Developmental Changes in the NAD Content in Sugar-Beet Root Mitochondria and Their Effect on the Oxidative Activity of These Organelles

The NAD content was determined in mitochondria isolated from sugar-beet roots at various stages of plant development. A high NAD content (7.6 ± 0.9 nmol/mg mitochondrial protein) was observed in the mitochondria of actively growing roots of 80–95-day-old plants, but it decreased ca. twofold by the end of the first year of plant development, before the roots were harvested for storage. The mitochondria isolated from roots stored at low temperature for two to three months and those after five to eight days of regrowth during the second year of plant development manifested an even lower NAD content (2.2 ± 0.4 and 2.0 ± 0.5 nmol/mg protein, respectively). A drastic decrease in the NAD content in mitochondria from stored roots did not result from the impairment of the inner membrane of these organelles and was evidently regulatory in its nature. The effect of developmental changes in the intramitochondrial NAD content on the malate oxidation pattern was studied. In the mitochondria of stored roots, the low NAD content limited the rate of malate oxidation in state 3, because the addition to the reaction mixture of exogenous NAD, which can be transported to the mitochondrial matrix, promoted malate oxidation by 30–50%. Rotenone inhibited malate oxidation in the stored-root mitochondria by more than 70%; in this case, the rate of rotenone-resistant malate oxidation in these organelles increased by several times in the presence of exogenous NAD. In the mitochondria of the growing root, exo-genous NAD did not affect the rate of malate oxidation, and rotenone inhibited it only by 25–35%. The analysis of the data obtained here and the published evidence suggests the existence of a universal mechanism of respiration control and the regulation of the functional activity of plant mitochondria. This mechanism acts through a change in the NAD content in the organelle matrix. This NAD can be used in the course of plant development, e.g., during the transition of sugar-beet-root cells in the dormant state, when the respiration rate must decline.     

Effect of KCl-medium on succinate oxidation and hydrogen peroxide generation in mitochondria of sugar beet taproot

The effect of substitution of KCl for sucrose in the reaction medium on succinate oxidation and hydrogen peroxide generation was investigated in the mitochondria isolated from stored taproots of sugar beet (Beta vulgaris L.). In a sucrose-containing medium, oxidation of succinate was inhibited by oxaloacetate; this inhibition was especially pronounced upon a decrease in substrate concentration and eliminated in the presence of glutamate, which removed oxaloacetate in the course of transamination. Irrespective of succinate concentration, substitution of KCl for sucrose in the medium considerably enhanced suppression of succinate oxidation apparently as a result of slow activation of succinate dehydrogenase (SDH) by its substrate. In this case, mitochondria showed the symptoms of uncoupling, lower values of membrane potential (ΔΨ), respiratory control (RC), and ADP/O induced by electrophoretic transport of potassium via K⁺ channel of mitochondria. KCl-dependent suppression of succinate oxidation by taproot mitochondria was accompanied by a considerable inhibition of H₂O₂ production as compared with the sucrose-containing medium. These results indicate that in the presence of potassium ions, ΔΨ dissipates, suppression of succinate oxidation by oxaloacetate increases, and succinate-dependent generation of ROS in sugar beet mitochondria is inhibited. A possible physiological role of oxaloacetate-restricted SDH activity in the suppression of respiration of storage organs protecting mitochondria from oxidative stress is discussed.     

Effect of salicylic acid on the metabolic activity of plant mitochondria

The effects of salicylic acid (SA) on mitochondrial respiration and generation of membrane potential across the inner membrane of mitochondria isolated from stored taproots of sugar beet (Beta vulgaris L.) and etiolated seedling cotyledons of yellow lupine (Lupinus luteus L.) were studied. When malate was oxidized in the presence of glutamate, low SA concentrations (lower than 1.0 mM) exerted predominantly uncoupling action on the respiration of taproot mitochondria: they activated the rate of oxygen uptake in State 4 (in the absence of ADP) and did not affect oxidation in State 3 (in the presence of ADP). In contrast, in lupine cotyledon mitochondria these SA concentrations inhibited oxygen uptake in the presence of ADP and much weaker activated substrate oxidation in State 4. Thus, SA (0.5 mM) reduced the respiratory control ratio according to Chance (RCR) by 25% in the taproots and 35% in cotyledons. When the concentration of phytohormone was increased (above 1.0 mM), malate oxidation in State 3 was inhibited and in State 4 — activated independently of the plant material used. In this case, the values of RCR and ADP/O were reduced by 50–60%. The effect of high SA concentrations (2 mM and higher) on malate oxidation depended on the duration of incubation and had a biphasic pattern: the initial activation of oxygen uptake was later replaced by its inhibition. The parallel studying the SA effect on the generation of membrane potential (ΔΨ) at malate oxidation in the mitochondria of beet taproots and lupine cotyledons showed that ΔΨ dissipation was observed because of SA uncoupling and inhibiting action on respiration. The degree of ΔΨ dissipation depended on the phytohormone concentration and duration on mitochondria treatment, especially at its high concentrations. In general, a correlation was found between the effects of SA on mitochondrial respiration and ΔΨ values in the coupling membranes. Furthermore, these results show that the responses of mitochondria to SA were determined not only by its concentration but also by treatment duration and evidently by the sensitivity to the phytohormone of mitochondria isolated from different plant tissues.     

Effect of phosphate and uncouplers on substrate transport and oxidation by isolated corn mitochondria

A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O₂ uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K⁺ permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V_max, probably reflecting a diffusion-limited process.     

Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris)

The present study compares the exogenous NAD(P)H oxidation and the membrane potential ([delta][psi]) generated in mitochondria isolated from different tissues of an important agricultural crop, sugar beet (Beta vulgaris}. We observed that mitochondria from taproots, cold-stored taproots, and in vitro-grown tissue cultures contain a functional NADH dehydrogenase, whereas only those isolated from tissue cultures displayed a functional NAD(P)H dehydrogenase. It is interesting that the NADH-dependent [delta][psi] of mitochondria from cold-stored taproots and from tissue cultures was not affected by free Ca2+ ions, whereas free Ca2+ was required for the mitochondrial NADPH oxidation by in vitro-grown cells and cytosolic NADH oxidation by mitochondria from fresh taproots. A tentative model accounting for the different response to Ca2+ ions of the NADH dehydrogenase in mitochondria from cold-stored taproots and tissue cultures of B. vulgaris is discussed.     

Oxaloacetate and malate transport by plant mitochondria

The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinländerin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10⁻³. From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport, probably linked to each other for the sake of charge compensation. (b) The transport of malate between the mitochondrial matrix and the cytosol is controlled by the oxaloacetate level in such a way that a redox gradient can be maintained between the NADH/NAD systems in the matrix and the cytosol. (c) The malate-oxaloacetate shuttle functions mainly in the export of malate from the mitochondria, whereas the import of malate as a respiratory substrate may proceed by the classical malate-phosphate antiport.     

Malate decarboxylation in isolated mitochondria from the crassulacean acid metabolism plant Sedum praealtum

Mitochondria isolated from the Crassulacean acid metabolism plant Sedum praealtum were demonstrated to decarboxylate added malate at basal rates of 30–50 μmol mg^?1 original chlorophyll h^?1. The basal rate could be stimulated markedly by the addition of ADP, oxaloacetic acid, an uncoupler of oxidative phosphorylation, or NAD, with maximum rates of 70–100 μmol mg^?1 original chlorophyll h^?1 observed. These observed rates were high enough to account for a large proportion of the estimated rate of malate decarboxylation in vivo. The major products of malate oxidation by the mitochondria in most cases were found to be pyruvate and CO₂, indicating that malate oxidation in these mitochondria proceeds mainly through NAD malic enzyme rather than NAD malate dehydrogenase. Under conditions employed little of the pyruvate formed was further oxidized, suggesting a fate other than oxidation (conversion to starch) for this pyruvate. Malate decarboxylation by mitochondria and by partially purified NAD malic enzyme was markedly inhibited by NaHCO₃. A possible physiological role is suggested for this inhibition as a feedback control on the enzyme.     

Effects of rotenoids on isolated plant mitochondria

The effects of several rotenoids have been studied on potato (Solanum tuberosum L.) tuber and etiolated mung bean (Phaseolus aureus Roxb.) hypocotyls mitochondria. The selective inhibition of mitochondrial complex I is characterized by several tests: (a) no effect can be observed on exogenous NADH or succinate oxidation; (b) malate oxidation is inhibited at pH 7.5; (c) one-third decrease of ADP/O ratio appears during malate oxidation at pH 6.5 or during α-ketoglutarate, citrate, or pyruvate oxidation at a pH about 7; (d) during malate oxidation at pH 6.5, a transient inhibition appears which can be maintained by addition of exogenous oxaloacetate; (e) in potato mitochondria, the inhibition of malate oxidation disappears at pH 6.5 when NAD⁺ is added. Then, a one-third decrease of the ADP/O ratio can be measured.

Such a selective inhibition of complex I is obtained with deguelin, tephrosin, elliptone, OH-12 rotenone, and almost all the rotenoids extracted from Derris roots. The presence of the rings A, B, C, D, E seems to be necessary for the selective inhibition. Opening of the E ring and hydroxylation of the 9 position (rot-2′-enoic acid) give a rotenoid derivative with multisite inhibitory activities on flavoproteins, which are quite comparable to those of common flavonoids such as kaempferol (Ravanel et al. 1982 Plant Physiol 69: 375-378).

     

The oxidation of glutamine and glutamate in relation to anion transport in enterocyte mitochondria.

The oxidation of L-glutamate and L-glutamine by enterocyte mitochondria was supported by malate. The stimulation of the rate of oxidation of the two amino acids by small amounts of added malate was 93% and 76% respectively. This could not be accounted for by the oxidation of the small amounts of malate added. Amino-oxyacetate added initially inhibited malate-supported oxidation of L-glutamate by 81% and that of L-glutamine by 38%. The inhibition of L-glutamate oxidation was partially reversed by L-glutamine. The dicarboxylate-carrier inhibitor 2-phenylsuccinate inhibited the malate-supported oxidation of both amino acids, but appeared to be slightly stimulatory to L-glutamine oxidation when added initially. The inhibition of L-glutamate oxidation was reversed by L-glutamine. The mitochondrial uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) inhibited malate-supported oxidation of L-glutamate by 78% when added initially. The oxidation of L-glutamine was completely inhibited. However, the uncoupler stimulated the oxidation of both amino acids when added finally. Pyruvate inhibited aspartate synthesis when either of these amino acids was the main substrate, alanine being synthesized. There was no effect on O2 uptake. Mitochondria did not swell in KCl solution, but swelled rapidly in water. Mitochondrial swelling in potassium phosphate and potassium acetate solutions was activated by valinomycin and to a lesser extent by the further addition of FCCP. With potassium malate, swelling was mainly activated by phosphate. The swelling of enterocyte mitochondria in potassium glutamate was slow. In glutamine solution, mitochondrial swelling was greater and appeared to be enhanced by the initial presence of small amounts of phosphate.     

Evidence for the role of malic enzyme in the rapid oxidation of malate by cod heart mitochondria

Mitochondria isolated from the heart of cod (Gadus morrhua callarias) oxidized malate as the only exogenous substrate very rapidly. Pyruvate only slightly increased malate oxidation by these mitochondria. This is in contrast with the mitochondria isolated from rat and rabbit heart which oxidized malate very slowly unless pyruvate was added. Arsenite and hydroxymalonate (an inhibitor of malic enzyme) inhibited the respiration rate of mitochondria isolated from cod heart, when malate was the only exogenous substrate. Inhibition caused by hydroxymalonate was reversed by the addition of pyruvate. In the presence of arsenite, malate was converted to pyruvate by cod heart mitochondria. Cod heart mitochondria incubated in the medium containing Triton X-100 catalyzed the reduction of NADP+ in the presence of L-malate and Mn2+ at relatively high rate (about 160 nmoles NADPH formed/min/mg mitochondrial protein). The oxidative decarboxylation of malate was also taking place when NADP+ was replaced by NAD+ (about 25 nmol NADH formed per min per mg mitochondrial protein). These results suggest that the mitochondria contain both NAD+- and NADP+-linked malic enzymes. These two activities were eluted from DEAE-Sephacel as two independent peaks. It is concluded that malic enzyme activity (presumably both NAD+- and NADP+-linked) is responsible for the rapid oxidation of malate (as the only external substrate) by cod heart mitochondria.     

The Effect of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Nicotinamide Adenine Dinucleotide-linked Substrates by Isolated Plant Mitochondria

The oxidation of malate, citrate, and α-ketoglutarate by cauliflower (Brassica oleacea L.) bud mitochondria was inhibited by rotenone. This inhibition was relieved upon addition of NAD⁺ to the medium, and ADP/O values were lowered to less than 2 when both rotenone and NAD⁺ were present. Dinitrophenol did not affect the relief of rotenone inhibition by exogenous NAD⁺.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208× and 66× less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.     

Control of mitochondrial Ca2+ retention by ADP-stimulated glutamic dehydrogenase

The protective effect of ADP on unspecific Ca²⁺ release and collapse of the transmembrane potential was analyzed in mitochondria from kidneys of rats. The presence of ADP in the incubation mixture prevents Ca²⁺ leakage and collapse of in sucrose-containing medium, but fails to do so in KCl medium. The effect of the adenine nucleotide in sucrose media correlates with an increase in the level of reduced pyridine nucleotides; the increase was due to a stimulatory effect on the activity of glutamic dehydrogenase. It also was observed that in KCl media, in the presence and in the absence of ADP the rate of NADH oxidation through the respiratory chain was higher than in sucrose; in this latter medium a high level of reduced pyridine nucleotides was found, in comparison to KCl media. It is proposed that the role of ADP is to increase glutamic dehydrogenase activity and in consequence to provoke a higher rate of formation of NADH which in turn controls Ca²⁺ release.     

The activation of non-phosphorylating electron transport by adenine nucleotides in Jerusalem-artichoke (Helianthus tuberosus) mitochondria.

In isolated plant mitochondria the oxidation of both succinate and exogenous NADH responded in the expected manner to the addition of ADP or uncoupling agents, and the uncoupled rate of respiration was often in excess of the rate obtained in the presence of ADP. However, the oxidation of NAD+-linked substrates responded in a much more complex manner to the addition of ADP or uncoupling agents such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone to mitochondria oxidizing pyruvate plus malate failed to result in a reliable stimulation; this uncoupled rate could be stimulated by adding AMP or ADP in the presence of oligomycin or bongkrekic acid. Spectrophometric measurements showed that the addition of AMP or ADP resulted in the simultaneous oxidation of endogenous nicotinamide nucleotide and the reduction of cytochrome b. ADP was only effective in bringing about these changes in redox state in the presence of Mg2+ whereas AMP did not require Mg2+. It was concluded that AMP activated the flow of electrons from endogenous nicotinamide nucleotide to cytochrome b, possible at the level of the internal NADH dehydrogenase.     

NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21-day-old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD⁺. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD⁺-IDH activity was about 2-fold higher than NADP⁺-IDH activity. The NAD⁺-IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis-Menten kinetics for NAD⁺ and Mg²⁺. The NADP⁺-IDH, in contrast, displayed lower K_m values. For NAD⁺-IDH the pH optimum was at 7.4, whereas NADP⁺-IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD⁺-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)⁺-IDH activities only at high concentrations.     

The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, ND_in(NADPH) catalyzes the rotenone-insensitive and Ca²⁺-dependent oxidation of NADPH and ND_in(NADH) catalyzes the rotenone-insensitive and Ca²⁺-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, ND_in(NADPH) and ND_in (NADH) activity with a K_i of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in K_i between the two ND_in made possible the use of DPI inhibition to estimate ND_in (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O₂ mg^?1 min^?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD⁺ or NADP⁺) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP⁺ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of ND_in(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.     

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.     

Changes in pyridine nucleotide levels alter oxygen consumption and extra-mitochondrial phosphates in isolated mitochondria: a 31P-NMR and NAD(P)H fluorescence study

Isolated rat-liver mitochondria were used to study the relation between mitochondrial NADH levels, oxygen consumption (QO2), and extra-mitochondrial phosphates. Alterations in NADH and QO2 were accomplished by incubating mitochondria with different substrates or varying amounts of exogenous ATPase while monitoring QO2 and NAD(P)H fluorescence. Two sets of conditions were studied: (1) in the presence of excess ADP and inorganic phosphate, an increase in NAD(P)H fluorescence was associated with a linear increase in QO2; (2) when QO2 was driven by the steady-state hydrolysis of ATP by exogenous ATPase, increases in QO2 were associated with proportional decreases in NAD(P)H fluorescence. For all substrates tested this relation was linear; however, the slope was substrate dependent. Different substrates were able to maintain different NAD(P)H levels at the same QO2. To investigate this further, effects of changing substrates at constant QO2 on NAD(P)H and extra-mitochondrial phosphates were determined. Addition of glutamate + malate to mitochondria respiring on citrate caused a 50% increase in NAD(P)H fluorescence, a 41% decrease in ADP, and a 30% decrease in inorganic phosphate. Similar changes for the substrate jump, pyruvate + malate to glutamate + malate were found. Finally, it was determined that a linear relation holds between increases in NAD(P)H fluorescence and increases in QO2 when substrates were varied at constant, physiologic levels of extra-mitochondrial ADP. These results indicate that QO2 depends on NAD(P)H levels as well as on extra-mitochondrial phosphates over a wide range of respiratory rates.