryl groups

1. 1. Cu²⁺ at a concentration of 10⁻⁴ M, when applied to the external side of the frog skin produces an increase in the short-circuit current (I_sc).

2. 2. This effect was studied in skins of Rana temporaria adapted to cold (5°C) and room temperature (20°C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda.

3. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu²⁺ than skins with high I_sc.

4. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high I_sc give good responses to Cu²⁺.

5. 5. 5,5′-dithiobis(2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu²⁺, and dithiothreitol an SH-reducing agent, reverses the effect of this ion.

6. 6. Cu²⁺ also induces an increase in the unidirectional K⁺ fluxes and unmasks a net outward potassium flux.

7. 7. The outward K⁺ flux induced by Cu²⁺ is sensitive to ouabain.

8. 8. It is concluded that Cu²⁺ increases the permeability of the external barrier of the frog skin to Na⁺ and K⁺, probably by reacting with SH groups.

Na+/Ca2+ exchange of isolated sarcolemmal membrane: effects of insulin,oxidants and insulin deficiency

In this study we prepared sarcolemmal fractions from bovine and rat hearts; their Na⁺K⁺ ATPase activities, measured in the presence of saponin to unmask latent Na⁺K⁺ ATPase, were 59.4 and 48.8 µ mol Pi/mg protein · h, respectively. The rate of Na⁺dependent Ca2⁺ uptake was linear for the first 10 s and a plateau was reached in 3 min. Oxidation by free radical generation either with H₂O₂, FeSO₄ plus DTT or xanthine oxidase plus hypoxanthine stimulated Na⁺/Ca²⁺ exchange in a time-dependent manner. The stimulation was abolished by deferoxamine or o-phenanthroline. By contrast, oxidation by HOCI inhibited Na⁺/Ca²⁺ exchange in proportion to its concentration, and this inhibition was antagonized by DTT. DTT alone had no effect on the exchange. Insulin stimulated Na⁺/Ca²⁺ exchange, its maximal effect was attained after 30min incubation with 100 µ units/ml. N-ethylmaleimide inhibited the exchange both in the presence and in the absence of insulin. Sarcolemmal fractions prepared from hearts of alloxan-treated, acutely diabetic rats showed a significant decrease in Na⁺/Ca²⁺ exchange. Addition of insulin in vitro significantly stimulated Na⁺/Ca²⁺ exchange of both diabetic and control groups. The results indicate that sarcolemmal Na⁺/Ca²⁺ exchange function is modulated by oxidation-reduction states and by the presence of insulin.     

Inhibitory effects of copper on bacteria related to the free ion concentration

Cu²⁺ ion determinations were carried out in complex and in inorganic salts-glycerol media, to which increasing amounts of Cu(II) had been added, with the ion-specific Cu(II)-Selectrode. Likewise, complexing capacity of bacterial suspensions was estimated by titration with CuSO₄.Copper-sensitive bacteria, e.g.,Klebsiella aerogenes, were inhibited in their growth and survival in the range of 10^–8–10^–6 M Cu²⁺ ion concentrations. In copper-buffered complex media, high copper loads could be tolerated, as growth proceeded with most of the copper bound to medium components. In low-complexing mineral salts media, in which high Cu²⁺ ion concentrations exist at low copper loads, there was competition of Cu²⁺ for binding sites of the cells. Total allowed copper was then determined by the ratio of copper to biomass.Copper-resistant bacteria could be isolated from a stock solution of CuSO₄, containing 100 ppm Cu(II). They were of thePseudomonas type and showed a much higher tolerance towards Cu²⁺, up to 10^–3 M.     

Protective effect of carboxyethylgermanium sesquioxide (Ge-132) on superoxide generation by60Co-irradiated leukocytes

The carboxyethylgermanium sesquioxide, Ge-132, is an organogermanium compound which has been shown to modulate leukocyte functions. In this study, we examined the effect of Ge-132 on the generation of superoxide radicals (O₂ ^–) either from leukocytes or in cell-free system, employing the highly sensitive 2-methyl-6-[p-methoxy-phenyl]-3,7-dihydroimidazo[1,2-alpha] pyrazin-3-one (MCLA) -dependent chemiluminescence method and the specific electron spin resonance/spin trapping method, respectively. In addition, thein vitro protective effect of Ge-132 on the leukocytes irradiated with⁶⁰Co was studied.The incubation with Ge-132 resulted in an increase in basal O₂ ^– release of intact leukocytes, but had no effect on OZ generation from leukocytes stimulated with phorbol myristate acetate (PMA). Irradiation with⁶⁰Co decreased the Oz generation from leukocytes in a dose-dependent manner. Ge-132 had no effect on basal O₂ ^– release from⁶⁰Co-irradiated leukocytes, but it prevented the decrease in PMA-stimulated O₂ ^– generation by irradiated leukocytes. Ge-132 itself had no superoxide scavenging activity in cell-free system. On the other hand, higher concentrations of Ge-132 had decreasing effects on both basal O₂ ^–1 release and PMA-stimulated O₂ ^– generation from leukocytes, but they did not affect leukocyte viability. Above results indicate that 1) Ge-132 can stimulate the basal O₂ ^– release from leukocytes, 2) Ge-132 can prevent the decrease of O₂ ^– generation by⁶⁰Co-irradiated leukocytes, 3) in higher concentrations, Ge-132 may have a membrane stabilizing effect.     

Evidence for a slow and oxygen-insensitive intra-molecular long range electron transfer from tyrosine residues to the semi-oxidized tryptophan 214 in human serum albumin: its inhibition by bound copper (II)

A slow, long range electron transfer (SLRET) in human serum albumin (HSA) is observed from an intact tyrosine (Tyr) residue to the neutral tryptophan (Trp) radical (Trp·) generated in pulse radiolysis. This radical is formed, at neutral pH, through oxidation with Br₂^·− radical anions of the single Trp 214 present. The SLRET rate constant of ~0.2 s⁻¹ determined is independent of HSA concentration and radiation dose, consistent with an intra-molecular process. This is the slowest rate constant so far reported for an intra-molecular LRET. In sharp contrast with the LRET reported for other proteins, the SLRET observed here is insensitive to oxygen, suggesting that the oxidized Trp is inaccessible to—or do not react with radiolytically generated O₂^·−. In N₂O-saturated solutions, the SLRET is inhibited by Cu²⁺ ions bound to the His 3 residue of the N-terminal group of HSA but it is partially restored in O₂-saturated solutions.     

Reaction of Mixed Valence State Cytochrome Oxidase with Oxygen in Plant Mitochondria: A STUDY BY LOW TEMPERATURE FLASH PHOTOLYSIS AND RAPID WAVELENGTH SCANNING OPTICAL SPECTROMETRY

The reaction of mixed valence state cytochrome oxidase (Cu_A²⁺a³⁺ · Cu_B⁺a₃²⁺) with O₂ at 173 K has been investigated in purified potato mitochondria by low temperature flash photolysis and rad wavelength scanning optical spectrometry in the visible region. The kinetics of the reaction have been analyzed simultaneously at six wavelength pairs (586-630, 590-630, 594-630, 604-630, 607-630, and 610-630 nanometers) by nonlinear optimization techniques, and found to proceed by a two-species sequential mechanism. The “pure” difference spectra of the two species, I_M and II_M, relative to unliganded mixed valence state cytochrome oxidase have been obtained. The difference spectrum of species I_M is characterized by a peak at 591 nanometers, with a shoulder at 584 nanometers and a trough at 602 nanometers, and that of species II_M by an α band split into a prominent peak at 607 nanometers and a small side peak at 594 nanometers. Evidence is presented to suggest that these two bands arise from O₂⁻ → Cu_B²⁺ and O₂⁻ → a₃²⁺ charge transfer transitions which would imply that O₂⁻ forms a bridging ligand between Cu_B and the iron atom of cytochrome a₃ in species II_M. The kinetics of the reaction and the spectral characteristics of species I_M and II_M obtained with the potato mitochondrial system are compared and contrasted with data in the literature on the beef heart mitochondrial system.     

Generation of hydrogen peroxide by brown adipose tissue mitochondria

This is the first report on the generation of H₂O₂ by brown adipose tissue mitochondria. Flavin dehydrogenase-linked substrates like succinate, glycerol-1-phosphate, and fatty acyl CoA were good substrates for the reaction, while NAD⁺-linked substrates were less effective. In cold-acclimated animals the activity showed a substantial increase (2.5-fold). TheK _m andV _max of the reaction were considerably lower than those of the respective dehydrogenase. Metal ions, particularly Cu²⁺ and Fe²⁺ were potent inhibitors of the reaction. Nucleoside diphosphates, which were inhibitors by themselves, potentiated the inhibitory action of Fe²⁺ ions. In most of the properties, the H₂O₂ generator of brown adipose tissue mitochondria resembled that of liver mitochondria.     

Salinity and Copper-Induced Oxidative Damage and Changes in the Antioxidative Defence Systems of Anabaena doliolum

Summary This study provides first-hand information on the salinity and copper-induced oxidative damage and its protection in Anabaena doliolum by the antioxidant defence system. Oxidative damage measured in terms of lipid peroxidation, electrolyte leakage and H₂O₂ production was induced by different concentrations of NaCl and Cu²⁺. A greater electrolyte leakage by NaCl than Cu²⁺ supported the hypothesis of salinity being more injurious than copper. To explore the survival strategies of A. doliolum under NaCl and Cu stress, enzymatic antioxidant activities e.g. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) and nonenzymatic antioxidant contents such as glutathione reduced (GSH), ascorbate, α-tocopherol, and carotenoid were measured. A general induction in SOD and APX activities as well as ascorbate and α-tocopherol contents was found under NaCl and Cu²⁺ stress. In contrast to this, an appreciable decline in GR activity, GSH pool and carotenoid content under Cu²⁺ and an increase under NaCl stress were observed. CAT activity was completely inhibited at high doses of NaCl but stimulated following Cu²⁺ treatment. The above results suggest the involvement of APX and CAT in the scavenging of H₂O₂ under Cu²⁺ stress. In contrast to this, only APX was involved in H₂O₂ scavenging under salt stress. Our postulate of Cu²⁺-mediated antagonism of salt stress can be explained by a conceivable reversion of Na⁺-induced disturbance of cellular homeostasis by redox active Cu²⁺.