Inhibition of the links between electron transfer and proton translocation in mitochondria

The mechanism by which proton extrusion is linked to electron transfer in mitochondria was investigated by means of the primary amine-specific reagent fluorescamine, and of compounds obtained from the reaction of fluorescamine with simple amines (e.g. benzylamine) and with the mycosamine-containing antibiotic amphotericin B. The effect of these 'modifiers' (i.e. fluorescamine transfer chain were assayed separately using specific inhibitors to block the action associated with the other site. Both types of modifiers inhibited the proton extrusion across the membrane to a significantly greater extent than the electron transfer process in both sites II and III. In contrast, the lactone derivative (or cyclic form) of the amine-fluorescamine compounds had no significant inhibitory effect on the proton extrusion and its associated electron transfer. These results are consistent with the hypothesis that the link between proton extrusion and electron transfer in mitochondria is indirect in nature. The results show that: (a) the links involved in sites II and III are identical or very similar in nature; (b) a covalent modification of primary amino groups in the inner membrane is not essential for the expression of these differential inhibitory effects; (c) specific structural features in the amine-fluorescamine compounds, and in the mitochondria-fluorescamine derivatives, are crucial for the expression of the inhibitory effects. Our results contradict the 'redox loop' model of Mitchell, and are compatible with the proton pump concept for the linked proton translocation in oxidative phosphorylation.     

Amine fluorescamine compounds inhibit oxidative phosphorylation in rat liver mitochondria

The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.     

Mutual regulation between mitochondrial ATPase and respiratory chain activities

Compounds made from the reaction of fluorescamine with simple primary amines and with mycosamine-containing macrolide antibiotics (e.g., amphotericin B) are used to investigate possible interactions between ATPase and respiration enzymes in rat liver mitochondria. The following observations have been made. (1) The acyclic form of the benzyl amine-fluorescamine compound stimulates the ATPase-linked inorganic phosphate formation, and this stimulation is not affected by rotenone, antimycin A, and potassium cyanide. In contrast, the respiratory inhibitors are able to prevent the stimulation of ATPase activity that is caused by conventional uncouplers e.g., 2,4-dinitrophenol. (2) The acyclic form of the amphotericin B-fluorescamine compound has no effect on ATPase-linked inorganic phosphate formation rate. However, in the presence of the antibiotic-fluorescamine compounds, the respiratory inhibitors are no longer able to prevent the uncoupler-stimulated ATPase activity. (3) The amine-fluorescamine modifiers have no effect on rotenone-sensitive NADH-cytochrome c reductase, on succinate-cytochrome c reductase, and on cytochrome oxidase in submitochondrial particles. (4) The amine-fluorescamine modifiers decrease the rate of the ATP-driven NAD⁺ reduction by succinate in submitochondrial particles. (5) The amine-fluorescamine modifiers inhibit the stimulation of respiration that is caused by conventional uncouplers, although the modifiers have no effect on the kinetics of the proton influx induced by uncouplers. The data are consistent with the hypothesis that the ATPase-linked and respiration-linked proton pumps may interact directly with each other, and this step establishes the mutual regulation between ATPase and respiratory activities.     

Differential inhibition of respiration and its dependent H+ extrusion by fluorescamine in rat liver mitochondria

Rat liver mitochondria were treated with varying amounts of fluorescamine ranging from 0 to 30 nmol/mg of protein. The biochemical activities of the modified mitochondria were analyzed. It was found that the respiration rate in the absence of ADP was not significantly affected, but that the state 3 respiration rate and the accompanying $\text{P}\text{O}$ ratio decreased as the labeling extent increased. It was also observed that the treatment inhibited the stimulation of respiration induced by the presence of uncouplers. However, the modification has no effect on the discharging rate of proton gradient by uncouplers. The intrinsic activities of NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome oxidase of the inner membrane were not affected by the modification. Measurement of the respiration-dependent proton extrusion (in the presence of valinomycin and potassium ion) with secondary ion movements inhibited, showed that the initial extrusion rate was reduced progressively. However, the observed amounts of proton extruded (ΔH⁺) and Δμ_H + were not affected. The observed reduction of the oxygen consumption rate was much less than that of the proton extrusion rate with increased labeling. These results suggest that some fluorescamine titratable primary amino groups may be involved in the controlling of the proton extrusion process. The implications on the mechanism of coupling in respirationdependent proton extrusion are discussed.     

Differential Inhibition of Tonoplast H-ATPase Activities by Fluorescamine and Its Derivatives

Corn (Zea mays L.) root tonoplast vesicles were treated with the primary-amine specific reagent, fluorescamine (FL). Modification by FL caused a differential inhibition to the coupled activities of tonoplast H⁺-ATPase. Within the range of 0 to 5 micromoles of FL per milligram of protein, the proton pumping rate was significantly reduced but ATP hydrolysis was only slightly affected. Yet, the membrane H⁺ leakage during the pumping stage increased only slightly. FL treatment resulted in (a) a decrease in amine containing phospholipids and (b) an insertion of multiple H-bonding moieties into the membrane. To test which of these two possible effects were responsible for inhibition, FL derivatives of benzylamine, butylamine, and phenylalanine were synthesized. It was found that the acyclic derivatives with high H-bonding potential at concentrations of 10 micromolar inhibited proton pumping by 50% without a significant effect on ATP hydrolysis. Cyclic derivatives were largely ineffectual. Proton leakage during pumping was not affected by these acyclic modifiers. Membrane fluidity, as measured by the polarization of diphenyl hexatriene, decreased upon treatment with either FL or its derivatives. The results suggest that the proton pumping is indirectly linked to ATP hydrolysis in the tonoplast vesicles, and the link between these processes is apparently weakened by the presence of acyclic fluorescamine derivatives in the membrane.     

Effects of borohydride-treated oligomycins on processes of energy transduction in mitochondria

Sodium borohydride in ethanol solution under mild conditions brings about the stepwise reduction of the 7-keto and the 11-keto groups of rutamycin and the oligomycins to the corresponding hydroxyl groups without further alterations of the macrocyclic lactone structure or other features of the molecule. The reduced compounds, as well as the parent antibiotics, inhibit the ADP-dependent (state 3) respiration, and the Pi formation and proton extrusion that are linked to ATP hydrolysis, but have no effect on other respiration-linked activities in intact rat liver mitochondria. Analogous inhibitory effects of borohydride-treated antibiotics are also observed in rat-liver submitochondrial particles. The reduced compounds are less potent inhibitors than the parent antibiotics. The reduced compounds are more efficient as inhibitors of Pi formation stimulated by conventional uncouplers (e.g. 2,4-dinitrophenol), than of Pi formation stimulated by certain amine-fluorescamine modifiers (e.g.) the benzylamine-fluorescamine compound. In contrast, the parent antibiotics are unable to discriminate between uncoupler-stimulated and modifier-stimulated Pi formation. It is suggested that rutamycin and the oligomycins bind to H+-ATPase as a result of hydrogen bonding to, at least, the 7-keto and/or the 11-keto groups of the antibiotics. When these keto groups are reduced to hydroxyl groups the hydrogen-bonding is less efficient due to the pronounced directional characteristic of hydrogen-bonding to keto groups.     

Metabolic network control of oxidative phosphorylation: multiple roles of inorganic phosphate

Phosphate (Pi) is a putative cytosolic signaling molecule in the regulation of oxidative phosphorylation. Here, by using a multiparameter monitoring system, we show that Pi controls oxidative phosphorylation in a balanced fashion, modulating both the generation of useful potential energy and the formation of ATP by F1F0-ATPase in heart and skeletal muscle mitochondria. In these studies the effect of Pi was determined on the mitochondria [NADH], NADH generating capacity, matrix pH, membrane potential, oxygen consumption, and cytochrome reduction level. Pi enhanced NADH generation and was obligatory for electron flow under uncoupled conditions. Pi oxidized cytochrome b (cyto-b) and reduced cytochrome c (cyto-c), potentially improving the coupling between the NADH free energy and the proton motive force. The apparent limitation in reducing equivalent flow between cyto-b and cyto-c in the absence of Pi was confirmed in the intact heart by using optical spectroscopic techniques under conditions with low cytosolic [Pi]. These results demonstrate that Pi signaling results in the balanced modulation of oxidative phosphorylation, by influencing both deltaGH+ generation and ATP production, which may contribute to the energy metabolism homeostasis observed in intact systems.     

Pyrophosphate import and synthesis by plant mitochondria

The matrix level of pyrophosphate (PPi) in mitochondria isolated from etiolated pea ( Pisum sativum L. cv. Alaska) stems was evaluated, on the basis of an enzymatic assay, to be approx. 0.2 m M . Pyrophosphate could enter from the cytoplasm to the mitochondria via adenine nucleotide translocase (ANT), because F^– and Ca²⁺ (two penetrating PPiase inhibitors) and atractylate (ANT inhibitor) inhibited PPiase activity in isolated mitochondria supplied with PPi. This result was also confirmed by measuring oxygen consumption and membrane potential (ΔΨ) in succinate-energized mitochondria. In a medium free of phosphate (Pi), the addition of PPi before the substrate rendered possible an ADP-stimulated oxygen consumption that was inhibited by F^– or Ca²⁺. In a similar experiment, ADP induced the dissipation of ΔΨ when it was added after the succinate-generated ΔΨ had reached a steady state and, again, F^– inhibited this dissipation. These results imply that PPi enters the mitochondria where it is hydrolyzed to 2 Pi which become available for the H⁺-ATPase (EC 3.6.1.34). In addition, PPi may be synthesized by the H⁺-PPiase (EC 3.6.1.1), acting as a synthase. This evidence arises from the observation that Pi stimulated an oxygen consumption (respiratory control ratio of 1.7) that was inhibited by F^– or Ca²⁺. The physiological role of the mitochondrial H⁺-PPiase is discussed in the light of the consideration that this enzyme can catalyse a readily reversible reaction.     

Thiols related to mitochondrial ATPase and transports: unmasking upon conformational changes supported by the comparative effects of ethacrynate and dihydroethacrynate.

Comparison between the effects on various rat liver mitochondrial functions of ethacrynate, a thiol reagent inhibitor of oxidative phosphorylations [3, 4] and those of dihydroethacrynate its saturated derivative which is not a thiol reagent, has been performed. Both, ethacrynate and dihydroethacrynate increase oxygen consumption by mitochondria in state 4 (succinate as substrate) in a concentration dependent way (from 1 to 5 X 10(-4) M EA or DHEA). This activation is followed, only with ethacrynate, by an inhibition appearing sooner with higher concentrations. After preincubation or mitochondria with ethacrynate (1 to 5 X 10(-4) M), the stimulation of respiration by (ADP + Pi) is completely inhibited whereas it is only weakly affected by dihydroethacrynate at the same concentrations. Ethacrynate and dihydroethacrynate provoke variations of intramitochondrial Mg2+ and K+ levels which need energy from the respiratory chain. These are affected by Pi or (Pi + ADP) in a different way with ethacrynate and with dihydroethacrynate. After preincubation with mitochondria, ethacrynate and to a smaller extent dihydroethacrynate, inhibit partially ADP translocation; ADP increases the inhibitory effect of EA on translocation and not that of dihydroethacrynate. Ethacrynate increases the oligomycin sensitive ATPase activity and dihydroethacrynate still more. After a ten minutes preincubation with mitochondria, ethacrynate and dihydroethacrynate hardly affect the 2.4 DNP stimulated ATPase activity. Preincubation with succinate or ADP strongly increases the ethacrynate inhibition whereas it decreases dihydroethacrynate inhibition. Ethacrynate and dihydroethacrynate do not affect the efflux of Pi produced by ATP hydrolysis but ethacrynate enforces the inhibitory effect of mersalyl (Mg2+ containing medium). After ten minutes of preincubation with mitochondria, ethacrynate binds 25 nmoles of -SH/mg protein (DTNB titration) and dihydroethacrynate has no effect. These results show an effect of ethacrynate on two types of thiols linked with energy conservation mechanisms and ADP translocation. These thiols could be unmasked or made accessible by conformational modifications of the inner membrane upon energization or addition of ADP.     

Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis.

The forward and reverse rates of the overall reaction catalyzed by the ATP synthase in intact rat heart mitochondria, as measured with 32P, were compared with the rates of two partial steps, as measured with 18O. Such rates have been measured previously, but their relationship to one another has not been determined, nor have the partial reactions been measured in intact mitochondria. The partial steps measured were the rate of medium Pi formation from bound ATP (in state 4 this also equals the rate of medium Pi into bound ATP) and the rate of formation of bound ATP from bound Pi within the catalytic site. The rates of both partial reactions can be measured by 31P NMR analysis of the 18O distribution in Pi and ATP released from the enzyme during incubation of intact mitochondria with highly labeled [18O]Pi. Data were obtained in state 3 and 4 conditions with variation in substrate concentrations, temperature, and mitochondrial membrane electrical potential gradient (delta psi m). Although neither binding nor release of ATP is necessary for phosphate/H2O exchange, in state 4 the rate of incorporation of at least one water oxygen atom into phosphate is approximately twice the rate of the overall reaction rate under a variety of conditions. This can be explained if the release of Pi or ATP at one catalytic site does not occur, unless ATP or Pi is bound at another catalytic site. Such coupling provides strong support for the previously proposed alternating site mechanism. In state 3 slow reversal of ATP synthesis occurs within the mitochondrial matrix and can be detected as incorporation of water oxygen atoms into medium Pi even though medium [32P]ATP does not give rise to 32Pi in state 3. These data can be explained by lack of translocation of ATP from the medium to the mitochondrial matrix. The rate of bound ATP formation from bound Pi at catalytic sites was over twice the rate of the overall reaction in both states 4 and 3. The rate of reaction at the catalytic site is considerably less sensitive to the decrease in membrane potential and the concentration of medium ADP than is the rate of medium ATP formation. This supports the view that the active catalytic site is occluded and proceeds at a rapid rate which is relatively independent of delta psi m and of media substrates.     

The proton leak across the mitochondrial inner membrane

The proton conductance of the mitochondrial inner membrane increases at high protonmotive force in isolated mitochondria and in mitochondria in situ in rat hepatocytes. Quantitative analysis of its importance shows that about 20-30% of the oxygen consumption by resting hepatocytes is used to drive a heat-producing cycle of proton pumping by the respiratory chain and proton leak back to the matrix. The flux control coefficient of the proton leak pathway over respiration rate varies between 0.9 and zero in mitochondria depending on the rate of respiration, and has a value of about 0.2 in hepatocytes. Changes in the proton leak pathway in situ will therefore change respiration rate. Mitochondria isolated from hypothyroid animals have decreased proton leak pathway, causing slower state 4 respiration rates. Hepatocytes from hypothyroid rats also have decreased proton leak pathway, and this accounts for about 30% of the decrease in hepatocyte respiration rate. Mitochondrial proton leak may be a significant contributor to standard metabolic rate in vivo.     

Transport of Ca and Mn by mitochondria from rat liver, heart and brain

The energy-dependent, respiration-supported uptake and the uncoupler- or Na+-induced release of Ca2+ and Mn2+ by mitochondria from rat liver, heart and brain were investigated, using as indicators radioisotopes (45Ca and 54Mn), proton ejection, oxygen consumption, nicotinamide nucleotide oxidation-reduction and, in the case of Ca2+, the metallochromic dye Arsenazo III. Ca2+ uptake in the presence of Pi was rapid in mitochondria from liver and brain, and less rapid in those from heart. Mn2+ uptake was much slower than that of Ca2+ in liver and heart, but only slightly slower in brain. When added together, Ca2+ accelerated the uptake of Mn2+, and Mn2+ retarded the uptake of Ca2+, by mitochondria from all three tissues. When Mn2+ was present during Ca2+ uptake, its own uptake remained accelerated even after Ca2+ uptake was terminated. Mg2+, which was not taken up, inhibited Ca2+ uptake by mitochondria from all three tissues, and, when present during Ca2+ uptake, accelerated the subsequent uptake of Mn2+. The uncoupler CCCP induced a release of both Ca2+ and Mn2+ from all three sources of mitochondria; yet, release of Mn2+ took place only in the absence of Pi. The release followed the same pattern as the uptake, i.e., Ca2+ accelerated the release of Mn2+ and Mn2+ retarded the release of Ca2+. Na+ induced a release of both Ca2+ and Mn2+ from heart and brain but not from liver mitochondria; again, Mn2+ release occurred only in the absence of Pi. The Na+-induced release of Ca2+ was inhibited by Mn2+, but the Na+-induced release of Mn2+ was not accelerated by Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.     

Tissue-specific depression of mitochondrial proton leak and substrate oxidation in hibernating arctic ground squirrels

A significant proportion of standard metabolic rate is devoted to driving mitochondrial proton leak, and this futile cycle may be a site of metabolic control during hibernation. To determine if the proton leak pathway is decreased during metabolic depression related to hibernation, mitochondria were isolated from liver and skeletal muscle of nonhibernating (active) and hibernating arctic ground squirrels (Spermophilus parryii). At an assay temperature of 37 degrees C, state 3 and state 4 respiration rates and state 4 membrane potential were significantly depressed in liver mitochondria isolated from hibernators. In contrast, state 3 and state 4 respiration rates and membrane potentials were unchanged during hibernation in skeletal muscle mitochondria. The decrease in oxygen consumption of liver mitochondria was achieved by reduced activity of the set of reactions generating the proton gradient but not by a lowered proton permeability. These results suggest that mitochondrial proton conductance is unchanged during hibernation and that the reduced metabolism in hibernators is a partial consequence of tissue-specific depression of substrate oxidation.     

Functional characterization of the mitochondrial uncoupling proteins from the white shrimp Litopenaeus vannamei

Mitochondrial uncoupling proteins (UCPs) play an essential role in dissipating the proton gradient and controlling the mitochondrial inner membrane potential. When active, UCPs promote proton leak across the inner membrane, oxidative phosphorylation uncoupling, oxygen uptake increase and decrease the ATP synthesis. Invertebrates possess only isoforms UCP4 and UCP5, however, the role of these proteins is not clear in most species since it may depend on the physiological needs of each animal. This study presents the first functional characterization of crustacean uncoupling proteins from the white shrimp Litopenaeus vannamei LvUCP4 and LvUCP5. Free radicals production in various shrimp organs/tissues was first evaluated, and mitochondria were isolated from shrimp pleopods. The oxygen consumption rate, membrane potential and proton transport of the isolated non-phosphorylating mitochondria were used to determine LvUCPs activation/inhibition. Results indicate that UCPs activity is stimulated in the presence of 4-hydroxyl-2-nonenal (HNE) and myristic acid, and inhibited by the purine nucleotide GDP. A hypoxia/re-oxygenation assay was conducted to determine whether UCPs participate in shrimp mitochondria response to oxidative stress. Isolated mitochondria from shrimp at re-oxygenation produced large quantities of hydrogen peroxide and higher levels of both LvUCPs were immunodetected. Results suggest that, besides the active response of the shrimp antioxidant system, UCP-like activity is activated after hypoxia exposure and during re-oxygenation. LvUCPs may represent a mild uncoupling mechanism, which may be activated before the antioxidant system of cells, to early control reactive oxygen species production and oxidative damage in shrimp.     

The dynamic behaviour of mitochondria in living zygotes during maturation and meiosis in Chlamydomonas reinhardtii

To understand fully the function of mitochondria during the development of cells and organs, it is important to elucidate the dynamics of their morphology. However, the detailed morphology of mitochondria during meiosis has not yet been studied in algae. We examined the mitochondrial morphology of Chlamydomonas reinhardtii and classified zygotes into seven types by mitochondrial morphology in order to analyse the morphological change in mature and meiotic zygotes. We also investigated the oxygen consumption of living zygotes and the effects of tubulin and actin polymerization inhibitors on mitochondria, using fluorescence microscopy and oxygen electrodes. During zygote maturation, mitochondria fragmented into small particles, with a large decrease in oxygen consumption. When mature zygotes were exposed to light, mitochondria became tubular and formed a network, and oxygen consumption gradually recovered. At the same time, particle-like mitochondrial nucleoids became stringy and produced new nucleoid particles. Tubular mitochondria accumulated around the cell nucleus and then spread throughout the cell. Cell division followed (first and second rounds), and the resultant daughter cells had tubular mitochondria in a mesh-like arrangement. An inhibitor of tubulin polymerization, demecolcine, inhibited the assembly of mitochondria around the cell nucleus, whereas an inhibitor of actin polymerization, latrunculin B, inhibited the formation of tubular mitochondria. These results suggest that microtubules are probably involved in mitochondrial accumulation around the cell nucleus, whereas microfilaments may maintain the tubular network of mitochondria.     

Generation of Radical Anions of Nifurtimox and Related Nitrofuran Compounds By Ascorbate

Nifurtimox analogues bearing triazol-4-yl, benzimidazol-1-yl, triazin-4-yl or related groups as counterpart of the (5-nitro-2-furfurylidene) amino group were reduced to their nitro anion radicals by ascorbate in anaerobic solutions at high pH. The ESR spectra of the radical anions showed hyperfine spin couplings restricted to the nitrofuran moiety. With these compounds, the spin density at the nitro group was greater than with nifurtimox, nitrofurazone and nitrofurantoin. At neutral pH, solutions containing ascorbate and nitrofuran derivatives consumed oxygen, the compounds bearing unsaturated nitrogen heterocycles being the most effective. Superoxide dismutase and catalase decreased the rate of oxygen consumption, thus demonstrating the production of superoxide and hydrogen peroxide, respectively. NMR spectra of the triazol-4-yl and triazin-4-yl nitrofuran derivatives showed a deshielding effect for the azomethine proton, which was undetectable with nifurtimox and nitrofurazone.     

Biochemical changes during sucrose deprivation in higher plant cells

The mobilization of stored carbohydrates (sucrose and starch) during sucrose starvation was studied with sycamore (Acer pseudoplatanus) cells. When sucrose was omitted from the nutrient medium, vacuolar sucrose was first consumed. When a threshold of intracellular sucrose concentration was attained the cytoplasmic phosphorylated compounds decreased whereas cytoplasmic Pi increased symmetrically. Such a situation triggered starch breakdown. When almost all the intracellular sucrose pool had disappeared, the cell respiration rates (normal and uncoupled) declined progressively. The decrease in the rate of respiration triggered by sucrose starvation was attributable neither to the availability of substrate for mitochondrial respiration nor to a decrease in the maximal rate of O2 consumption by mitochondria expressed in terms of nanomole of O2 consumed per min/mg of mitochondrial protein. In fact, the uncoupled respiration rates decreased in parallel with the decrease in total intracellular cardiolipin or cytochrome aa3. These results demonstrate therefore that after a long period of sucrose starvation the progressive decrease in the uncoupled rate of O2 consumption by sycamore cells was attributable to a progressive diminution of the number of mitochondria/cell.     

Mitochondrial hydrogen peroxide production alters oxygen consumption in an oxygen-concentration-dependent manner

Metabolic responses of mammalian cells toward declining oxygen concentration are generally thought to occur when oxygen limits mitochondrial ATP production. However, at oxygen concentrations markedly above those limiting to mitochondria, several mammalian cell types display reduced rates of oxygen consumption without energy stress or compensatory increases in glycolytic ATP production. We used mammalian Jurkat T cells as a model system to identify mechanisms responsible for these changes in metabolic rate. Oxygen consumption was 31% greater at high oxygen (150–200 μM) compared to low oxygen (5–10 μM). Hydrogen peroxide was implicated in the response as catalase prevented the increase in oxygen consumption normally associated with high oxygen. Cell-derived hydrogen peroxide, predominately from the mitochondria, was elevated with high oxygen. Oxygen consumption related to intracellular calcium turnover was shown, through EDTA chelation and dantrolene antagonism of the ryanodine receptor, to account for 70% of the response. Oligomycin inhibition of oxygen consumption indicated that mitochondrial proton leak was also sensitive to changes in oxygen concentration. Our results point toward a mechanism in which changes in oxygen concentration influence the rate of hydrogen peroxide production by mitochondria, which, in turn, alters cellular ATP use associated with intracellular calcium turnover and energy wastage through mitochondrial proton leak.     

Action of a new compound, derived from NEM and DTE, on anionic translocation in mitochondria

The activity of translocating system which mediates the transport of Pi and citric cycle intermediates in mitochondria, has been determined with the multi-layer centrifugation technique. Contrary to all expectation it has been found that NEM, which binds tightly to SH groups, and DTE which reacting with disulphides increases the number of thiol groups of mitochondrial membrane, both inhibited the Pi leads to OH- and increased the initial rate of succinate leads to Pi exchange diffusion reactions. Identical results were obtained when mitochondria were preincubated with both NEM and DTE. The possibility that in these last conditions the effect on the translocator could be not determined by NEM and DTE per se but by a compound derived from their interaction, has been tested. Indeed solutions of NEM and DTE added in the concentration ratio of 2 to 1 and in absence of mitochondria, promoted the formation of a new compound, indicated as DTS, evidentiable by following the disappearance of both the absorbance of NEM at 302 nm and the free SH groups of DTE. Succinate leads to Pi and Pi leads to OH- exchange reactions were respectively stimulated and inhibited by DTS with a behaviour comparable to that observed in presence of NEM and/or DTE. The results are interpreted as a further and decisive support to the hypothesis that SH groups cannot be considered as functional active sites of the translocating system.