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Preparation of 1-acyl-2-succinyl glycero-3-phosphorylcholine and evidence against its involvement in succinate dehydrogenase action.

1-Acyl-2-succinyl glycero-3-phosphorylcholine (GPC) was synthesized and its properties described. Although 1-acyl-2-succinyl GPC is a good substrate for succinate dehydrogenase, experiments on the incorporation of [2,3-14C] succinate into mitochondrial lipids gave no evidence to indicate that it is an intermediate in the enzymic oxidation of succinate to fumerate, as has been suggested earlier.

Animals↗

Succinate transport inhibition by valproate in rat renal mitochondria.

Sodium valproate is an antiepileptic drug which may have side effects on different organs. Its mechanism of action, as yet unclear, may involve an effect on membranes. One possibility, an effect on mitochondrial membranes via an inhibition of oxidative phosphorylation, has been studied here by observing the transmembranal transport of a respiratory substrate, succinate, in rat kidney mitochondria incubated with valproate, or from rats injected with valproate. Succinate transport was inhibited in both conditions, which suggests that the effect was probably due to a direct effect of valproate rather than to an action of a valproate metabolite. For the valproate-incubated mitochondria, inhibition, described by a bell-shaped curve, started at a valproate concentration of 10(-7) M and was maximum at valproate 10(-5)M. Valproate's effect on mitochondrial transmembranal succinate transport can be compared to other evidence for membranal actions of valproate, actions which may clarify certain therapeutic or toxic properties of this drug.

Animals↗

Developmental changes in succinate dehydrogenase activity in muscle fibers from normal and dystrophic mice.

The question of whether or not the development of dystrophic muscles is similar to that of normal muscles, prior to the manifestations of the symptoms of the disease, is investigated here. The developmental change in the activity of succinate dehydrogenase was therefore measured in individual fibers of prospectively dystrophic muscles from 10- to 28-day-old mice (strain C57Bl/6J dy2j) and compared with that of muscles from normal mice of the same age. It was found that up to 10 days of age, muscle fibers from normal and prospective dystrophic animals had low succinate dehydrogenase activities, and were all more or less uniform. Thereafter in the normal muscle the overall activity of the enzyme increased and the fibers became more heterogeneous with age. By 21 days the extensor digitorum longus muscle resembled that of the adult. At that time, fibers from prospectively dystrophic muscles had lower succinate dehydrogenase activities and were more homogeneous. Thus fibers from prospectively dystrophic muscles fail to achieve their adult characteristics by 21 days. On the basis of these results, it is suggested that muscle maturation is retarded in dystrophic animals.

Animals↗

Mesocestoides corti: a GC/MS study of the effects of oxaloacetate and iodoacetate on the isotope ratios in succinate and lactate excreted in the presence of NaH13CO3.

The current study was undertaken to gain further insight into the carbohydrate metabolism of the proliferating larvae of Mesocestoides corti by studying the effect of the metabolic inhibitors oxaloacetate and iodoacetate on the incorporation of label from NaH13CO3 into the excretory products lactate and succinate. These products were converted to their methyl derivatives and analyzed by gas chromatography/mass spectrometry (GC/MS) to determine the isotope ratios. The percentage of [1-13C]lactate, [1-13C]succinate, and [1,4-13C2]succinate decreased in the presence of oxaloacetate; iodoacetate did not significantly influence the relative amount of the former, but markedly increased percentages of the latter two. The data provide information on the relative rates of the pathways and steps within them and the influence of the inhibitors on these pathways.

Animals↗

Occurrence of two distinct succinate thiokinases in animal tissues.

Although succinate thiokinase from mammalian sources has hitherto been described as showing substrate specificity for guanine nucleotide, a range of mammalian tissues has here been found to display succinate thiokinase activity with both guanine and adenine nucleotides as substrates. Evidence is presented for the existence of two distinct succinate thiokinases and this is confirmed by their separation by affinity chromatography. Each enzyme is specific for one nucleotide and is inhibited by the non-substrate nucleotide. The physiological roles of the two enzymes is yet to be established.

Adenosine Diphosphate↗

Processing of Bacillus subtilis succinate dehydrogenase and cytochrome b-558 polypeptides. Lack of covalently bound flavin in the Bacillus enzyme expressed in Escherichia coli.

The DNA sequence of the Bacillus subtilis sdh operon coding for the two succinate dehydrogenase subunits and cytochrome b-558 (the membrane anchor protein) has recently been established. We have now determined the extent of N-terminal processing of each polypeptide by radiosequence analysis. At the same time, direct evidence for the correctness of the predicted reading frames has been obtained. The cytochrome showed a ragged N-terminus, with forms lacking one residue, and is inserted across the membrane without an N-terminal leader-peptide. Covalently bound flavin was not detectable in B. subtilis succinate dehydrogenase expressed in Escherichia coli despite normal N-terminal processing of the apoprotein. This provides an explanation to why the succinate dehydrogenase synthesized in E. coli is not functional and demonstrates that host-specific factors regulate the coenzyme attachment.

Amino Acid Sequence↗

Identification of the axial heme ligands of cytochrome b556 in succinate: ubiquinone oxidoreductase from Escherichia coli.

Electron paramagnetic resonance (EPR) and near-infrared magnetic circular dichroism (MCD) have been used to identify the ligands to the cytochrome b556 component of succinate: ubiquinone oxidoreductase (succinate dehydrogenase) from Escherichia coli. The 'highly axial low spin' (HALS) EPR spectrum suggests bis(histidine) ligation of the heme with the histidines in a staggered configuration. The near-infrared MCD spectrum exhibits a low energy maximum at 1600 nm which is also clearly indicative of bis(histidine) ligation of the heme iron. The data unambiguously demonstrate that the heme b556 is ligated to E. coli succinate dehydrogenase via two histidines.

Binding Sites↗

Distribution of phosphate-activated glutaminase, succinic dehydrogenase, pyruvate dehydrogenase and gamma-glutamyl transpeptidase in post-mortem brain from Huntington's disease and agonal cases.

The activity of phosphate-activated glutaminase was reduced throughout the brain of cases with longstanding illnesses (agonal controls) compared to cases dying suddenly. The reduction was less marked in cortical than sub-cortical areas, with the caudate nucleus occupying an intermediate position. In control brains succinic dehydrogenase and pyruvate dehydrogenase were little affected by the ante-mortem clinical state. Of 9 brain areas studied, only the caudate nucleus showed a reduction of phosphate-activated glutaminase and succinic dehydrogenase in Huntington's disease greater than in agonal controls. The levels of succinic dehydrogenase and pyruvate dehydrogenase were highly correlated in frontal cortex and in caudate nucleus of Huntington's disease and control brains. There was a significant reduction in pyruvate dehydrogenase mean activity and a significant increase in gamma-glutamyl transpeptidase mean activity in Huntington's disease caudate nucleus. The level of pyruvate dehydrogenase significantly decreased and the level of gamma-glutamyl transpeptidase significantly increased with increasing duration of illness, possibly due to a progressive loss of neurons and increase in the density of glia in Huntington's disease caudate nucleus.

Adult↗

Defect in succinate oxidation by isolated muscle mitochondria in a patient with symmetrical lesions in the basal ganglia.

A 3-year-old boy was referred for evaluation of psychomotor retardation. He had a waddling gait with proximal hypotonia and paresis. Computed tomography (CT scan) and magnetic resonance imaging (MRI) of the brain demonstrated symmetrical lesions in the basal ganglia suggesting bilateral necrosis. Lactate and pyruvate levels in blood and cerebrospinal fluid were persistently elevated. A biopsy of the quadriceps muscle showed normal light microscopic findings except for a slightly raised number of lipid droplets. Electron microscopy confirmed this and also showed a rather large number of subsarcolemmal mitochondria without crystalline inclusions. Biochemical studies showed a normal carnitine level and normal mitochondrial enzyme activities in muscle homogenate, including succinate-cytochrome c reductase. However, intact isolated mitochondria failed to oxidize succinate. An explanation for this paradoxical finding is a deficiency in that part of the coenzyme Q (CoQ) that is reduced by the succinate dehydrogenase complex. The differential diagnosis between Leigh's syndrome and infantile bilateral striatal necrosis (IBSN) is discussed. The role of neuroradiology in prompting complementary investigations is stressed.

Basal Ganglia Diseases↗

Age-dependent modifications of rat heart succinate dehydrogenase.

The activity of rat heart succinate dehydrogenase (SDH) increases 1.7-fold in old animals (Vitorica, J., Cano, J., Satrústegui, J. and Machado, A., Mech. Ageing Dev., 16 (1981) 105-116). This increase is due to an increase in enzyme protein itself because: (a) the activation state of the enzyme does not vary with age; and (b) the increase of activity is paralleled by an increase in immunoprecipitable SDH in old rat heart mitochondria. SDH from old rat heart mitochondria differs in a number of ways from that of young animals: (a) The km value for succinate increases with age. (b) The thermostability decreases, and the activation energy in the 20-40 degrees C interval is higher in old animals. (c) The breaking points of the Arrhenius plots of SDH are shifted to higher values. (d) Reactivity towards N-ethylmaleimide in succinate protected mitochondria decreases with age.

Aging↗

Differential expression of two succinate dehydrogenase subunit-B genes and a transition in energy metabolism during the development of the parasitic nematode Haemonchus contortus.

The carbohydrate metabolism of free-living and parasitic stages of the sheep nematode Haemonchus contortus was studied, and it was demonstrated that during development a switch occurred from Krebs-cycle activity towards a more fermentative metabolism. During this switch a transition might take place in complex II of the respiratory chain. In the free-living (L3) and early parasitic (XL3) stages, complex II catalyses the oxidation of succinate to fumarate via the Krebs cycle, whereas in adults complex II functions in the reverse reaction, the reduction of fumarate to succinate. L3 and XL3 were shown to already possess a large anaerobic capacity. They survived well in the absence of oxygen or in the presence of cyanide, which completely blocked respiration. Krebs-cycle activity, however, was only partially inhibited by cyanide; the XL3s in particular produced in the presence of cyanide large amounts of propanol, the production of which probably functions as an alternative electron sink. For further investigation of the observed metabolic switch, complex II of the respiratory chain, a key enzyme involved in this switch, was studied. The B subunit of complex II was cloned and sequenced. These clones all showed sequences similar to the B subunit of succinate dehydrogenase from other species, and included the amino-terminal signal sequence for importation into mitochondria. Two genes were identified, types 1 and 2, based on the DNA and amino acid sequences and on the lack of cross-reaction to each other when used as probes on Southern blots. On Northern blots, the two genes showed a different expression pattern during the development of the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Sequence comparison between the flavoprotein subunit of the fumarate reductase (complex II) of the anaerobic parasitic nematode, Ascaris suum and the succinate dehydrogenase of the aerobic, free-living nematode, Caenorhabditis elegans.

Complex II in adult mitochondria of the parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron-transport observed in these organelles. In the present study, cDNAs for the flavoprotein (Fp) subunits of complex II have been isolated, cloned and sequenced from both A. suum and the aerobic, free-living nematode, Caenorhabditis elegans. Additional sequence at the 3' end of the mRNAs was determined by the Rapid Amplification of cDNA Ends (RACE). Nucleotide sequence analysis of the A. suum cDNAs revealed a 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1935 nucleotides and a 3' untranslated region of 616 nucleotides including a poly (A) tail from a polyadenylation signal (AATAAA). The open reading frame encoded a 645 amino acid sequence, including a 30 amino acid mitochondrial presequence. The amino acid sequences for the Fp subunits from both organisms were very similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme a succinate dehydrogenase. The ascarid sequence was much less similar to the Escherichia coli fumarate reductase. The sensitivity of other Fp subunits to sulfhydryl reagents appears to reside in a cysteine immediately preceding a conserved arginine in the putative active site. In both nematode sequences, this cysteine is replaced by serine even though the succinate dehydrogenase activity of both enzymes is still sensitive to sulfhydryl inhibition. A cysteine six residues upstream of the serine may be involved in the sulfhydryl sensitivity of the nematode enzymes. Surprisingly, in contrast to succinate dehydrogenase activity, the fumarate reductase activity of the ascarid enzyme was not sensitive to sulfhydryl inhibition, suggesting that the mechanism of the two reactions involves separate catalytic processes.

Amino Acid Sequence↗

A double-blind, placebo-controlled, multicenter trial of lithium succinate ointment in the treatment of seborrheic dermatitis. Efalith Multicenter Trial Group.

BACKGROUND: Seborrheic dermatitis, an inflammatory dermatosis that principally affects sebaceous areas, may be related to the presence of the yeast Pityrosporum ovale. Topical therapy with corticosteroids, although in general effective, may be associated with several unwanted effects. The development of alternative non-steroid-based therapies may enable patients to avoid the use of topical steroids and help elucidate the pathogenesis of the disease. OBJECTIVE: This study aimed to assess the efficacy and safety of an ointment containing 8% lithium succinate in the treatment of seborrheic dermatitis. METHODS: The study consisted of a multicenter placebo-controlled clinical trial in 227 adult patients. RESULTS: Lithium succinate ointment was significantly more effective than placebo in treating all the symptoms of seborrheic dermatitis. No specific unwanted effects were associated with its use. Relapse was slow when treatment was stopped. CONCLUSION: The effectiveness of lithium succinate ointment in the treatment of seborrheic dermatitis adds to the evidence implicating P. ovale in the pathogenesis of that condition.

Adolescent↗

HNMR of succinate binding to aspartate transcarbamylase. A comparison of results in D2O and H2O.

The interaction of succinate with asparatete transcarbamylase from Escherichia coli has been studied by magnetic resonance relaxation measurements of the dicarboxylic acid methylene protons in H2O solutions. The pH and temperature dependence of the relaxation in the presence of either native asparte transcarbamylase or its catalytic subunit in H2O solutions is qualitatively very similar to the corresponding situation utilizing D2O as the solvent. From previous result of measurements in D2O[C.B. Beard and P.G. Schmidt, Biochemistry 12(1973)2255] a mechanism was proposed involving 2 protonated groups affecting succinate binding and titratable over the pH range 7-10. Quantitatively, fitting the data from H2O solutions to the mechanism yeilds values of the fitting parameters generally in good agreement with the D2O experiments. The main exceptions are the pKa values calculated for the two titratable groups. For these species the values obtained in the presence of the catalytic subunit are 6.7 and 7.8 in H2O solutions versus 7.3 and 8.6 in D2O solutions. In the presence of native enzyme the corresponding values are 6.8 and 8.3 in H2O versus 7.6 and 9.2 in D2O. These observed differences are consistent with differences in ionization constants of weak acids in D2O relative to H2O. The results imply that succinate interaction with the enzyme active site is similar in the two solvents.

Aspartate Carbamoyltransferase↗

Effect of succinate on aminopyrine N-demethylase activity.

Succinate stimulated the aminopyrine N-demethylase activity in the presence of rate-limiting concentrations of NADPH in the crude mitochondrial preparation, as well as in a reconstituted system containing microsomes and microsome-free mitochondria. The increase in enzyme activity was accompanied by a decrease of the apparent Km of NADPH. The stimulating effect of succinate was counteracted by malonate, rotenone and pentachlorophenol. No significant formaldehyde oxidation was exhibited by the crude mitochondrial preparation under the conditions of N-demethylase assay. Enhancement of the N-demethylase activity by succinate is supposed to be due to a mitochondrial-microsomal interaction which may play a role in the regulation of drug metabolism.

Aminopyrine N-Demethylase↗

Apparent ATP-linked succinate thiokinase activity and its relation to nucleoside diphosphate kinase in mitochondrial matrix preparations from rabbit.

The relative abilities of ATP and GTP to support succinyl-CoA synthesis by mitochondrial matrix fractions prepared from rabbit heart and liver mitoplasts were investigated. The activity supported by ATP in rabbit heart preparations was less than 15% of that obtained with GTP, while no ATP-supported activity was observed in rabbit liver preparations. However, the addition of 30 micromolar GDP to matrix fractions from either heart or liver stimulated the ATP-supported activity to 40% of that observed with GTP, and the further addition of bovine liver nucleoside diphosphate kinase in the presence of 8 microM added GDP increased the activity to near that observed with GTP. The specific activity of nucleoside diphosphate kinase assayed directly in mitochondrial matrix from heart was about 15% of the specific activity of ATP-supported succinate thiokinase induced upon adding GDP. Evidence for a complex between nucleoside diphosphate kinase and succinate thiokinase in mitochondrial matrix from rabbit heart was obtained by glycerol density gradient centrifugation. It is proposed that binding of nucleoside diphosphate kinase to succinate thiokinase activates the former enzyme, accounts for the ATP-supported succinyl-CoA synthetase activity observed, and is involved in the channeling of high energy phosphate from GTP produced in the Krebs cycle to the ATP pool.

Adenosine Triphosphate↗

Characterization of oxygen-resistant Chinese hamster ovary cells. III. Relative resistance of succinate and alpha-ketoglutarate dehydrogenases to hyperoxic inactivation.

We have recently shown that exposure of Chinese hamster ovary (CHO) cells to a toxic dose of normobaric hyperoxia (98% O2 for 3 days) caused a disturbance of cellular energy metabolism, that is, respiratory failure followed by stimulation of glycolytic activity and a net depletion of ATP. Respiratory failure was correlated with a selective inactivation of three mitochondrial enzymes, that is, partial inactivation of NADH dehydrogenase and virtually complete inactivation of succinate and alpha-ketoglutarate dehydrogenase activities (Schoonen et al., 1990). To elucidate the biochemical basis of resistance to hyperoxia in a previously described oxygen-resistant substrain of Chinese hamster ovary (CHO) cells, we compared the resistant cells with wildtype CHO cells with respect to several key parameters of oxidative and glycolytic energy metabolism. The two cell types were critically different in that the succinate and alpha-ketoglutarate dehydrogenases of the oxygen-resistant cells were relatively resistant to inactivation by hyperoxia, which may at least partly explain their enhanced capacity to respire and survive under hyperoxic conditions. Although the biochemical basis for the observed enzyme resistance to hyperoxic inactivation remains to be elucidated, the present data underscore the importance of succinate and alpha-ketoglutarate dehydrogenases as critical targets in hyperoxic killing of wildtype CHO cells.

Adenosine Triphosphate↗

Solid-state 27Al and cross-polarization 13C magic-angle spinning nuclear magnetic resonance characterisation of aluminas derived from basic aluminum succinate.

Solid-state 27Al and cross-polarization (CP) 13C magic-angle spinning nuclear magnetic resonance (MAS NMR) was used to characterise aluminas obtained by calcining basic aluminium succinate at different temperatures. In basic aluminium succinate, aluminium is in the tetrahedral coordination only. However, in both X-ray amorphous and gamma-aluminas aluminium is in the tetrahedral and octahedral coordination. The intensity of the aluminium peaks is at a maximum for samples calcined at 700 degrees C. 13C CP MAS NMR indicates the bonding of Al with carbon of the carboxylate group in basic aluminium succinate. Samples calcined at 400-870 degrees C do not give 13C spectra.

Aluminum↗