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.     

Mode of Action of the Antibiotic Siccanin on Intact Cells and Mitochondria of Trichophyton mentagrophytes

Siccanin at 3 mug/ml completely inhibited the growth of Trichophyton mentagrophytes. The primary site of action of siccanin on T. mentagrophytes is succinate dehydrogenase in the terminal electron transport system. At a concentration of siccanin giving 50% inhibition of growth (0.3 mug/ml), respiration of intact cells was inhibited more strongly than any other cellular functions tested, including the syntheses of cellular ribonucleic acid, deoxyribonucleic acid, phospholipid, protein, and cell wall fractions. In addition, at the same concentration siccanin did not cause any detectable damage in the permeability of the cells. Furthermore, the oxidation of succinate in mitochondrial preparation is more sensitive to the antibiotic than respiration in intact cells. Oxidation of other substrates tested was less sensitive to siccanin than that of succinate. The antibiotic inhibited both phosphorylation and oxidation, without causing changes in the P:O ratio. Siccanin at 0.03 mug/ml, which caused 50% inhibition of succinate oxidation in mitochondria, had effect neither on the exchange reaction between inorganic phosphate (P(i)) and adenosine triphosphate (ATP) nor on that between adenosine diphosphate and ATP. An ATP phosphohydrolase activity was also insensitive to the antibiotic. At very high concentrations, however, the antibiotic slightly inhibited the P(i)-ATP exchange reaction. From those results, it was concluded that siccanin inhibits fungal growth by inhibiting the respiratory electron transport system.     

Evidence of a phosphate-transporter system in the inner membrane of isolated mitochondria

1. The organic mercurial sodium mersalyl, formaldehyde, dicyclohexylcarbodiimide and tributyltin each blocked respiratory-chain-linked ATP synthesis in rat liver mitochondria. 2. Mersalyl and formaldehyde also blocked a number of other processes dependent on the entry of inorganic phosphate into mitochondria, including mitochondrial respiration and swelling stimulated by cations and phosphate, the substrate-level phosphorylation reaction of the citric acid cycle, and swelling in ammonium phosphate. 3. Dicyclohexylcarbodi-imide and tributyltin did not inhibit the entry of phosphate into mitochondria. 4. Mersalyl and formaldehyde had a relatively slight effect on succinate oxidation and swelling stimulated by cations when phosphate was replaced by acetate, on succinate oxidation stimulated by uncoupling agents, and on swelling in solutions of ammonium salts other than phosphate or arsenate. 5. Formaldehyde blocked the oxidation of NAD-linked substrates in mitochondria treated with 2,4-dinitrophenol and the ATP-dependent reduction of NAD by succinate catalysed by ox heart submitochondrial particles. Both these effects appear to be due to an inhibition by formaldehyde of the NAD-flavin region of the respiratory chain. 6. Concentrations of dicyclohexylcarbodiimide or tributyltin sufficient to abolish ADP-stimulated respiration blocked the dinitrophenol-stimulated adenosine triphosphatase activity, whereas mersalyl and formaldehyde caused only partial inhibition of ATP hydrolysis. 7. When mitochondria were incubated with dinitrophenol and ATP, less than 10% of the total inorganic phosphate liberated was recovered in the mitochondria and no swelling occurred. In the presence of mersalyl or formaldehyde at least 80% of the total inorganic phosphate liberated was retained in the mitochondria and extensive swelling was observed. This swelling was inhibited by oligomycin but not by antimycin or rotenone. 8. The addition of mersalyl to mitochondria swollen by treatment with valinomycin, K(+) and phosphate blocked the contraction induced by dinitrophenol and caused an increase in the phosphate content of the mitochondria, but had no effect on the contraction of mitochondria when phosphate was replaced by acetate. 9. It is concluded that mitochondria contain a phosphate-transporter system, which catalyses the movement of phosphate in either direction across the mitochondrial membrane, and that this system is inactivated by organic mercurials and by formaldehyde. Evidence is presented that the phosphate-transporter system is situated in the inner membrane of rat liver mitochondria and is also present in other types of mammalian mitochondria.     

Hydrogen peroxide generation by higher plant mitochondria oxidizing complex I or complex II substrates.

The generation of H2O2 by isolated pea stem mitochondria, oxidizing either malate plus glutamate or succinate, was examined. The level of H2O2 was almost one order of magnitude higher when mitochondria were energized by succinate. The succinate-dependent H2O2 formation was abolished by malonate, but unaffected by rotenone. The lack of effect of the latter suggests that pea mitochondria were working with a proton motive force below the threshold value required for reverse electron transfer. The activation by pyruvate of the alternative oxidase was reflected in an inhibition of H2O2 formation. This effect was stronger when pea mitochondria oxidized malate plus glutamate. Succinate-dependent H2O2 formation was ca. four times lower in Arum sp. mitochondria (known to have a high alternative oxidase) than in pea mitochondria. An uncoupler (FCCP) completely prevented succinate-dependent H2O2 generation, while it only partially (40-50%) inhibited that linked to malate plus glutamate. ADP plus inorganic phosphate (transition from state 4 to state 3) also inhibited the succinate-dependent H2O2 formation. Conversely, that dependent on malate plus glutamate oxidation was unaffected by low and stimulated by high concentrations of ADP. These results show that the main bulk of H2O2 is formed during substrate oxidation at the level of complex II and that this generation may be prevented by either dissipation of the electrochemical proton gradient (uncoupling and transition state 4-state 3), or preventing its formation (alternative oxidase). Conversely, H2O2 production, dependent on oxidation of complex I substrate, is mainly lowered by the activation of the alternative oxidase.     

The inhibition of mitochondrial dicarboxylate transport by inorganic phosphate, some phosphate esters and some phosphonate compounds

1. P(i) competitively inhibited succinate oxidation by intact uncoupled mitochondria in the presence of sufficient N-ethylmaleimide to block the phosphate carrier, with a K(i) of 2.5mm. 2. Of a large number of phosphate esters and phosphonate compounds, phenyl phosphate and phenylphosphonate were found to inhibit competitively uncoupled succinate oxidation by intact but not broken mitochondria. By comparison, benzoate was a relatively weak competitive inhibitor of succinate oxidation by intact mitochondria but a relatively potent inhibitor of succinate dehydrogenase. 3. Phenyl phosphate and phenylphosphonate were non-penetrant, and inhibited P(i)-dependent swelling of mitochondria suspended in isosmolar ammonium malate in a manner non-competitive with P(i). The inhibitors did not affect mitochondrial swelling when tested with P(i) alone. 4. It is concluded that: (i) phenyl phosphate and phenylphosphonate behaved as non-penetrant analogues of P(i), since their inhibitory properties were in strict contrast with those of benzoate; (ii) phenyl phosphate and phenylphosphonate interacted with the dicarboxylate carrier but not with the phosphate carrier; (iii) P(i) was effective as a competitive inhibitor of succinate oxidation because of its being either an alternative substrate for the dicarboxylate carrier or competitive with succinate for the intramitochondrial cations as proposed by Harris & Manger (1968).     

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.     

The effects of acetylcolletotrichin on the mitochondrial respiratory chain

1. Acetylcolletotrichin is a phytotoxic compound that has been isolated from the culture medium of the fungus Colletotrichum capsici (Grove et al., 1966). 2. With isolated liver and kidney mitochondria acetylcolletotrichin markedly inhibited the oxidation of succinate and those substrates with NAD-linked dehydrogenases, but did not inhibit the oxidation of ascorbate in the presence of tetramethyl-p-phenylenediamine. In this respect its action was similar to that of antimycin A. 3. Acetylcolletotrichin differed from antimycin in that, even at high concentrations which produced a maximal inhibitory effect, its action was partially reversed by uncoupling agents. Also acetylcolletotrichin had no detectable effect on the oxidative activity of blowfly flight-muscle mitochondria and was not very effective with heart mitochondria. 4. Acetylcolletotrichin inhibited the oxidative activity of liver mitochondria more markedly when respiration was stimulated by ADP together with phosphate and was less effective when respiration was stimulated by uncoupling agents. 5. There was an unusual interaction between the succinate oxidation system and the oxidation of glutamate together with malate. Thus, glutamate together with malate, even in the presence of rotenone, markedly decreased the effectiveness of acetylcolletotrichin in inhibiting succinate oxidation. 6. These effects were paralleled in the observed redox changes of cytochrome c. 7. The unusual behaviour of the cytochromes b in the presence of acetylcolletotrichin is described, and it is suggested tentatively that this inhibitor acts between cytochromes b with absorption maxima at 30 degrees C of approximately 560 and 565nm.     

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.     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase.

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH4+-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibted by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitos of respiration or an uncoupler but not in ther absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition. On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibted by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Evidence That a Malate/Inorganic Phosphate Exchange Translocator Imports Carbon across the Leucoplast Envelope for Fatty Acid Synthesis in Developing Castor Seed Endosperm

In this study we examined the processes by which malate and pyruvate are taken up across the leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH₄⁺-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate.

1. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue.

2. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibited by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate.

3. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitors of respiration or an uncoupler but not in their absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition.

On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibited by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Effect of exogenous nucleotides on the candicidin fermentation.

Addition of cyclic-AMP (c-AMP) to Streptomyces griseus fermentations inhibited candicidin formation. In a phosphate-free resting cell system, c-AMP inhibited net candicidin formation and incorporation of labeled propionate and p-aminobenzoic acid into the antibiotic but did not inhibit protein synthesis. All nucleotides tested, regardless of the position of the phosphate ester, were effective inhibitors; nucleosides and free bases were not. Inhibition occurred whether the nucleotide was added early or late. The results indicate that inhibition of antibiotic formation by exogenous nucleotides, including cyclic nucleotides, is similar to the effect produced by inorganic phosphate.     

Oxaloacetate inhibition of succinate oxidation in tightly coupled liver mitochondria with ferricyanide as an electron acceptor

When ferricyanide is used as an artificial electron acceptor, succinate oxidation by tightly coupled liver mitochondria becomes inhibited after 1–3 min. No inhibition occurs in the presence of rotenone or glutamate establishing that oxaloacetate causes the inhibtion. Oxygen consumption by mitochondria oxidizing succinate does not become inhibited in the absence of rotenone suggesting that oxaloacetate accumulates to a greater extent when ferricyanide is added than when oxygen is the terminal acceptor. Higher levels of oxaloacetate in the ferricyanide reaction are apparently due to an increased rate of synthesis rather than a decreased rate of removal. Thus it appears that when succinate is the substrate and oxygen the terminal acceptor a control mechanism exists which blocks oxidation of malate. When ferricyanide is added as an artificial electron acceptor this control is lost and oxaloacetate accumulates to inhibit succinate oxidation.     

Oxidation of Alanine, Acetate, Glutamate, and Succinate by Digitonin-Permeabilized Leishmania major Promastigotes

Leishmania major promastigotes were treated with digitonin and the rates at which [1 -¹⁴C]acetate, [1,4-¹⁴C]succinate, [1-¹⁴C]glutamate, and [U-¹⁴C]alanine are oxidized were measured in the presence of suitable cofactors. Acetate was oxidized at the lowest rate of the four substrates examined, even in the presence of added NAD, CoA, ADP and acetyl-CoA synthase. Its rate of oxidation was negligible if the permeabilized cells were washed before the cofactors were added, indicating the requirement for an as yet unknown factor. Succinate was oxidized at a rate much higher than the very slow rate at which it is oxidized by intact cells. Its rate of oxidation was strongly inhibited by antimycin A, but that of glutamate was scarcely affected. Fumarate inhibited the rate of oxidation of acetate, glutamate, and succinate, but increased that of alanine, Ca⁺⁺ inhibited the rates of oxidation of alanine and succinate, but not of acetate or glutamate. Increasing the osmolality by addition of mannitol partially inhibited the rate of oxidation of alanine but had little effect on that of glutamate. These results show that appreciable transaminase activity remains in the permeabilized cells and support earlier data indicating the presence of a branched NAD-to-cytochrome oxidase system. These results also provide preliminary information on the sensitivity of the two branches to Ca⁺⁺, hyperosmolality, and Krebs cycle intermediates     

Contracted state as an energy source for ca binding and ca + inorganic phosphate accumulation by corn mitochondria

An investigation has been made of the possibility of utilizing the potential energy of the contracted state of corn mitochondria to drive Ca + inorganic phosphate accumulation. Contraction was obtained with succinate or NADH oxidation. In the succinate experiments the mitochondria were contracted in buffered KCl layered over sucrose in centrifuge tubes and centrifuged down through distinct wash, reactive and isotope exchange layers. In the NADH experiments, ion accumulation was initiated upon exhaustion of the substrate. The results show that mitochondria in the contracted state will actively bind some ⁴⁵Ca, but no real accumulation occurs until inorganic phosphate is available. Substrate powered contraction in the presence of inorganic phosphate also provides a potential for accumulation upon subsequent reaction of the mitochondria with Ca. It is deducted that contraction is due to X~I formation, to which Ca will bind. Subsequent reaction with inorganic phosphate produces CaX~P, which is the transport moiety. When X~P is formed first, Ca also reacts to produce CaX~P. Hence it is immaterial which ion reacts first with the contracted state. Contraction is believed to result from the action of a mechanoenzyme, presumably I~. The stability of CaX~I must be low for the mitochondria swell very rapidly upon exhaustion of NADH or blocking of succinate oxidation by cyanide.     

Phosphate inhibition of secondary metabolism in Streptomyces hygroscopicus and its reversal by cyclic AMP

Inorganic phosphate inhibited the biosynthesis of the macrolide antibiotic turimycin in different strains of Streptomyces hygroscopicus. In the wild type strain a depression was observed with increasing phosphate concentrations. A total inhibition was found at 0.1 M phosphate. In a high producing mutant a minimum of turimycin production occured when the phosphate concentration was between 5 mM and 10 mM. Above this concentration the antibiotic synthesis increased again but the production period shifted to a later period of cultivation. Addition of inorganic phosphate resulted in an initial increase of intracellular cyclic AMP content. But a second elevation characterizing the normal level of cyclic AMP throughout the growth phase was prevented by phosphate. Exogenous cyclic AMP as well as positive effectors of the adenylyl cyclase system were able to overcome the phosphate suppression. Cyclic AMP abolished the reduction of protein synthesis following phosphate addition and caused the reappearance of a protein band which may be responsible for the turimycin biosynthesis.     

The Respiratory Chain of Plant Mitochondria: XI. Electron Transport from Succinate to Endogenous Pyridine Nucleotide in Mung Bean Mitochondria

Energy-linked reverse electron transport from succinate to endogenous NAD in tightly coupled mung bean (Phaseolus aureus) mitochondria may be driven by ATP if the two terminal oxidases of these mitochondria are inhibited, or may be driven by the free energy of succinate oxidation. This reaction is specific to the first site of energy conservation of the respiratory chain; it does not occur in the presence of uncoupler. If mung bean mitochondria become anaerobic during oxidation of succinate, their endogenous NAD becomes reduced in the presence of uncoupler, provided that both inorganic phosphate (P_i) and ATP are present. No reduction occurs in the absence of P_i, even in the presence of ATP added to provide a high phosphate potential. If fluorooxaloacetate is present in the uncoupled, aerobic steady state, no reduction of endogenous NAD occurs on anaerobiosis; this compound is an inhibitor of malate dehydrogenase. This result implies that endogenous NAD is reduced by malate formed from the fumarate generated during succinate oxidation. The source of free energy is most probably the endogenous energy stores in the form of acetyl CoA, or intermediates convertible to acetyl CoA, which removes the oxaloacetate formed from malate, thus driving the reaction towards reduction of NAD.     

Nutritional effects on mitochondrial bioenergetics. Alterations in oxidative phosphorylation by rat liver mitochondria.

Rats malnourished since birth and fed on a protein-free diet for 2 weeks showed a 23-27% decrease in the State-3 oxidation of glutamate, succinate and ascorbate + NNN' N'-tetramethyl-p-phenylenediamine by liver mitochondria compared with control fed animals. ATP synthesis and the respiratory control index were diminished at the three coupling sites, but significant alterations were not observed in ADP/O ratios. Vmax. for NADH oxidation in electron-transport particles was 40% lower. Mitochondrial cytochromes b and c1 remained unchanged, but cytochrome c was increased by 26%. Cytochromes a + a3 were diminished by 22%. Vmax. for mitochondrial ATPase was 23% lower. These results suggest that the lower content of cytochrome a + a3 at the rate-controlling step of oxidative phosphorylation in malnourished rats might be mainly responsible for the decrease in substrate oxidations as well as ATP synthesis at the three coupling sites. The decreased synthesis and hydrolysis of ATP suggests that other energy-dependent mitochondrial processes could be decreased during malnutrition.     

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.     

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.