Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.     

The redistribution of carbon label by the reactions involved in glycolysis, gluconeogenesis and the tricarboxylic acid cycle in rat liver

A scheme is presented that shows how the reactions involved in gluconeogenesis, glycolysis and the tricarboxylic acid cycle are linked in rat liver. Equations are developed that show how label is redistributed in aspartate, glutamate and phosphopyruvate when it is introduced as specifically labelled pyruvate or glucose either at a constant rate (steady-state theory) or at a variable rate (non-steady-state theory). For steady-state theory the fractions of label introduced as specifically labelled pyruvate that are incorporated into glucose and carbon dioxide are also given, and for both theories the specific radioactivities of aspartate and glutamate relative to the specific radioactivity of the substrate. The theories allow for entry of label into the tricarboxylic acid cycle via both oxaloacetate and acetyl-CoA, for (14)CO(2) fixation and for loss of label from the tricarboxylic acid cycle in glutamate, but not for losses in citrate. They also allow for incomplete symmetrization of label in oxaloacetate due to incomplete equilibration with fumarate both in the extramitochondrial part of the cell and in the mitochondrion on entry of oxaloacetate into the tricarboxylic acid cycle. In the latter case failure both of oxaloacetate to equilibrate with malate and of malate to equilibrate with fumarate are considered.     

Nonfunctional Tricarboxylic Acid Cycle and the Mechanism of Glutamate Biosynthesis in Acetobacter suboxydans

Acetobacter suboxydans does not contain an active tricarboxylic acid cycle, yet two pathways have been suggested for glutamate synthesis from acetate catalyzed by cell extracts: a partial tricarboxylic acid cycle following an initial condensation of oxalacetate and acetyl coenzyme A. and the citramalate-mesaconate pathway following an initial condensation of pyruvate and acetyl coenzyme A. To determine which pathway functions in growing cells, acetate-1-(14)C was added to a culture growing in minimal medium. After growth had ceased, cells were recovered and fractionated. Radioactive glutamate was isolated from the cellular protein fraction, and the position of the radioactive label was determined. Decarboxylation of the C5 carbon removed 100% of the radioactivity found in the purified glutamate fraction. These experiments establish that growing cells synthesize glutamate via a partial tricarboxylic acid cycle. Aspartate isolated from these hydrolysates was not radioactive, thus providing further evidence for the lack of a complete tricarboxylic acid cycle. When cell extracts were analyzed, activity of all tricarboxylic acid cycle enzymes, except succinate dehydrogenase, was demonstrated.     

Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.     

Metabolic compartmentation of pyruvate in the isolated perfused rat heart.

1. Prompted by the finding of markedly differing specific radioactivities of tissue alanine and lactate in isolated rat hearts perfused with [1-14C]pyruvate, a more detailed study on the cytosolic subcompartmentalization of pyruvate was undertaken. Isolated rat hearts were perfused by the once-through Langendorff technique under metabolic and isotopic steady-state conditions but with various routes of radioactive label influx, and the specific radioactivities of pyruvate, lactate and alanine were determined. An enzymic method was devised to determine the specific radioactivity of C-1 of pyruvate. 2. Label introduction as [1-14C]pyruvate resulted in a higher specific radioactivity of tissue alanine and mitochondrial pyruvate than of lactate, and a higher specific radioactivity of perfusate lactate than of tissue lactate. Label introduction as [1-14C]lactate resulted in a roughly similar isotope dilution into the tissue and perfusate pyruvate and the tissue alanine. Label introduction as [3,4-14C]glucose resulted in the same specific radioactivity of tissue lactate and alanine and a roughly similar specific radioactivity of mitochondrial pyruvate. 3. The results can be reconciled with a metabolic model containing two cytosolic functional pyruvate pools. One pool (I) communicates more closely with the glycolytic system, whereas the other (II) communicates with extracellular pyruvate and intracellular alanine. Pool II is in close connection with intramitochondrial pyruvate. The physical identity of the cytosolic subcompartments of pyruvate is discussed.     

The interaction of glycolysis, gluconeogenesis and the tricarboxylic acid cycle in rat liver in vivo

1. The equations derived by Heath (1968) were applied to data from experiments on rats in four metabolic states: fed, post-absorptive, starved and 2hr. after an eventually lethal injury. The data used were: (a) The fractions of label injected as C1-, C2- and C3-pyruvate (where the prefix indicates the position of labelling) that are incorporated into carbon dioxide and glucose in post-absorptive and injured rats (yields). Yields could be corrected to yields on label taken up by the liver. (b) The (C5-label in glutamate)/(total label in glutamate) ratio in the liver after C2-pyruvate in rats in all four states. (c) The distribution of label within glutamate after C2-pyruvate or C2-alanine in the livers of fed, post-absorptive and starved rats. (d) The distribution of label within glucose after C2-lactate or C2-pyruvate in starved rats. (e) The relative specific radioactivities of pyruvate, aspartate, glutamate and (in two states only) of glucose 6-phosphate after injection of [U-(14)C]glucose into rats in all four states. These data were previously published, except those after (e) and some after (b) above, which are given in this paper. 2. In addition the concentrations of pyruvate, citrate, glutamate and aspartate in the livers of post-absorptive and injured rats were found. Injury decreased glutamate and citrate concentrations and to a smaller extent aspartate and pyruvate concentrations. 3. Non-steady-state theory showed that most of the data could be used without serious error in steady-state theory. Steady-state theory correlated all but one observation (the relative yields of (14)CO(2) from C2- and C3-pyruvate) listed after (a)-(e) above within the experimental errors, and gave rough estimates of the rates of pyruvate carboxylation, conversion of pyruvate and fat into acetyl-CoA and utilization of glutamate. The main conclusions were: (a) symmetrization of label in oxaloacetate both in the mitochondrion and in the cytoplasm was far from complete, because oxaloacetate did not equilibrate with fumarate in either. From this and other findings it was deduced: (b) that malate or fumarate or both left the mitochondrion, and not oxaloacetate; (c) that there was a loss from the mitochondrion of a fraction of the malate or fumarate or both formed from succinate, and (d) the resulting deficiency of oxaloacetate for the perpetuation of the tricarboxylic acid cycle was made up from pyruvate in fed and post-absorptive rats, but (e) in the starved rat could only be made up by utilization of glutamate. (f) In the fed rat the tricarboxylic acid cycle ran mostly on pyruvate, but in the post-absorptive and starved rat mostly on fat. (g) In the injured rat the tricarboxylic acid cycle was slowed, label in oxaloacetate was completely symmetrized (cf. conclusion a), and the tricarboxylic acid cycle utilized glutamate. (h) The conclusions were not invalidated by isotopic exchange, i.e. flux of label without net flux of compound, nor by interaction with lipogenic processes. (i) In the kidneys interaction between the tricarboxylic acid cycle and gluconeogenesis was different from in the liver, and was much less. The effects on the theory were roughly assessed, and were small. 4. The experiments and optimum experimental conditions required to check the theory are listed, and several predictions, open to experimental confirmation, are made.     

13C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver

13C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the 13C enrichments at the individual carbons of glutamate when [3-13C]alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of 13C label into fatty acids was monitored in this liver preparation. Livers were perfused under steady-state conditions with labeled substrates that are converted to either [2-13C]acetyl-CoA or [1-13C]acetyl-CoA, which in the de novo synthesis pathway label alternate carbons in fatty acids. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by [1-13C]acetyl-CoA (from [2-13C]pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by [3-13C]alanine plus [2-13C]ethanol, which are converted to [2-13C]acetyl-CoA. Thus, measurement of 13C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 13C-n.m.r. investigation of the metabolism of amino acids in renal proximal convoluted tubules of normal and streptozotocin-treated rats and rabbits.

13C-n.m.r. spectroscopy was used to determine the metabolic fate of alanine and aspartate in rat and rabbit kidney proximal tubules. The main purpose of the present study was to investigate the effect of streptozotocin-induced diabetes on the influx of 13C label from [3-13C]alanine into the tricarboxylic acid cycle and through the fructose-1,6-bisphosphatase pathway. This influx was calculated from the relative enrichment of 13C in the various glutamate and glutamine carbon atoms. The relative proportion of 13C label which entered the tricarboxylic acid cycle via pyruvate carboxylase relative to the proportion that entered via pyruvate dehydrogenase was 1.92 +/- 0.02 in fed control rats and 2.27 +/- 0.04 in streptozotocin-treated rats. However, streptozotocin-induced diabetes did not significantly affect this ratio in rabbit proximal convoluted tubular cells. Only in rat proximal convoluted tubular cells did we observe an increase in flux through the fructose-1,6-bisphosphatase pathway by streptozotocin treatment compared with fed controls. The data suggest that streptozotocin-induced diabetes in rats causes the same metabolic changes as does chronic acidosis.     

Factors Affecting the Pathways of Glucose Catabolism and the Tricarboxylic Acid Cycle in Pseudomonas natriegens

Less than 50% of theoretical oxygen uptake was observed when glucose was dissimilated by resting cells of Pseudomonas natriegens. Low oxygen uptakes were also observed when a variety of other substrates were dissimilated. When uniformly labeled glucose-(14)C was used as substrate, 56% of the label was shown to accumulate in these resting cells. This material consisted, in part, of a polysaccharide which, although it did not give typical glycogen reactions, yielded glucose after its hydrolysis. Resting cells previously cultivated on media containing glucose completely catabolized glucose and formed a large amount of pyruvate within 30 min. Resting cells cultivated in the absence of glucose catabolized glucose more slowly and produced little pyruvate. Pyruvate disappeared after further incubation. In this latter case, experimental results suggested (i) that pyruvate was converted to other acidic products (e.g., acetate and lactate) and (ii) that pyruvate was further catabolized via the tricarboxylic acid cycle. Growth on glucose repressed the level of key enzymes of the tricarboxylic acid cycle and of lactic dehydrogenase. Growth on glycerol stimulated the level of these enzymes. A low level of isocitratase, but not malate synthetase, was noted in extracts of glucose-grown cells. Isocitric dehydrogenase was shown to require nicotinamide adenine dinucleotide phosphate (NADP) as cofactor. Previous experiments have shown that reduced NADP (NADPH(2)) cannot be readily oxidized and that pyridine nucleotide transhydrogenase could not be detected in extracts. It was concluded that acetate, lactate, and pyruvate accumulate under growing conditions when P. natriegens is cultivated on glucose (i) because of a rapid initial catabolism of glucose via an aerobic glycolytic pathway and (ii) because of a sluggishly functioning tricarboxylic acid cycle due to the accumulation of NADPH(2) and to repressed levels of key enzymes.     

Pyruvate metabolism in Halobacterium salinarium studied by intracellular 13C nuclear magnetic resonance spectroscopy.

13C nuclear magnetic resonance spectroscopy was used to study the metabolism of [2-13C]pyruvate in intact cells of Halobacterium salinarium. The spectra of these cells show that pyruvate is reduced to lactic acid and transaminated to alanine. The intensity of C-2 lactate is higher under anaerobic conditions than under aerobic conditions. When cells are grown in the absence of glucose, the level of C-2 lactate intensity is lower. In extracts of these cells, the level of NADH-dependent lactate dehydrogenase activity is lower than that of cells grown in the presence of glucose. A C-5 glutamate resonance suggests the entry of pyruvate into the tricarboxylic acid cycle through acetyl-coenzyme A. In addition, the label is also observed at C-3 and C-4 of glutamate, signifying a pyruvate carboxylase-type reaction and scrambling of label at the fumarate-succinate stage plus malic enzyme operation, respectively. Citrate synthase and malic enzyme activity appear to be controlled by the growth conditions of H. salinarium.     

A model for measurement of lactate disappearance with isotopic tracers in the steady state.

1. The irreversible disappearance of lactate carbon from the body (RdL) is commonly calculated from data obtained with a continuous infusion of isotopically labelled lactate tracer. The tracer infusion rate divided by the steady-state lactate specific radioactivity in blood is taken to give the rate of lactate disappearance. 2. Measurement of lactate disappearance is complicated by the fact that it is reversibly converted into pyruvate as well as being irreversibly removed from the system. 3. We analysed a four-compartment model of lactate metabolism, representing blood lactate, tissue lactate and pyruvate carbon pools. 4. The standard method of calculating RdL from the lactate tracer infusion rate divided by the specific radioactivity of lactate was not validated. 5. We found that RdL can be calculated from the infusion rate and the pyruvate specific radioactivity, multiplied by the fraction of the total carbon flow out of pyruvate that goes to lactate. 6. Therefore, if almost all of the pyruvate carbon flows back to lactate, then RdL approaches the tracer infusion rate divided by the pyruvate specific radioactivity. On the other hand, if the rate of oxidation is large in relation to the rate of pyruvate conversion into lactate, than RdL is overestimated when calculated from the pyruvate specific radioactivity. 7. Calculation of RdL with the arterial lactate specific radioactivity results in an underestimate of the true RdL.     

Influence of tricarboxylic acid cycle intermediates and related metabolites on the biosynthesis of aflatoxin by resting cells of Aspergillus flavus.

Resting cells of Aspergillus flavus synthesized aflatoxin from acetate as the sole carbon source after 36 h of incubation. Addition of pyruvate (5.5 mg/m) as cosubstrate to [1-14C]acetate and unlabeled acetate considerably reduced toxin production but increased the radioactivity on the tricarboxylic acid intermediates. This suggests that high tricarboxylic acid activity drastically affected toxin synthesis.     

Metabolism of propionate by sheep liver: Pathway of propionate metabolism in aged homogenate and mitochondria

Experiments were conducted with aged nuclear-free homogenate of sheep liver and aged mitochondria in an attempt to measure both the extent of oxidation of propionate and the distribution of label from [2-¹⁴C]propionate in the products. With nuclear-free homogenate, propionate was 44% oxidized with the accumulation of succinate, fumarate, malate and some citrate. Recovery of ¹⁴C in these intermediates and respiratory carbon dioxide was only 33%, but additional label was detected in endogenous glutamate and aspartate. With washed mitochondria 30% oxidation of metabolized propionate occurred, and proportionately more citrate and malate accumulated. Recovery of ¹⁴C in dicarboxylic acids, citrate, α-oxoglutarate, glutamate, aspartate and respiratory carbon dioxide was 91%. The specific activities of the products and the distribution of label in the carbon atoms of the dicarboxylic acids were consistent with the operation solely of the methylmalonate pathway together with limited oxidation of the succinate formed by the tricarboxylic acid cycle via pyruvate. In a final experiment with mitochondria the label consumed from [2-¹⁴C]propionate was entirely recovered in the intermediates of the tricarboxylic acid cycle, glutamate, aspartate, methylmalonate and respiratory carbon dioxide.     

The pathway of glycollate utilization in Chlorella pyrenoidosa

1. Exogenous glycollate was rapidly metabolized in both the light and the dark by photoautotrophically grown Chlorella pyrenoidosa. 2. The incorporation of (14)C from [1-(14)C]glycollate by these cells was inhibited by the tricarboxylic acid-cycle inhibitors monofluoroacetate, diethylmalonate and arsenite, and also by alpha-hydroxypyrid-2-ylmethanesulphonate and isonicotinylhydrazine. 3. Short-term kinetic experiments showed over 80% of the total (14)C present in the soluble fraction from the cells to be in glycine and serine after 10s. This percentage decreased with time whereas the percentage radioactivity in glycerate increased for up to 30s then remained steady. The percentage of the total radioactivity present in citrate increased over the experimental period. Malate was the only other tricarboxylic acid-cycle intermediate to become labelled. 4. The kinetic and inhibitor experiments supported the following pathway of glycollate incorporation: glycollate --> glyoxylate --> glycine --> serine --> hydroxypyruvate --> glycerate --> 3-phosphoglycerate --> 2-phosphoglycerate --> phosphoenolpyruvate --> pyruvate --> acetyl-CoA. 5. The specific activities of the enzymes catalysing this metabolic sequence in cell-free extracts were great enough to account for the observed rate of glycollate metabolism of 0.25mumol/h per mg dry wt. of cells in the light.     

A study on the precursors of the acetyl moiety of acetylcholine in brain slices. Observations on the compartmentalization of the acetyl-coenzyme A pool

1. A method was devised for the determination of the specific radioactivity of the acetyl moiety of acetylcholine synthesized from various (14)C-labelled substrates. 2. The precursor for the acetyl moiety of acetylcholine was studied in slices of striatum and cerebral cortex from rat and guinea-pig brain. Incorporation of radioactivity into acetylcholine was determined after incubating the slices in the presence of [2-(14)C]acetate, [(14)C]bicarbonate, [1,5-(14)C]citrate, dl-[1- or 5-(14)C]glutamate or [1- or 2-(14)C]pyruvate. 3. After incubation for 1h, acetylcholine was accumulated significantly in both striatum slices (4.1nmol/mg of protein) and cerebral-cortex slices (0.57nmol/mg of protein) from the rat. Final concentrations were about 11 and 5 times respectively the initial values. 4. With slices from rat striatum, rat cerebral cortex and guinea-pig cerebral cortex, the specific radioactivity of acetylcholine derived from [2-(14)C]pyruvate was very high, reaching approx. 30, 20 and 6% respectively of the initial specific radioactivity of added pyruvate in the medium. With the striatum slices this high value was reached after incubation for 15min. Incorporation of radioactivity from [2-(14)C]acetate was only 1.25, 5.3 and 19.7% of that from [2-(14)C]pyruvate in rat striatum, rat cerebral-cortex and guinea-pig cerebral-cortex slices respectively. A small but definite incorporation was found from [5-(14)C]glutamate. No incorporation was found from the other substrates. The findings suggest that pyruvate is the most important precursor for the synthesis of the acetyl moiety of acetylcholine in brain slices. 5. The specific radioactivity of acetylcholine relative to that of citrate when [2-(14)C]pyruvate was used compared with that obtained when [2-(14)C]acetate was used. A marked difference was found in all slices, suggesting metabolic compartmentation of the acetyl-CoA pool.     

Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.     

In vivo 13C NMR study of the bidirectional reactions of the Wood-Werkman cycle and around the pyruvate node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici

This study used in vitro 13C NMR spectroscopy to directly examine bidirectional reactions of the Wood-Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-13C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 micromol min(-1) g(-1) (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood-Werkman cycle was evident from the formation of intermediates such as [3-13C]pyruvate and [3-13C]malate and of products like [2-13C]acetate from [2-13C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-13C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.     

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.     

Pyruvate carboxylase: affinity labelling of the pyruvate binding site.

The active-site-directed reagent, bromopyruvate has been used to covalently label the pyruvate binding site of pyruvate carboxylase (E.C.6.4.1.1.) isolated from sheep liver. Oxalo-acetate proved to be the most effective reaction component in protecting the enzyme against inactivation; pyruvate was less effective although its efficiency was enhanced by the presence of acetyl CoA. The other reaction components, MgATP^2? and HCO₃^? failed to protect the enzyme against inactivation. Using bromo[2¹⁴C]pyruvate, it was shown that at 100% inactivation, 1.5 pyruvyl residues were bound per mole of biotin and when the reaction was carried out in the presence of acetyl CoA, this ratio was reduced to 1.0. Analysis of pronase digests of the enzyme revealed that more than 90% of the radioactivity was present as carboxy-hydroxyethyl cysteine.     

The CO2 assimilation via the reductive tricarboxylic acid cycle in an obligately autotrophic,aerobic hydrogen-oxidizing bacterium,Hydrogenobacter thermophilus

The incorporation of ¹⁴CO₂ by the cell suspensions of an extremely thermophilic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus was studied. After short time incubation of the cell suspensions with ¹⁴CO₂, the radiactivity was initially present in aspartate, glutamate, succinate, phosphorylated compounds, citrate, malate and fumarate. All of these compounds except phosphorylated compounds were related to the members of the tricarboxylic acid cycle. The proportion of labelled aspartate onglutamate in total radioactivity on each chromatogram decreased with incubation time, while the percentage of the radioactivity incorporated in phosphorylated compounds increased with time up to 10 s. These indicated that aspartate and glutamate is derived from primary products of CO₂ fixation.In cell-free extracts of Hydrogenobacter thermophilus, the two key enzymes in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase could not be detected. The key enzymes of the reductive tricarboxylic acid cycle, fumarate reductase and ATP citrate lyase were present. Activities of phosphoenolpyruvate synthetase and pyruvate carboxylase were also detected. The referse reactions (dehydrogenase reactions) of -ketoglutarate synthase and pyruvate synthase could be detected by using methyl viologen as an electron acceptor.These findings strongly suggested that a new type of the reductive tricarboxylic acid cycle operated as the CO₂ fixation pathway in Hydrogenobacter thermophilus.