Biochemical effects of mild iron deficiency and cold acclimatization on rat skeletal muscle

Most of the previous studies on the effects of iron deficiency on skeletal muscle respiratory capacity and work performance have been investigated in severe or moderate iron-deficiency anemia. We report here that even in mild iron deficiency where the hemoglobin concentration was 10 g/dl and the iron stores in livers and spleen were not completely depleted, a marked reduction in succinate dehydrogenase was observed in skeletal muscles but not in heart. Similarly, cytochrome oxidase activities were reduced. Although no significant change in glycerophosphate dehydrogenase was detected in the iron-deficient rats, exposure to cold in this group greatly reduced this enzyme activity. As cold acclimatization accelerates marrow erythropoiesis (20) which in turn, demands more iron, it seems that in the iron-insufficient state, this iron demand for marrow activity may persist at the expense of the tissue iron pool, resulting in a marked reduction in glycerophosphate dehydrogenase activities. Since succinate dehydrogenase plays a significant role in the impairment of mitochondrial function and early fatigue of iron-deficient muscle (11), the present study shows that even in mild iron deficiency, some loss of muscle functions could result as succinate dehydrogenase activities were greatly reduced.     

Tricarboxylic acid cycle enzymes following thiamine deficiency

Thiamine (Vitamin B1) deficiency (TD) leads to memory deficits and neurological disease in animals and humans. The thiamine-dependent enzymes of the tricarboxylic acid (TCA) cycle are reduced following TD and in the brains of patients that died from multiple neurodegenerative diseases. Whether reductions in thiamine or thiamine-dependent enzymes leads to changes in all TCA cycle enzymes has never been tested. In the current studies, the pyruvate dehydrogenase complex (PDHC) and all of enzymes of the TCA cycle were measured in the brains of TD mice. Non-thiamine-dependent enzymes such as succinate dehydrogenase (SDH), succinate thiokinase (STH) and malate dehydrogenase (MDH) were altered as much or more than thiamine-dependent enzymes such as the alpha-ketoglutarate dehydrogenase complex (KGDHC) (-21.5%) and PDHC (-10.5%). Succinate dehydrogenase (SDH) activity decreased by 27% and succinate thiokinase (STH) decreased by 24%. The reductions in these other enzymes may result from oxidative stress because of TD or because these other enzymes of the TCA cycle are part of a metabolon that respond as a group of enzymes. The results suggest that other TCA cycle enzymes should be measured in brains from patients that died from neurological disease in which thiamine-dependent enzymes are known to be reduced. The diminished activities of multiple TCA cycle enzymes may be important in our understanding of how metabolic lesions alter brain function in neurodegenerative disorders.     

Succinate-dependent lipid peroxidation and its prevention by reduced ubiquinone in beef heart submitochondrial particles.

When succinate and ADP-Fe3+ chelate were added to beef heart submitochondrial particles pretreated with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase of the mitochondrial respiratory chain, the formation of malondialdehyde was observed. No formation was observed without the pretreatment. Oxaloacetate competitively inhibited the malondialdehyde formation with an apparent Ki of 3.4 microM. The malondialdehyde formation seemed to be initiated at the location between the p-hydroxymercuribenzoate-sensitive site and the 2-thenoyltrifluoroacetone-sensitive site of the succinate dehydrogenase because it was inhibited by the mercurial. Ubiquinone-10 was rapidly destroyed during the malondialdehyde-forming reaction when it was in the oxidized form, while the ubiquinone was not destroyed and the malondialdehyde formation was abolished when about 50% of the ubiquinone in the particles was in the reduced state. These observations suggest that the succinate-dependent peroxidation is strongly controlled by the redox state of ubiquinone.     

Perturbation of mitochondrial composition in muscle by iron deficiency. Implications regarding regulation of mitochondrial assembly

It has been reported that the mitochondrial cytochromes and citrate cycle enzymes occur in constant proportions to each other and increase or decrease roughly in parallel in response to various stimuli. The purpose of this study was to determine whether this proportionality is an obligatory consequence of the way in which mitochondria are assembled. Severe iron deficiency was used to bring about decreases of the iron-containing constituents of the mitochondrial respiratory chain in skeletal muscle. Cytochrome c concentration and cytochrome oxidase activity were decreased approximately 50%, while succinate dehydrogenase and NADH dehydrogenase activities were decreased by 78% in iron-deficient muscle. On electron microscopic examination, mitochondria in iron-deficient muscles had relatively sparse numbers of cristae. The iron deficiency had little or no effect on the levels of a range of mitochondrial matrix enzymes, including citrate synthase, isocitrate dehydrogenase, fumarase, aspartate aminotransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacid-CoA transferase, and acetoacetyl-CoA thiolase. These results show that the usual constant proportions between the constituents of the mitochondrial respiratory chain and matrix enzymes are not obligatory; they provide evidence that mitochondrial matrix enzymes and respiratory chain constituents can be incorporated into mitochondria independently and that the ratios between them can vary within wide limits.     

L-Pyroglutamic Acid Inhibits Energy Production and Lipid Synthesis in Cerebral Cortex of Young Rats In Vitro

In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and -glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO₂ production and lipid synthesis from [U-¹⁴C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO₂ production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5–3.0 mM and cytochrome c oxidase activity by 22–30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases.     

Inborn errors of complex II--unusual human mitochondrial diseases.

The succinate dehydrogenase consists of only four subunits, all nuclearly encoded, and is part of both the respiratory chain and the Krebs cycle. Mutations in the four genes encoding the subunits of the mitochondrial respiratory chain succinate dehydrogenase have been recently reported in human and shown to be associated with a wide spectrum of clinical presentations. Although a comparatively rare deficiency in human, molecularly defined succinate dehydrogenase deficiency has already been found to cause encephalomyopathy in childhood, optic atrophy or tumor in adulthood. Because none of the typical housekeeping genes encoding this respiratory chain complex is known to present tissue-specific isoforms, the tissue-specific involvement represents a quite intriguing question, which is mostly addressed in this review. A differential impairment of electron flow through the respiratory chain, handling of oxygen, and/or metabolic blockade possibly associated with defects in the different subunits that can be advocated to account for tissue-specific involvement is discussed.     

Inborn errors of complex II – Unusual human mitochondrial diseases

The succinate dehydrogenase consists of only four subunits, all nuclearly encoded, and is part of both the respiratory chain and the Krebs cycle. Mutations in the four genes encoding the subunits of the mitochondrial respiratory chain succinate dehydrogenase have been recently reported in human and shown to be associated with a wide spectrum of clinical presentations. Although a comparatively rare deficiency in human, molecularly defined succinate dehydrogenase deficiency has already been found to cause encephalomyopathy in childhood, optic atrophy or tumor in adulthood. Because none of the typical housekeeping genes encoding this respiratory chain complex is known to present tissue-specific isoforms, the tissue-specific involvement represents a quite intriguing question, which is mostly addressed in this review. A differential impairment of electron flow through the respiratory chain, handling of oxygen, and/or metabolic blockade possibly associated with defects in the different subunits that can be advocated to account for tissue-specific involvement is discussed.     

Inhibition of mitochondrial function in isolated rat liver mitochondria by azole antifungals

Ketoconazole is an imidazole oral antifungal agent with a broad spectrum of activity. Ketoconazole has been reported to cause liver damage, but the mechanism is unknown. However, ketoconazole and a related drug, miconazole, have been shown to have inhibitory effects on oxidative phosphorylation in fungi. Fluconazole, another orally administered antifungal azole, has also been reported to cause liver damage despite its supposedly low toxicity profile. The primary objective of this study was to evaluate the metabolic integrity of adult rat liver mitochondria after exposure to ketoconazole, miconazole, fluconazole, and the deacetylated metabolite of ketoconazole by measuring ADP-dependent oxygen uptake polarographically and succinate dehydrogenase activity spectrophotometrically. Ketoconazole, N-deacetyl ketoconazole, and miconazole inhibited glutamate-malate oxidation in a dose-dependent manner such that the 50% inhibitory concentration (I₅₀ was 32, 300, and 110 μM, respectively. In addition, the effect of ketoconazole, miconazole, and fluconazole on phosphorylation coupled to the oxidation of pyruvate/malate, ornithine/malate, arginine/malate, and succinate was evaluated. The results demonstrated that ketoconazole and miconazole produced a dose-dependent inhibition of NADH oxidase in which ketoconazole was the most potent inhibitor. Fluconazole had minimal inhibitory effects on NADH oxidase and succinate dehydrogenase, whereas higher concentrations of ketoconazole were required to inhibit the activity of succinate dehydrogenase. N-deacetylated ketoconazole inhibited succinate dehydrogenase with an I₅₀ of 350 μM. In addition, the reduction of ferricyanide by succinate catalyzed by succinate dehydrogenase demonstrated that ketoconazole caused a dose-dependent inhibition of succinate activity (I₅₀ of 74 μM). In summary, ketoconazole appears to be the more potent mitochondrial inhibitor of the azoles studied; complex I of the respiratory chain is the apparent target of the drug's action. © 1997 John Wiley & Sons, Inc.     

Properties of bovine heart mitochondrial cytochrome b560

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565.     

The relationship of 4-hydroxybenzoic acid to lysine and methionine formation in Escherichia coli

1. A multiple aromatic mutant, Escherichia coli 156:53D2, required 4-hydroxybenzoic acid for rapid aerobic growth on a number of carbon sources. 2. In the absence of 4-hydroxybenzoic acid aerobic growth was stimulated by a mixture of lysine and methionine and by succinate. The influence of the amino acids is attributed to a sparing of succinyl-CoA. 3. Low activities of both alpha-oxoglutarate dehydrogenase and fumarate reductase were found in organisms grown aerobically without 4-hydroxybenzoate and consequently both mechanisms known for the formation of succinate were impaired. 4. The low fumarate-reductase activity in these organisms was due to repression of enzyme synthesis by aeration and not to enzyme inactivation. In contrast lactate dehydrogenase and ethanol dehydrogenase were induced. This is interpreted as the appearance of alternative routes of NADH oxidation when electron transfer to oxygen is impaired. 5. The activities of the other tricarboxylic acid-cycle enzymes tested were little influenced by 4-hydroxybenzoate deficiency, although anaerobiosis resulted in a fall in activity.     

Pentachlorophenol inhibition of succinate oxidation by the respiratory chain in submitochondrial particles from the bovine heart

Study on the effect of pentachlorophenol on the succinate oxidase activity of submitochondrial particles and on the reduction level of cytochromes b revealed that the Ki value for PCP is equal to 2-4 microM. The succinate-DCPIP-reductase activity is noncompetitively inhibited with PCP (by 75-85%) (Ki = 3.6 microM). In the case of the succinate-PMS-reductase activity PCP at micromolar concentrations decreases the value of V only by 40% (C50 = 2 microM) with a simultaneous increase of the Km value for PMS. The identity of Ki values for PCP under these conditions suggests that the effect of PCP is due to the inhibitor interaction with the same component of the succinate dehydrogenase complex. The type of action of PCP on the succinate-acceptor-reductase activities indicates that the inhibiting effect of PCP on succinate oxidations is similar to that exerted by traditional inhibitors of succinate dehydrogenase--tenoyltrifluoroacetone and carboxins. Since PCP inhibits succinate dehydrogenase at low concentrations, it seems likely that the biological (pesticidal) effect of PCP is provided for not only by its uncoupling action but also by the inhibition of succinate oxidation in the respiratory chain.     

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.     

Myeloperoxidase-mediated inhibition of microbial respiration: damage to Escherichia coli ubiquinol oxidase

A microbicidal system, mediated by neutrophil myeloperoxidase, inhibits succinate-dependent respiration in Escherichia coli at rates that correlate with loss of microbial viability. Succinate dehydrogenase, the initial enzyme of the succinate oxidase respiratory pathway, catalyzes the reduction of ubiquinone to ubiquinol, which is reoxidized by terminal oxidase complexes. The steady-state ratio of ubiquinol to total quinone (ubiquinol + ubiquinone) reflects the balance between dehydrogenase-dependent ubiquinone reduction and terminal oxidase-dependent ubiquinol oxidation. Myeloperoxidase had no effect on total quinone content of E. coli but altered the steady-state ratio of ubiquinol to total quinone. The ratio doubled for organisms incubated with the myeloperoxidase system for 10 min, suggesting decreased ubiquinol oxidase activity, which was confirmed by observation of a 50% decrease in oxidation of the ubiquinol analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol. Despite inhibition of ubiquinol oxidase, overall succinate oxidase activity remained unchanged, suggesting that succinate dehydrogenase activity was preserved and that the dehydrogenase was rate limiting. Microbial viability was unaffected by early changes in ubiquinol oxidase activity. Longer (60 min) exposure of E. coli to the myeloperoxidase system resulted in only modest further inhibition of the ubiquinol oxidase, but the ubiquinol to total quinone ratio fell to 0%, reflecting complete loss of succinate dehydrogenase activity. Succinate oxidase activity was abolished, and there was extensive loss of microbial viability. Early myeloperoxidase-mediated injury to ubiquinol oxidase appeared to be compensated for by higher steady-state levels of ubiquinol which sustained electron turnover by mass effect. Later myeloperoxidase-mediated injuries eliminated succinate-dependent ubiquinone reduction, through inhibition of succinate dehydrogenase, with loss of succinate oxidase activity, effects which were associated with, although not clearly causal for, microbicidal activity.     

Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi

A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265-271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes.     

Rhodanese-Mediated sulfur transfer to succinate dehydrogenase.

The interaction of the sulfurtransferase rhodanese (EC 2.8.1.1) with succinate dehydrogenase (EC 1.3.99.1), yeast alcohol dehydrogenase (EC 1.1.1.1) and bovine serum albumin was studied. Succinate dehydrogenase incorporates the sulfane sulfur of [35S]rhodanese and, in the presence of unlabelled rhodanese, also incorporates that of [35S]thiosulfate. Rhodanese releases most of its transferable sulfur and is re-loaded in the presence of thiosulfate. Rhodanese undergoes similar modifications with yeast alcohol dehydrogenase but this latter does not bind 35S in amounts comparable to those incorporated in succinate dehydrogenase: nearly all the 35S released by [35S]rhodanese is with low-molecular-weight compounds. Bovine serum albumin also binds very little sulfur and [35S]rhodanese present in the reaction mixture does not discharge its radioactive sulfur nor does it take up sulfur from thiosulfate. Sulfur release from rhodanese appears to depend on the presence of - SH groups in the acceptor protein. Sulfur incorporated into succinate dehydrogenase was analytically determined as sulfide. A comparison of the optical spectra of succinate dehydrogenase preparations incubated with or without rhodanese indicates that there is an effect of the sulfurtransferase on the iron-sulfur absorption of the flavorprotein. The interaction of rhodanese with succinate dehydrogenase greatly decreases the catalytic activity of rhodanese with respect to thiocyanate formation. This is attributed to modifications in rhodanese associated with the reduction of sulfane sulfur to sulfide. Thiosulfate in part protects from this deactivation. The reconstitutive capacity of succinate dehydrogenase increased in parallel with sulfur incorporated in that enzyme following its interaction with rhodanese.     

An antimycin-insensitive succinate-cytochrome c reductase activity in pure reconstitutively active succinate dehydrogenase

Antimycin-insensitive succinate-cytochrome c reductase activity has been detected in pure, reconstitutively active succinate dehydrogenase. The enzyme catalyzes electron transfer from succinate to cytochrome c at a rate of 0.7 mumole succinate oxidized per min per mg protein, in the presence of 100 microM cytochrome c. This activity, which is about 2% of that of reconstitutive (the ability of succinate dehydrogenase to reconstitute with coenzyme ubiquinone-binding proteins (QPs) to form succinate-ubiquinone reductase) or succinate-phenazine methosulfate activity in the preparation, differs from antimycin-insensitive succinate-cytochrome c reductase activity detected in submitochondrial particles or isolated succinate-cytochrome c reductase. The Km for cytochrome c for the former is too high to be measured. The Km for the latter is about 4.4 microM, similar to that of antimycin-sensitive succinate-cytochrome c activity in isolated succinate-cytochrome c reductase, suggesting that antimycin-insensitive succinate-cytochrome c activity of succinate-cytochrome c reductase probably results from incomplete inhibition by antimycin. Like reconstitutive activity of succinate dehydrogenase, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase is sensitive to oxygen; the half-life is about 20 min at 0 degrees C at a protein concentration of 23 mg/ml. In the presence of QPs, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase disappears and at the same time a thenoyltrifluoroacetone-sensitive succinate-ubiquinone reductase activity appears. This suggests that antimycin-insensitive succinate-cytochrome c reductase activity of succinate dehydrogenase appears when succinate dehydrogenase is detached from the membrane or from QPs. Reconstitutively active succinate dehydrogenase oxidizes succinate using succinylated cytochrome c as electron acceptor, suggesting that a low potential intermediate (radical) may be involved. This suggestion is confirmed by the detection of an unknown radical by spin trapping techniques. When a spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), is added to a succinate oxidizing system containing reconstitutively active succinate dehydrogenase, a PBN spin adduct is generated. Although this PBN spin adduct is identical to that generated by xanthine oxidase, indicating that a perhydroxy radical might be involved, the insensitivity of this antimycin-insensitive succinate-cytochrome c reductase activity to superoxide dismutase and oxygen questions the nature of this observed radical.     

Ziram, a sulfhydryl reagent and specific inhibitor of yeast mitochondrial dehydrogenases.

The fungicide zinc dimethyldithiocarbamate (ziram) is a sulfhydryl reagent which inhibits specifically the growth of the yeast Saccharomyces cerevisiae on nonfermentable substrates. In isolated mitochondria, the uncoupled as well as the state 3 oxidations of succinate, α-ketoglutarate, ethanol, and malate plus pyruvate are sensitive to ziram concentrations of 10 to 30 μm. The oxidations of isocitrate, of external NADH, of α-glycerophosphate, and of ascorbate plus tetramethylphenylenediamine exhibit a lower sensitivity to ziram. Succinate, α-ketoglutarate, and pyruvate dehydrogenases activities are 50% inhibited by concentration of ziram lower than 10 μm. At the same concentrations, neither the mitochondrial transports of succinate, ADP, or phosphate nor oxidative phosphorylation and adenosine triphosphatase activities are modified. The kinetic study of the inhibition by ziram of succinate dehydrogenase activity shows that ziram is noncompetitive with succinate and produces sigmoidal inhibitions of state 3 and of uncoupled oxidation of succinate by intact mitochondria. Inhibition of succinate:phenazine methosulfate oxidoreductase activity yields exponential kinetics. However sigmoidal-type inhibition is observed when succinate dehydrogenase activity is stimulated by ATP.     

Interactions between carbon and nitrogen metabolism in Fibrobacter succinogenes S85: a 1H and 13C nuclear magnetic resonance and enzymatic study

The effect of the presence of ammonia on [1-13C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by 13C and 1H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The 13C enrichment of succinate and acetate was precisely quantified by 13C-filtered spin-echo difference 1H-NMR spectroscopy. The presence of ammonia did not modify the 13C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the 13C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by 13C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and 1H NMR. When cells were incubated in vivo with [1-13C]glucose, only 13C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.     

Succinate oxidase and fumarate reductase systems of filarial parasite Setaria digitata

Activities of succinate oxidase, fumarate reductase (FR) and succinate dehydrogenase (SDH) under a set of defined conditions were determined in the mitochondrial isolate from Setaria digitata, the filarial parasite from the cattle Bos indicus. Presence of only two activities namely SDH and succinate--UQ reductase of the succinate oxidase system could be detected in S. digitata. In the absence of cytochromes, the 3rd enzyme of the complex namely cytochrome oxidase is absent and it is proposed that an alternative oxidase is responsible for completing the succinate oxidation expressed as succinate oxidase activity. Though SDH and FR catalyse reverse reactions, they responded differently to modulators such as oxaloacetate, aspartate, alanine, pyruvate and fumarate. The degree of response of the two activities against inhibitors of electron transport was also different. Interestingly fumarate caused only 50% inhibition of succinate oxidation, while the effect against FR was more convincing.