2-Oxoglutarate: Oxidation and Role as a Potential Precursor of Cytosolic Acetyl-CoA for the Synthesis of Acetylcholine in Rat Brain Synaptosomes

The possibility that 2-oxoglutarate may supply acetyl units for the cytosolic synthesis of acetylcholine in rat brain synaptosomes was investigated. The contribution of [14C]2-oxoglutarate to the synaptosomal synthesis of [14C]acetylcholine was found to be negligible despite evidence for its uptake and oxidation. The activity of the enzymes NADP-isocitrate dehydrogenase (EC 1.1.1.42), aconitate hydratase (EC 4.2.1.3), and ATP citrate-lyase (EC 4.1.3.8) were measured in the synaptosol. NADP-isocitrate dehydrogenase and aconitate hydratase are present at three- to 1.5-fold higher activities than ATP citrate-lyase. It seems likely that these enzymes contribute to the metabolism of citrate and prevent detectable formation of cytosolic acetyl-CoA from exogenously added 2-oxoglutarate (or citrate). The data further suggest that ATP citrate-lyase may in part be associated with the mitochondrial fraction.     

Oxidation of γ-Hydroxybutyrate to Succinic Semialdehyde by a Mitochondrial Pyridine Nucleotide-Independent Enzyme

An antibody that inhibits over 95% of the cytosolic NADP+-dependent gamma-hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1-14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1-14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADP+ and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+-dependent cytosolic oxidoreductase which catalyzes this reaction.     

Pyruvate metabolism in rat brain mitochondria.

1. Oxidation of pyruvate by rat brain mitochondria was stimulated in state 3 by malate or succinate up to 250 nmoles O2/mg protein/min. Oxidation of malate, succinate, 2-oxoglutarate or glutamate as the sole substrates, was 1/4 - 1/5 that observed with pyruvate. 2. Maximum oxygen consumption in state 3 was observed at pH 6.90 - 7.20, whereas in state 4 it was not affected by changes in pH. 3. In state 4, in the absence of exogenous acceptor or acetyl residues, acetate was the main oxidation product, corresponding to about 80% of the amount of pyruvate utilized. Malate did not affect the rate of pyruvate utilization but lowered acetate concentration and raised concentration of citrate and 2-oxoglutarate. 4. In state 3, pyruvate and malate were converted mainly to 2-oxoglutarate, its concentration being three times as high as that of citrate. 5. Formation of citrate, 2-oxoglutarate and acetate from pyruvate in brain is considered as a function of availability of the acceptor of acetyl residues and the energy state of mitochondrion.     

Control of the tricarboxylate cycle and its interactions with glycolysis during acetate utilization in rat heart

1. Transient and steady-state changes caused by acetate utilization were studied in perfused rat heart. The transient period occupied 6min and steady-state changes were followed in a further 6min of perfusion. 2. In control perfusions glucose oxidation accounted for 75% of oxygen utilization; the remaining 25% was assumed to represent oxidation of glyceride fatty acids. With acetate in the steady state, acetate oxidation accounted for 80% of oxygen utilization, which increased by 20%; glucose oxidation was almost totally suppressed. The rate of tricarboxylate-cycle turnover increased by 67% with acetate perfusion. The net yield of ATP in the steady state was not altered by acetate. 3. Acetate oxidation increased muscle concentrations of acetyl-CoA, citrate, isocitrate, 2-oxoglutarate, glutamate, alanine, AMP and glucose 6-phosphate, and lowered those of CoA and aspartate; the concentrations of pyruvate, ATP and ADP showed no detectable change. The times for maximum changes were 1min, acetyl-CoA, CoA, alanine and AMP; 6min, citrate, isocitrate, glutamate and aspartate; 2-4min, 2-oxoglutarate. Malate concentration fell in the first minute and rose to a value somewhat greater than in the control by 6min. There was a transient and rapid rise in glucose 6-phosphate concentration in the first minute superimposed on the slower rise over 6min. 4. Acetate perfusion decreased the output of lactate, the muscle concentration of lactate and the [lactate]/[pyruvate] ratio in perfusion medium and muscle in the first minute; these returned to control values by 6min. 5. During the first minute acetate decreased oxygen consumption and lowered the net yield of ATP by 30% without any significant change in muscle ATP or ADP concentrations. 6. The specific radioactivities of cycle metabolites were measured during and after a 1min pulse of [1-(14)C]acetate delivered in the first and twelfth minutes of acetate perfusion. A model based on the known flow rates and concentrations of cycle metabolites was analysed by computer simulation. The model, which assumed single pools of cycle metabolites, fitted the data well with the inclusion of an isotope-exchange reaction between isocitrate and 2-oxoglutarate+bicarbonate. The exchange was verified by perfusions with [(14)C]bicarbonate. There was no evidence for isotope exchange between citrate and acetyl-CoA or between 2-oxoglutarate and malate. There was rapid isotope equilibration between 2-oxoglutarate and glutamate, but relatively poor isotope equilibration between malate and aspartate. 7. It is concluded that the citrate synthase reaction is displaced from equilibrium in rat heart, that isocitrate dehydrogenase and aconitate hydratase may approximate to equilibrium, that alanine aminotransferase is close to equilibrium, but that aspartate transamination is slow for reasons that have yet to be investigated. 8. The slow rise in citrate concentration as compared with the rapid rise in that of acetyl-CoA is attributed to the slow generation of oxaloacetate by aspartate aminotransferase. 9. It is proposed that the tricarboxylate cycle may operate as two spans: acetyl-CoA-->2-oxoglutarate, controlled by citrate synthase, and 2-oxoglutarate-->oxaloacetate, controlled by 2-oxoglutarate dehydrogenase; a scheme for cycle control during acetate oxidation is outlined. The initiating factors are considered to be changes in acetyl-CoA, CoA and AMP concentrations brought about by acetyl-CoA synthetase. 10. Evidence is presented for a transient inhibition of phosphofructokinase during the first minute of acetate perfusion that was not due to a rise in whole-tissue citrate concentration. The probable importance of metabolite compartmentation is stressed.     

THE EFFECT OF ASPARTATE ON CITRATE METABOLISM IN THE CYTOSOLIC FRACTION OF BRAIN UNDER CONDITIONS OF NORMOXIA, HYPOXIA AND ANAESTHESIA

—The utilization of citrate by the cytoplasmic fraction of rat brain is inhibited in hypoxia and remains unaltered in anaesthesia. The addition of exogenous aspartate to the cytosolic fraction isolated from brains of hypoxic animals increases the rate of citrate removal. The level of cytosolic aspartate gradually decreases when the exposure period to low oxygen tension is increased and reaches a minimum after 30 min. The levels of mitochondrial aspartate and of cytoplasmic carbamyl aspartate remain constant. The low level of cytosolic aspartate is accompanied by an increase in the concentration of cytosolic urea and increase in the aspartate level in blood serum. It is suggested that the oxidation of citrate by the cytoplasmic fraction of brain is inhibited in hypoxia owing to the decrease in endogenous aspartate. The decrease in the level of cytoplasmic aspartate is caused by the diversion of this substrate toward urea synthesis and by the increased leakage across the cell/blood barrier to the blood stream. Anaesthesia prevents the changes induced by hypoxia.     

Coordinated regulation of ammonium assimilation and carbon catabolism by glyoxylate in Saccharomyces cerevisiae

The activities of citrate synthase (EC 4.1.3.7) and NADP+-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.4) of Saccharomyces cerevisiae were inhibited in vitro by glyoxylate. In the presence of glyoxylate, pyruvate and glyoxylate pools increased, suggesting that glyoxylate was efficiently transported and catabolized. Pyruvate accumulation also indicates that citrate synthase was inhibited. A decrease in the glutamate pool was also observed under these conditions. This can be attributed to an increased transamination rate and to the inhibitory effect of glyoxylate on NADP+-dependent GDH. Furthermore, the increase in the ammonium pool in the presence of glyoxylate suggests that NADP+-dependent GDH was being inhibited in vivo, since the activity of glutamine synthetase did not decrease under these conditions. We propose that the inhibition of both citrate synthase and NADP+-dependent GDH could form part of a mechanism that regulates the internal 2-oxoglutarate concentration.     

Oxidation of acetate through reactions of the citric acid cycle by Geobacter sulfurreducens in pure culture and in syntrophic coculture

Geobacter sulfurreducens strain PCA oxidized acetate to CO2 via citric acid cycle reactions during growth with acetate plus fumarate in pure culture, and with acetate plus nitrate in coculture with Wolinella succinogenes. Acetate was activated by succinyl-CoA:acetate CoA-transferase and also via acetate kinase plus phosphotransacetylase. Citrate was formed by citrate synthase. Soluble isocitrate and malate dehydrogenases NADP+ and NAD+, respectively. Oxidation of 2-oxoglutarate was measured as benzyl viologen reduction and strictly CoA-dependent; a low activity was also observed with NADP+. Succinate dehydrogenase and fumarate ductase both were membrane-bound. Succinate oxidation was coupled to NADP+ reduction whereas fumarate reduction was coupled to NADPH and NADH Coupling of succinate oxidation to NADP+ or cytochrome(s) reduction required an ATP-dependent reversed electron transport. Net ATP synthesis proceeded exclusively through electron transport phosphorylation. During fumarate reduction, both NADPH and NADH delivered reducing equivalents into the electron transport chain, which contained a menaquinone. Overall, acetate oxidation with fumarate proceeded through an open loop of citric acid cycle reactions, excluding succinate dehydrogenase, with fumarate reductase as the key reaction for electron delivery, whereas acetate oxidation in the syntrophic coculture required the complete citric acid cycle.     

Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.     

Metabolite fluxes across the inner membrane of plant mitochondria — inhibition by phthalonic acid

Transport and oxidation-reduction of citrate, 2-oxoglutarate and oxaloacetate by mitochondria isolated from thermogenic (Arum maculatum, Sauromatum guttatum spadices), green leaf (Pisum sativum) or etiolated (Phaseolus aureus, Helianthus tuberosus) plant tissues was found to be inhibited by phthalonic acid. No inhibition was found for NADH oxidation, glutamate, succinate or glycine transport and oxidation and malate transport. The much greater sensitivity of citrate oxidation to phthalonate inhibition compared with that of 2-oxoglutarate indicated that different carriers were involved, neither of which appeared to be rate-limiting for oxidation. Fluxes of oxaloacetate, and their sensitivity to phthalonate, indicated that this keto acid may use either the same carrier as 2-oxoglutarate or an oxaloacetate-specific carrier.Abbreviation PTA phthalonic acid     

REGIONAL AND SUBCELLULAR DISTRIBUTION OF AMINOTRANSFERASES IN RAT BRAIN

Abstract— Aminotransferase activity was measured in various areas of the nervous system of the rat (cortical grey matter, midbrain, corpus callosum, spinal cord and sciatic nerve) and in subcellular fractions of rat brain (nuclei, mitochondria and cytosol). Activity was low or absent in the sciatic nerve relative to that in the other areas, with the exception of incubation of glutamate with oxaloacetate (25 per cent of the activity found in brain) and of asparagine with 2-oxoglutarate (65 per cent of the activity found in brain). The distribution of enzymic activity was not homogeneous; alanine-2-oxoglutarate aminotransferase was highest in cortical grey matter; leucine- and GABA-2-oxoglutarate aminotransferases were highest in midbrain. Incubation of phenylalanine or tyrosine with 2-oxoglutarate gave similar activities in grey matter and midbrain. Activity generally was higher in the grey matter than in corpus callosum or spinal cord. However, incubations of methionine with 2-oxoglutarate, or glutamine with glyoxylate, gave similar activities in the three areas studied from the brain, whereas incubations of glutamate with glyoxylate gave highest activity in the corpus callosum. Only incubations of asparagine with 2-oxoglutarate, and glutamate with glyoxylate, gave significant activity in the nuclear subcellular fraction. Aminotransferase activity of phenylalanine, tyrosine or GABA with 2-oxoglutarate, or ornithine or glutamine with glyoxylate, was localized to mitochondria. The remaining reactions studied (glutamate with oxaloacetate; leucine, alanine, methionine or asparagine with 2-oxoglutarate and glutamate with glyoxylate) demonstrated activity in both the mitochondrial fraction and the soluble supernatant fraction.     

Studies on the control of 4-aminobutyrate metabolism in ''synaptosomal'' and free rat brain mitochondria.

1. The specific activities of 4-aminobutyrate aminotransferase (EC 2.6.1.19) and succinate semialdehyde dehydrogenase (EC 1.2.1.16) were significantly higher in brain mitochondria of non-synaptic origin (fraction M) than those derived from the lysis of synaptosomes (fraction SM2). 2. The metabolisms of 4-aminobutyrate in both 'free' (non-synaptic, fraction M) and 'synaptic' (fraction SM2) rat brain mitochondria was studied under various conditions. 3. It is proposed that 4-aminobutyrate enters both types of brain mitochondria by a non-carrier-mediated process. 4. The rate of 4-aminobutyrate metabolism was in all cases higher in the 'free' (fraction M) brain mitochondria than in the synaptic (fraction SM2) mitochondria, paralleling the differences in the specific activities of the 4-aminobutyrate-shunt enzymes. 5. The intramitochondrial concentration of 2-oxoglutarate appears to be an important controlling parameter in the rate of 4-aminobutyrate metabolism, since, although 2-oxoglutarate is required, high concentrations (2.5 mM) of extramitochondrial 2-oxoglutarate inhibit the formation of aspartate via the glutamate-oxaloacetate transaminase. 6. The redox state of the intramitochondrial NAD pool is also important in the control of 4-aminobutyrate metabolism; NADH exhibits competitive inhibition of 4-aminobutyrate metabolism by both mitochondrial populations with an apparent Ki of 102 muM. 7. Increased potassium concentrations stimulate 4-aminobutyrate metabolsim in the synaptic mitochondria but not in 'free' brain mitochondria. This is discussed with respect to the putative transmitter role of 4-aminobutyrate.     

Inhibition by oxalomalate of rat liver mitochondrial and extramitochondrial aconitate hydratase

1. The effect of oxalomalate on the oxidation of citrate and cis-aconitate in rat liver mitochondria, and on the activity of mitochondrial and cytoplasmic aconitate hydratase, has been investigated. 2. Oxalomalate that was added to intact rat liver mitochondria at high concentrations (2mm) produced complete inhibition of citrate and cis-aconitate oxidation, but lower concentrations (0.1-0.25mm) inhibited oxidation of citrate more than that of cis-aconitate. 3. Aconitate hydratase that was either extracted from mitochondria or soluble in the cytoplasm, was strongly inhibited by low concentrations of oxalomalate (0.01-0.2mm), the mitochondrial enzyme being more sensitive than the soluble one. 4. Oxalomalate, when added together with citrate, produced competitive inhibition; the K(i) values calculated were 1x10(-6)m for the mitochondrial and 2.5x10(-6)m for the cytoplasmic enzyme. 5. With both the enzymic preparations oxalomalate added together with the substrates inhibited the initial rate of the reaction citrate-->cis-aconitate more than that of the reaction isocitrate-->cis-aconitate. 6. After 2min of preincubation of the inhibitor with either of the enzymic preparations the inhibition increased tenfold and became irreversible; under these conditions both the reactions were inhibited to the same extent. 7. The inhibition by oxalomalate of aconitate hydratase appeared to be similar in many respects to that produced by fluorocitrate on the same enzyme.     

RELATIONSHIP BETWEEN THE 4-AMINOBUTYRATE BYPATH AND THE OXIDATION OF 2-OXOGLUTARATE IN RAT BRAIN MITOCHONDRIA

Abstract— A method is described to evaluate simultaneously the contributions of 2-oxoglutarate oxidation and the GABA bypath to succinate production in isolated rat brain mitochondria.
2-Oxoglutarate oxidation is under respiratory control whereas the activity of the GABA shunt is but slightly affected by the mitochondrial energy state.
The oxidation of GABA is half-maximal with 5m m -GABA. GABA does not affect 2-oxoglutarate oxidation. 1 m m -2-oxoglutarate is optimal for GABA oxidation, whereas higher concentrations inhibit the shunt activity.
The rate of GABA oxidation observed in vitro (5 nmol/min.mg mitochondrial protein) is comparable to the activity of the shunt under in viuo conditions.
The control and the compartmentation of GABA oxidation are discussed.     

The regulation and glutamate metabolism by tricarboxylic acid-cycle activity in rat brain mitochondria

1. The interrelationship of metabolism of pyruvate or 3-hydroxybutyrate and glutamate transamination in rat brain mitochondria was studied. 2. If brain mitochondria are incubated in the presence of equimolar concentrations of pyruvate and glutamate and the K(+) concentration is increased from 1 to 20mm, the rate of pyruvate utilization is increased 3-fold, but the rate of production of aspartate and 2-oxoglutarate is decreased by half. 3. Brain mitochondria incubated in the presence of a fixed concentration of glutamate (0.87 or 8.7mm) but different concentrations of pyruvate (0 to 1mm) produce aspartate at rates that decrease as the pyruvate concentration is increased. At 1mm-pyruvate, the rate of aspartate production is decreased to 40% of that when zero pyruvate was present. 4. Brain mitochondria incubated in the presence of glutamate and malate alone produce 2-oxoglutarate at rates stoicheiometric with the rate of aspartate production. Both the 2-oxoglutarate and aspartate accumulate extramitochondrially. 5. Externally added 2-oxoglutarate has little inhibitory effect (K(i) approx. 31mm) on the production of aspartate from glutamate by rat brain mitochondria. 6. It is concluded that the inhibitory effect of increased C(2) flux into the tricarboxylic acid cycle on glutamate transamination is caused by competition for oxaloacetate between the transaminase and citrate synthase. 7. Evidence is provided from a reconstituted malate-aspartate (or Borst) cycle with brain mitochondria that increased C(2) flux into the tricarboxylic acid cycle from pyruvate may inhibit the reoxidation of exogenous NADH. These results are discussed in the light of the relationship between glycolysis and reoxidation of cytosolic NADH by the Borst cycle and the requirement of the brain for a continuous supply of energy.     

Kinetic properties of NADP-specific isocitrate dehydrogenase from bovine adrenals

At low concentrations of Mg2+ or Mn2+ the reaction catalyzed by isocitrate dehydrogenase from bovine adrenal cortex proceeds with a lag period which disappears as a result of the enzyme saturation with Mn2+ or Mg2+. The nu o versus D,L-isocitrate concentration curve is non-hyperbolic, which may be interpreted either by the presence of two active sites with different affinity for the substrate (K'mapp = 2.3 and 63 microM) within the enzyme molecule or by the "negative" cooperativity of these sites. The apparent Km value for NADP lies within the range of 3.6-9 microM. High concentrations of NADP inhibit isocitrate dehydrogenase (Ki = 1.3 mM). NADP.H inhibits the enzyme in a mixed manner with respect to NADP (Ki = 0.32 mM). In the presence of NADP.H the curve nu o dependence on NADP concentration shows a "negative" cooperativity between NADP binding sites. The reverse enzyme-catalyzed reaction of reductive carboxylation of 2-oxoglutarate does not exhibit any significant deviations from the Michaelis-Menten kinetics. The Km value for 2-oxoglutarate is 120 microM, while that for NADP.H is 10 microM.     

Analysis of the kinetic mechanism of halophilic NADP-dependent glutamate dehydrogenase

The amination of 2-oxoglutarate catalyzed by NADP-specific glutamate dehydrogenase (EC 1.4.1.4, L-glutamate:NADP+ oxidoreductase (deaminating)) from Halobacterium halobium has been analyzed by initial rate, graphical analysis, and product and competitive inhibition studies. Initial rate and graphical analysis reveal that a B term (representing 2-oxoglutarate) is not statistically necessary for an initial rate equation. However, the absence of a B term does not distinguish between ordered and random binding of NADPH and ammonia. The patterns of product inhibition by NADP+ and L-glutamate, and competitive inhibition by hydroxylamine and succinate permit deduction of the kinetic mechanism as ordered, with NADPH, 2-oxoglutarate and ammonia added in that order, and L-glutamate release preceding NADP+ release.     

Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively.     

2-Oxoglutarate:NADP(+) oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction

The conversion of [(14)C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying beta-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated. With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol min(-1) mg(-1). 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH ( approximately 10% rate); in contrast NADH did not serve as an electron donor. Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NADP(+), and NADPH:acceptor oxidoreductase. KGOR was purified by a 76-fold enrichment. The enzyme had a molecular mass of 290 +/- 20 kDa and was composed of three subunits of 63 (gamma), 62 (alpha), and 37 (beta) kDa in a 1:1:1 ratio, suggesting an (alphabetagamma)(2) composition. The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol). KGOR from A. evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NADP(+) and oxidized viologens as electron acceptors; in contrast NAD(+) was not reduced. These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle. Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NADP(+), depending on the cellular needs. N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum. We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation. The alpha-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the beta-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the gamma-subunit is supposed to harbor the CoA binding site. This is the first study of an NADP(+)-specific KGOR. A similar NADP(+)-specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.     

A method of determining 2-oxoglutarate dehydrogenase activity in intact mitochondria

A rather simple method is suggested for measuring the activity of 2-oxoglutarate dehydrogenase of intact mitochondria. The method is based on the determination of the rate of exogenic 2-oxoglutarate decrease in the mitochondrial suspension. Experiments with sodium arsenite and comparison of kinetic parameters of the 2-oxoglutarate, dehydrogenase reaction and transport of 2-oxoglutarate to mitochondria have shown that the measurable exogenic 2-oxoglutarate oxidation rate corresponds to the 2-oxoglutarate dehydrogenase activity in intact mitochondria. The method made it possible to establish the stimulating effect of ADP on the 2-oxoglutarate dehydrogenase activity of intact mitochondria and the absence of such an effect in destructed mitochondria.     

THE EFFECTS OF INTOXICATING DOSES OF ETHANOL UPON INTERMEDIARY METABOLISM IN RAT BRAIN

Abstract— The effect of acute (8-min) and prolonged (13-h) exposures to high doses of ethanol upon the intermediary metabolites of rat brain has been studied, with the use of a new freezing technique which minimizes post-mortem changes. Injection of ethanol (80 mmol/kg body wt) produced general anaesthesia within 8 min after administration. At this time there were increases in the brain contents of glucose, glucose-6-phosphate and citrate; there was no change in arterial pCO₂. Rats under ethanol anaesthesia for 13 h showed increases in brain contents of glycogen, glucose and glucose 6-phosphate; and decreases in lactate, pyruvate, α-oxoglutarate and malate. Under similar experimental conditions, arterial pCO₂, increased from 37 to 51 Torr. The changes in levels of metabolites after injection of ethanol were similar to those after administration of many volatile anaesthetic agents or elevation of brain CO₂ by other means. Although brain levels of malate and α-oxoglutarate decreased after prolonged exposure to ethanol, the mitochondrial redox state was maintained. Accordingly, the levels of glutamate and aspartate fell in accordance with the law of mass action. The maintenance of the cytoplasmic and mitochondrial redox states in the brain during ethanol intoxication was in marked contrast to the effects on the liver. We suggest that the different effects observed in brain and liver result from the action of ethanol upon the nerve cell membrane in brain, whereas the primary target in liver is alcohol dehydrogenase.