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.     

Heterotrophic Carbon Metabolism by Beggiatoa alba

The assimilation and metabolism of CO(2) and acetate by Beggiatoa alba strain B18LD was investigated. Although B. alba was shown to require CO(2) for growth, the addition of excess CO(2) (as NaHCO(3)) to the medium in a closed system did not stimulate growth. Approximately 24 to 31% of the methyl-labeled acetate and 38 to 46% of the carboxyl-labeled acetate were oxidized to (14)CO(2) by B. alba. The apparent V(max) values for combined assimilation and oxidation of [2-(14)C]acetate by B. alba were 126 to 202 nmol min(-1) mg of protein(-1) under differing growth conditions. The V(max) values for CO(2) assimilation by heterotrophic and mixotrophic cells were 106 and 131 pmol min(-1) mg of protein(-1), respectively. The low V(max) values for CO(2) assimilation, coupled with the high V(max) values for acetate oxidation, suggested that the required CO(2) was endogenously produced from acetate. Moreover, exogenously supplied acetate was required by B. alba for the fixation of CO(2). From 61 to 73% of the [(14)C]acetate assimilated by washed trichomes was incorporated into lipid. Fifty-five percent of the assimilated [2-(14)C]acetate was incorporated into poly-beta-hydroxybutyric acid. This was consistent with chemical data showing that 56% of the heterotrophic cell dry weight was poly-beta-hydroxybutyric acid. Succinate and CO(2) were incorporated into cell wall material, proteins, lipids, nucleic acids, and amino and organic acids, but not into poly-beta-hydroxybutyric acid. Glutamate and succinate were the major stable products after short-term [1-(14)C]acetate assimilation. Glutamate and aspartate were the first stable (14)CO(2) fixation products, whereas glutamate, a phosphorylated compound, succinate, and aspartate were the major stable (14)CO(2) fixation products over a 30-min period. The CO(2) fixation enzymes isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate; reversed) and malate dehydrogenase (nicotinamide adenine dinucleotide phosphate; decarboxylating) were found in cell-free extracts of both mixotrophically grown and heterotrophically grown cells. The data indicate that the typical autotrophic CO(2) fixation mechanisms are absent from B. alba B18LD and that the CO(2) and acetate metabolism pathways are probably linked.     

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 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 metabolism of glyoxylate by cell-free extracts of Pseudomonas sp

1. Extracts of Pseudomonas sp. grown on butane-2,3-diol oxidized glyoxylate to carbon dioxide, some of the glyoxylate being reduced to glycollate in the process. The oxidation of malate and isocitrate, but not the oxidation of pyruvate, can be coupled to the reduction of glyoxylate to glycollate by the extracts. 2. Extracts of cells grown on butane-2,3-diol decarboxylated oxaloacetate to pyruvate, which was then converted aerobically or anaerobically into lactate, acetyl-coenzyme A and carbon dioxide. The extracts could also convert pyruvate into alanine. However, pyruvate is not an intermediate in the metabolism of glyoxylate since no lactate or alanine could be detected in the reaction products and no labelled pyruvate could be obtained when extracts were incubated with [1-¹⁴C]glyoxylate. 3. The ¹⁴C was incorporated from [1-¹⁴C]glyoxylate by cell-free extracts into carbon dioxide, glycollate, glycine, glutamate and, in trace amounts, into malate, isocitrate and α-oxoglutarate. The ¹⁴C was initially incorporated into isocitrate at the same rate as into glycine. 4. The rate of glyoxylate utilization was increased by the addition of succinate, α-oxoglutarate or citrate, and in each case α-oxoglutarate became labelled. 5. The results are consistent with the suggestion that the carbon dioxide arises by the oxidation of glyoxylate via reactions catalysed respectively by isocitratase, isocitrate dehydrogenase and α-oxoglutarate dehydrogenase.     

One-carbon metabolism in methanogenic bacteria: analysis of short-term fixation products of 14CO2 and 14CH3OH incorporated into whole cells.

Methanobacterium thermoautotrophicum, M. ruminantium, and Methanosarcina barkeri were labeled with 14CO2 (14CO2 + H14CO3- + 14CO32-) for from 2 to 45 s. Radioactivity was recovered in coenzyme M derivatives, alanine, aspartate, glutamate, and several unidentified compounds. The properties of one important structurally unidentified intermediate (yellow fluorescent compound) displayed UV absorbance maxima at pH 1 of 290 and 335 nm, no absorbance in the visible region, and a fluorescence maximum at 460 nm. Label did not appear in organic phosphates until after 1 min. 14CH3OH was converted by M. barkeri primarily into coenzyme M derivatives at 25 s. [2-14C]acetate was assimilated by M. thermoautotrophicum mainly into alanine and succinate during 2 to 240 s, but not into coenzyme M derivatives or yellow fluorescent compound. Cell-free extracts of M. thermoautotrophicum lacked ribulose 1,5-bisphosphate carboxylase activity. The data indicated the absence of the Calvin, serine, and hexulose phosphate paths of C1 assimilation in the methanogens examined and indicated that pyruvate was an early intermediate product of net CO2 fixation. The in vivo importance of coenzyme M derivatives in methanogenesis was demonstrated.     

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

The tricarboxylic acid cycle in Dictyostelium discoideum. Metabolite concentrations, oxygen uptake and 14c-labelled amino acid labelling patterns.

Some aspects of tricarboxylic acid-cycle activity during differentiation and aging in Dictyostelium discoideum were examined. The concentrations of glutamate, aspartate, alanine, citrate, 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, pyruvate and acetyl-CoA were determined at four stages over the course of differentiation. The rate of O2 utilization was also determined over differentiation. In addition, experiments are described in which the specific radioactivities of citrate, 2-oxoglutarate, succinate, fumarate and malate were determined during a 30 min labelling of cells from the preculmination stage of development with [14C]glutamate, [14C]aspartate or [14C]alanine. A similar experiment was also performed with cells from the aggregation stage of development using [14C]glutamate.     

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.     

Pyruvate metabolism by lymphocytes: evidence for an additional ketogenic tissue

The rate of utilization of pyruvate (at various concentrations) was measured in lymphocytes prepared from rat mesenteric lymph nodes. The quantitative contribution of pyruvate to CO2, lactate, aspartate, alanine, citrate, acetate, acetyl-CoA and ketone bodies accounted for the pyruvate metabolized. Pyruvate utilization was depressed by increasing concentrations of pyruvate. The maximum catalytic activities and selected intracellular distributions of the following enzymes of pyruvate, citrate and acetyl-CoA metabolism were measured: citrate synthase, ATP-citrate lyase, lactate dehydrogenase, acetyl-CoA hydrolase, acetylcarnitine transferase, NAD+- and NADP+- isocitrate dehydrogenases, HMG-CoA lyase, HMG-CoA synthase, Pyruvate dehydrogenase, acetoacetyl-CoA thiolase, 3-oxoacid-CoA transferase, 3-hydroxybutyrate dehydrogenase and pyruvate carboxylase. Acetyl-CoA formed from pyruvate did not contribute to the respiratory energy metabolism of resting lymphocytes. Instead acetyl-CoA was converted to acetoacetate by reactions which may favour the pathway catalyzed by acetoacetyl-CoA thiolase and 3-oxoacid-CoA transferase. Acetate, acetyl- and palmitoyl-carnitine inhibited the decarboxylation of [1-14C] pyruvate. These observations may be connected with the suppression of pyruvate utilization by increased pyruvate substrate concentration. Only very small amounts of either pyruvate or acetate were incorporated into lipids in resting lymphocytes. The amounts incorporated were partitioned in approximately the same pattern into FFA, T.G., cholesterol and cholesterol esters. Taken together the data show that pyruvate metabolism is directed inter alia at the formation of acetoacetate which may serve as a lipid synthesis precursor. When pyruvate utilization and metabolism was enhanced by concanavalin A, then acetoacetate formation was not favoured and from this it is proposed that the acetyl units may then be directed into lipid synthesis and may also make a contribution to the energy metabolism of the activated lymphocyte.     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.     

Amino acid oxidation and alanine production in rat hemidiaphragm in vitro. Effects of dichloroacetate.

Dichloroacetate (an activator of pyruvate dehydrogenase) stimulates 14CO2 production from [U-14C]glucose, but not from [U-14C]glutamate, [U-14C]aspartate, [U-14C]- and [1-14C]-valine and [U-14C]- and [1-14C]-leucine. It is concluded (1) that pyruvate dehydrogenase is not rate-limiting in the oxidation to CO2 of amino acids that are metabolized to tricarboxylic acid-cycle intermediates, and (2) that carbohydrate (and not amino acids) is the main carbon precursor in alanine formation in muscle.     

Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barkeri and Methanobacterium thermoautotrophicum

The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

Effects of ischaemia on metabolite concentrations in rat liver

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.     

The conversion of alanine into glutamine in guinea-pig renal cortex. Essential role of pyruvate carboxylase.

1. The metabolism of L-alanine was studied in isolated guinea-pig kidney-cortex tubules. 2. In contrast with previous conclusions of Krebs [(1935) Biochem. J. 29, 1951-1969], glutamine was found to be the main carbon and nitrogenous product of the metabolism of alanine (at 1 and 5 mM). Glutamate and ammonia were only minor products. 3. At neither concentration of alanine was there accumulation of glucose, glycogen, pyruvate, lactate, aspartate or tricarboxylic acid-cycle intermediates. 4. Carbon-balance calculations and the release of 14CO2 from [U-14C]alanine indicate that oxidation of the alanine carbon skeleton occurred at both substrate concentrations. 5. A pathway involving alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, pyruvate carboxylase and enzymes of the tricarboxylic acid cycle is proposed for the conversion of alanine into glutamine. 6. Strong evidence for this pathway was obtained by: (i) suppressing alanine removal by amino-oxyacetate, and inhibitor of transaminases, (ii) measuring the release of 14CO2 from [1-14C]alanine, (iii) the use of L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which induced a large increase in ammonia release from alanine, and (iv) the use of fluoroacetate, an inhibitor of aconitase, which inhibited glutamine synthesis with concomitant accumulation of citrate from alanine. 7. In this pathway, the central role of pyruvate carboxylase, which explains the discrepancy between our results and those of Krebs (1935), was also demonstrated.     

ALTERATION OF TRICARBOXYLIC ACID CYCLE METABOLISM IN RAT BRAIN SLICES BY HALOTHANE

Abstract—

• 1 Metabolism of [2-¹⁴C]pyruvate, [1-¹⁴C]acetate and [5-¹⁴C]citrate in the rat cerebral cortex slices was studied in the presence of halothane. Metabolites assayed include acetylcholine (ACh), citrate, glutamate, glutamine, γ-aminobutyrate (GABA) and aspartate. The trichloroacetic acid soluble extract, the trichloroacetic acid insoluble precipitate and its lipid extract were also studied.
• 2 In control experiments, pyruvate preferentially labelled ACh, citrate, glutamate, GABA and aspartate. Acetate labeled ACh, but to a lesser extent than pyruvate. Acetate also labeled lipids and glutamine. Citrate labeled lipids but not ACh and served as a preferential precursor for glutamine. These data support a three-compartment model for cerebral tricarboxylic acid cycle metabolism.
• 3 Halothane caused increases in GABA and aspartate contents and a decrease in ACh content. It has no effect on the contents of citrate, glutamate and glutamine.
• 4 Halothane preferentially inhibited the metabolic transfer of radioactivity from pyruvate into almost all metabolites, an effect probably not related to pyruvate permeability. This is interpreted as halothane depression of the‘large metabolic compartment’ which includes the nerve endings.
• 5 Halothane increased the metabolic transfer of radioactivity from acetate into lipids but did not alter such a transfer into the trichloracetic acid extract.
• 6 Halothane increased the metabolic transfer of radioactivity from citrate into the trichloroacetic acid precipitate, lipids and especially glutamine. Transfer of citrate radioactivity into GABA was somewhat decreased.
• 7 The differential effects of halothane on acetate and citrate utilization suggest that the ‘small metabolic compartment’ should be subdivided. Therefore, at least three metabolic compartments are demonstrated.
• 8 Halothane did not interfere with the dicarboxylic acid portion of the tricarboxylic acid cycle.

     

Carbohydrate and amino acid metabolism in Tuber borchii mycelium during glucose utilization: a (13)C NMR study

The metabolism of [1-13C]glucose in the vegetative mycelium of the ectomycorrhizal ascomycete Tuber borchii was studied in order to characterize the biochemical pathways for the assimilation of glucose and amino acid biosynthesis. The pathways were characterized using nuclear magnetic resonance spectroscopy in conjunction with [1-13C]glucose labeling. The enzymes of mannitol cycle and ammonium assimilation were also evaluated. The majority of the 13C label was incorporated into mannitol and this polyol was formed via a direct route from absorbed glucose. Amino acid biosynthesis was also an important sink of assimilated carbon and 13C was mainly incorporated into alanine and glutamate. From this intramolecular 13C enrichment, it is concluded that pyruvate, arising from [1-13C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase and pyruvate carboxylase before entering the Krebs cycle. The transfer of 13C-labeled mycelium on [12C]glucose showed that mannitol, alanine, and glutamate carbon were used to synthesize glutamine and arginine that likely play a storage role.     

Pathway of carbon flow during fatty acid synthesis from lactate and pyruvate in rat adipose tissue

The metabolism of pyruvate and lactate by rat adipose tissue was studied. Pyruvate and lactate conversion to fatty acids is strongly concentration-dependent. Lactate can be used to an appreciable extent only by adipose tissue from fasted-refed rats. A number of compounds, including glucose, pyruvate, aspartate, propionate, and butyrate, stimulated lactate conversion to fatty acids. Based on studies of incorporation of lactate-2-(3)H and lactate-2-(14)C into fatty acids it was suggested that the transhydrogenation sequence of the "citrate-malate cycle"(1) was not providing all of the NADPH required for fatty acid synthesis from lactate. An alternative pathway for NADPH formation involving the conversion of isocitrate to alpha-ketoglutarate via cytosolic isocitrate dehydrogenase was proposed. Indirect support for this proposal was provided by the rapid labeling of glutamate from lactate-2-(14)C by adipose tissue incubated in vitro, as well as the demonstration that glutamate can be readily metabolized by adipose tissue via reactions localized largely in the cytosol. Furthermore, isolated adipose tissue mitochondria convert alpha-ketoglutarate to malate, or in the presence of added pyruvate, to citrate. Glutamate itself can not be metabolized by these mitochondria, a finding in keeping with the demonstration of negligible levels of NAD-glutamate dehydrogenase activity in adipose tissue mitochondria. Pyruvate stimulated alpha-ketoglutarate and malate conversion to citrate and reduced their oxidation to CO(2). It is proposed that under conditions of excess generation of NADH malate may act as a shuttle carrying reducing equivalents across the mitochondrial membrane. Malate at low concentrations increased pyruvate conversion $$Word$$ citrate and markedly decreased the formation of CO(2) by isolated adipose tissue mitochondria. Malate also stimulated citrate and isocitrate metabolism by these mitochondria, an effect that could be blocked by 2-n-butylmalonate. This potentially important role of malate in the regulation of carbon flow during lipogenesis is underlined by the observation that 2-n-butylmalonate inhibited fatty acid synthesis from pyruvate, but not from glucose and acetate, and decreased the stimulatory effect of pyruvate on acetate conversion to fatty acids.