Participation of the adenine nucleotide translocator in the regulation of pyruvate oxidation in heart mitochondria

A regulatory role of adenine nucleotide translocator (ANT) was determined by titration of mitochondrial respiration (state 3) with carboxyatractyloside. It was shown that ANT regulates pyruvate oxidation: the control strength is more pronounced after depletion of endogenous substrates or after the increase in extramitochondrial ATP/ADP. The rate of succinate oxidation is controlled mainly by succinate dehydrogenase, while ANT does not participate in its regulation.     

Proapoptotic action of alpha-tocopheryl succinate in rat thymocytes is conditioned by the inhibition of mitochondrial succinate dehydrogenase

It has been established that alpha-tocopheryl succinate in concenrations 10-100 microM inhibits in a dose-dependent manner the viability of primary culture rats thymocytes and causes the DNA internucleosomal degradation that testifies to apoptotic way of thymocytes destruction. These effects were accompanied by an enhanced production of intracellular superoxide. This is the first report demonstrating that apoptosis induced by alpha-tocopheryl succinate was accompanied by a dose-dependent inhibition of mitochondrial succinate dehydrogenase. Known apoptosis inducers--actinomicin D, staurosporin and hydrogen peroxide decreased a cell survival but neither induced any significant changes in succinate dehydrogenase activity which means that this effect is characteristic only of alpha-tocopheryl succinate and seems to be an important event triggering the apoptotic response by it. It was supposed that alpha-tocopheryl succinate might appear as a pseudosubstrate for mitochondrial succinate dehydrogenase leading to its inhibition, dysfunction of the mitochondrial electron transport chain, generation of reactive oxygen species and iduction of apoptosis.     

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.     

A possible mechanism for the increased oxidation of choline after chronic ethanol ingestion

An attempt has been made to determine the location of the site at which the metabolism of ethanol interacts with that of choline to produce an increase in the oxidation of choline. The first enzyme in the oxidation pathway for choline, choline dehydrogenase, was assayed using a newly developed spectro-photometric assay and freshly isolated intact rat liver mitochondria. No changes were observed in either the ‘apparent’ V or the ‘apparent’ K_m values of choline dehydrogenase for choline after ethanol ingestion. However, when the choline oxidase system was assayed, a 28% decrease in ‘apparent’ K_m for choline and a 53% increase in ‘apparent’ V was observed. The effects of ATP on choline oxidase were studied further, and a 29.4% decrease was observed in mitochondrial ATP levels from freshly isolated mitochondria from the ethanoltreated rats. In vitro aging of mitochondria further decreased the level of ATP, and the rate of decrease was considerably faster during the first hour in the mitochondria from the ethanol-treated animals. The decreases in ATP from both control and experimental mitochondria were accompanied by increases in choline oxidase activity. The initial decrease in ATP was correlated with an increase in mitochondrial ATPase activity which may be related to an increase in mitochondrial Mg²⁺. Because chronic ethanol ingestion has resulted in decreased oxidation rates of succinate and β-hydroxybutyrate while at the same time increasing the oxidation rates of choline, the studies reported here suggest that the effect of chronic ethanol ingestion is primarily on a step that is unique to choline and which probably exists prior to the electron transport chain.     

A possible mechanism for the increased oxidation of choline after chronic ethanol ingestion.

An attempt has been made to determine the location of the site at which the metabolism of ethanol interacts with that of choline to produce an increase in the oxidation of choline. The first enzyme in the oxidation pathway for choline, choline dehydrogenase, was assayed using a newly developed spectrophotometric assay and freshly isolated intact rat liver mitochondria. No changes were observed in either 'apparent' V or the 'apparent' Km values of choline dehydrogenase for choline after ethanol ingestion. However, when the choline oxidase system was assayed, a 28% decrease in 'apparent' Km for choline and a 53% increase in 'apparent' V was observed. The effects of ATP on choline oxidase were studied further, and a 29.4% decrease was observed in mitochondrial ATP levels from freshly isolated mitochondria from the ethanol-treated rats. In vitro aging of mitochondria further decreased the level of ATP, and the rate of decrease was considerably faster during the first hour in the mitochondria from the ethanol-treated animals. The decreases in ATP from both control and experimental mitochondria were accompanied by increases in choline oxidase activity. The initial decrease in ATP was correlated with an increase in mitochondrial ATPase activity which may be related to an increase in mitochondria Mg2+. Because chronic ethanol ingestion has resulted in decreased oxidation rates of succinate and beta-hydroxybutyrate while at the same time increasing the oxidation rates of choline, the studies reported here suggest that the effect of chronic ethanol ingestion is primarily on a step that is unique to choline and which probably exists prior to the electron transport chain.     

Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate

3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.     

Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets

Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.     

Regulation of succinate oxidation by NAD+ in mitochondria purified from potato tubers

NAD⁺ supplied to purified Solanum tuberosum mitochondria caused progressive inhibition of succinate oxidation in State 3. This inhibition was especially pronounced at alkaline pH and at low succinate concentrations. Glutamate counteracted the inhibition. NAD⁺ promoted oxaloacetate accumulation in State 3; supplied oxaloacetate inhibited O₂ uptake in the presence of succinate much more severely in State 3 than in State 4. NAD reduction linked to succinate oxidation by ATP-dependent reverse electron transport was likewise inhibited by oxaloacetate. We conclude that NAD⁺-induced inhibition of succinate oxidation is due to an inhibition of succinate dehydrogenase resulting from increased accumulation of oxaloacetate generated from malate oxidation via malate dehydrogenase. The results are discussed in the context of the known regulatory characteristics of plant succinate dehydrogenase.     

Relation of phospholipase D activity to the decay of succinate dehydrogenase and of covalently bound flavin in yeast cells undergoing glucose repression

The disappearance of succinate dehydrogenase activity and of protein-bound histidyl flavin were studied in aerobic yeast cells incubated with high glucose concentrations. The decay of succinate dehydrogenase activity, covalently bound flavin, and of respiration is prevented by cycloheximide but not by chloramphenicol. During this decay there is a large increase in mitochondrial phospholipase D activity; the appearance of this enzyme is also prevented by cycloheximide. It seems possible, therefore, that the formation of phospholipase D may be important in triggering the disappearance of covalently bound flavin, succinate dehydrogenase, and of other mitochondrial enzymes during glucose repression of aerobic yeast cells.     

New insights into the regulation of plant succinate dehydrogenase. On the role of the protonmotive force

Regulation of succinate dehydrogenase was investigated using tightly coupled potato tuber mitochondria in a novel fashion by simultaneously measuring the oxygen uptake rate and the ubiquinone (Q) reduction level. We found that the activation level of the enzyme is unambiguously reflected by the kinetic dependence of the succinate oxidation rate upon the Q-redox poise. Kinetic results indicated that succinate dehydrogenase is activated by both ATP (K(1/2) approximately 3 microm) and ADP. The carboxyatractyloside insensitivity of these stimulatory effects indicated that they occur at the cytoplasmic side of the mitochondrial inner membrane. Importantly, our novel approach revealed that the enzyme is also activated by oligomycin (K(1/2) approximately 16 nm). Time-resolved kinetic measurements of succinate dehydrogenase activation by succinate furthermore revealed that the activity of the enzyme is negatively affected by potassium. The succinate-induced activation (+/-K(+)) is prevented by the presence of an uncoupler. Together these results demonstrate that in vitro activity of succinate dehydrogenase is modulated by the protonmotive force. We speculate that the widely recognized activation of the enzyme by adenine nucleotides in plants is mediated in this manner. A mechanism that could account for such regulation is suggested and ramifications for its in vivo relevance are discussed.     

T-2 toxin inhibits mitochondrial function in yeast

T-2 toxin inhibits oxygen consumption of whole cells and purified mitochondria of Saccharomyces cerevisiae. Inhibition of mitochondrial respiration is not relieved by 2, 4-dinitrophenol, indicating that T-2 toxin inhibits mitochondrial function at the level of the electron transport chain. T-2 toxin inhibition of state 3 respiration (with succinate) is overcome by N, N, N', N'-tetramethyl-p-phenylenediamine, indicating inhibition of site II of the electron transport chain. T-2 toxin inhibits mitochondrial succinate dehydrogenase activity and increases mitochondrial NADH dehydrogenase activity.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Alterations in membrane function in an Escherichia coli mutant tolerant to colicins Ia and Ib.

An Escherichia coli mutant (tolI) previously shown to be tolerant to colicins Ia and Ib is defective in several functions of the bacterial cytoplasmic membrane. When compared with its parental strain, X36, whole cells of tolI show reduced rates of respiration with succinate, malate, or lactate as the substrate but near-normal rates with glucose or glycerol. Cell membrane preparations prepared from tolI cells exhibit reduced succinate and D-lactate oxidase activity but elevated levels of reduced-form nicotinamide adenine dinucleotide (NADH) oxidase. tolI cells have reduced levels of succinate and D-lactate dehydrogenase but normal levels of NADH dehydrogenase. Glycerol-grown tolI cells and membrane vesicles prepared from such cells are defective in the active transport of several amino acids and thiomethyl-beta-D-galactoside; however, they accumulate higher levels of alpha-methylglucoside when compared with X36 whole cells or vesicles. Although tolI cells adsorb less colicin Ia at high colicin concentrations than do X36 cells, it is shown that the adsorption of an Ia molecule to tolI cells has a lower probability of eliciting cell death than does Ia adsorption to strain X36 cells. It is concluded that a single mutation can lead to an alteration in several aspects of cytoplasmic membrane function and colicin I sensitivity.     

Mechanism of the Increase in Succinate Dehydrogenase Activity in Wounded Sweet Potato Root Tissue

The large subunit (mol wt: 65,000) of sweet potato succinatedehydrogenase was isolated by SDS-polyacrylamide gel electrophoresisof a succinate dehydrogenase preparation, which had been partiallypurified from root mitochondria by solubilizing the enzyme withEmulgen 810, DEAE-cellulose column chromatography, and polyacrylamidegel electrophoresis. Antibody to the purified large subunitwas produced in a rabbit, and the antiserum obtained was judgedto be specific to the large subunit based on the results ofdouble immunodiffusion tests and immunoelectrophoresis. Rocketimmunoelectrophoresis with the antiserum showed that the increasein succinate dehydrogenase activity during the ageing of sliced,sweet potato root tissue was due to an increase in the amountof enzyme protein. Both the increases in the activity of succinatedehydrogenase and in the amount of the large subunit proteinwere inhibited by cycloheximide or chloramphenicol. We proposethat synthesis of the large subunit of succinate dehydrogenaseon cytoplasmic ribosomes is controlled by a mitochondrial translationproduct(s). ¹ This work was supported in part by a research fund from TheIshida Foundation, Nagoya, Japan. (Received November 28, 1981; Accepted February 17, 1982)     

Presence in Dry Pea Cotyledons of Soluble Succinate Dehydrogenase That Is Assembled into the Mitochondrial Inner Membrane during Seed Imbibition

SUCCINATE DEHYDROGENASE (SUCCINATE: phenazine methosulfate oxidoreductase, EC 1.3.99.1) activity in crude mitochondrial fraction from pea (var. Alaska) cotyledons increased during seed imbibition to reach a maximum after about 12 hours. The increase was not inhibited by either cycloheximide or d(-)threo-chloramphenicol. The postmicrosomal fraction from dry cotyledons, but not that from fully imbibed ones, contained a soluble form of succinate dehydrogenase. The soluble enzyme was partially purified by ammonium sulfate fractionation and diethylaminoethyl-cellulose and Sepharose 6B column chromatography. The enzyme showed no succinate-coenzyme Q oxidoreductase activity and had a molecular mass of about 100,000 daltons. The soluble enzyme seemed to differ only slightly from succinate dehydrogenase solubilized from the mitochondrial inner membrane from fully imbibed cotyledons by a detergent. It is proposed that the soluble succinate dehydrogenase is associated with an inert mitochondrial inner membrane in dry cotyledons to form an active one during seed imbibition.     

Association between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase

The kinetic parameters of the individual reaction of pig heart alpha-ketoglutarate dehydrogenase complex, succinate thiokinase and the alpha-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system were studied. The KCoAm of alpha-ketoglutarate dehydrogenase complex and the K-succinyl CoAm of succinate thiokinase decreased in the coupled system when compared to those of the individual enzyme reactions. This phenomenon can be explained by the interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. By means of poly(ethylene glycol) precipitation, ultracentrifugation and gel chromatography we were able to detect a physical interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. Of the seven investigated proteins only succinate thiokinase showed association with alpha-ketoglutarate dehydrogenase complex. On the other hand, succinate thiokinase did not associate with other high molecular weight mitochondrial enzymes such as pyruvate dehydrogenase complex and glutamate dehydrogenase. On this basis, the interaction between succinate thiokinase and alpha-ketoglutarate dehydrogenase complex was assumed to be specific. These in vitro data raise the possibility that a portion of the citric acid cycle enzymes exists as a large multienzyme complex in the mitochondrial matrix.     

Soluble succinate dehydrogenase from the halophilic archaebacterium, Halobacterium halobium

Succinate dehydrogenase activity was found in both the cytoplasmic and the membrane fractions from disrupted Halobacterium halobium cells. The cytoplasmic enzyme was found to be soluble in aqueous media and had an apparent molecular weight of 90,000. The enzyme activity of the cytoplasmic succinate dehydrogenase was salt dependent, with preference for KCl over KNO3. The Km values for succinate of the soluble and the membrane-bound succinate dehydrogenases from H. halobium were 2.3 +/- 0.3 and 0.7 +/- 0.1 mM, respectively. The soluble succinate dehydrogenase was obtained from two different strains of H. halobium and was obtained independently of the method used to disrupt the bacteria. Thus, the archaebacterium, H. halobium, contains a succinate dehydrogenase which differs from the succinate dehydrogenase in most eucaryotic and eubacterial cells, where the enzyme is tightly membrane-bound.