Short-term effects of carbon dioxide on carnation callus cell respiration

The addition of potassium bicarbonate to the electrode cuvette immediately stimulated the rate of dark O₂ uptake of photomixotrophic and heterotrophic carnation (Dianthus caryophyllus L.) callus, of Elodea canadensis (Michx) leaves, and of other plant tissues. This phenomenon occurred at pH values lower than 7.2 to 7.8, and the stimulation depended on the concentration of gaseous CO₂ in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO₂ again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO₂/bicarbonate dependent stimulation of respiration did not occur in animal tissues such as rat diaphragm and isolated hepatocytes, and was inhibited by salicylhydroxamic acid in carnation callus cells and E. canadensis leaves. This suggested that the alternative oxidase was engaged during the stimulation in plant tissues. The cytochrome pathway was severely inhibited by CO₂/bicarbonate either in the absence or in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone. The activity of cytochrome c oxidase of callus tissue homogenates was also inhibited by CO₂/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO₂) partially inhibited the cytochrome pathway (apparently at the oxidase level), and this block in electron transport elicited a large transient engagement of the alternative oxidase when present uninhibited.     

Bicarbonate-dependent,carbonate radical anion-driven tocopherol-mediated human LDL peroxidation: an in vitro and in vivo study

Abstract

We have here investigated possible occurrence of bicarbonate-dependent, carbonate radical anion (CO₃^??)-driven tocopherol-mediated human LDL peroxidation (TMP) in vitro and in vivo. CO₃^??, generated in vitro by the SOD1/H₂O₂/bicarbonate system, readily promoted TMP, which was dependent on α-tocopherol and bicarbonate concentrations, and was inhibited by the CO₃^?? scavenger ethanol; moreover, TMP induced in vitro by the SOD1/H₂O₂/bicarbonate system occurred in the presence of α-tocopherol that typically underwent slow oxidative consumption. In the in vivo clinical setting, we showed that, compared to controls, hypertensive patients with diuretic-induced metabolic alkalosis and heightened blood bicarbonate concentration had lipid hydroperoxide burden and decreased α-tocopherol content in the LDL fraction, with direct significant correlation between the LDL levels of α-tocopherol and those of lipid hydroperoxides; remarkably, after resolution of metabolic alkalosis, together with normalization of blood bicarbonate concentration, the LDL content of lipid hydroperoxides was decreased and that of α-tocopherol augmented significantly. These findings suggest bicarbonate-dependent, CO₃^??-driven LDL TMP in vivo. In conclusion, the present study highlights the occurrence of bicarbonate-dependent, CO₃^??-driven human LDL TMP, the role of which in pathological conditions such as atherosclerosis warrants, however, further investigation.     

Vanadate-stimulated NADH oxidation in microsomes

Addition of vanadate, stimulated oxidation of NADH by rat liver microsomes. The products were NAD⁺ and H₂O₂. High rates of this reaction were obtained in the presence of phosphate buffer and at low pH values. The yellow-orange colored polymeric form of vanadate appears to be the active species and both ortho- and meta-vanadate gave poor activities even at mM concentrations.The activity as measured by oxygen uptake was inhibited by cyanide, EDTA, mannitol, histidine, ascorbate, noradrenaline, adriamycin, cytochrome c, Mn²⁺, superoxide dismutase, horseradish peroxidase and catalase. Mitochondrial outer membranes possess a similar activity of vanadate-stimulated NADH oxidation. But addition of mitochondria and some of its derivative particles abolished the microsomal activity. In the absence of oxygen, disappearance of NADH measured by decrease in absorbance at 340 nm continued at nearly the same rate since vanadate served as an electron acceptor in the microsomal system. Addition of excess catalase or SOD abolished the oxygen uptake while retaining significant rates of NADH disappearance indicating that the two activities are delinked. A mechanism is proposed wherein oxygen receives the first electron from NAD radical generated by oxidation of NADH by phosphovanadate and the consequent reduced species of vanadate (V^iv) gives the second electron to superoxide to reduce it H₂O₂. This is applicable to all membranes whereas microsomes have the additional capability of reducing vanadate.     

The direct oxidation of ethanol by a catalase- and alcohol dehydrogenase-free reconstituted system containing cytochrome P-4501.

Ethanol oxidation activity has been reconstituted in a system composed of NADPH-cytochrome c reductase, synthetic dilauroylglycerol-3-phosphorylcholine and cytochrome P-450 purified from liver microsomes of phenobarbital-treated rats. This system is free of alcohol dehydrogenase and catalase activities. Furthermore, sodium azide (1 mm), a catalase inhibitor, is without effect on ethanol metabolism. There is a requirement for both NADPH-cytochrome c reductase and cytochrome P-450 and a partial requirement for phospholipid for ethanol oxidation by the reconstituted system. In addition, both NADPH and O₂ are required for catalysis. Under optimal reaction conditions, the rate of acetaldehyde formation if 25 to 50 nmol/min/nmol of cytochrome P-450. Cytochrome P-450 from other sources, including the homogeneous P-450LM₂ from phenobarbital-treated rabbits, have also been found to catalyze ethanol oxidation in reconstituted systems. Antibody prepared against cytochrome P-450 inhibits ethanol metabolism in the reconstituted system consistent with a cytochrome P-450-mediated reaction. Furthermore, cumene hydroperoxide can replace both NADPH and NADPH-cytochrome c reductase in ethanol oxidation and catalysis can be demonstrated in a system composed of only cytochrome P-450, lipid, ethanol, and cumene hydroperoxide. These data implicate cytochrome P-450 in the direct oxidation of ethanol by this system.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes. Evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 × g particulate fraction of the postnuclear supernatant obtained from guinea-pig phagocytosing polymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O^?₂ generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH.2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1 : 1.3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O^?₂ generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation.4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O^?₂ formed during the oxidation of NADPH.5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH.6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH.7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible.8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase.9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O₂; an enzymatic, apparently non-univalent, O₂ reduction and a non-enzymatic chain reaction.10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O₂ is optimal at pH 7.0 with 1 mM NADPH.     

A novel phenomenon of burst of oxygen uptake during decavanadate-dependent oxidation of NADH

Oxidation of NADH by decavanadate, a polymeric form vanadate with a cage-like structure, in presence of rat liver microsomes followed a biphasic pattern. An initial slow phase involved a small rate of oxygen uptake and reduction of 3 of the 10 vanadium atoms. This was followed by a second rapid phase in which the rates of NADH oxidation and oxygen uptake increased several-fold with a stoichiometry of NADH: O₂ of 11. The burst of NADH oxidation and oxygen uptake which occurs in phosphate, but not in Tris buffer, was prevented by SOD, catalase, histidine, EDTA, MnCl₂ and CuSO₄, but not by the hydroxyl radical quenchers, ethanol, methanol, formate and mannitol. The burst reaction is of a novel type that requires the polymeric structure of decavanadate for reduction of vanadium which, in presence of traces of H₂O₂, provides a reactive intermediate that promotes transfer of electrons from NADH to oxygen.     

Characterization of oxygen free radicals generated during vanadate-stimulated NADH oxidation

The oxidation of NADH and accompanying reduction of oxygen to H₂O₂ stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H₂O₂ all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H₂0₂-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.     

Evaluation of activity of selected antioxidants on proteins in solution and in emulsions

Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox, or the carotenoids astaxanthin and canthaxanthin were incubated with BSA or BSA/LnMe and oxidation was initiated either with the water-soluble azo-initiator 2,2′ azo-bis-(2-amidinopropane) hydrochloride (AAPH), or FeCl₃ and ascorbate, or the Fenton system using FeCl₂/EDTA/H₂O₂, or with the singlet oxygen generating species anthracene-9,10-dipropionic acid disodium 1,4 endoperoxide (NDPO₂).

The results show that all the antioxidants tested were inefficient in the system with FeCl₃/ascorbate. However, with the other initiating agents, the hydrophilic antioxidant, Trolox, was the most effective in preventing both protein and lipid oxidation. In contrast the lipophilic antioxidants were ineffective in preventing oxidation of BSA in aqueous solution, but did show some moderate antioxidative activity on protein and lipid in the BSA/LnMe system. Using the singlet oxygen generating system it was also demonstrated that Trolox always provided better protection of the protein than tocopherol and the carotenoids in both the BSA and the BSA/LnMe systems. In conclusion, prevention of protein oxidation using a water-soluble antioxidant has a protective effect on the lipid fraction and this approach deserves further attention in complex biological systems.     

Generation of allantoin from the oxidation of urate by cytochrome c and its possible role in Reye's syndrome

Allantoin in the presence of calcium ions has been implicated as a potential toxic agent in Reye's syndrome. An investigation of possible alternative sources of allantoin in humans, which lack the enzyme uricase, has been initiated. Urate is a strong reducing agent which can reduce cytochrome c nonenzymatically, with the concomitant production of CO₂ and H⁺. The stoichiometries measured for the various reactants and products were 1 urate:2 cytochrome c:1 H⁺:1 CO₂. The initial reaction rate depended on the concentrations of both urate and cytochrome c, with reaction kinetics that were first order with respect to urate and second order with respect to cytochrome c. The participation of molecular oxygen in this reaction could not be detected. The pH and ionic strength optima for this reaction were determined to be 9.5–10.5 and 10⁻⁵m, respectively. Based on the results reported here, the following balanced equation can be written: urate⁻² + 2 cytochrome c⁺³ + 2 H₂O → allantoin + 2 cytochrome c⁺² + H⁺ + HCO₃⁻. We propose that allantoin can be generated from the oxidation of urate by cytochrome c⁺³, and that this is a potential source of allantoin in human tissues.     

Effect of trifluoperazine on skeletal muscle mitochondrial respiration

The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca²⁺-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the $\text{ADP}\text{O}$ and $\text{Ca}{}^{\mathrm{2+}}\text{O}$ ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca²⁺-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c₁ and cytochrome c₁-aa₃ segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c₁ by 92% with succinate as substrate, and of cytochrome c and cytochrome aa₃ by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.     

The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c₅₅₂, is similar to a number of c-type cytochromes from the α-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c₅₅₂ revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

Bicarbonate-catalyzed hydrogen peroxide oxidation of cysteine and related thiols

The effect of bicarbonate on the rates of the H₂O₂ oxidation of cysteine, gluthathione, and N-acetylcysteine to the corresponding disulfides was investigated. The relative oxidation rates at pH 8 for the different thiols are inversely related to the pK_a values of the thiol groups, and the reactive nucleophiles are identified as the thiolate anions or their kinetic equivalents. The second-order rate constants at 25 °C for the reaction of the thiolate anions with hydrogen peroxide are 17 ± 2 M⁻¹ s⁻¹ for all three substrates. In the presence of bicarbonate (>25 mM), the observed rate of thiolate oxidation is increased by a factor of two or more, and the catalysis is proposed to be associated with the formation of peroxymonocarbonate from the equilibrium reaction of hydrogen peroxide with bicarbonate (via CO₂). The calculated second-order rate constants for the direct reaction of the three thiolate anions with peroxymonocarbonate fall within the range of 900-2000 M⁻¹ s⁻¹. Further oxidation of disulfides by peroxymonocarbonate results in the formation of thiosulfonate and sulfonate products. These results strongly suggest that peroxymonocarbonate should be considered as a reactive oxygen species in aerobic metabolism with relevance in thiol oxidations.     

Influence of buffer system and pH on the amount of oxygen exchanged between solvent H2O and the CO2 produced in the aerobic oxidation of Cypridina luciferin catalyzed by Cypridina luciferase.

Incorporation of ¹⁸O into CO₂ was measured under various buffer conditions when the bioluminescent oxidation of Cypridina luciferin, catalyzed by luciferase, was carried out either in H₂¹⁶O medium with ¹⁸O₂ gas, or in H₂¹⁸O medium with ¹⁶O₂ gas. The results indicate that (1) the exchange of oxygen between CO₂ and solvent H₂O is significantly influenced by the kind of buffer as well as by pH, (2) the exchange of oxygen between solvent H₂O and CO₂ produced from luciferin in a neutral buffer can be reasonably well estimated from the exchange that takes place when the same amount of CO₂ gas is introduced into the same buffer by the presently employed method, and (3) in the Cypridina bioluminescent reaction, one of two oxygens of O₂ is quantitatively incorporated into the product CO₂ prior to the exchange of oxygen between CO₂ and solvent H₂O.     

Redox state of cytochrome C in the presence of the 6-hydroxydopamine/oxygen couple: Oscillations dependent on the presence of hydrogen peroxide or superoxide

The reduction of ferricytochrome c in the presence of 6-hydroxydopamine/O₂ mixtures was examined under various reaction conditions. As the autoxidation of 6-hydroxy-dopamine progressed to completion, there were fluctuations in the net redox reactivity between reducing and oxidizing steady states. This was reflected in a sequence of damped oscillations in the redox state of cytochrome c. Corresponding to the time when 6-hydroxydopamine was 75–100% exhausted, reoxidation of the ferrocytochrome c occurred (prevented by catalase or catalase plus Superoxide dismutase). After the H₂O₂, in turn, was mostly consumed, the next phase commenced in which the cytochrome c became reduced for a second time. This reductive phase was 52% inhibited by superoxide dismutase. In the subsequent and final phase of the process, a progressive oxidation of cytochrome c lasting at least 24 h was observed. Of the initial reduction of ferricytochrome c, at most 37% can be attributed to direct reduction by 6-hydroxydopamine or its semiquinone. This initial net reduction of cytochrome c was inhibited 51% by superoxide dismutase and 41% by catalase. However, since either catalase or superoxide dismutase inhibited the autoxidation of 6-hydroxydopamine by at least as much as it slowed the reduction of cytochrome c, their effects in slowing the reduction of cytochrome c resulted largely from the decreased production of those free radicals which reduce ferricytochrome c, and only in part from accelerated removal. Elimination of the actions of transition metal ions (whether by passage of the buffer solutions through Chelex 100 resins or by addition of desferrioxamine to the reaction medium) slowed both the reoxidation and rereduction by up to 96%. Addition of mannitol decreased the rate of the first reoxidation by 25% and increased the rate of the rereduction by 7%. In general, the oscillations are explicable in terms of changes in the steady state levels of O₂ and H₂O₂, with metal ions playing a major role and hydroxyl radicals a minor role in both the reoxidation and rereduction.     

Succinate oxidation by coupled potato tuber mitochondria followed by rapid scan spectrometry

Oxidation of succinate by potato tuber mitochondria has been investigated from aerobiosis to complete anuerobiosis. Difference spectra of the various steps were recorded by a rapid scan spectrometer delivering averaged spectra every 3 s in the range 380 to 630 mm. The transitions between state 3 and 4 resulted in large redox changes, essentially for the b cytochromes, and in significant changes in the spectral baseline (light scattering). At anaerobiosis the cytochromes c, c₁ and a were reduced while cytochrome a, remained oxidized. – Addition of uncouplers in aerobiosis induced oxidation of the b cytochromes, and when anaerobiosis occurred cytochromes c, c₁a and a₃ were reduced simultaneously. When uncouplers were added in anaerobiosis a partial oxidation of the b cytochromes and the reduction of cytochrome a₃ were observed. These results are interpreted as the building up of a membrane potential, maximal in state 4 and stable after anaerobiosis. The cytochromes buried in the membrane equilibrate with the membrane potential, and their redox states are sensitive to the changes. Variations of membrane potential also induce changes in the light scattering by the mitochondrial membrane.     

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O₂) conditions. The system measures the concentrations of O₂ and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O₂ concentration and electron turnover of the enzyme. At a high O₂ concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O₂. At low O₂ (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O₂ consumption. At both high and low O₂ concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.     

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa₃ in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230).2. The K_m for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 μM in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation.3. The apparent K_m for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts “competitively” towards oxygen.4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H⁺ gradients modify the oxidase activity in reconstituted vesicles.     

Chromium and human low-density lipoprotein oxidation

Chromium is a catalytic metal able to foster oxidant damage, albeit its capacity to induce human LDL oxidation is to date unkown. Thus, we have investigated whether trivalent and hexavalent chromium, namely Cr(III) and Cr(VI), can induce human LDL oxidation. Cr(III) as CrCl₃ is incapable of inducing LDL oxidation at pH 7.4 or 4.5. However, Cr(III), specifically at physiological pH of 7.4 and in the presence of phosphates, causes an absorbance increase at 234 resembling a spectrophotometric kinetics of LDL oxidation with a lag- and propagation-like phase. In this regard, it is conceivable that peculiar Cr(III) forms such as Cr(III) hydroxide and, especially, Cr(III) polynuclear hydroxocomplexes formed at pH 7.4 interact with phosphates generating species with an intrinsic absorbance at 234 nm, which increases over time resembling a spectrophotometric kinetics of LDL oxidation. Cr(VI), as K₂Cr₂O₇, can instead induce substantial human LDL oxidation at acidic pH such as 4.5, which is typical of the intracellular lysosomal compartment. LDL oxidation is related to binding of Cr(VI) to LDL particles with quenching of the LDL tryptophan fluorescence, and it is inhibited by the metal chelators EDTA and deferoxamine, as well as by the chain-breaking antioxidants butylated hydroxytoluene and probucol. Moreover, Cr(VI)-induced LDL oxidation is inhibited by mannitol conceivably by binding Cr(V) formed from LDL-dependent Cr(VI) reduction and not by scavenging hydroxyl radicals (OH); indeed, the OH scavengers sodium formate and ethanol are ineffective against Cr(VI)-induced LDL oxidation. Notably, heightened LDL lipid hydroperoxide levels and decreased LDL tryptophan fluorescence occur in Cr plating workers, indicating Cr-induced human LDL oxidation in vivo. The biochemical, pathophysiological and clinical implications of these novel findings on chromium and human LDL oxidation are discussed.