Effects of phthalate esters on the respiration of rat liver mitochondria.

The effects of phthalate esters on the oxidation of succinate, glutamate, beta-hydroxybutyrate and NADH by rat liver mitochondria were examined and it was found that di-n-butyl phthalate (DBP) strongly inhibited the succinate oxidation by intact and sonicated rat mitochondria, but did not inhibit the State 4 respiration with NAD-linked substrates such as glutamate and beta-hydroxybutyrate. However, oxygen uptake accelerated by the presence of ADP and substrate (State 3) was inhibited and the rate of oxygen uptake decreased to that without ADP (State 4). It was concluded that phthalate esters were electron and energy transport inhibitors but not uncouplers. Phthalate esters also inhibited NADH oxidation by sonicated mitochondria. The degree of inhibition depended on the carbon number of alkyl groups of phthalate esters, and DBP was the most potent inhibitor of respiration. The activity of purified beef liver glutamate dehydrogenase [EC 1.4.1.3] was slightly inhibited by phthalate esters.     

Stimulation by quinones of cyanide-resistant respiration in rat liver and heart mitochondria

It was shown that hydrophilic benzo- and naphthoquinones stimulate the cyanide-resistant respiration in liver and muscle mitochondria when succinate or NADH and glutamate or malate are used as oxidation substrates. The substrate-dependent oxygen uptake in the presence of cyanide is initiated by menadione, vicasol, 1.2-naphthoquinone, coenzyme Q0 and duroquinone. Rotenone and antimycin A do not inhibit the cyanide-resistant respiration. Oxidation of glutamate and malate in the course of CN-resistant respiration is inhibited by ortho- and bathophenanthroline and p-chloromercurybenzoate, whereas succinate oxidation by tenoyltrifluoroacetone, carboxin and pentachlorophenol. Superoxide dismutase, Cu2+ and catalase inhibit the CN-resistant respiration in the presence of quinones. Addition of catalase to the experimental cell causes O2 release.     

The action of tellurite, a reagent for thiol groups, on mitochondria oxidative processes

Tellurite, a reagent for thiol groups, added to rat kidney, or liver mitochondria at the concentration of 1 mM, selectively inhibits the oxidation of NAD dependent substrates (pyruvate, α-ketoglutarate, glutamate, etc.) without affecting the oxidation of succinate, α-glycerophosphate and ascorbate. Moreover NADH oxidation by rat heart or by aged liver mitochondria is not affected by tellurite.     

The effect of 4-hydroxy-2, 3-trans-pent-en-1-al and maleylaldehyde on some mitochondrial functions

Low concentrations of HPE and MLA inhibited state 3 respiration of rat liver mitochondria in the presence of different NAD⁺-dependent substrates. MLA appeared to be more active than HPE. High aldehyde concentrations inhibited the state 3 respiration with succinate. The restraint of succinate oxidation by HPE and MLA and of glutamate plus malate oxidation by MLA correlated with the inhibition of succinate and glutamate dehydrogenase activites, respectively. HPE inhibited glutamate dehydrogenase at concentrations higher than those affecting glutamate oxidation. Malate dehydrogenase activity was slightly sensitive to HPE and MLA. Both aldehydes inhibited NADH oxidation by freeze-thawed mitochondria. These results suggest the existence of a site particularly sensitive to aldehydes in the electron transport chain between the specific NAD⁺-linked dehydrogenases and ubiquinone.     

Glucagon treatment of rats activates the respiratory chain of liver mitochondria at more than one site

The rate of reduction of ferricyanide in the presence and absence of antimycin and ubiquinone-1 was measured using liver mitochondria from control and glucagon treated rats. Glucagon treatment was shown to increase electron flow from both NADH and succinate to ubiquinone, and from ubiquinone to cytochrome c. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was shown to inhibit the oxidation of glutamate + malate to a much greater extent than that of succinate or duroquinol. Spectral and kinetic studies confirmed that electron flow between NADH and ubiquinone was the primary site of action but that the interaction of the ubiquinone pool with complex 3 was also affected. The effects of various respiratory chain inhibitors on the rate of uncoupled oxidation of succinate and glutamate + malate by control and glucagon treated mitochondria were studied. The stimulation of respiration seen in the mitochondria from glucagon treated rats was maintained or increased as respiration was progressively inhibited with DCMU, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO) and colletotrichin, but greatly reduced when inhibition was produced with malonate or antimycin. These data were also shown to support the conclusion that glucagon treatment may cause some stimulation of electron flow through NADH dehydrogenase, succinate dehydrogenase and through the bc1 complex, probably at the point of interaction of the complexes with the ubiquinone pool. The effects of glucagon treatment on duroquinol oxidation and the inhibitor titrations could not be mimicked by increasing the matrix volume, nor totally reversed by aging of mitochondria. These are both processes that have been suggested as the means by which glucagon exerts its effects on the respiratory chain (Armston, A.E., Halestrap, A.P. and Scott, R.D., 1982, Biochim. Biophys. Acta 681, 429-439). It is concluded that an additional mechanism for regulating electron flow must exist and a change in lipid peroxidation of the inner mitochondrial membrane is suggested.     

The inhibition of pyruvate and ls(+)-isocitrate oxidation by succinate oxidation in rat liver mitochondria

1. The effects of succinate oxidation on pyruvate and also isocitrate oxidation by rat liver mitochondria were studied. 2. Succinate oxidation was without effect on pyruvate and isocitrate oxidation when respiration was maximally activated with ADP. 3. When respiration was partially inhibited by atractylate, succinate oxidation severely inhibited the oxidation of pyruvate and isocitrate. 4. This inhibitory effect of succinate was associated with a two- to three-fold increase in the reduction of mitochondrial NAD(+) but no change in the reduction of cytochrome b. 5. It is concluded that, in the partially energy-controlled state, respiration is more severely inhibited at the first phosphorylating site than at the other two. 6. The effects of succinate oxidation are compared with those of palmitoylcarnitine oxidation. It is concluded that a rapid flow of electrons directly into the respiratory chain at the level of cytochrome b is in itself inadequate to inhibit the oxidation of intramitochondrial NADH. 7. The effects of succinate oxidation on pyruvate oxidation were similar in rat heart and liver mitochondria.     

Failure of exogenous NADH and cytochrome c to support energy-dependent swelling of mitochondria

The possibility of direct oxidation of external NADH in rat liver mitochondria and of the inner membrane potential generation in this process is still not clear. In the present work, the energy-dependent swelling of mitochondria in the medium containing valinomycin and potassium acetate was measured as one of the main criteria of the proton-motive force generation by complex III, complex IV, and both complexes III and IV of the respiratory chain. Mitochondria swelling induced by external NADH oxidation was compared with that induced by succinate or ferrocyanide oxidation, or by electron transport from succinate to ferricyanide. Mitochondria swelling, nearly equal to that promoted by ferrocyanide oxidation, was observed under external NADH oxidation, but only after the outer mitochondrial membrane was ruptured as a result of the swelling-contraction cycle, caused by succinate oxidation and its subsequent inhibition. In this case, significantly accelerated intermembrane electron transport and well-detected inner membrane potential generation, in addition to mitochondria swelling, were also observed. Presented results suggest that exogenous NADH and cytochrome c do not support the inner membrane potential generation in intact rat liver mitochondria, because the external NADH-cytochrome c reductase system, oriented in the outer mitochondrial membrane toward the cytoplasm, is inaccessible for endogenous cytochrome c reduction; as well, the inner membrane cytochrome c oxidase is inaccessible for exogenous cytochrome c oxidation.     

Carvedilol inhibits the exogenous NADH dehydrogenase in rat heart mitochondria

There are several reports on the oxidation of external NADH by an exogenous NADH dehydrogenase in the outer leaflet of the inner membrane of rat heart mitochondria. Until now, however, little was known about its physiological role in cellular metabolism. The present work shows that carvedilol (?1-[carbazolyl-(4)-oxy]-3-[2-methoxyphenoxyethyl)amino]-pro - panol-(2)?) is a specific inhibitor of an exogenous NADH dehydrogenase in rat heart mitochondria. Carvedilol does not affect oxygen consumption linked to the oxidation of succinate and internal NADH. It is also demonstrated that the inhibition of exogenous NADH dehydrogenase by carvedilol is accompanied by the inhibition of alkalinization of the external medium. In contrast to the addition of glutamate/malate or succinate, exogenous NADH does not generate a membrane potential in rat heart mitochondria, as observed with a TPP(+) electrode. It is also demonstrated that the oxygen consumption linked to NADH oxidation is not due to permeabilized mitochondria, but to actual oxidase activity in the inner membrane. The enzyme has a K(m) for NADH of 13 microM. Carvedilol is a noncompetitive inhibitor of this external NADH dehydrogenase with a K(i) of 15 microM. Carvedilol is the first inhibitor described to this organospecific enzyme. Since this enzyme was demonstrated to play a key role in the cardiotoxicity of anticancer drugs of the anthracycline family (e.g., adriamycin), we may suggest that the administration of carvedilol to tumor patients treated with adriamycin might be of great help in the prevention of the cardioselective toxicity of this antibiotic.     

Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NAD+-linked substrates by MPP+

1-methyl-4-phenylpyridine (MPP+), a major product of the oxidation of the neurotoxic amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been postulated to be the compound responsible for destruction of nigrostriatal neurons in man and primates and for inhibition of mitochondrial NADH oxidation which leads to cell death. We have confirmed that 0.5 mM MPP+ inhibits extensively the oxidation of NAD+-linked substrates in intact liver mitochondria in State 3 and after uncoupling, while succinate oxidation is unaffected. However, in inverted mitochondria, inner membrane preparations, and Complex I NADH oxidation is not significantly affected at this concentration of MPP+, nor are malate and glutamate dehydrogenases or the carriers of these substrates inhibited. We report here the discovery of an uptake system for MPP+ in mitochondria which is greatly potentiated by the presence of malate plus glutamate and inhibited by respiratory inhibitors, suggesting an energy-dependent carrier. A 40-fold concentration of MPP+ in the mitochondria occurs in ten minutes. This might account for the inhibition of malate and glutamate oxidation in intact mitochondria.     

MDH2 produced OAA is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle

The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD⁺ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD⁺ redox stress have not been elucidated. The recent discovery that complex I (NADH dehydrogenase), and not complex II (Succinate dehydrogenase), can assemble with other respiratory chain complexes to form functional entities called respirasomes, led to the assumption that this supramolecular organization would favour NADH oxidation. Unexpectedly, characterization of heart and liver mitochondria demonstrates that the RC systematically favours electrons provided by the ‘respirasome free’ complex II. Our results demonstrate that the preferential succinate driven respiration is tightly controlled by OAA levels, and that OAA feedback inhibition of complex II rewires RC fuelling increasing NADH oxidation capacity. This new regulatory mechanism synergistically increases RC's NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA, preventing malate production from succinate to favour oxidation of cytosolic malate. This regulatory mechanism synergistically adjusts RC and TCA fuelling in response to extramitochondrial malate produced by the MAS.     

Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart mitochondria.

The saturation kinetics of NADH and succinate oxidation for Coenzyme Q (CoQ) has been re-investigated in pentane-extracted lyophilized beef heart mitochondria reconstituted with exogenous CoQ10. The apparent 'Km' for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The Km value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinate oxidation is close to the content of respiratory chain enzymes.     

Suppression of the mitochondrial oxidation of (-)-palmitylcarnitine by the malate-aspartate and alpha-glycerophosphate shuttles.

Palmitylcarnitine oxidation by isolated liver mitochondria has been used to investigate the interaction of fatty acid oxidation with malate, glutamate, succinate, and the malate-aspartate shuttle. Mitochondria preincubated with fluorocitrate were added to a medium containing 2mM ATP and ATPase. This system, characterized by a high energy change, allowed titration of respiration to any desired rate between States 4 and 3 (Chance, B., and Williams, G. R. (1956) Adv. Enzymol. Relat. Areas Mol. Biol. 17, 65-134). When respiration (reference, with palmitylcarnitine and malate as substrates) was set at 75% of State 3, the oxidation of palmitylcarnitine was limited by acetoacetate formation. The addition of malate or glutamate approximately doubled the rate of beta oxidation. Malate circumvented this limitation by citrate formation, but the effect of glutamate apparently was due to enhancement of the capacity for ketogenesis. The rate of beta oxidation was curtailed when malate and glutamate were both present. This curtailment was more pronounced when the malate-aspartate shuttle was fully reconstituted. Among the oxidizable substrates examined, succinate was most effective in inhibiting palmitylcarnitine oxidation. Mitochondrial NADH/NAD+ ratios were correlated positively with suppression of beta oxidation. The degree of suppression of beta oxidation by the malate-aspartate shuttle (NADH oxidation) or by succinate oxidation was dependent on the respiratory state. Both substrates extensively reduced mitochondrial NAD+ and markedly suppressed beta oxidation as respiration approached State 4. Calculations of the rates of flux of hydrogen equivalents through beta oxidation show that the suppression of beta oxidation by glutamate or by the malate-aspartate shuttle is accounted for by increased flux of reducing equivalents through mitochondrial malic dehydrogenase. This increased Flux is accompanied by an increase in the steady state NADH/NAD+ ratio and a marked decrease in the synthesis of citrate. The alpha-glycerophosphate shuttle was reconstituted with mitochondria isolated from rats treated with L-thyroxine. This shuttle was about equal to the reconstructed malate-aspartate shuttle in supression of palmitylcarnitine oxidation. This interaction could not be demonstrated in euthyroid animals owing to the low activity of the mitochondrial alpha-glycerol phosphate dehydrogenase. It is concluded that beta oxidation can be regulated by the NADH/NAD+ ratio. The observed stimulation of flux through malate dehydrogenase both by glutamate and by the malate-aspartate shuttle results in an increased steady state NADH/NAD+ ratio, and is linked to a stoichiometric outward transport of aspartate. We suggest, therefore, that some of the reducing pressure exerted by the malate-aspartate shuttle and by glutamate plus malate is provided through the energy-linked, electrogenic transport of aspartate out of the mitochondria. These results are discussed with respect to the mechanism of the genesis of ethanol-induced fatty liver.     

Activation of electron transport at the terminal site of the respiratory chain by the submitochondrial fluid from the rat and guinea pig liver

Supermitochondrial liquid (SL) of rat and guinea-pig liver increases the activity of 2, 3, 5 triphenyltetrasolium chloride (TPC) and tetrasolium violet (TV) reduction at succinate, NADH and NADPH oxidation by mitochondria (MC). SL contains an activating factor A, being evidently of a protein nature and factor B, increasing the activating activity of factor A. NAD, NADP, NADH and NADP at 5 x 10(-5)-1 x 10(-4) M concentration activate the TPC and TV reduction at succinate oxidation by mitochondria. TPC and TV reduction at succinate and NADP oxidation by mitochondria makes antimicin and cyanide sensitive. SL does not influence succinate dehydrogenase activity, when used as electron acceptors of ferricyanide, blue Vurster, cytochrome C, blue and violet nitrotetrasolium. Activation of electron transfer chair between cytochrome C and oxygen is supposed to be responsible for such an effect.     

Two separate pathways underlie NADH and succinate oxidation in swine heart mitochondria: Kinetic evidence on the mobile electron carriers

We have investigated NADH and succinate aerobic oxidation in frozen and thawed swine heart mitochondria. Simultaneous oxidation of NADH and succinate showed complete additivity under a variety of experimental conditions, suggesting that the electron fluxes originating from NADH and succinate are completely independent and do not mix at the level of the so-called mobile diffusible components. We ascribe the results to mixing of the fluxes at the level of cytochrome c in bovine mitochondria: the Complex IV flux control coefficient in NADH oxidation was high in swine mitochondria but very low in bovine mitochondria, suggesting a stronger interaction of cytochrome c with the supercomplex in the former. This was not the case in succinate oxidation, in which Complex IV exerted little control also in swine mitochondria. We interpret the data in swine mitochondria as restriction of the NADH flux by channelling within the I-III₂-IV supercomplex, whereas the flux from succinate shows pool mixing for both Coenzyme Q and probably cytochrome c. The difference between the two types of mitochondria may be ascribed to different lipid composition affecting the cytochrome c binding properties, as suggested by breaks in Arrhenius plots of Complex IV activity occurring at higher temperatures in bovine mitochondria.     

Impaired substrate utilization in mitochondria from strain 129 dystrophic mice

Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca²⁺/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg²⁺-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD⁺ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD⁺ in the case of NAD⁺-linked substrates.     

The inhibition of NADH oxidase by the lower homologs of coenzyme Q.

The Coenzyme Q homologs having short isoprenoid chains are much less efficient than the higher homologs in restoring NADH oxidation in pentane-extracted lyophilized beef heart mitochondria; they have however high restoring activity for succinate oxidation. The same pattern is observed in pentane extracted submitochondrial particles ETP only if the quinones are added to detergent-treated membranes, showing that in ETP there is a decreased accessibility of the long chain quinones in comparison with the lower homologs. In intact mitochondria and ETP, CoQ₃ inhibits NADH oxidation while leaving succinate oxidation unaffected; the inhibition of NADH oxidation by CoQ₃ is not reversed by serum albumin but is reversed by CoQ₇, particularly when the membrane has been previously “opened” with deoxycholate. CoQ₃ may accept electrons from NADH in cyanide-inhibited ETP, allowing coupling at the first phosphorylation site as shown by the quenching of the fluorescence of atebrine. The mechanism of CoQ₃ inhibition is probably related to its insufficient rate of reoxidation by the following segment of the respiratory chain when it has been reduced by NADH dehydrogenase.     

2,3-butanedione monoxime unmasks Ca(2+)-induced NADH formation and inhibits electron transport in rat hearts

We used 2,3-butanedione monoxime (BDM) to suppress work by the perfused rat heart and to investigate the effects of calcium on NADH production and tissue energetics. Hearts were perfused with buffer containing BDM and elevated perfusate calcium to maintain the rates of cardiac work and oxygen consumption at levels similar to those of control perfused hearts. BDM plus calcium hearts displayed higher levels of NADH surface fluorescence, indicating calcium activation of mitochondrial dehydrogenases. These hearts, however, displayed 20% lower phosphocreatine levels. BDM suppressed the rates of state 3 respiration of isolated mitochondria. Uncoupled respiration was suppressed to a lesser degree, and the state 4 respiration rates were not affected. Double-inhibitor experiments with liver mitochondria using BDM and carboxyatractyloside (CAT) were used to identify the site of inhibition. BDM at low levels (0-5 mM) suppressed respiration. In the presence of CAT at levels that inhibit respiration by 60%, low levels of BDM were without effect. Because these effects were not additive, BDM does not inhibit adenine nucleotide transport. This was supported by an assay of adenine nucleotide transport in liver mitochondria. BDM did not inhibit ATP hydrolysis by submitochondrial particles but strongly suppressed reversed electron transport from succinate to NAD(+). Oxidation of NADH by submitochondrial particles was inhibited by BDM but oxidation of succinate was not. We conclude that BDM inhibits electron transport at site 1.     

Inhibition of mitochondrial electron transport by hydrophilic metal chelators. Determination of dehydrogenase topography

The topography of the inner mitochondrial membrane was investigated using inhibitors of electron transport on preparations of beef heart mitochondria and electron transport particles of opposite orientation. Reductions of juglone, ferricyanide, indophenol, coenzyme Q, duroquinone, and cytochrome c by NADH are inhibited to different extents on both sides of the membrane by the impermeant hydrophilic chelators bathophenanthroline sulfonate and orthophenanthroline. The extent of inhibition for each acceptor increased in the order given. At least two chelator-sensitive sites are present on each membrane face between the flavoprotein and coenzyme Q and a chelator-sensitive site is present on the matrix face between the sites of coenzyme Q and duroquinone interaction. Duroquinol oxidation in mitochondria only is stimulated by bathophenanthroline sulfonate. Juglone reduction is stimulated in electron transport particles (only) by p-hydroxymercuribenzenesulfonate, but after mercurial treatment, juglone reduction in both particles and mitochondria is more sensitive to bathophenanthroline sulfonate.Succinate dehydrogenase components are inhibited by hydrophilic orthophenanthroline or bathophenanthroline sulfonate in mitochondria only. Electron flow between the dehydrogenases of succinate and NADH occurs via a chelator-sensitive site located on the matrix face of the membrane. Inter-complex electron flow is prevented by rotenone or thenoyltrifluoroacetone. The lack of succinate-indophenol reductase inhibition by bathophenanthroline sulfonate in the presence of rotenone or thenoyltrifluoroacetone indicates that the rotenone-sensitive site may be located on the matrix face and demonstrates that electrons flow between the NADH and succinate dehydrogenases via a hydrophilic chelator and rotenone-thenoyltrifluoroacetone-sensitive site on the matrix face of the membrane. Inhibition by hydrophilic chelators only in mitochondria indicates that succinate dehydrogenase as well as NADH dehydrogenase has a transmembranous orientation.     

The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals.

1. Studies on the cytochrome spectra of liver mitochondria from control and glucagon-treated rats in State 4, State 3 and in the presence of uncoupler are reported. 2. The stimulation of electron flow between cytochromes c1 and c observed previously [Halestrap (1978) Biochem. J. 172, 399-405] was shown to be an artefact of Ca2+-induced swelling of mitochondria. 3. When precautions were taken to prevent such swelling, glucagon treatment was shown to enhance the reduction of cytochromes c, c1 and b558 in both State 3 and uncoupled conditions with either succinate or glutamate + malate as substrate. An increase in the reduction of cytochromes b562 and b566 was also seen in some, but not all, experiments. 4. In State 4 with succinate but not glutamate + malate as substrate, cytochromes c, c1, b558, b562 and b566 showed increased reduction. 5. Glucagon stimulated oxidation of duroquinol and palmitoylcarnitine by intact mitochondria and of NADH by disrupted mitochondria. 6. No effect of glucagon on succinate dehydrogenase activity or the temperature-dependence of succinate oxidation could be detected. 7. Glucagon enhanced the inhibition of the respiratory chain by colletotrichin, but not antimycin or 8-heptyl-4-hydroxyquinoline N-oxide. 8. These results are interpreted in terms of a primary stimulation by glucagon of the 'Q cycle' [Mitchell (1976) J. Theor. Biol. 62, 827-367] within Complex III (ubiquinol:cytochrome c oxidoreductase) and a secondary site of action involving stimulation of electron flow into Complex III from the ubiquinone pool. 9. Ageing of mitochondria, hyperosmotic treatment or addition of 20 mM-benzyl alcohol opposed the effects of glucagon treatment on cytochrome spectra and colletotrichin inhibition of respiration. 10. These results support the hypothesis that glucagon exerts its effects on the mitochondria by perturbing the membrane structure.     

Pyruvate metabolism in castor-bean mitochondria.

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.