Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z

Actinobacillus sp. 130Z fermented glucose to the major products succinate, acetate, and formate. Ethanol was formed as a minor fermentation product. Under CO₂-limiting conditions, less succinate and more ethanol were formed. The fermentation product ratio remained constant at pH values from 6.0 to 7.4. More succinate was produced when hydrogen was present in the gas phase. Actinobacillus sp. 130Z grew at the expense of fumarate and l-malate reduction, with hydrogen as an electron donor. Other substrates such as more-reduced carbohydrates (e.g., d-sorbitol) resulted in higher succinate and/or ethanol production. Actinobacillus sp. 130Z contained the key enzymes involved in the Embden-Meyerhof-Parnas and the pentose-phosphate pathways and contained high levels of phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, pyruvate kinase, pyruvate formate-lyase, phosphotransacetylase, acetate kinase, malic enzyme, and oxaloacetate decarboxylase. The levels of PEP carboxykinase, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. 130Z than in Escherichia coli K-12 and accounted for the differences in succinate production. Key enzymes in end product formation in Actinobacillus sp. 130Z were regulated by the energy substrates. Received: 2 September 1996 / Accepted: 10 January 1997     

Pathway of propionate formation in Desulfobulbus propionicus

Whole cells of Desulfobulbus propionicus fermented [1-¹³C]ethanol to [2-¹³C] and [3-¹³C]propionate and [1-¹³C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate     

Pathway and sites for energy conservation in the metabolism of glucose by Selenomonas ruminantium.

On the basis of enzyme activities detected in extracts of Selenomonas ruminantium HD4 grown in glucose-limited continuous culture, at a slow (0.11 h-1) and a fast (0.52 h-1) dilution rate, a pathway of glucose catabolism to lactate, acetate, succinate, and propionate was constructed. Glucose was catabolized to phosphoenol pyruvate (PEP) via the Emden-Meyerhoff-Parnas pathway. PEP was converted to either pyruvate (via pyruvate kinase) or oxalacetate (via PEP carboxykinase). Pyruvate was reduced to L-lactate via a NAD-dependent lactate dehydrogenase or oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA) and CO2 by pyruvate:ferredoxin oxidoreductase. Acetyl-CoA was apparently converted in a single enzymatic step to acetate and CoA, with concomitant formation of 1 molecule of ATP; since acetyl-phosphate was not an intermediate, the enzyme catalyzing this reaction was identified as acetate thiokinase. Oxalacetate was converted to succinate via the activities of malate dehydrogenase, fumarase and a membrane-bound fumarate reductase. Succinate was then excreted or decarboxylated to propionate via a membrane-bound methylmalonyl-CoA decarboxylase. Pyruvate kinase was inhibited by Pi and activated by fructose 1,6-bisphosphate. PEP carboxykinase activity was found to be 0.054 mumol min-1 mg of protein-1 at a dilution rate of 0.11 h-1 but could not be detected in extracts of cells grown at a dilution rate of 0.52 h-1. Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase. Possession of these five sites for energy conservation may explain the high yields reported here (56 to 78 mg of cells [dry weight] mol of glucose-1) for S. ruminantium HD4 grown in glucose-limited continuous culture.     

Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitata.

Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.     

Channeling of TCA cycle intermediates in cultured Saccharomyces cerevisiae

Oxidation of [3-13C]propionate was studied in cultured yeast cells, and the distribution of label in the 2- and 3-positions of alanine was detected by 13C NMR. [3-13C]Propionate forms [2-13C]succinyl-CoA in the mitochondria which then enters the citric acid cycle and forms malate through two symmetrical intermediates, succinate and fumarate. If these symmetrical intermediates randomly diffuse from one enzyme to the next in mitochondria as is normally assumed, then 13C labeling in malate C2 and C3 must be equal. However, any direct transfer of metabolites from site to site between succinate thiokinase, succinate dehydrogenase, and fumarase would result in an uneven distribution of 13C in malate C2 and C3 and any molecules derived from malate. Since pyruvate may be derived from malate via the malic enzyme and subsequently converted into alanine by transamination, any 13C asymmetry in alanine C2 and C3 must directly reflect the 13C distribution in the malate pool. During oxidation of [3-13C]propionate, we detect a significant quantity of labeled alanine, where 13C enrichment in C3 is significantly higher than that in C2. Inhibition of succinate dehydrogenase with malonate or creating conditions that increase the chances of a back-reaction (from malate to fumarate) result in a significant decrease in the asymmetric labeling of alanine. Ubiquinone-deficient yeast cells (having only 10% of the oxidative capacity of wild-type cells) could slowly oxidize propionate, but in this case the 13C labeling was equal in the C2 and C3 of alanine, showing that isotope randomization had occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactate Metabolism by Veillonella parvula

A strain of Veillonella parvula M4, which grows readily in lactate broth without a requirement for carbon dioxide, has been isolated from the oral cavity. Anaerobic, washed cells of this organism fermented sodium lactate to the following products (moles/100 moles of lactate): propionate, 66; acetate, 40; carbon dioxide, 40; and hydrogen, 14. Cells grew readily in tryptone-yeast extract broth with pyruvate, oxaloacetate, malate, and fumarate, but poorly with succinate. The fermentation of pyruvate, oxaloacetate, or lactate plus oxaloacetate by washed cells resulted in the formation of propionate and acetate in ratios significantly lower than those observed with lactate as the sole carbon source. This was primarily due to increased acetate production. Cell-free extracts were unable to degrade lactate but metabolized lactate in the presence of oxaloacetate, indicating the presence of malic-lactic transhydrogenase in this organism. Lactic dehydrogenase activity was not observed. Evidence is presented for oxaloacetate decarboxylase and malic dehydrogenase activities in extracts.     

Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo

1. The aim of this work was to investigate the role of phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) in the conversion of fat to sugar by the cotyledons of seedlings of Cucurbita pepo. 2. The enzyme was partially purified from the cotyledons of 5-day-old seedlings. The Michaelis constants for oxaloacetate and ATP were 56 and 119 micron, respectively. The decarboxylation reaction was optimum at pH 7.4. A range of intermediary metabolites did not affect the activity of the enzyme, but 3-mercaptopicolinic acid at micron concentrations was an effective inhibitor. 3. Centrifugation of extracts of 5-day-old cotyledons sedimented appreciable proportions of the ribuloseibisphosphate carboxylase, isocitrate lyase and fumarate hydratase present but very little of the phosphoenolpyruvate carboxykinase. 4. Measurements of phosphoenolpyruvate carboxykinase of cotyledons during germination showed that the maximum catalytic activity exceeded, and changed coincidently with, the rate of gluconeogenesis. 5. 3-Mercaptopicolinic acid inhibited gluconeogenesis from [1-14C]- and [2-14C]acetate supplied to excised cotyledons. The detailed distribution of 14C indicated inhibition of the conversion of oxaloacetate to phosphoenolpyruvate. 6. It is concluded that in marrow cotyledons phosphoenolpyruvate carboxykinase is in the soluble phase of the cytoplasm and catalyses a component reaction of gluconeogenesis.     

Regulation of gluconeogenesis and lipogenesis. The regulation of mitochondrial pyruvate metabolism in guinea-pig liver synthesizing precursors for gluconeogenesis

1. The carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase in guinea-pig liver mitochondria was determined by measuring the amount of (14)C from H(14)CO(3) (-) fixed into organic acids in the presence of pyruvate, ATP, Mg(2+) and P(i). The main products of pyruvate carboxylation were malate, fumarate and citrate. Pyruvate utilization, metabolite formation and incorporation of (14)C from H(14)CO(3) (-) into these metabolites in the presence and the absence of ATP were examined. The synthesis of phosphoenolpyruvate from pyruvate and bicarbonate is minimal during continued oxidation of pyruvate. Larger amounts of phosphoenolpyruvate are formed from alpha-oxoglutarate than from pyruvate. Addition of glutamate, alpha-oxoglutarate or fumarate did not appreciably increase formation of phosphoenolpyruvate when pyruvate was used as substrate. With alpha-oxoglutarate as substrate addition of fumarate resulted in increased formation of phosphoenolpyruvate, whereas addition of succinate inhibited phosphoenolpyruvate formation. In the presence of added oxaloacetate guinea-pig liver mitochondria synthesized phosphoenolpyruvate in amount sufficiently high to play an appreciable role in gluconeogenesis. 2. Addition of fatty acids of increasing carbon chain length caused a strong inhibition of pyruvate oxidation and phosphoenolpyruvate formation, and greatly promoted carbon dioxide fixation and malate, citrate and acetoacetate accumulation. The incorporation of (14)C from H(14)CO(3) (-), [1-(14)C]pyruvate and [2-(14)C]pyruvate into organic acids formed was examined. 3. It is concluded that guinea-pig liver pyruvate carboxylase contributes significantly to gluconeogenesis and that fatty acids and metabolites play an important role in its regulation.     

RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

The effect of glucagon on the carbon flux from palmitate into glucose, lactate and ketone bodies, studied with isolated hepatocytes

Glucagon induced a rapid (within 3 min) increase in glucose radioactivity and a decrease in the labeling of ketone bodies when isolated hepatocytes were incubated in the presence of [1-14C]palmitate. Simultaneously, the hormone induced a decrease in the levels of pyruvate and Krebs cycle intermediates and an increase in the level of phosphoenolpyruvate (PEP). The glucagon-induced increase in glucose radioactivity was much larger than the simultaneous decrease in lactate labeling. A comparison of the incorporation of labeled carbon from [1-14C]palmitate and [U-14C]palmitate into glucose and CO2 indicates a selective stimulatory action of glucagon on the flux through the phosphoenolpyruvate carboxykinase (PEPCK) reaction.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.     

Effect of phenylephrine on pyruvate dehydrogenase in fasting rat livers

Previous estimates of flux through the pyruvate-dehydrogenase complex were made by measuring 14CO2 generated from oxidation of [1-14C]pyruvate, assuming a 1:1 stoichiometry. However, this method fails to discriminate between 14CO2 produced from pyruvate dehydrogenase and 14CO2 generated from phospho-enolpyruvate carboxykinase and citric-acid-cycle dehydrogenases. While some previous reports have attempted to correct for the additional 14CO2 production by comparing 14CO2 generated by [1-14C]pyruvate with [2-14C]pyruvate or [3-14C]pyruvate, the estimates are flawed by failure to determine the radioactivity and distribution of the 14C label in the oxalacetate pool. The present method circumvents these problems by utilizing [1,4-14C]succinate to radiolabel the oxalacetate pool and by directly measuring the specific radioactivity of malate. The results demonstrate that flux through the pyruvate-dehydrogenase complex is negligible compared to the other reactions which generate 14CO2 from [1-14C]lactate in the fasted state. Phenylephrine did not significantly alter this result in the fasted state. However, 14CO2 production via the pyruvate-dehydrogenase complex is large (approximately 11.5 nmol.min-1.mg mitochondrial protein-1) compared to 14CO2 production via phosphoenolpyruvate carboxykinase and citric-acid-cycle dehydrogenases (approximately 6.4 nmol.min-1.mg-1) when the pyruvate-dehydrogenase complex is activated, in the fed state with 1 mM dichloroacetate.     

Physiological and biochemical properties of the alkaliphilic anaerobic hydrolytic bacterium <Emphasis Type="Italic">Alkaliflexus imshenetskii</Emphasis>

The growth of Alkaliflexus imshenetskii and concentrations of metabolites produced by this microorganism during growth on various organic substrates were studied. It was shown that, although the composition and quantitative ratios of the fermentation products depended on the substrates utilized, acetate and succinate were always the major metabolites, while only minor amounts of formate were produced. During growth on xylan and starch, diauxy was observed caused by the successive decomposition of oligosaccharides and monosaccharides. It was demonstrated that, when grown on cellobiose, A. imshenetskii is capable of succinate fermentation mediated by phosphoenolpyruvate carboxykinase, pyruvate kinase, fumarate reductase, pyruvate ferredoxin oxidoreductase, malate dehydrogenase, and methylmalonyl-CoA decarboxylase. Succinate may be both the intermediate and final product of the A. imshenetskii metabolism, being fermented to propionate by methylmalonyl-CoA decarboxylase.     

Changes in photosynthetic carbon flow in transgenic rice plants that express C4-type phosphoenolpyruvate carboxykinase from Urochloa panicoides

A cDNA encoding phosphoenolpyruvate carboxykinase (PCK) of Urochloa panicoides (a PCK-type C4 plant) was expressed in rice (Oryza sativa cv Tsukinohikari) plants under the control of the promoter of a maize (Zea mays) gene for phosphoenolpyruvate carboxylase or pyruvate, orthophosphate dikinase with the transit peptide of the small subunit of Rubisco. Crude extracts prepared from the green leaves of transgenic plants had high PCK activity and the newly expressed PCK was localized in chloroplasts. In labeling experiments with (14)CO(2) up to 20% of the radioactivity was incorporated into 4C compounds (malate, oxaloacetate, and aspartate) in excised leaves of transgenic plants, as compared with about 1% in excised leaves of control plants. There was a positive correlation between PCK activity and the extent of labeling of 4C compounds. When L-[4-(14)C]malate was fed to excised leaves the extent of incorporation of radioactivity into sucrose was 3-fold greater in transgenic plants than in control plants and the level of radiolabeled aspartate was significantly lower in transgenic plants. These results indicate that the ectopic expression of PCK in rice chloroplasts was able partially to change the carbon flow in mesophyll cells into a C4-like photosynthetic pathway. Such a strategy appears to provide a possible method for enhancing the photosynthetic capacity of C3 plants.     

Intracellular distribution of some enzymes of the glutamine utilisation pathway in rat lymphocytes

In lymphocytes of the rat, pyruvate kinase, phosphoenolpyruvate carboxykinase and NADP+-linked malate dehydrogenase (decarboxylating) are distributed almost exclusively in the cytosol whereas pyruvate carboxylase is distributed almost entirely in the mitochondria. For NAD+-linked malate dehydrogenase and aspartate aminotransferase approximately 80% and 40%, respectively, are in the cytosolic compartment. Since glutaminase is present in the mitochondria, glutamine is converted to malate within the mitochondria but further metabolism of the malate is likely to occur in the cytosol. Hence pyruvate produced from this malate, via oxaloacetate and phosphoenolpyruvate carboxykinase, may be rapidly converted to lactate, so restricting the entry of pyruvate into the mitochondria and explaining why very little glutamine is completely oxidised in these cells despite a high capacity of the Krebs cycle.     

Formation of formate and hydrogen, and flux of reducing equivalents and carbon in Ruminococcus flavefaciens Fd-1

A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H₂ in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture. PEP was converted to acetate and CO₂ (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase). Lactate was not formed even during rapid growth (batch culture, µ = 0.35/h). H₂ was formed by a hydrogenase rather than by cleavage of formate, and ¹³C-NMR and¹⁴ C-exchange reaction data indicated that formate was produced by CO₂ reduction, not by a cleavage of pyruvate. The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range. However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the presence of H₂. This shift suggested that reducing equivalents could be balanced through formate or H₂ production without affecting the yields of the major carbon-containing fermentation endproducts.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides

A method has been developed for rapidly preparing bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase-type C4 plant. These cells catalyzed both HCO3(-)- and oxaloacetate-dependent oxygen evolution; oxaloacetate-dependent oxygen evolution was stimulated by ATP. For this activity oxaloacetate could be replaced by aspartate plus 2-oxoglutarate. Both oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution were accompanied by PEP production and both were inhibited by 3-mercaptopicolinic acid, an inhibitor of PEP carboxykinase. The ATP requirement for oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution could be replaced by ADP plus malate. The increased oxygen evolution observed when malate plus ADP was added with oxaloacetate was accompanied by pyruvate production. These results are consistent with oxaloacetate being decarboxylated via PEP carboxykinase. We suggest that the ATP required for oxaloacetate decarboxylation via PEP carboxykinase may be derived by phosphorylation coupled to malate oxidation in mitochondria. These bundle sheath cells apparently contain diffusion paths for the rapid transfer of compounds as large as adenine nucleotides.     

Intermediary carbon metabolism of Azospirillum brasilense.

Azospirillum brasilense Sp 7 grew rapidly in AZO medium containing reduced nitrogen and succinate as an energy source, with a doubling time of 43 min. No growth was measured with glucose as the sole carbon source. In contrast, Azospirillum lipoferum Sp 59b could grow in media containing either succinate or glucose with a doubling time of 69 min and 223 min, respectively. Warburg-Barcroft respirometry showed that the rate of oxygen consumption by A. brasilense Sp 7 on glucose medium (0.034 mumol of O2 min-1 mg-1 of cell protein) was only one-quarter of that on succinate medium (0.14 mumol of O2 min-1 mg-1). Radioisotopic labeling showed that very little glucose was assimilated by A. brasilense Sp 7 as compared to succinate. High respiration rates were measured on A. lipoferum Sp 59b with either succinate (0.15 mumol of O2 min-1 mg-1) or glucose (0.13 mumol of O2 min-1 mg-1) as the sole carbon source. The pattern of CO2 evolution from differentially labeled succinate indicated that A. brasilense Sp 7 had a complete tricarboxylic acid cycle. Assimilation of most of the radioactivity from labeled succinate, pyruvate, and acetate into lipids suggested a strong anabolic metabolism and the presence of an active malic enzyme of phosphoenolpyruvate carboxykinase. The distribution of radioactivity from differentially labeled pyruvate showed that gluconeogenesis competed with pyruvate dehydrogenase. Uptake and incorporation of labeled acetate also indicated the presence of a glyoxylate cycle in A. brasilense Sp 7.     

Isolation of dicarboxylic acid- and glucose-binding proteins from Pseudomonas aeruginosa.

Inducible binding proteins for C4-dicarboxylic acids (DBP) and glucose (GBP) were isolated from Pseudomonas aeruginosa by extraction of exponential-phase cells with 0.2 M MgC12 (pH 8.5) and by an osmotic shock procedure without affecting cell viability. DBP synthesis was induced by growth on aspartate, alpha-ketoglutarate, succinate, fumarate, malate, and malonate but not by growth on acetate, citrate, pyruvate, or glucose. Binding of succinate by DBP was competitively inhibited by 10-fold concentrations of fumarate and malate but not by a variety of related substances. GBP synthesis and transport of methyl alpha-glucoside by whole cells were induced by growth on glucose or pyruvate plus galactose, 2-deoxyglucose, or methyl alpha-glucoside but not by growth on gluconate, succinate, acetate, or pyruvate. The binding of radioactive glucose by GBP was significantly inhibited by 10-fold concentrations of glucose, galactose, and glucose-1-phosphate but not by the other carbohydrates tested. The binding of glucose by GBP or succinate by DBP did not result in any chemical alteration of the substrates.     

Regulation of pyruvate metabolism via pyruvate carboxylase in rat brain mitochondria

1. The fixation of CO(2) by pyruvate carboxylase in isolated rat brain mitochondria was investigated. 2. In the presence of pyruvate, ATP, inorganic phosphate and magnesium, rat brain mitochondria fixed H(14)CO(3) (-) into tricarboxylic acid-cycle intermediates at a rate of about 250nmol/30min per mg of protein. 3. Citrate and malate were the main radioactive products with citrate containing most of the radioactivity fixed. The observed rates of H(14)CO(3) (-) fixation and citrate formation correlated with the measured activities of pyruvate carboxylase and citrate synthase in the mitochondria. 4. The carboxylation of pyruvate by the mitochondria had an apparent K(m) for pyruvate of about 0.5mm. 5. Pyruvate carboxylation was inhibited by ADP and dinitrophenol. 6. Malate, succinate, fumarate and oxaloacetate inhibited the carboxylation of pyruvate whereas glutamate stimulated it. 7. The results suggest that the metabolism of pyruvate via pyruvate carboxylase in brain mitochondria is regulated, in part, by the intramitochondrial concentrations of pyruvate, oxaloacetate and the ATP:ADP ratio.