Reduction of superoxide production by mitochondria oxidizing NADH in the presence of organic acids

Effects of multiple substrates on oxygen uptake and superoxide production by mitochondria isolated from the pericarp tissue of green bell pepper (Capsicum annuum L.) were studied. Mitochondria isolated from peppers stored at 4 °C for 5 and 6 days had higher rates of oxygen uptake and were less sensitive to cyanide than mitochondria isolated from freshly harvested peppers. Succinate enhanced state 2 and state 4 rates of oxygen uptake with exogenous NADH in the absence of cytochrome path inhibitors, but not state 3 rates by mitochondria isolated from either freshly harvested or cold-stored bell peppers. The sensitivity of NADH oxidation to cyanide was reduced by both malate and succinate in mitochondria from cold-stored bell peppers, whereas only succinate was effective in mitochondria from freshly harvested peppers.Mitochondria isolated from both freshly harvested peppers and those stored at 4 °C for 5 and 6 days produced superoxide in the absence of exogenous substrates. Superoxide production by mitochondria from freshly harvested bell peppers increased when the mitochondria were supplied with malate, succinate or NADH, but only NADH enhanced superoxide production by mitochondria from cold-stored peppers. Both succinate and malate reduced the production of superoxide by mitochondria isolated from cold-stored bell peppers. Succinate and malate as second substrates also reduced the production of superoxide with NADH by mitochondria from both freshly harvested and cold-stored bell peppers. Malonate, a competitive inhibitor of succinate dehydrogenase, was inhibitory to oxygen uptake and to superoxide production.Mitochondria isolated from cold-stored bell peppers converted succinate to pyruvate at 25 °C at considerably higher rates than those of mitochondria from freshly harvested bell peppers. Since pyruvate has been shown to activate the alternative oxidase and the presence of pyruvate is essential for continued alternative oxidase activity, we suggest that pyruvate limits superoxide production by enhancing the flow of electrons through the alternative path. A direct scavenging of superoxide by succinate, malate and pyruvate, however, cannot be ruled out.     

Multiple-site inhibition by colloidal elemental sulfur (S°) of respiration by mitochondria from young dormant α spores of Phomopsis viticola

Beffa, T., Pezet, R. and Turian, G. 1987. Multiple-site inhibition by colloidal elemental sulfur (S°) of respiration by mitochondria from young dormant α spores of Phomopsis viticola. Mitochondria from young dormant α spores of Phomopsis viticola Sacc. (ATCC 44940) were isolated by grinding and differential centrifugation. They presented a good integrity of their inner and outer membranes as measured by biochemical assays. Electron microscopic analysis revealed an homogenous population. The highest respiratory activities were observed with NADH and ascorbate + tetra-methyl-p-phenylenediamine (TMPD). Malate stimulated the oxidation of pyruvate, citrate or α-ketoglutarate. The coupling of respiration to oxidative phosphorylation appeared at the time of spore germination. The respiratory activities of mitochondria isolated from young dormant α spores of P. viticola were strongly inhibited by S°. The sensitivity of mitochondrial oxidation of different substrates (NADH, pyruvate + malate, succinate and ascorbate + TMPD) to S° was heterogenous and indicated multiple-site action. Thus preincubation of mitochondria with 30 μM S° before addition of substrates fully prevented NADH oxidation (>98%), and strongly inhibited oxidation of pyruvate + malate (85%), succinate (60%) and ascorbate + TMPD (74%). S° inhibited more rapidly the oxidation of succinate than that of other substrates. In the presence of dithiothreitol (DTT), S°-inhibited oxidation of all substrates (except ascorbate + TMPD) could only be transiently and weakly reestablished. The inhibitory action of S° on the oxidation of NADH, pyruvate + malate and succinate was higher than that observed with sulfhydryl group reagents such as mersalyl, Hg-acetate or p - chloromercuribenzoate. In contrast to S° these SH-group reagents could not inhibit oxidation of ascorbate + TMPD. S°, by its dual capacity to oxidize the SH-groups and to self-reduce, probably at the level of cytochrome c oxidase, could produce a modification of the oxidation state of the respiratory complexes thereby disturbing the electron flux.     

Some Reactions of Isolated Corn Mitochondria Influenced by Juglone

The effects of juglone on the uptake of O₂ by excised corn roots (Zea mays L., Wf9 cms- T × M14) and isolated corn mitochondria arc reported. The O₂ uptake by excised corn roots, as measured by an O₂ electrode, was inhibited more than 90% after a one-hour treatment of 500 μM juglone. Lesser inhibitions were observed with 50 μM and 250 μM juglone. In a KC1 reaction medium in the absence of inorganic phosphate (Pi), juglone stimulated the rate of O₂ uptake by isolated mitochondria oxidizing NADH, succinate, or malate + pyruvate. In the presence of Pi, juglone concentrations of 3 μM and greater inhibited the state 3 oxidation rates of succinate and malate + pyruvate, lowered respiratory control and ADP/O ratios obtained from the oxidation of NADH, malate + pyruvate, or succinate, and reduced the coupled deposition of calcium phosphate within isolated mitochondria driven, by the oxidation of malate + pyruvate. The inhibition of state 3 O₂ uptake by isolated mitochondria, an oxidative state in which electron transfer is coupled to ATP production, is seen to correlate with the inhibition affected by juglone when applied to tissues in vivo.     

Properties of substantially chlorophyll-free pea leaf mitochondria prepared by sucrose density gradient separation

Mitochondria isolated from pea leaves (Pisum sativum L. var Massey Gem) and purified on a linear sucrose density gradient were substantially free of contamination by Chl and peroxisomes. They showed high respiratory rates and good respiratory control and ADP/O ratios. Malate, glutamate, succinate, glycine, pyruvate, α-ketoglutarate, NADH, and NADPH were oxidized but little or no oxidation of citrate, isocitrate, or proline was detected. The oxidation of NADPH by the purified mitochondria did not occur via a transhydrogenase or phosphatase converting it to NADH. NADPH oxidation had an absolute requirement for added Ca²⁺, whereas NADH oxidation proceeded in its absence. In addition, oxidation of the two substrates showed different sensitivities to chelators and sulfhydryl reagents, and faster rates of O₂ uptake were observed with both substrates than with either alone. This indicates that the NADPH dehydrogenase is distinct from the exogenous NADH dehydrogenase.     

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.     

The Effect of Cadmium on Electron and Energy Transfer Reactions in Corn Mitochondria

The effects of cadmium on isolated corn shoot mitochondria were determined. In the absence of phosphate cadmium stimulated the oxidation of exogenous NADH optimally at 0.025 mM, but was inhibitory at 0.1 mM and above. The presence of phosphate negated the cadmium stimulation of exogenous NADH oxidation and permitted inhibitions only at higher cadmium concentrations. Succinate or malate + pyruvate oxidation in the absence of phosphate was inhibited to a greater extent by cadmium than when phosphate was present. ADP/O and respiratory control ratios were reduced by cadmium but generally were less sensitive to cadmium than state 4 or minus phosphate respiration. The data suggest that the site of cadmium effect is likely to be early in electron transport. Cadmium had a pronounced effect on mitochondrial swelling under either passive or active conditions. When succinate or exogenous NADH were being oxidized swelling occurred at 0.05 mM cadmium, but with malate + pyruvate the cadmium concentration had to exceed 1.0 mM. Phosphate (2 mM) prevented the swelling. Dithiothreitol, a SH group protector, prevented any effect of cadmium on swelling or respiration which suggests that sulfhydryl groups are likely involved in the cadmium-membrane interaction.     

Characterization of the respiratory activity of mitochondria isolated from an insect cell line CP-1268Laspeyresia pomonella

Summary Mitochondria have been isolated from the codling mothLaspeyresia pomonella, CP-1268 cell line. The mitochondrial fraction was isolated from pooled 4 d, exponential growth phase, cultures. The mitochondria were determined to be intact based on the demonstration of respiratory control, the effects of 2,4 dinitrophenol and oligomycin on respiration, the inability to oxidize NADH, and the inability of cytochromec to enhance respiration. The isolated mitochondria were able to oxidize succinate, pyruvate, malate, α-ketoglutarate, and α-glycerophosphate efficiently. Of the substrates tested, the CP-1268 mitochondria oxidized succinate most efficiently. The respiratory control ratios ranged from a high of 4.6 for pyruvate to a low of 1.7 with α-glycerophosphate. These findings confirm that the mitochondria were tightly coupled. The data also confirm the presence of three sites of oxidative phosphorylation because NAD-linked substrates had ADP-to-O ratios approaching 3 and flavoprotein linked substrates had values approaching 2.     

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity.     

Respiratory metabolism of a "petite negative"yeast Schizosaccharomyces pombe 972h-

The respiratory metabolism of Schizosaccharomyces pombe 972h(-), a fission, haplontic, "petite negative" yeast, was studied. Glucose and glycerol are good growth substrates and are oxidized under appropriate conditions. l-Lactate, ethanol, malate, and succinate are oxidized but are poor substrates for growth. d-Lactate and pyruvate are neither oxidized nor used for growth. Limited growth was observed under anaerobic conditions. The addition of 0.3% KNO(3) to a rich medium relieves the oxygen requirement. A continuous increase of cell respiration during growth on repressive concentration of glucose was observed, suggesting the presence of glucose repression of respiration. Reduced nicotinamide adenine dinucleotide (NADH), succinate, alpha-glycerophosphate, and ascorbate plus tetramethyl-p-phenylenediamine are oxidized by a mitochondrial fraction. NADH and succinate oxidations are inhibited by antimycin A and NaCN but not by rotenone, suggesting the absence of the phosphorylation site I and the presence of sites II and III. The effects of several mitochondrial inhibitors on growth and respiration indicate that the requirement of an oxidant for growth is related neither to the functioning of the respiratory electron transport chain nor to the formation of respiratory energy. The previously suggested correlations between the nonviability of vegetative "petites" mutants, the absence of repression of respiration by glucose, and the incapacity to grow under anaerobic conditions are thus not strictly valid for S. pombe.     

The Interaction between Exogenous NADH Oxidase and Succinate Oxidase in Jerusalem Artichoke (Helianthus tuberosus) Mitochondria

Most isolated plant mitochondria oxidize exogenous NADH viaan electron transport pathway which is resistant to piericidinA and coupled to the synthesis of two molecules of ATP. Resultspresented show that succinate can inhibit this oxidation ofadded NADH. The inhibition was most marked in the absence ofADP (state 4), less obvious in the presence of added ADP (state3), and absent in the presence of a weak acid uncoupling agent.The presence of malonate prevented the inhibition. The degreeof inhibition was dependent on the concentration of succinateand appeared to be non-competitive in nature. The inhibitionwas shown not to be the result of the reversed flow of electronsfrom succinate to NAD^$. The presence of external NADH appearednot to alter the rate of oxidation of succinate.     

Oxidation of NADH via an "external" pathway in skeletal-muscle mitochondria and its possible role in the repayment of lactacid oxygen debt

Mitochondria isolated from skeletal muscle of rat catalyse oxidation of the external NADH (in the presence of rotenone, antimycin A and cytochrome c) at a rate of 15 natoms O2/min/mg protein by a pathway sensitive to mersalyl. In a medium supplemented with commercial lactate dehydrogenase, or when mitochondria were incubated in the presence of a cytoplasm, the NADH oxidation could be arrested by pyruvate. The inhibitory effect of pyruvate could be released by lactate. In the presence of NAD and cytochrome c, the reconstructed system containing skeletal muscle mitochondria plus cytoplasmic fraction was active in oxidation of L-lactate despite of the presence of rotenone and antimycin A. The lactate oxidation was sensitive to mersalyl and cyanide.     

Characterization of the respiratory activity of mitochondria isolated from an insect cell line CP-1268 Laspeyresia pomonella

Mitochondria have been isolated from the codling moth Laspeyresia pomonella, CP-1268 cell line. The mitochondrial fraction was isolated from pooled 4 d, exponential growth phase, cultures. The mitochondria were determined to be intact based on the demonstration of respiratory control, the effects of 2,4 dinitrophenol and oligomycin on respiration, the inability to oxidize NADH, and the inability of cytochrome c to enhance respiration. The isolated mitochondria were able to oxidize succinate, pyruvate, malate, alpha-ketoglutarate, and alpha-glycerophosphate efficiently. Of the substrates tested, the CP-1268 mitochondria oxidized succinate most efficiently. The respiratory control ratios ranged from a high of 4.6 for pyruvate to a low of 1.7 with alpha-glycerophosphate. These findings confirm that the mitochondria were tightly coupled. The data also confirm the presence of three sites of oxidative phosphorylation because NAD-linked substrates had ADP-to-O ratios approaching 3 and flavoprotein linked substrates had values approaching 2.     

Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori

Abstract Oxygen uptake by Helicobacter pylori cells and membranes was determined. Cells from stirred broth cultures or agar plates, suspended in buffer, possessed a variable and apparently endogenous respiration which could be sustained for several hours. In contrast, oxygen consumption by cells from statically incubated broth cultures, in the absence of added substrate, was transient or undetectable. These latter cells, however, oxidised ethanol, fumarate, glucose, d-lactate, pyruvate and succinate, though glucose-oxidising ability declined rapidly. The K _m s for d-lactate, pyruvate and succinate metabolism were low (≤20 μM) and oxygen uptake was approximately 1.5, 2 and 2 mol per mol substrate respectively, indicating metabolism beyond acetate plus CO₂ and implying the presence of tricarboxylic acid cycle activity. Cell membranes oxidised fumarate, d-lactate, NADH, NADPH and succinate. NADPH oxidation was six times more rapid than that of NADH. Rates of oxygen uptake by cells suspended in buffer with metabolisable substrate were < 20% of those for cells suspended in a brain heart infusion medium. Uninoculated medium consumed significant quantities of oxygen.     

Glyceollin: a site-specific inhibitor of electron transport in isolated soybean mitochondria

The glyceollin inhibition of electron transport by isolated soybean and corn mitochondria was similar to that of rotenone, acting at site I between the internal NADH dehydrogenase and coenzyme Q. Coupled state 3 malate oxidation was inhibited by glyceollin and rotenone with apparent K_i values of about 15 and 5 micromolar, respectively. Carbonylcyanide m-chlorophenyl hydrazone uncoupled state 4 malate oxidation was also inhibited by glyceollin and rotenone, but uncoupled succinate and exogenous NADH state 4 oxidation was only slightly inhibited by both compounds. Glyceollin also inhibited ferricyanide reduction with malate as the electron donor, with an apparent K_i of 5.4 micromolar, but failed to inhibit such reduction with succinate or externally added NADH as electron donors. Glyceollin did not inhibit state 4 oxidation of malate, succinate, or exogenous NADH. Glyceollin did not act as a classical uncoupler or as an inhibitor of oxidative phosphorylation.     

Alternative Oxidase Activity and the Ubiquinone Redox Level in Soybean Cotyledon and Arum Spadix Mitochondria during NADH and Succinate Oxidation

In Arum and soybean (Glycine max L.) mitochondria, the dependence of the alternative oxidase activity on the redox level of ubiquinone, with NADH and succinate as substrates, was studied, using a voltametric procedure to measure the ubiquinone redox poise in the mitochondrial membrane. The results showed that when the enzyme was activated by pyruvate the relationship between the alternative oxidase rate and the redox state of the ubiquinone pool was the same for both NADH and succinate oxidations. In the absence of pyruvate the alternative oxidase had an apparent lower affinity for ubiquinol. This was more marked with NADH than with succinate and was possibly due to pyruvate production during succinate oxidation or to an activation of the alternative oxidase by succinate itself. In Arum spadix (unlike soybean cotyledon) mitochondria, succinate oxidation via the alternative oxidase maintained the ubiquinone pool in a partially reduced state (60%), whereas NADH oxidation kept it almost completely reduced. Previous data comparing mitochondria from thermogenic and nonthermogenic tissues have not examined the full range of ubiquinone redox levels in both tissues, leading to the suggestion that the activity of alternative oxidase for Arum was different from nonthermogenic tissues. When the complete range of redox states of ubiquinone is used and the oxidase is fully activated, the alternative oxidase from thermogenic tissue (Arum) behaves similarly to that of nonthermogenic tissue (soybean).     

Pyridine Nucleotide-Linked Lactate Dehydrogenase of Tetrahymena: Evidence for D- and L-Enzymes in the Mitochondria

An NAD-linked lactate dehydrogenase (LDH) in a crude mitochondrial fraction obtained from Tetrahymena homogenates was previously reported by this laboratory. This fraction contains the NADH and succinate oxidase system as well as the mitochondrial cytochromes and carries out oxidative phosphorylation. The preparation catalyzes the oxidation of D- and L-lactate linked only to certain analogs of NAD; it has not been possible to demonstrate NAD-dependent D- or L-lactate oxidation nor is there any evidence that either of these enzymes is a flavoprotein as indicated by their inability to reduce directly certain artificial electron acceptors. A lactate racemase is not present.     

Changes in bioenergetics of skeletal muscle mitochondria during experimental trichinosis in rats

Changes in bioenergetics were studied in mitochondria isolated from skeletal muscles of rats in the course of experimental trichinosis. In fortified media supporting oxidative phosphorylation, oxygen uptake with succinate was greatly enhanced, while the oxidation of glutamate and of malate plus pyruvate was manifold inhibited. The protective effect of BSA added was negligible in this case.     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

Oxidations of various substrates and effects of the inhibitors on purified mitochondria isolated from <Emphasis Type="Italic">Kalanchoë pinnata</Emphasis>

Kalanchoë pinnata mitochondria readily oxidized succinate, malate, NADH, and NADPH at high rates and coupling. The highest respiration rates usually were observed in the presence of succinate. The high rate of malate oxidation was observed at pH 6.8 with thiamine pyrophosphate where both malic enzyme (ME) and pyruvate dehydrogenase were activated. In CAM phase III of K. pinnata mitochondria, both ME and malate dehydrogenase (MDH) simultaneously contributed to metabolism of malate. However, ME played a main function: malate was oxidized via ME to produce pyruvate and CO₂ rather than via MDH to produce oxalacetate (OAA). Cooperative oxidation of two or three substrates was accompanied with the dramatic increase in the total respiration rates. Our results showed that the alternative (Alt) pathway was more active in malate oxidation at pH 6.8 with CoA and NAD⁺ where ME operated and was stimulated, indicating that both ME and Alt pathway were related to malate decarboxylation during the light. In K. pinnata mitochondria, NADH and NADPH oxidations were more sensitive with KCN than that with succinate and malate oxidations, suggesting that these oxidations were engaged to cytochrome pathway rather than to Alt pathway and these capacities would be desirable to supply enough energy for cytosol pyruvate orthophosphate dikinase activity.     

Isolation and oxidative properties of intact mitochondria isolated from spinach leaves

A procedure was described for preparing intact mitochondria from spinach (Spinacia oleracea L.) leaves. These mitochondria oxidized succinate, malate, pyruvate, α-ketoglutarate, and NADH with good respiratory control and ADP/O ratios comparable to those observed with mitochondria from other plant tissues. Glycine was oxidized by the preparations. This oxidation linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport and phosphorylation inhibitors.