Oxidation of reduced nicotinamide adenine dinucleotide phosphate by plant mitochondria.

Mitochondria isolated from various plant tissues (leaves, etiolated shoots and hypocotyls, and stem tubers) oxidize exogenous NADPH with respiratory control values and ADP:O ratios similar to those obtained with exogenous NADH as substrate. In all the mitochondria investigated, the electron-transfer inhibitors rotenone and amytal each had the same effect on the oxidation of NADPH as they had on the oxidation of NADH. The oxidation of exogenous NADPH by white potato tuber mitochondria was much more sensitive to inhibition by citrate or ethylene glycol bis-(beta-aminoethyl ether)-N,N-tetraacetic acid than was the oxidation of NADH. Mitochondria isolated from aged beetroot slices showed an increased capacity for the oxidation of exogenous NADH (compared with mitochondria from fresh tissue) but no such increase in the capacity to oxidize exogenous NADPH. These results suggest that exogenous NADPH and NADH are oxidized via different flavoproteins in plant mitochondria.     

The inhibition of exogenous NAD(P)H oxidation in plant mitochondria by chelators and mersalyl as a function of pH

The oxidation of exogenous NADH by Jerusalem artichoke ( Helianthus tuberosus L.) tuber mitochondria was strongly inhibited at pH 7.2 by EDTA, EGTA and mersalyl and by chlorotetracycline in the presence of Ca²⁺. This inhibition disappeared at pH 5.5 where about 50% activity was found as compared to controls at pH 7.2. The rate of oxidation of NADPH at pH 5.5 was the same as for NADH but it was inhibited by 50% by both EDTA and mersalyl.
Mitochondria from Arum maculatum spadices oxidised NADH and NADPH with pH optima of 7.2 and 6.5, respectively. In the presence of EDTA the optima shifted to 6.7 and 5.9, respectively, due to an inhibition at higher pH and a lack of inhibition at lower pH. At pH 6.7 NADH oxidation was completely insensitive to both EDTA and mersalyl whereas the oxidation of NADPH was inhibited by more than 50%. The inhibition of NAD(P)H oxidation by chelators at neutral pH was due to the removal of Ca²⁺ from the membranes in both types of mitochondria. The differences observed in the properties of NADH and NADPH oxidation suggest that two different dehydrogenases are involved. Because of the strong pH-dependence and the changes in chelator-sensitivity in the physiological pH-range 6–8 it is suggested that the properties of NAD(P)H oxidation provide the cell with important means of metabolic regulation.     

Modulation of the Access of Exogenous NAD(P)H to the Alternative Pathway in Potato Tuber Callus Mitochondria with Triton X-100

Alternative oxidase activity in potato tuber (Solanum tuberosum L. cv Bintje) callus mitochondria with exogenous NAD(P)H as substrate is inhibited by low concentrations of the detergent Triton X-100. Alternative oxidase activity with succinate or malate as substrate is not affected by these low concentrations of Triton X-100. Cytochrome pathway activity was not influenced under these conditions, neither with endogenous nor with exogenous substrate. Washing of Triton X-100-treated mitochondria did partially restore both uninhibited and CN-resistant NADH oxidation, indicating that under these conditions Triton X-100 does not permanently remove major components from the mitochondrial membrane. Apparently, it is possible to manipulate mitochondria in such a way that the access of exogenous NADH to the alternative pathway is blocked while access to the cytochrome pathway is uninhibited. It is suggested that membrane conditions have a regulatory function (possibly via influencing the diffusion path) in the oxidation of exogenous NADH via the alternative pathway.     

On the presence of a nicotinamide nucleotide transhydrogenase in mitochondria from potato tuber

Mitochondria isolated from potato (Solanum tuberosum L.) tuber were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Submitochondrial particles derived from these mitochondria by sonication catalyzed a reduction of NAD⁺ or 3-acetylpyridine-NAD⁺ by NADPH, which showed a maximum of about 50 to 150 nanomoles/minute·milligram protein at pH 5 to 6. The K_m values for 3-acetylpyridine-NAD⁺ and NADPH were about 24 and 55 micromolar, respectively. Intact mitochondria showed a negligible activity in the absence of detergents. However, in the presence of detergents the specific activity approached about 30% of that seen with submitochondrial particles. The potato mitochondria transhydrogenase activity was sensitive to trypsin and phenylarsine oxide, both agents that are known to inhibit the mammalian transhydrogenase. Antibodies raised against rat liver transhydrogenase crossreacted with two peptides in potato tuber mitochondrial membranes with a molecular mass of 100 to 115 kilodaltons. The observed transhydrogenase activities may be due to an unspecific activity of dehydrogenases and/or to a genuine transhydrogenase. The activity contributions by NADH dehydrogenases and transhydrogenase to the total transhydrogenase activity were investigated by determining their relative sensitivities to trypsin. It is concluded that, at high or neutral pH, the observed transhydrogenase activity in potato tuber submitochondrial particles is due to the presence of a genuine and specific high molecular weight transhydrogenase. At low pH an unspecific reaction of an NADH dehydrogenase with NADPH contributes to the total trans-hydrogenase activity.     

Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. I. Pre-steady-state kinetics with NADPH

The reduction of NADH:Q oxidoreductase by NADPH occurring in submitochondrial particles has been studied with the freeze-quench technique. It was found that 50% of the Fe-S clusters 2, 3 and 4 could be reduced by NADPH within 30 ms at pH 6.5. The remainder of the clusters, including cluster 1, were reduced slowly and incompletely; it was concluded that these clusters play no role in the NADPH oxidase activity. Nearly the same results were obtained at pH 8 under anaerobic conditions, demonstrating that the rate of reaction of NADPH with the enzyme was essentially the same at both pH values. The rate and extent of reduction of half of the clusters 2 by NADPH at pH 8 were not affected by the presence of O2 of rotenone. This implies a pH-dependent oxidation of the enzyme as the cause for the absence of the NADPH oxidase activity at this pH. A dimeric model of the enzyme is proposed in which one protomer, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, is responsible for NADH oxidation at pH 8. This protomer cannot react with NADPH. The other protomer, containing only FMN and the clusters 2, 3 and 4, is supposed to catalyse the oxidation of NADPH. The oxidation of this protomer by ubiquinone is expected to be strongly dependent on pH. This protomer might also catalyse NADH oxidation at pH 6-6.5.     

Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. I. Pre-steady-state kinetics with NADPH

The reduction of NADH:Q oxidoreductase by NADPH occurring in submitochondrial particles has been studied with the freeze-quench technique. It was found that 50% of the Fe-S clusters 2, 3 and 4 could be reduced by NADPH within 30 ms at pH 6.5. The remainder of the clusters, including cluster 1, were reduced slowly and incompletely; it was concluded that these clusters play no role in the NADPH oxidase activity. Nearly the same results were obtained at pH 8 under anaerobic conditions, demonstrating that the rate of reaction of NADPH with the enzyme was essentially the same at both pH values. The rate and extent of reduction of half of the clusters 2 by NADPH at pH 8 were not affected by the presence of O₂ of rotenone. This implies a pH-dependent oxidation of the enzyme as the cause for the absence of the NADPH oxidase activity at this pH. A dimeric model of the enzyme is proposed in which one protomer, containing FMN and the Fe-S clusters 1–4 in stoichiometric amounts, is responsible for NADH oxidation at pH 8. This protomer cannot react with NADPH. The other protomer, containing only FMN and the clusters 2, 3 and 4, is supposed to catalyse the oxidation of NADPH. The oxidation of this protomer by ubiquinone is expected to be strongly dependent on pH. This protomer might also catalyse NADH oxidation at pH 6–6.5.     

Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD.

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.     

Properties of the oxidation of exogenous NADH and NADPH by plant mitochondria. Evidence against a phosphatase or a nicotinamide nucleotide transhydrogenase being responsible for NADPH oxidation

(1) The optimum pH for the oxidation of exogenous NADH by mitochondria from both Jerusalem artichoke (Helianthus tuberosus) tubers and Arum maculatum spadices was 7.0–7.1. NADPH oxidation had a lower optimum pH of 6.6 in Arum and 6.0 in Jerusalem artichoke mitochondria. In both types of mitochondria the rates of NADH and NADPH oxidation were identical below pH 6.0–5.5. (2) It is shown conclusively that neither a phosphatase converting NADPH to NADH nor a nicotinamide nucleotide transhydrogenase was involved in the oxidation of NADPH by these mitochondria. (3) Palmitoyl-CoA, an inhibitor of transhydrogenase activity in mammalian mitochondria, inhibits both NADH and NADPH oxidation by plant mitochondria with a K_i of about 10 μM. (4) It is concluded that the known properties of NAD(P)H oxidation are best explained by assuming the presence of a second dehydrogenase specific for NADPH. At low pH, electron flow from the two dehydrogenases to oxygen shares a common rate-limiting step.     

ATP synthesis during exogenous NADH oxidation. A reappraisal

This paper reports a reinvestigation on the pathway for mitochondrial oxidation of exogenous NADH and on the related ATP synthesis, first reported 30 years ago (Lehninger, A.L. (1951) J. Biol. Chem. 190, 345-359). NADH oxidation, both in intact and in water-treated mitochondria, is 90% inhibited by mersalyl, an inhibitor of the outer membrane NADH-cytochrome b5 reductase, and 10% inhibited by rotenone. The mersalyl-sensitive, but not the rotenone-sensitive, portion of NADH oxidation is stimulated by exogenous cytochrome c. Part of ATP synthesis is independent of exogenous NADH and cytochrome c, and is inhibited by rotenone and antimycin A, and is therefore due to oxidation of endogenous substrates. Another part of ATP synthesis is dependent on exogenous NADH and cytochrome c, is insensitive to rotenone and antimycin A, and is due to operation of cytochrome oxidase. It is concluded that (i) oxidation of exogenous NADH in the presence of cytochrome c proceeds mostly through NADH-cytochrome b5 reductase and cytochrome b5 on the outer membrane and then through cytochrome oxidase via the cytochrome c shuttle, and (ii) ATP synthesis during oxidation of exogenous NADH is partly due to oxidation of endogenous substrates and partly to operation of cytochrome oxidase receiving electrons from the outer membrane via cytochrome c.     

Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria.

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.     

Vanadate-dependent oxidation of pyridine nucleotides in rat liver microsomal membranes

An enzymatic Na₃VO₄-dependent system for the oxidation of reduced pyridine nucleotides in purified rat liver microsomes was characterized. The system has a pH optimum of 6.5, and appears to be specific for vanadate, since activity in the presence of a related transition metal, molybdate, was not detected. Vanadate-dependent oxidation occurred with a concomitant consumption of O₂ and, contrary to previous reports, preferred NADPH over NADH. At pH 6.5, the NADPH/NADH oxidase activity ratio was greater than 2:1. Sodium vanadate-dependent oxidation of NADH was inhibited by rotenone, antimycin A, NaN₃, and NaCN. Conversely, Na₃VO₄-dependent NADPH oxidation was slightly affected by rotenone, but was insensitive to antimycin A, NaN₃, NaCN, or quinacrine. Vanadate-dependent oxidation of either pyridine nucleotide was inhibited by the addition of either Superoxide dismutase or catalase, indicating that both superoxide and hydrogen peroxide may be intermediates in the process. Linear sucrose gradient purification of the microsomes showed that the vanadate-dependent system for NADPH oxidation resides primarily in the endoplasmic reticulum. These studies indicate the existence of separate and distinct enzymatic systems for vanadate-stimulated oxidation of NADPH and NADH in mammalian microsomal membranes, and argue against an exclusive role of endogenous Superoxide in the process.     

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.     

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 pathway of electron transfer in NADH:Q oxidoreductase

The pre-steady-state reduction by NADPH of NADH:Q oxidoreductase, as present in submitochondrial particles, has been further investigated with the rapid-mixing, rapid-freezing technique. It was found that trypsin treatment, that had previously been used to inactivate the transhydrogenase activity (Bakker, P.T.A. and Albracht, S.P.J. (1986) Biochim. Biophys. Acta 850, 413-422), considerably affected the stability at pH 6.2 of the NAD(P)H oxidation activity of submitochondrial particles. Use of the inhibitor butadione circumvented this problem, thus allowing a more careful investigation of the kinetics at pH 6.2. In the presence of the inhibitor rotenone it was found that 50% of the Fe-S clusters 3 and all of the Fe-S clusters 2 and 4 could be reduced by NADPH within 30 ms at pH 6.2. The remainder of the Fe-S clusters 3 and all of the Fe-S clusters 1 were reduced slowly (complete reduction only after more than 60 s). It was concluded that these latter Fe-S clusters play no role in the NADPH oxidation activity. In the absence of rotenone at pH 6.2 only 50% of the Fe-S clusters 2-4 could be reduced within 30 ms, while Fe-S cluster 1 was again not reduced. This difference was attributed to the fast reoxidation of part of the Fe-S clusters 2 and 4 by ubiquinone. At pH 8.0, where the NADPH oxidation activity is almost zero, 50% of the Fe-S clusters 2-4 could still be reduced by NADPH within 30 ms, while Fe-S cluster 1 was not reduced. The presence of rotenone had no effect on this reduction. From these observations it is concluded that the Fe-S clusters 2 and 4, which were rapidly reduced by NADPH and reoxidised by ubiquinone at pH 6.2, could not be reduced by NADPH at 8.0. This provides an explanation why NADH:Q oxidoreductase was not able to oxidise NADPH at pH 8.0, while part of the Fe-S clusters were still rapidly reduced. As a working hypothesis a dimeric structure for NADH:Q oxidoreductase is proposed. One protomer (B) contains FMN and Fe-S clusters 1-4 in equal amounts; the other protomer (A) is identical except for the absence of Fe-S cluster 1. NADH is able to react with both protomers, while NADPH only reacts with protomer A. A pH-dependent electron transfer from protomer A to protomer B is proposed, which would allow the reduction of Fe-S clusters 2 and 4 of protomer B by NADPH at pH 6.2, which is required for NADPH:Q oxidoreductase activity.     

The oxidation of exogenous NADH by mitochondria of Euglena gracilis

A novel oxidase activity of external NADH was found in mitochondria of a streptomycin-bleached mutant and the wild strain of Euglena gracilis. In contrast to higher plants the oxidation of external NADH in mitochondria of E. gracilis is sensitive to rotenone and yields the same phosphorylation efficiency as the matrix pool of NADH. Simulation of this activity by the classic complex I of the matrix side of the mitochondrial membrane, as a result of preparation-generated artefacts, is excluded. The external NADH-dehydrogenase activity is bound to the inner mitochondrial membrane with its active side facing the cytosol. State-4 enzyme activity is only slightly influenced by pH in the physiological range, whereas state-3 oxidation indicates an optimum in the physiological pH, as expected from a limitation by the ATPase. The external redox potential of NADH does not control enzyme activity. The results are discussed with respect to the metabolic status of the cells at the time of harvesting.     

Demonstration of the existence of an organo-specific NADH dehydrogenase in heart mitochondria

Experimental evidence is presented showing the existence of an NADH-consuming enzyme in heart mitochondria, in addition to the NADH--ubiquinone oxidase of complex I. In contrast to the latter, the novel enzyme is accessible from the extramitochondrial space. Removal of the outer membranes from intact mitochondria had no influence on exogenous NADH consumption, indicating its location at the cytosolic face of the inner membrane. The enzyme could be solubilized from this membrane and purified by sedimentation through preformed sucrose gradients. Liver mitochondria exhibited no oxidation of external NADH, suggesting that the enzyme is organo-specific. The "exogenous NADH dehydrogenase" of heart mitochondria was found to introduce reducing equivalents into the respiratory chain before the rotenone block, indicating that the enzyme is associated with complex I. The enzyme was also demonstrated to be involved in electron flow from the respiratory chain to exogenous electron acceptors, including NAD+. This permitted us to elicit the existence of an energy-dependent reversed electron flow from complex II to complex I. The redox shuttle established by the novel enzyme could be of significance for the regulation of cellular NADH and the metabolic activation of foreign compounds such as adriamycin.     

Membrane-Associated NAD-Dependent Isocitrate Dehydrogenase in Potato Mitochondria

The oxidation isotherms for citrate and isocitrate by potato (Solanum tuberosum var. Russet Burbank) mitochondria in the presence of NAD differ markedly. Citrate oxidation shows positively cooperative kinetics with a sigmoid isotherm, whereas isocitrate oxidation shows Michaelis-Menten kinetics at concentrations up to 3 millimolar, and cooperative kinetics thereafter up to 30 millimolar. In the absence of exogenous NAD, the isocitrate isotherm is sigmoid throughout. The dual isotherm for isocitrate oxidation in the presence of exogenous NAD reflects the operation of two forms of isocitrate dehydrogenase, one in the matrix and one associated with the inner mitochondrial membrane. Whereas in intact mitochondria the activity of the membrane-bound enzyme is insensitive to rotenone, and to butylmalonate, an inhibitor of organic acid transport, isocitrate oxidation by the soluble matrix enzyme is inhibited by both. The membrane-bound isocitrate dehydrogenase does not operate through the NADH dehydrogenase on the outer face of the inner mitochondrial membrane, and is thus considered to face inward. The regulatory potential of isocitrate dehydrogenase in potato mitochondria may be realized by the apportionment of the enzyme between its soluble and bound forms.     

L-lactate metabolism in potato tuber mitochondria

We investigated the metabolism of L-lactate in mitochondria isolated from potato tubers grown and saved after harvest in the absence of any chemical agents. Immunologic analysis by western blot using goat polyclonal anti-lactate dehydrogenase showed the existence of a mitochondrial lactate dehydrogenase, the activity of which could be measured photometrically only in mitochondria solubilized with Triton X-100. The addition of L-lactate to potato tuber mitochondria caused: (a) a minor reduction of intramitochondrial pyridine nucleotides, whose measured rate of change increased in the presence of the inhibitor of the alternative oxidase salicyl hydroxamic acid; (b) oxygen consumption not stimulated by ADP, but inhibited by salicyl hydroxamic acid; and (c) activation of the alternative oxidase as polarographically monitored in a manner prevented by oxamate, an L-lactate dehydrogenase inhibitor. Potato tuber mitochondria were shown to swell in isosmotic solutions of ammonium L-lactate in a stereospecific manner, thus showing that L-lactate enters mitochondria by a proton-compensated process. Externally added L-lactate caused the appearance of pyruvate outside mitochondria, thus contributing to the oxidation of extramitochondrial NADH. The rate of pyruvate efflux showed a sigmoidal dependence on L-lactate concentration and was inhibited by phenylsuccinate. Hence, potato tuber mitochondria possess a non-energy-competent L-lactate/pyruvate shuttle. We maintain, therefore, that mitochondrial metabolism of L-lactate plays a previously unsuspected role in the response of potato to hypoxic stress.     

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.

     

Enzymatic properties of the membrane-bound NADH oxidase system in the aerobic respiratory chain of Bacillus cereus

Membranes prepared from Bacillus cereus KCTC 3674, grown aerobically on a complex medium, oxidized NADH exclusively, whereas deamino-NADH was little oxidized. The respiratory chain-linked NADH oxidase exhibited an apparent K(m) value of approximately 65 microM for NADH. The maximum activity of the NADH oxidase was obtained at about pH 8.5 in the presence of 0.1 M KCl (or NaCl). Respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited the activity of the NADH oxidase by about 90% at a concentration of 40 microM. Interestingly, rotenone and capsaicin inhibited the activity of the NADH oxidase by about 60% at a concentration of 40 microM and the activity was also highly sensitive to Ag(+).