The glucose metabolism of adult Ostertagia circumcincta, in vitro

Adult Ostertagia circumcincta from freshly killed sheep were incubated at 39 degrees C in a medium containing inorganic salts, antibiotics and D-[U-14C] glucose. The worms appeared healthy even after incubation for as long as 72 h. All the radioactivity was recovered either within the worms or in the incubation vessel in the form of metabolic products or unmetabolized glucose. Incubations were carried out at low oxygen tension except for those in which CO2 was measured. These were either aerobic or anaerobic. In terms of both quantity and radioactivity the main metabolic products of glucose were CO2, propan-1-ol, acetate and propionate. Smaller amounts of ethanol, lactate and succinate were formed. The results are compared with those found for the similar nematode Haemonchus contortus.     

Metabolism of propionate to acetate in the cockroach Periplaneta americana

Carbon-13 NMR and radiotracer studies were used to determine the precursor to methylmalonate and to study the metabolism of propionate in the cockroach Periplaneta americana. [3,4,5-13C3]Valine labeled carbons 3, 4, and 26 of 3-methylpentacosane, indicating that valine was metabolized via propionyl-CoA to methylmalonyl-CoA and served as the methyl branch unit precursor. Potassium [2-13C]propionate labeled the odd-numbered carbons of hydrocarbons and potassium [3-13C]propionate labeled the even-numbered carbons of hydrocarbons in this insect. This labeling pattern indicates that propionate is metabolized to acetate, with carbon-2 of propionate becoming the methyl carbon of acetate and carbon-3 of propionate becoming the carboxyl carbon of acetate. In vivo studies in which products were separated by HPLC showed that [2-14C]propionate was readily metabolized to acetate. The radioactivity from sodium [1-14C]propionate was not incorporated into succinate nor into any other tricarboxylic acid cycle intermediate, indicating that propionate was not metabolized via methylmalonate to succinate. Similarly, [1-14C]propionate did not label acetate. An experiment designed to determine the subcellular localization of the enzymes involved in converting propionate to acetate showed that they were located in the mitochondrial fraction. Data from both in vivo and in vitro studies as a function of time indicated that propionate was converted directly to acetate and did not first go through tricarboxylic acid cycle intermediates. These data demonstrate a novel pathway of propionate metabolism in insects.     

Anaerobic incorporation of radioactivity from 2,3-14C-succinic acid into citric acid cycle intermediates and related compounds in the sea musselMytilus edulis L.

Summary Sea mussels were exposed to nitrogen for various periods (0, 1, 3 and 6 days) and subsequently injected with 2,3-¹⁴C-succinic acid. After 2.5 h anaerobic incubation concentrations of succinate, some amino acids and volatile fatty acids were determined as well as the distribution of radioactivity.Conversion of the precursor decreased from 80 to 40%, due to increased dilution with endogenous succinate, accumulated during the anaerobic preincubation period.More than 80% of the activity of the converted 2,3-¹⁴C-succinic acid was incorporated into malate, aspartate, glutamate, alanine and propionate. This indicates that succinate is not only an end product of anaerobic glycogen breakdown, but remains an active intermediate of the tricarboxylic acid cycle, which can still operate under anaerobic conditions.Concentration and radioactivity of propionate were markedly increased after prolonged anoxia, which gives evidence that succinate is actively converted to propionate during anaerobiosis.Observed accumulation of glutamate during anoxia is explained by incomplete oxidation of pyruvate, which leaves the tricarboxylic acid cycle at the stage of 2-ketoglutarate.     

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.     

Carbohydrate catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The plerocercoids of S. solidus possess a complete sequence of glycolytic and tricarboxylic acid cycle enzymes. The presence of phosphoenolpyruvate carboxykinase and fumarate reductase activity and the relatively low activities of aconitase and isocitrate dehydrogenase suggest that carbon dioxide fixation is an important pathway in this parasite. Carbon balances show that glycogen is the main energy source under both aerobic and anaerobic conditions and there is only a slight Pasteur effect. Aerobically 22·5% of the glycogen catabolized is excreted as acetate and propionate (4:1), anaerobically 70% of the glycogen utilized can be accounted for as acetate and propionate (1:3). The results indicate that anaerobically the plerocercoids fix carbon dioxide and have a partial reversed tricarboxylic acid cycle, whilst under aerobic conditions at least part of the carbohydrate may be oxidized via a functional tricarboxylic acid cycle.     

High-level heterologous production of propionate in engineered Escherichia coli

A propanologenic (i.e., 1-propanol-producing) bacterium Escherichia coli strain was previously derived by activating the genomic sleeping beauty mutase (Sbm) operon. The activated Sbm pathway branches out of the tricarboxylic acid (TCA) cycle at the succinyl-CoA node to form propionyl-CoA and its derived metabolites of 1-propanol and propionate. In this study, we targeted several TCA cycle genes encoding enzymes near the succinyl-CoA node for genetic manipulation to identify the individual contribution of the carbon flux into the Sbm pathway from the three TCA metabolic routes, that is, oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt. For the control strain CPC-Sbm, in which propionate biosynthesis occurred under relatively anaerobic conditions, the carbon flux into the Sbm pathway was primarily derived from the reductive TCA branch, and both succinate availability and the SucCD-mediated interconversion of succinate/succinyl-CoA were critical for such carbon flux redirection. Although the oxidative TCA cycle normally had a minimal contribution to the carbon flux redirection, the glyoxylate shunt could be an alternative and effective carbon flux contributor under aerobic conditions. With mechanistic understanding of such carbon flux redirection, metabolic strategies based on blocking the oxidative TCA cycle (via ∆sdhA mutation) and deregulating the glyoxylate shunt (via ∆iclR mutation) were developed to enhance the carbon flux redirection and therefore propionate biosynthesis, achieving a high propionate titer of 30.9 g/L with an overall propionate yield of 49.7% upon fed-batch cultivation of the double mutant strain CPC-Sbm∆sdhA∆iclR under aerobic conditions. The results also suggest that the Sbm pathway could be metabolically active under both aerobic and anaerobic conditions.     

Anaerobic Growth of Corynebacterium glutamicum via Mixed-Acid Fermentation

Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD₆₀₀) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO₂ is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions.     

Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate

Most reported efforts to enhance production of the industrially valuable specialty chemical succinate have been done under anaerobic conditions, where E. coli undergoes mixed-acid fermentation. These efforts have often been hampered by the limitations of NADH availability, poor cell growth, and slow production. An aerobic succinate production system was strategically designed that allows E. coli to produce and accumulate succinate efficiently and substantially as a product under absolute aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhAB, icd, iclR) and acetate pathways (poxB, ackA-pta) of E. coli were created to construct the glyoxylate cycle for aerobic succinate production. Experiments in flask studies showed that 14.28 mM of succinate could be produced aerobically with a yield of 0.344 mole/mole using 55 mM glucose. In aerobic batch reactor studies, succinate production rate was faster, reaching 0.5 mole/mole in 24 h with a concentration of 22.12 mM; further cultivation showed that succinate production reached 43 mM with a yield of 0.7. There was also substantial pyruvate and TCA cycle C(6) intermediate accumulation in the mutant. The results suggest that more metabolic engineering improvements can be made to this system to make aerobic succinate production more efficient. Nevertheless, this aerobic succinate production system provides the first platform for enhancing succinate production aerobically in E. coli based on the creation of a new aerobic central metabolic network.     

The Products and Pathways of Glucose Catabolism in Herpetomonas muscarum ingenoplastis and Herpetomonas muscarum muscarum

ABSTRACT. The products and pathways of glucose catabolism in the insect trypanosomatids Herpetomonas muscarum ingenoplastis and Herpetomonas muscarum muscarum have been studied with the aim of elucidating how both organisms are able to proliferate well under aerobic and anaerobic conditions. When incubated in medium containing glucose as the only exogenous carbon source, catabolism was found to be fermentative in both cases. Acetate was a major product of both organisms while H. m. ingenoplastis produced more ethanol and propionate and less succinate than H. m. muscarum . Ethanol production by H. m. ingenoplastis decreased both under anaerobic conditions and in the presence of elevated CO₂ concentrations, whereas succinate and propionate release by this organism were greater in high CO₂ and anoxia, respectively. Succinate production by H. m. muscarum was greatest under anaerobic conditions in elevated CO₂ whereas propionate was only a minor product. The same four products were released during growth of the organisms in complex medium, but the relative proportions differed suggesting that other substrates were being used. Both organisms contained enzymes of the glycolytic and pentose phosphate pathways, but while all activities of the TCA cycle were present in H. m. muscarum . NAD-linked isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate CoA synthase and succinate dehydrogenase were not detected in H. m. ingenoplastis . Fumarate reductase activity was present in both organisms. The data presented suggest that CO₂-fixation and reverse flux through the TCA cycle may be important factors that enable the organisms to undergo anaerobiosis.     

Glucose metabolism of adult Schistosoma japonicum as revealed by nuclear magnetic resonance spectroscopy with D-[13C6]glucose

Excretory end-products of adult Schistosoma japonicum, fed D-[13C6]glucose in vitro under aerobic and anaerobic conditions, were studied using 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy. The glucose in the medium is degraded to produce lactate and alanine aerobically and succinate and acetate as well as lactate and alanine anaerobically. Succinate and acetate have not been previously recorded as excretory products resulting from the metabolism of glucose for schistosomes. The presence of [13C3] and [2,3-13C2]lactate, and [1,2,2'-13C3] and [2,2'-13C2]succinate as end-products suggests that a partial reversed tricarboxylic acid (TCA) cycle is active in adult Schistosoma japonicum under anaerobic conditions. The physiological role of this pathway in adult schistosomes remains obscure.     

Aerobic and anaerobic metabolism of the freshwater oligochaete Tubifex sp.

The freshwater oligochaete Tubifex shows several mechanisms of metabolic adaptations, enabling the worms to occupy saprobial habitats of extremely variable oxygen content. Under normoxic conditions the metabolism of the worms is mainly aerobic with a respiratory ratio of 0.7. Under hypoxic conditions, metabolism of energy sources via aerobic and anaerobic pathways is observed. During complete anoxia acetate and propionate are the main products of glycogen degradation and they are excreted in constant rates into the water. A retransfer of the worms to aerobic conditions enables them to regain aerobic metabolic state within about 60 min.In two Tubifex habitats, which we have characterized, concentrations of dissolved organic material (DOM) were low in the surface water, but high in the interstitial water from sediments. The short-chain fatty acids acetate and propionate reached concentrations up to 1 mmole/liter. Employing radioisotope techniques, we demonstrated that Tubifex can achieve an integumentary uptake of acetate and propionate from artificial tap water at naturally occurring concentrations of 5 to 1000 M. Levels of uptake (600 to 800 nmoles/g wwt.hr) and transport characteristics are very similar to those of marine invertebrates associated with detritus rich sediments. The uptake is susceptible to inhibition by structurally analogous compounds and to metabolic inhibition. Furthermore, DOM uptake in Tubifex is susceptible to inhibition by oxygen depletion, ouabain and Na⁺-depletion. The results may suggest that a carrier system for DOM transport exists in the integument of the worms. The uptake system is highly specific for aliphatic C2 and C3 carboxylic acids. The absorbed volatile fatty acids are rapidly metabolized. Only 15 min after absorption, a considerable amount of radioactivity is present in the glycogen storage of the animals. Depending on the substrate concentration assumed to be available for uptake, up to 40 per cent of the oxidative requirement of the worms may be attained by using dissolved organic material from the interstitial water of their habitat.Supported by the Deutsche Forschungsgemeinschaft (Ho 631/9-9).     

Glutamine- vs glucose-supported motor activity in Schistosoma mansoni: physiological relevance of aerobic metabolism

The ability of Schistosoma mansoni to generate energy through aerobic metabolic processes was examined in adult parasites in vitro. Parasite catabolism of radiolabeled glucose, glutamine, and other amino acids to CO2 and Krebs cycle intermediates was measured under a variety of incubation conditions. L-Glutamine was metabolized to CO2 via the intermediates glutamate, alpha-ketoglutaramate, and alpha-ketoglutarate in worms incubated in a balanced salts solution containing this amino acid as the only organic constituent. Of the other amino acids tested, CO2 production was detected from L-glutamate and L-asparagine. The catabolism of L-glutamine to CO2 was reduced by the respiratory inhibitor antimycin A. The motility of schistosomes in culture was maintained for at least 24 hr when L-glutamine was the only carbon source available to the worms. Under these conditions, motility was reduced when parasites were exposed to a respiratory inhibitor such as KCN, antimycin A, rotenone, or oligomycin, but it was completely restored by the addition of glucose to the medium. These results suggest that while the schistosome is capable of limited aerobic energy-generating processes under certain conditions, survival is not contingent upon these processes in the presence of glucose.     

Isolated rat heart mitochondria are able to metabolize pent-4-enoate to tricarboxylic acid-cycle intermediates.

The metabolism of four short-chain odd-number-carbon fatty acids, pentanoate, pent-4-enoate, propionate and acrylate, was studied in isolated rat heart mitochondria incubated in [14C]bicarbonate buffer. Under these conditions pentanoate was metabolized with a concomitant accumulation of malate and incorporation of 14CO2 into non-volatile compounds. The metabolism of propionate to tricarboxylic acid-cycle intermediates required the addition of ATP and oligomycin. After addition of a small amount of rotenone to the incubation medium, pent-4-enoate was metabolized with an increase in malate from less than 3 nmol/mg of protein to 34.0 +/- 1.5 nmol/mg in 40 min, during which time the amount of 14CO2 fixed in acid-stable compounds increased from 1.56 +/- 0.30 to 41.1 +/- 2.6 nmol/mg of protein. Acrylate was not metabolized under any of the conditions tested. The results show that cardiac mitochondria must have an enzyme system that is capable of reducing the double bond of either pent-4-enoate or its metabolities. That the metabolism of pent-4-enoate occurs through a reductive step and energy-dependent carboxylation is evident from the requirement for NAD+ reduction by partial inhibition of the mitochondrial respiratory chain and the presence of ATP and CO2. The results do not enable us to say whether the compound reduced is pent-4-enoyl-CoA or acryloyl-CoA.     

Succinate synthesis and excretion by Penicillium simplicissimum under aerobic and anaerobic conditions

Succinate is an interesting chemical for industries producing food and pharmaceutical products, surfactants, detergents and biodegradable plastics. Succinate is produced mainly by a mixed-acid fermentation process using anaerobically growing bacteria. However, succinate excretion is also widespread among fungi. In this article we report results on the intracellular concentration and the excretion of succinate by Penicillium simplicissimum under aerobic and anaerobic conditions. The intracellular concentration of succinate increased slightly with the specific growth rate and strongly if the respiratory chain was inhibited by sodium azide or anaerobic conditions (N(2)). A strong increase of succinate excretion was observed if the respiratory chain was inhibited. It is suggested that succinate synthesis under functional (sodium azide) or environmental (N(2)) anaerobic conditions occurs via the reductive part of the tricarboxylic acid cycle. Succinate is then excreted because the oxidative part of the tricarboxylic acid cycle is inactive. A possible role of succinate synthesis in the regeneration of NAD ('fumarate respiration') is discussed.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.     

Microbial mineralization of diisopropanolamine.

Diisopropanolamine (DIPA) is a "sweetening agent" used to remove hydrogen sulfide from sour natural gas, and it is a contaminant at some sour gas treatment facilities in western Canada. To investigate the biodegradation of this alkanolamine, 14C-DIPA was used in anaerobic and aerobic mineralization studies. Between 3 and 78% of the radioactivity from this compound was released as 14CO2 in sediment-enrichment cultures incubated under nitrate-reducing conditions. Similarly, 12-78% of the label was converted to 14CO2 in sediment-enrichment cultures incubated under Mn(IV)-reducing conditions. These activities were observed at 8 degrees C, a typical groundwater temperature in western Canada, and at 28 degrees C. In contrast, DIPA-degrading activity was difficult to sustain under Fe(III)-reducing conditions, and < 25% of the radioactive label from 14C-DIPA was liberated as 14CO2. Two mixed cultures and two isolates (both irregular, non-sporeforming, Gram-positive rods) were used to assess aerobic mineralization of 14C-DIPA. The aerobic mixed cultures released 73 and 79% of the radioactive label as 14CO2, whereas the pure cultures liberated only 39 and 47% as 14CO2. Between one-third and one-half of the nitrogen from DIPA was found as ammonium-N in aerobic batch cultures. These results clearly demonstrate that DIPA is mineralized under a variety of incubation conditions.     

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.     

Carbohydrate Metabolism in Leukocytes VII. Metabolism of Glucose, Acetate, and Propionate by Human Plasma Cells

Plasma cells obtained from the peripheral blood of a patient with multiple myeloma was incubated in serum and Krebs-Ringer bicarbonate buffer with (14)C-labeled glucose, acetate, and propionate. Glucose utilization by these cells amounted to 0.5 mumole per hr per 10(8) cells and was mainly via the Embden-Meyerhof pathway, and only 6% or less traversed the hexose monophosphate shunt. The presence of Krebs cycle activity was demonstrated by direct isolation of several labeled intermediates after incubation with either (14)C-acetate or (14)C-propionate. The distribution of (14)C in lactate, succinate, fumarate, malate, aspartate, and glutamate indicate a complete Krebs cycle. Acetate was metabolized via the Krebs cycle to the extent of 0.15 mumoles per hr per 10(8) cells, and the rate of propionate utilization was 0.17 mumoles per hr per 10(8) cells.     

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.