Nature of the activation of succinate dehydrogenase by various effectors and in hypobaria and hypoxia

1. Hepatic mitochondrial succinate dehydrogenase (succinate:(acceptor)oxidoreductase, EC 1.3.99.1) was activated by preincubation of mitochondria with four diverse classes of compounds, the dicarboxylic acids, nitrophenols, quinols (and ubiquinols) and pyrophosphates. Of the various compounds tested malonate, oxaloacetate and pyrophosphate, well-known competitive inhibitors of the enzyme, and also hydroquinone and ubiquinols were effective even at low concentrations and showed maximal stimulation in 2 min.2. Activation of succinate dehydrogenase by ubiquinol-9 and ubiquinol-10 was comparable to succinate activation in fresh mitochondria, and was much higher in the aged samples.3. Preincubation of mitochondria with succinate, 2,4-dinitrophenol, pyrophosphate and ATP also stimulated the succinate-2,2′,5,5′-tetraphenyl-3,3′-(4,4′-biphenylene) ditetrazolium chloride (NT) reductase activity, whereas malonate, hydroquinone and ubiquinol-9 were ineffective. A differential activation of the flavoprotein by the oxidized and reduced forms of ubiquinone-9 was observed, the former stimulating the reduction of NT and the latter of phenazine methosulphate-2,6-dichlorophenolindophenol.4. Repeated washing of the activated mitochondrial samples with the sucrose homogenizing medium, partially reversed the activation by effectors other than succinate. Further washing of the activated preparations after a second preincubation with succinate reverted the enzyme activity to the basal level in the case of malonate, ATP and pyrophosphate but not that of hydroquinone and ubiquinol-9.5. Increase in the activity of hepatic mitochondrial succinate dehydrogenase, but not of succinate-NT reductase, known to occur in rats exposed to hypobaria was also observed in hypoxia indicating that it is an effect of lowered O₂ tension. The enzyme activity in these “partially activated” preparations was stable to washing with the sucrose homogenizing medium and could be fully activated to the same level as in the controls showing thereby the qualitative nature of the change. On washing these succinate-activated preparations further with the medium, the “hypobaric activation” was not reversed to the basal level, whereas the “hypoxic activation” was reversed. These results suggest that the effectors responsible for the activation of succinate dehydrogenase under hypobaric and hypoxic conditions are probably different; the former may be of the ubiquinol type and the latter of the malonate type.     

Activation of succinate dehydrogenase by bicarbonate

Succinate dehydrogenase (SD) of mitochondria from rat liver or kidney is to a large extent in the active form as isolated, whereas SD activity of heart and skeletal muscle mitochondria can be activated as much as ten-fold over the basal activity when isolated. Incubation of the latter at 37° with bicarbonate resulted in more extensive activation of SD than when succinate was the activator. Activation by bicarbonate was not readily reversed by washing unless succinate was also present. The data indicate that bicarbonate and succinate share the same site for activation of SD. A physiological role for bicarbonate in regulation of SD activity in muscle is suggested.     

Inhibition of 2-oxoglutarate oxidation in plant mitochondria by pyruvate

Oxidation of 2-oxoglutarate (in the presence of malonate) by mitochondria isolated from turnip, pea leaf and cauliflower tissue was dramatically inhibited by micromolar concentrations of pyruvate. Pyruvate, however, had little or no effect on 2-OG oxidation when carried out in the absence of malonate. The inhibition was reversed by alpha-cyano-4-hydroxycinnamic acid, indicating pyruvate uptake into the matrix was required for the inhibitory effect. In contrast, pyruvate had no effect on 2-oxoglutarate oxidation by mitochondria isolated from rat heart. The possible significance of the effect in terms of the control of 2-oxoglutarate dehydrogenase activity during the operation of a malate/aspartate shuttle in plant mitochondria is discussed.     

A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver.

The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.     

The induction of the beta state of the comuton regulation of mitochondrial oxidative phosphorylation by 2,4-DNP and malonate

The effect of 2,4-DNP and malonate on tissue-specific uncoupling of oxidative phosphorylation (OP) of rat liver and kidney mitochondria by homologous comutons has been studied. The addition of 2,4-DNP in the presence of comuton induced beta state of comuton regulation. Transfer of liver mitochondria from alpha to beta state also resulted from partial inhibition of succinate dehydrogenase activity of addition of 0.25-0.35 mM malonate. This suggests that the transfer to beta state may be caused by de-energization of mitochondria.     

Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: activity, properties, and subcellular localization

The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.     

Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium

Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue protectant and Tetranitro BT as final electron acceptor. Control media lacked either substrate or substrate and coenzyme. Controls were also performed by adding malonate (a competitive inhibitor of succinate dehydrogenase), pyruvate (a non-competitive inhibitor of lactate dehydrogenase), oxalate (a competitive inhibitor of lactate dehydrogenase) or N-ethylmaleimide (a blocker of SH groups). A specific malonate-sensitive linear test minus control response for succinate dehydrogenase activity was obtained in liver (1.6 mumol H2cm-3 min-1) and tracheal epithelium (0.8 mumol H2cm-3 min-1) but not in heart muscle. All variations in the incubation conditions tested did not result in a linear test minus control response in the latter tissue. Because the reaction was sensitive to malonate, it was concluded that the initial reaction rate was the specific rate of succinate dehydrogenase activity in heart muscle (9.1 mumol H2 cm-3 min-1). Test minus control reactions for lactate dehydrogenase activity were distinctly non-linear for all tissues tested. This appeared to be due to product inhibition by pyruvate generated during the reaction and therefore it was concluded that the appropriate control reaction was the test reaction in the presence of 20 mM pyruvate. The initial rate of the test minus this control was the true rate of lactate dehydrogenase activity. The lactate dehydrogenase activity thus found in liver parenchyma was 5.0 mumol of H2 generated per cm3 liver tissue per min.     

Tight binding of oxaloacetate to succinate dehydrogenase

[¹⁴C]Oxaloacetate forms a stable complex with succinate dehydrogenase which withstands repeated Sephadex filtration. Oxidized glutathione, 2-thenoyltrifluoroacetone, KCN and ageing at +4° at neutral pH do not prevent the enzyme to bind oxaloacetate. The binding is prevented by succinate or malonate but the complex, once formed, can not be split by these compounds, although the enzyme activity can be restored; the reconstitutive property of succinate dehydrogenase is, however, irreversibly lost. Bound oxaloacetate does not exchange with added oxaloacetate, but can be released by perchloric acid. Sonic particles of beef heart mitochondria can also bind oxaloacetate. However, this complex can be split by succinate or malonate.     

Localization of PTP-1B, SHP-2, and Src exclusively in rat brain mitochondria and functional consequences

An immunodetection study of protein tyrosine phosphatase 1B (PTP-1B), SHP-2, and Src in isolated mitochondria from different rat tissues (brain, muscle, heart, liver, and kidney) revealed their exclusive localization in the brain. Given this result, we sought whether mitochondria respond to ATP and to the general tyrosine phosphatase inhibitor orthovanadate and found little or no change in the tyrosine phosphorylation profile of mitochondria from muscle, heart, liver, and kidney. In contrast, ATP induced an enhancement in the tyrosine-phosphorylated protein profile of brain mitochondria, which was further greatly enhanced with orthovanadate and which disappeared when Src was inhibited with two inhibitors: PP2 and PP1. Importantly, we found that in brain mitochondria, ATP addition induced Src autophosphorylation at Tyr-416 in its catalytic site, leading to its activation, whereas the regulatory Tyr-527 site remained unphosphorylated. Functional implications were addressed by measurements of the enzymatic activity of each of the oxidative phosphorylation complexes in brain mitochondria in the presence of ATP. We found an increase in complex I, III, and IV activity and a decrease in complex V activity, partially reversed by Src inhibition, demonstrating that the complexes are Src substrates. These results complemented and reinforced our initial study showing that respiration of brain mitochondria was partially dependent on tyrosine phosphorylation. Therefore, the present data suggest a possible control point in the regulation of respiration by tyrosine phosphorylation of the complexes mediated by Src auto-activation.     

Modification of Brain Mitochondrial Enzymes by the Oxygen Analogue of Ronnel at Various Stages of Development and Aging

This paper describes the effect of the organophosphorus compound, the oxygen analogue of ronnel (OAR), on the activity of some membrane-bound enzyme systems in the brain mitochondria of developing, young-adult, and old rats. Age-related changes were noted in the cholesterol-to-protein ratio, whereas the phospholipid content in mitochondria showed little change during development as well as aging. The results obtained suggest that development of brain succinate dehydrogenase may consist in a decrease of Km and increase of Vmax values. In aged rats an altered, perhaps inhibited form of the enzyme is produced. The oxygen analogue of ronnel caused a mixed-type inhibition of the succinate dehydrogenase derived from brains of 4-day-old, 16-day-old and 2-month-old animals. In the case of enzyme from the brain of 18-month-old rats, a typical competitive-type inhibition was observed. Mechanisms responsible for inhibition of the succinate: cytochrome c reductase from brains of developing animals are similar to those for succinate dehydrogenase. In aged rats (18 months old), however, a noncompetitive mechanism of inhibition of succinate: cytochrome c reductase was revealed. The experiments reported here provide evidence that lipid-soluble molecules of OAR may interact with membrane phospholipids and lead to modification of membrane architecture and also of enzyme kinetic behaviour. It may be also concluded, that the sensitivity of the enzyme systems studied to inhibition by OAR is an age-dependent phenomenon. Modification of membrane by development or aging alters the kinetics as well as the sensitivity of enzymes to inhibitors.     

Redistribution of the flux-control coefficients in mitochondrial oxidative phosphorylations in the course of brain edema

This work describes the control exerted by dicarboxylate carrier and succinate dehydrogenase activities on the oxidative phosphorylations in rabbit brain mitochondria as an edema develops. Vasogenic edema leads to an uncompetitive inhibition of succinate dehydrogenase activity and to a large decrease of oxidative phosphorylations linked to succinate utilisation. Naftidrofuryl treatment in vivo restores both a high succinate dehydrogenase activity and a normal respiratory rate. In order to quantify the control of oxidative phosphorylations by the succinate dehydrogenase step, we applied the control analysis (Kacser, H. and Burns, J.A. (1973) in Rate Control of Biological Processes (Davies, D.D., ed.), pp. 65-104, Cambridge University Press, London; Heinrich, R. and Rapoport, T.A. (1974) Eur. J. Biochem. 42, 89-95). By using two inhibitors, one (phenylsuccinate) acting only on the dicarboxylate carrier and another (malonate) acting on both the dicarboxylate carrier and the succinate dehydrogenase, a method was developed to calculate the control coefficients of these two steps. The main result is that in mitochondria isolated from normal tissue succinate dehydrogenase exerted no control, but in the course of edema this enzymatic step became a controlling one: a transition from zero to a high control coefficient (0.5) was observed from the onset of intracellular edema for the threshold value of water/dry-weight tissue of 4.6.     

Activation of succinate oxidase in the inner membrane of rat liver mitochondria

It is shown that the process of activation of succinate oxidase from inner membranes of the rat liver mitochondria by succinate and malonate is specific for the succinate dehydrogenase component of oxidase. These activation constants are comparable with those found by other authors in activation of succinate dehydrogenase and succinate oxidase from oxaloacetate-preincubated submitochondrial fragments of the bull heart. Probably, the 4-fold activation of succinate oxidase from inner membranes of the liver mitochondria reported in this paper depends on separation of endogenous oxaloacetate from the succinate dehydrogenase component of oxidase.     

The influence of storage temperature on the transition, activation enthalpy, and activity of enzymes associated with inner mitochondrial membranes

The effects of storage at low temperature on the transition in enzyme function, Tf*, and the Arrhenius activation energy, Ea, were determined for several enzymes associated with the inner membrane of rat liver mitochondria. The enzymes studied were succinate:cytochrome c reductase, cytochrome c oxidase, beta-hydroxybutyrate dehydrogenase, and oligomycin-sensitive, Mg2+-activated ATPase. For freshly isolated mitochondria the Tf*, for succinate:cytochrome c reductase and cytochrome c oxidase, occurred at approximately 23 degrees C and was coincident with a transition in structure, Ts*, determined as the change in temperature coefficient of motion for a spin label intercalated with the membrane lipids. This suggest that the change in thermal response of the membrane-associated enzymes is related to a change in molecular ordering of the membrane lipids. When mitochondria were stored at -12 degrees C, the specific activities of succinate:cytochrome c reductase and cytochrome c oxidase decreased. Concomitant with these changes the Ea, above Tf*, increased. After 100 days storage at -12 degrees C, Ea above Tf* approached the value for Ea below Tf* such that the transition in thermal response could no longer be detected. In contrast, for mitochondria stored at -196 degrees C, although the specific activity declined over the 100 days storage, no changes in either Ea or Tf* were evident. The results indicate a need for caution in evaluating comparative studies of Tf and Ea, for membrane-associated enzymes, using mitochondria which have been frozen and stored.     

Activation of liver succinate dehydrogenase in rats exposed to hypobaric conditions

1. On brief exposure of rats to hypobaric conditions, the activity of hepatic mitochondrial succinate dehydrogenase was raised from the basal state to a ;partially activated state'. This was further raised to ;fully activated state' by preincubation of mitochondria with succinate, as was the activity in mitochondria from normal rats. 2. On washing mitochondria with the homogenizing sucrose medium the activity excess obtained on preincubation with succinate was lost in mitochondria from both normal and treated rats. 3. The enzyme in the ;partially activated state' from animals exposed to hypobaric conditions was stable to the washing procedure but was labilized and reverted to a low basal state of activity on freezing and thawing of the isolated mitochondria. 4. The results suggest that activation of succinate dehydrogenase under hypobaric conditions represents a conformational change leading to a stable, partially activated, form of the enzyme system: this is the first evidence of physiological modulation of this rate-limiting step in the control of the rate of oxidation of succinate.     

Partial purification and characterization of citrate synthase from Dictyostelium discoideum.

The effect of thyroidectomy on oxidative metabolism of rat liver, kidney, and brain mitochondria has been examined. The respiration in liver, kidney, and brain mitochondria was affected differentially after thyroidectomy, the common effect in all the tissues being the impairment in state 3 as well as state 4 rates of succinate oxidation. Thyroidectomy did not have any effect on $\text{ADP}\text{O}$ ratios; however, compared to normal, respiratory control indexes were, in general, somewhat higher. Thyroidectomy also did not alter total ATPase activity of liver, kidney, and brain mitochondria, although the basal ATPase activity had decreased significantly under these conditions. The cytochrome content of the mitochondria also showed tissue-specific changes after thyroidectomy; however, no significant changes in the absorption characteristics of the cytochromes were seen. The succinate and glutamate dehydrogenase activities of mitochondria from liver, kidney, and brain were not affected by thyroidectomy, thereby ruling out the possibility that the decrease in substrate oxidation may be due to alterations in the primary dehydrogenase levels. It is concluded that thyroid hormone(s) may have a tissue-specific role in regulating the metabolic functions of mitochondria.     

Dissociation constants of succinate dehydrogenase complexes with succinate, fumarate and malonate

The rates of the oxidized (Eox) and reduced (Ered) (by NAD . H through the ubiquinone pool) succinate dehydrogenase inhibition by N-ethyl-maleimide are equal and obey pseudo-first order kinetics. The protection of the enzyme against irreversible alkylation was used to quantitate the dissociation constants for Eox and Ered complexes with fumarate, succinate and malonate under conditions when no intramolecular redox reactions might occur. the membrane-bound succinate dehydrogenase catalyzes the succinate : phenazine-methosulphate reductase reaction in the presence of thenoyltrifluoroacetone by a Slater-Bonner mechanism. A comparison of the constants measured by the protection with those derived from the steady-state kinetics shows that succinate affinity for Eox is about 10 times higher than that for Ered; the reverse relations were found for fumarate, whereas the affinity for malonate only slightly depends on the redox state of the enzyme. The data obtained suggest that the dicarboxylate binding at the active site induces changes in the enzyme redox potential. The surface charge does not contribute significantly to the energy of the dicarboxylate binding to the active site of the membrane-bound enzyme.     

Enzyme, electron microscopic and polarographic characteristics of isolated rat brain mitochondria. III. Quantitative assessment of their distribution in fractions of the homogenate]

Activities of succinate dehydrogenase, succinate- and NAD-H-cytochrome c--reductases, and cytochrome c--oxidase was compared in 1 g tissue homogenate and homogenate fractions made from 1 g brain tissue using various solutions. Fractionation resulted in the increased activities of NADH- and succinate cytochrome reductases, and in the loss of succinate dehydrogenase activity, cytochrome oxidase was less influenced. These phenomena are regarded as signs of the interrelation between mitochondria and other constituents of brain cell within homogenates. Maximal quantity of mitochondria isolated from homogenates is no more than 20% of all the mitochondrial homogenates (according enzyme data). The electronogram of the brain mitochondrial preparation isolated in the Krebs--Ringer solution without glucose pointed out to a high homogeneity of mitochondria in the residue.     

The role of succinate in the respiratory chain of Trypanosoma brucei procyclic trypomastigotes.

Trypanosoma brucei procyclic trypomastigotes were made permeable by using digitonin (0-70 micrograms/mg of protein). This procedure allowed exposure of coupled mitochondria to different substrates. Only succinate and glycerol phosphate (but not NADH-dependent substrates) were capable of stimulating oxygen consumption. Fluorescence studies on intact cells indicated that addition of succinate stimulates NAD(P)H oxidation, contrary to what happens in mammalian mitochondria. Addition of malonate, an inhibitor of succinate dehydrogenase, stimulated NAD(P)H reduction. Malonate also inhibited intact-cell respiration and motility, both of which were restored by further addition of succinate. Experiments carried out with isolated mitochondrial membranes showed that, although the electron transfer from succinate to cytochrome c was inhibitable by antimycin, NADH-cytochrome c reductase was antimycin-insensitive. We postulate that the NADH-ubiquinone segment of the respiratory chain is replaced by NADH-fumarate reductase, which reoxidizes the mitochondrial NADH and in turn generates succinate for the respiratory chain. This hypothesis is further supported by the inhibitory effect on cell growth and respiration of 3-methoxyphenylacetic acid, an inhibitor of the NADH-fumarate reductase of T. brucei.     

Effect of Free Malonate on the Utilization of Glutamate by Rat Brain Mitochondria

Malonate is an effective inhibitor of succinate dehydrogenase in preparations from brain and other organs. This property was reexamined in isolated rat brain mitochondria during incubation with L-glutamate. The biosynthesis of aspartate was determined by a standard spectrofluorometric method and a radiometric technique. The latter was suitable for aspartate assay after very brief incubations of mitochondria with glutamate. At a concentration of 1 mM or higher, malonate totally inhibited aspartate biosynthesis. At 0.2 mM, the inhibitory effect was still present. It is thus possible that the natural concentration of free malonate in adult rat brain of 192 nmol/g wet weight exerts an effect on citric acid cycle reactions in vivo. The inhibition of glutamate utilization by malonate was readily overcome by the addition of malate which provided oxaloacetate for the transamination of glutamate. The reaction was accompanied by the accumulation of 2-oxoglutarate. The metabolism of glutamate was also blocked by inclusion of arsenite and gamma-vinyl-gamma-aminobutyric acid but again added malate allowed transamination to resume. When arsenite and gamma-vinyl-gamma-aminobutyric acid were present, the role of malonate as an inhibitor of malate entry into the mitochondrial interior could be determined without considering the inhibition of succinate dehydrogenase. The apparent Km and Vmax values for uninhibited malate entry were 0.01 mM and 100 nmol/mg protein/min, respectively. Malonate was a competitive inhibitor of malate transport (Ki = 0.75 mM).     

Androgenic control of hepatic mitochondrial metabolism in an apoda, Gegenophis carnosus (Beddome).

In vivo administration of testosterone significantly stimulated the activities of cytochrome oxidase, alpha-glycerophosphate dehydrogenase (alpha-GPDH), succinate dehydrogenase (SDH) and adenosine triphosphatase (Mg2+ ATPase), in mitochondria isolated from the liver of G. carnosus. Administration of dehydroepiandrosterone and androstenedione while significantly stimulated the activities of cytochrome oxidase and alpha-GPDH, did not change that of SDH and Mg2+ ATPase. Simultaneous injections of testosterone and actinomycin D or chloramphenicol prevented the testosterone-stimulated activities of all the oxidative enzymes studied. The results clearly document the important stimulatory role of androgens in the regulation of hepatic mitochondrial metabolism in G. carnosus.