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.     

Oxalate accumulation from citrate by Aspergillus niger. I. Biosynthesis of oxalate from its ultimate precursor.

Carbon-14 was incorporated from citrate-1,5-14C, glyoxylate-14C(U), or glyoxylate-1-14C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-14C(U) was superior to glyoxylate-1-14C and citrate-1,5-14C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC1.1.3.1; glyoxylate oxidase, EC1.2.3.5;NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC1.2.1.7) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.     

Organic acid metabolism in Sclerotinia sclerotiorum

Summary Citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), NADP specific isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2) and malate dehydrogenase (EC 1.1.1.37) were detected in cell-free preparations of Sclerotinia sclerotiorum (Lib.) D By. grown on liquid glucose-salts medium in stationary culture. Isocitrate lyase (EC 4.1.3.1) was present when the fungus grew on a carbohydrate-free medium but was not detected when the cultures grew on the glucose-salts medium. The amount of oxalate in the culture filtrate declined as the specific activity of citrate synthase and malate dehydrogenase in the mycelium declined. Increasing the initial pH of the medium resulted in an increase of the dicarboxylic acids in the culture filtrate and the specific activity of malate dehydrogenase in the mycelium. The specific reaction(s) leading to oxalic acid formation were not identified.     

Regulation of biosynthesis of secondary metabolites. I. Biosynthesis of chlortetracycline and tricarboxylic acid cycle activity

In the course of submerged cultivation of low-production and industrial production strains of Streptomyces aureofaciens, the activity of enzymes of the tricurboxylic acid cycle was studied. The activities of citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37) were estimated spectrophotometrically in cell-free preparations. In the growth phase, mainly the initial reactions of the cycle were active with both strains. In production-phase, the activities of enzymes in the low-production strain were 2–5 × higher than in the production strain. Benzylthioeyanate, at a concentration of 5 × l0^?5M, stimulated chlortetracycline production of both strains with accompanying decrease in activity of the enzymes of the tricarboxylic acid cycle. The role of the tricarboxylic acid cycle in control of chlortetracycline biosynthesis is discussed.     

Factors involved in changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of acetyl-CoA carboxylase (EC 6.4.1.2), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), extramitochondrial aconitate hydratase (EC 4.2.1.3) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) have been measured in the livers of developing rats from late foetal life to maturity. 2. The effect of altering the weaning time on some enzymes associated with lipogenesis has been studied. Weaning rats at 15 days of age instead of 21 days results in an immediate increase in the activity of ;malic' enzyme (EC 1.1.1.40) whereas the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and ATP citrate lyase (EC 4.1.3.8) did not increase until 4-5 days and acetyl-CoA carboxylase 2-3 days after early weaning. Weaning rats on to an artificial-milk diet led to complete repression of the rise in activity of hepatic enzymes associated with lipogenesis normally found on weaning, except for ;malic' enzyme, which increased in activity after 20 days of age. 3. The effect of intraperitoneal injections of glucagon, cortisol, growth hormone and thyroxine on the same hepatic enzymes has been investigated. Only thyroxine had any effect on enzyme activities and caused a 20-fold increase in ;malic' enzyme activity and a twofold increase in ATP citrate lyase activity. 4. The activities of hepatic glucose 6-phosphate dehydrogenase and ;malic' enzyme are higher in adult female than in adult male rats and it has been shown that this sex difference in enzyme activities is due to both male and female sex hormones. 5. Hepatic malate, citrate, pyruvate, glucose 6-phosphate and phosphoenolpyruvate concentrations have been measured throughout development. 6. The results are discussed in relation to the dietary and hormonal control of hepatic enzyme activities during development.     

Characterization of aconitate hydratase from mitochondria and cytoplasm of ascites tumor cells

This paper describes the characterization of aconitate hydratase (EC 4.2.1.3) in cytoplasmic and mitochondrial extracts from Ehrlich ascites tumor cells carried by BALB/C mice. The results show a similar distribution of aconitate hydratase in both extracts, with specific activities much lower than those found in pig and mouse tissues. Mitochondrial aconitate hydratase shows a substrate inhibition by citrate with a Km similar to that found in cytoplasm (Km = 1.0 mM and 0.9 mM, respectively). Oxalacetate produces a mixed type of inhibition in both cytoplasmic and mitochondrial aconitate hydratases with different inhibition constants (Ki = 0.3 mM and 1.0 mM, respectively). Moreover, the specific activities of aconitate hydratase in both cytoplasm and mitochondria decrease when the tumor progresses in the peritoneum of BALB/C mice, as well as the percentage of aconitate hydratase activity in the presence of oxalacetate as the inhibitor. These results indicate that the activity and kinetics of aconitate hydratase are markedly altered by neoplastic transformation as occurs in Ehrlich ascites tumor cells. Since aconitate hydratase is not a key enzyme, these unexpected data are of interest in the study of cancer biochemistry.     

Regulation of aconitate hydratase activity from rat kidney cortex by bicarbonate.

1. The increase in pH value and bicarbonate concentration stimulated citrate synthesis from pyruvate and malate, inhibiting simultaneously conversion of isocitrate to citrate. 2. Bicarbonate inhibited competitively the activity of aconitate hydratase, probably binding with the two active sites of the enzyme. The Ki values for the cytoplasmic and mitochondrial enzyme were, respectively, 27 and 38 mM. The pH optimum for both forms of the enzyme in Tris-HCl buffer was in the range 7.8-8.6, and in bicarbonate buffer varied from 7.2 to 8.0, depending on the form of the enzyme and the substrate used. 3. Only free, completely dissociated citrate anion acts as a substrate for aconitate hydratase. 4. The role of aconitate hydratase as a factor controlling the rate of citrate metabolism in kidney in metabolic alkalosis is discussed.     

Oxalate accumulation from citrate by Aspergillus niger

Carbon-14 was incorporated from citrate-1,5-¹⁴C, glyoxylate-¹⁴C(U), or glyoxylate-1-¹⁴C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-¹⁴C(U) was superior to glyoxylate-1-¹⁴C and citrate-1,5-¹⁴C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC 3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC 1.1.3.1; glyoxylate oxidase, EC 1.2.3.5; NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC 1.2.1.17) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.Abbreviations Used DCPIP 2,6-dichlorophenolindophenol     

Citrate Cycle and Related Metabolism of Listeria monocytogenes

The growth response of Listeria monocytogenes strains A4413 and 9037-7 to carbohydrates was determined in a defined medium. Neither pyruvate, acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, nor malate supported growth. Furthermore, inclusion of any of these carbohydrates in the growth medium with glucose did not increase the growth of Listeria over that observed on glucose alone. Resting cell suspensions of strain A4413 oxidized pyruvate but not acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, or malate. Cell-free extracts of strain A4413 contained active citrate synthase, aconitate hydratase, isocitrate dehydrogenase, malate dehydrogenase, fumarate hydratase, fumarate reductase, pyruvate dehydrogenase system, and oxidases for reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. The alpha-ketoglutarate oxidation system, succinate dehydrogenase, isocitrate lyase, and malate synthase were not detected. Cytochromes were not detected. The data suggest that strain A4413, under these conditions, utilizes a split noncyclic citrate pathway which has an oxidative portion (citrate synthase, aconitate hydratase, and isocitrate dehydrogenase) and a reductive portion (malate dehydrogenase, fumarate hydratase, and fumarate reductase). This pathway is probably important in biosynthesis but not for a net gain in energy.     

Control of the citric acid cycle by glyoxylate. The mechanism of inhibition of oxoglutarate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase

1. The effects of glyoxylate on partially purified preparations of aconitate hydratase, isocitrate dehydrogenase and oxoglutarate dehydrogenase were compared with those of oxalomalate and hydroxyoxoglutarate (obtained by condensation of glyoxylate with oxaloacetate and pyruvate respectively). 2. Glyoxylate (1mm) did not affect aconitate hydratase and isocitrate dehydrogenase, whereas oxalomalate (1mm) inhibited the enzyme activities completely. 3. Glyoxylate (0.025mm) inhibited oxoglutarate dehydrogenase irreversibly, whereas the same concentrations of oxalomalate and hydroxyoxoglutarate were ineffective. This inhibitory effect was prevented if oxoglutarate, pyruvate or oxaloacetate was mixed with the enzyme before the glyoxylate. 4. Incubation of oxoglutarate dehydrogenase with radioactive glyoxylate produced radioactive carbon dioxide; radioactivity was also recovered in the portion of the enzyme identified with thiamin pyrophosphate. 5. The behaviour of glyoxylate in producing multiple inhibitions of the citric acid cycle, either by direct interaction with oxoglutarate dehydrogenase, or by means of its condensation compounds which inhibit aconitate hydratase and isocitrate dehydrogenase, is discussed.     

Metabolic adaptations during lactogenesis. Fatty acid synthesis in rabbit mammary tissue during pregnancy and lactation

1. Mammary tissue was obtained from rabbits at various stages of pregnancy and lactation and used for tissue-slice incubations (to measure the rate of fatty acid synthesis and CO(2) production) and to determine relevant enzymic activities. A biphasic adaptation in fatty acid synthetic capacity during lactogenesis was noted. 2. The first lactogenic response occurred between day 15 and 24 of pregnancy. Over this period fatty acid synthesis (from acetate) increased 14-fold and the proportions of fatty acids synthesized changed to those characteristic of milk fat (77-86% as C(8:0)+C(10:0) acids). 3. The second lactogenic response occurred post partum as indicated by increased rates of fatty acid synthesis and CO(2) production (from acetate and glucose) and increased enzymic activities. 4. Major increases in enzymic activities between mid-pregnancy and lactation were noted for ATP citrate lyase (EC 4.1.3.8), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA carboxylase (EC 6.4.1.2), fatty acid synthetase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). Smaller increases in activity occurred with glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) and NADP(+)-isocitrate dehydrogenase (EC 1.1.1.42) and the activity of NADP(+)-malate dehydrogenase (EC 1.1.1.40) was negligible at all periods tested. 5. During pregnancy and lactation there was a close temporal relationship between fatty acid synthetic capacity and the activities of ATP citrate lyase (r=0.94) and acetyl-CoA carboxylase (r=0.90).     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.     

Fluorocitrate inhibition of aconitate hydratase and the tricarboxylate carrier of rat liver mitochondria

1. The effect of biologically synthesized and purified fluorocitrate on the metabolism of tricarboxylate anions by isolated rat liver mitochondria was investigated, in relation to the claim by Eanes et al. (1972) that this fluoro compound inhibits the tricarboxylate carrier at concentrations at which it has little effect on the aconitate hydratase activity. 2. That the inhibitory action of fluorocitrate is at the level of the aconitate hydratase and not at the level of the tricarboxylate carrier is indicated by the following findings. Although the oxidation of citrate and cis-aconitate, but not that of isocitrate, was inhibited by fluorocitrate, the exchange of internal citrate for external citrate or l-malate was not. Had the tricarboxylate carrier been affected, these latter exchange reactions would have been inhibited. 3. By using aconitate hydratase solubilized from mitochondria it was found that with citrate as substrate the inhibition by fluorocitrate was partially competitive (K(i)=3.4x10(-8)m), whereas with cis-aconitate as substrate the inhibition was partially non-competitive (K(i)=3.0x10(-8)m).     

Does the pentose cycle play a major role for NADPH supply in the heart?

It was attempted to determine the substrate flux through the pentose cycle in isolated rat hearts which performed pressure-volume work employing 14CO2 production from [1-14C]glucose (Kühn & Scholz (1982) Eur. J. Biochem. 124, 611-617). Even under conditions of increased NADPH requirements (infusion of tert-butylhydroperoxide) and a diminished 14CO2 production from glucose via the citrate cycle (in the presence of oleate as additional substrate) or enhanced activity of glucose-6-phosphate dehydrogenase (pretreatment with isoproterenol), a substrate flux through the pentose cycle was not detectable. The lower limit of detection is 0.01 mumol/(min X g). The increase in 14CO2 production from [1-14C]- and [6-14C]glucose and the acceleration in the washout when tert-butylhydroperoxide was present suggest an increase of substrate flux through the citrate cycle; therefore it is concluded that NADPH required for the removal of peroxides via the glutathione system is derived from the isocitrate dehydrogenase reaction.     

[14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate.

Previous attempts to account for the labelling in vivo of liver metabolites associated with the citrate cycle and gluconeogenesis have foundered because proper allowance was not made for the heterogeneity of the liver. In the basal state (anaesthetized after 24h starvation) this heterogeneity is minimal, and we show that labelling by [14C]bicarbonate can be interpreted unambiguously. [14C]Bicarbonate was infused to an isotopic steady state, and measurements were made of specific radioactivities of blood bicarbonate, alanine, glycerol and lactate, of liver alanine and lactate, and of individual carbon atoms in blood glucose and liver aspartate, citrate and malate. (Existing methods for several of these measurements were extensively modified.) The results were combined with published rates of gluconeogenesis, uptake of gluconeogenic precursors by the liver, and citrate-cycle flux, all measured under similar conditions, and with estimates of other rates made from published data. To interpret the results, three ancillary measurements were made: the rate of CO2 exchange by phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) under conditions that simulated those in vivo; the 14C isotope effect in the pyruvate carboxylase (EC 6.4.1.1) reaction (14C/12C = 0.992 +/- 0.008; S.E.M., n = 8); the ratio of labelling by [2-14C]- to that by [1-14C]-pyruvate of liver glutamate 1.5 min after injection. This ratio, 3.38, is a measure of the disequilibrium in the mitochondria between malate and oxaloacetate. The data were analysed with due regard to experimental variance, uncertainties in values of fluxes measured in vitro, hepatic heterogeneity and renal glucose output. The following conclusions were reached. The results could not be explained if CO2 fixation was confined to pyruvate carboxylase and there was only one, well-mixed, pool of oxaloacetate in the mitochondria. Addition of the other carboxylation reactions, those of PEPCK, isocitrate dehydrogenase (EC 1.1.1.42) and malic enzyme (EC 1.1.1.40), was not enough. Incomplete mixing of mitochondrial oxaloacetate had to be assumed, i.e. that there was metabolic channelling of oxaloacetate formed from pyruvate towards gluconeogenesis. There was some evidence that malate exchange across the mitochondrial membrane might also be channelled, with incomplete mixing with that in the citrate cycle. Calculated rates of exchange of CO2 by PEPCK were in agreement with those measured in vitro, with little or no activation by Fe2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)     

The inhibition by fluorocitrate of rat liver mitochondrial and extramitochondrial aconitate hydratase

1. The effects of synthetic fluorocitrate were studied on: (a) the oxidation of citrate and cis-aconitate by rat liver mitochondria; (b) the activity of the aconitate hydratase found in the liver cell sap; (c) the activity of the aconitate hydratase solubilized from liver mitochondria. 2. Fluorocitrate was found to be a potent inhibitor of oxidation of citrate but only a weak inhibitor of oxidation of cis-aconitate: 6.7mum-fluorocitrate (containing 4% of the inhibitory isomer) caused 94% inhibition of the oxidation of citrate (2mm) whereas 1.0mm-fluorocitrate was necessary to provoke the same inhibition when cis-aconitate (2mm) was the substrate. The degree of inhibition varied in relation to the respiratory state of mitochondria when fluorocitrate was added. The inhibition could be partially reversed by cis-aconitate. 3. The aconitate hydratase extracted from the mitochondria was much less inhibited by fluorocitrate than was the mitochondria-bound enzyme, and the aconitate hydratase found in the cell sap was even less sensitive. 0.3mm-Fluorocitrate was required to cause 50% inhibition of the reaction citrate-->cis-aconitate, catalysed by the aconitate hydratase extracted from the mitochondria, and 1.2m-fluorocitrate for the extramitochondrial enzyme. For both enzymes the reaction citrate-->cis-aconitate was 2-3 times more sensitive to fluorocitrate than was the reaction isocitrate-->cis-aconitate. The inhibition was of the competitive type for both reactions.     

Catalytic Properties of Cytoplasmic and Mitochondrial Aconitate Hydratase from Rat Cardiomyocytes

Enzymatic activity of aconitate hydratase (aconitase, EC 4.2.1.3) from the rat heart is localized in the cytoplasm (65%) and mitochondria (35%). Cytoplasmic and mitochondrial forms of aconitate hydratase were separated by ion-exchange chromatography on CM-Cellulose and CM-Sephadex. The two forms have similar molecular weight, optimal pH range, and spectral properties; however, they have different chromatography properties, K _m for citrate and isocitrate, as well as sensitivity to Fe²⁺ ions.     

Analysis of tricarboxylic acid-cycle metabolism of hepatoma cells by comparison of 14CO2 ratios.

The CO2-ratios method is applied to the analysis of abnormalities of TCA (tricarboxylic acid)-cycle metabolism in AS-30D rat ascites-hepatoma cells. This method utilizes steady-state 14CO2-production rates from pairs of tracers of the same compound to evaluate TCA-cycle flux patterns. Equations are presented that quantitatively convert CO2 ratios into estimates of probability of flux through TCA-cycle-related pathways. Results of this study indicated that the ratio of 14CO2 produced from [1,4-14C]succinate to 14CO2 produced from [2,3-14C]succinate was increased by the addition of glutamine (5 mM) to the medium. An increase in the succinate CO2 ratio is quantitatively related to an increased flux of unlabelled carbon into the TCA-cycle-intermediate pools. Analysis of 14C distribution in [14C]citrate derived from [2,3-14C]succinate indicated that flux from the TCA cycle to the acetyl-CoA-derived carbons of citrate was insignificant. Thus the increased succinate CO2 ratio observed in the presence of glutamine could only result from an increased flux of carbon into the span of the TCA cycle from citrate to oxaloacetate. This result is consistent with increased flux of glutamine to alpha-oxoglutarate in the incubation medium containing exogenous glutamine. Comparison of the pyruvate CO2 ratio, steady-state 14CO2 production from [2-14C]pyruvate versus [3-14C]pyruvate, with the succinate 14CO2 ratio detected flux of pyruvate to C4 TCA-cycle intermediates in the medium containing glutamine. This result was consistent with the observation that [14C]aspartate derived from [2-14C]pyruvate was labelled in C-2 and C-3. 14C analysis also produced evidence for flux of TCA-cycle carbon to alanine. This study demonstrates that the CO2-ratios method is applicable in the analysis of the metabolic properties of AS-30D cells. This methodology has verified that the atypical TCA-cycle metabolism previously described for AS-30D-cell mitochondria occurs in intact AS-30D rat hepatoma cells.     

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.     

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger.

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of Pi and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added 14CO2 was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this was oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.