Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes

At the pyruvate branch point, the fermentative and oxidative metabolic routes diverge. Pyruvate can be transformed either into lactate in mammalian cells or into ethanol in yeast, or transported into mitochondria to fuel ATP production by oxidative phosphorylation. The recently discovered mitochondrial pyruvate carrier (MPC), encoded by MPC1, MPC2, and MPC3 in yeast, is required for uptake of pyruvate into the organelle. Here, we show that while expression of Mpc1 is not dependent on the carbon source, expression of Mpc2 and Mpc3 is specific to fermentative or respiratory conditions, respectively. This gives rise to two alternative carrier complexes that we have termed MPC_FERM and MPC_OX. By constitutively expressing the two alternative complexes in yeast deleted for all three endogenous genes, we show that MPC_OX has a higher transport activity than MPC_FERM, which is dependent on the C‐terminus of Mpc3. We propose that the alternative MPC subunit expression in yeast provides a way of adapting cellular metabolism to the nutrient availability.     

Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde

Biotransformation of benzaldehyde and pyruvate into (R)-phenylacetylcarbinol (PAC) catalysed by Candida utilis pyruvate decarboxylase (PDC) at low buffer concentration (20 mM MOPS) was enhanced by maintenance of neutral pH through acetic acid addition. PDC was very stable in this buffer (half-life 138 h at 6 degrees C), however a benzaldehyde emulsion (400 mM) caused rapid deactivation. The inclusion of 2M glycerol did not protect PDC from inactivation by benzaldehyde but initial rates were increased by 50% and the final PAC level was enhanced from 40 to 51 g l(-1). Low levels of by-products acetaldehyde (0.1-0.15 g l(-1)) and acetoin (1.1-1.3 g l(-1)) were formed in both the presence and absence of 2 M glycerol. Interestingly PDC was more stable towards benzaldehyde when pyruvate was present: no activity was lost during the first hour of biotransformation (2 M glycerol, benzaldehyde concentration decreased from 400 to 345 mM, pyruvate from 480 to 420 mM) but PDC was completely inactivated in less than 30 min when exposed to the same concentrations of benzaldehyde in the absence of pyruvate. Thus the enzyme in catalytic action was more stable than the resting enzyme.     

Transpulmonary pyruvate kinetics

Shuttling of intermediary metabolites, such as pyruvate, contributes to the dynamic energy and biosynthetic needs of tissues. Tracer kinetic studies offer a powerful tool to measure the metabolism of substrates like pyruvate that are simultaneously taken up from and released into the circulation by organs. However, we understood that during each circulatory passage, the entire cardiac output transits the pulmonary circulation. Therefore, we examined the transpulmonary pyruvate kinetics in an anesthetized rat model during an unstimulated (Con), lactate clamp (LC), and epinephrine infusion (Epi) conditions using a primed-continuous infusion of [U-13C]pyruvate. Compared with Con and Epi stimulation, LC significantly increased mixed central venous ([v]) and arterial ([a]) pyruvate concentrations (P < 0.05). We hypothesized that the lungs, specifically the pulmonary capillary beds are sites of simultaneous production and removal of pyruvate and contributes significantly to whole body carbohydrate intermediary metabolism. Transpulmonary net pyruvate balances were positive during all three conditions, indicating net pyruvate uptake. Net balance was significantly greater during epinephrine stimulation compared with the unstimulated control (P < 0.05). Tracer-measured pyruvate fractional extraction averaged 42.8 ± 5.8% for all three conditions and was significantly higher during epinephrine stimulation (P < 0.05) than during either Con or LC conditions, that did not differ from each other. Pyruvate total release (tracer measured uptake - net balance) was significantly higher during epinephrine stimulation (400 ± 100 μg/min) vs. Con (30 ± 20 μg/min) (P < 0.05). These data are interpreted to mean that significant pyruvate extraction occurs during circulatory transport across lung parenchyma. The extent of pulmonary parenchymal pyruvate extraction predicts high expression of monocarboxylate (lactate/pyruvate) transporters (MCTs) in the tissue. Western blot analysis of whole lung homogenates detected three isoforms, MCT1, MCT2, and MCT4. We conclude that a major site of circulating pyruvate extraction resides with the lungs and that during times of elevated circulating lactate, pyruvate, or epinephrine stimulation, pyruvate extraction is increased.     

Inhibition of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase by glyoxylate

The overall reaction catalyzed by the pyruvate dehydrogenase complex from rat epididymal fat tissue is inhibited by glyoxylate at concentrations greater than 10 μm. The inhibition is competitive with respect to pyruvate; K_i was found to be 80 μm. Qualitatively similar results were observed using pyruvate dehydrogenase from rat liver, kidney, and heart. Glyoxylate also inhibits the pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat, with the inhibition being readily detectable using 50 μm glyoxylate. These effects of glyoxylate are largely reversed by millimolar concentrations of thiols (especially cysteine) because such compounds form relatively stable adducts with glyoxylate. Presumably these inhibitions by low levels of glyoxylate had not been previously observed, because others have used high concentrations of thiols in pyruvate dehydrogenase assays. Since the inhibitory effects are seen with suspected physiological concentrations, it seems likely that glyoxylate partially controls the activity of pyruvate dehydrogenase in vivo.     

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.     

Identity of kynurenine: pyruvate aminotransferase with histidine: pyruvate aminotransferase.

Kynurenine pyruvate aminotransferase was purified from rat kidney. The purified enzyme had an isoelectric point of pH 5.2 and a pH optimum of 9.3. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors. L-Amino acids were effective in the following order of activity: histidine greather than phenylalanine greater than kynurenine greater than tyrosine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values were about 0.63 mM, 1.4 mM and 0.09 mM for histidine, kynurenine and phenylalanine, respectively. Km values for pyruvate were 5.5 mM with histidine as amino donor, 1.3 mM with kynurenine and 8.5 mM with phenylalanine. Kynurenine pyruvate aminotransferase activity of the enzyme was inhibited by the addition of histidine or phenylalanine. The molecular weights determined by gel filtration and sucrose density gradient centrifugation were approximately 76000 and 79000, respectively. On the basis of purification ratio, substrate specificity, inhibition by common substrates, subcellular distribution, isoelectric focusing and polyacrylamide-gel electrophoresis, it is suggested that kynurenine pyruvate aminotransferase is identical with histidine pyruvate aminotransferase and also with phenylalanine pyruvate aminotransferase. The physiological significance of the enzyme is discussed.     

Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase

The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His-Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.     

The pyruvate branch point in fish liver mitochondria: Effects of acylcarnitine oxidation on pyruvate dehydrogenase and pyruvate carboxylase activities

Summary Palmitoyldl-carnitine inhibits¹⁴CO₂ production from 1-[¹⁴C]-pyruvate and from 1-[¹⁴C]-alanine by mitochondria from rainbow trout liver. The inhibitory effect occurs in both respiratory states III and IV. Fixation of H¹⁴CO ₃ ^– into acid-stable products by intact mitochondria requires pyruvate and ATP and is inhibited by sodium arsenite. This inhibitory effect is completely abolished by acetyldl-carnitine. It is proposed that under these conditions, oxidation of palmitoyldl-carnitine results in inhibition of pyruvate dehydrogenase while oxidation of acetyldl-carnitine results in activation of pyruvate carboxylase in intact rainbow trout liver mitochondria.     

Characterization of the inhibition of Escherichia coli pyruvate dehydrogenase complex by pyruvate

The E. coli pyruvate dehydrogenase complex was inhibited by pyruvate in absence of its cofactor, NAD+. The inhibition was found to increase with pH and phosphate concentration of the buffer and decrease with its ionic strength. The inhibition profile was different with MOPS buffer. No radioactivity was found in the enzyme, when the latter was incubated with 2-14C-pyruvate. The results suggest that covalent adduct formation is not necessary for the observed inhibition.     

Nonenzymatic decarboxylation of pyruvate

Triton X-100, retinol, retinoic acid, retinal, hexane, dithiothreitol, mercaptoethanol, and some other commercially available chemicals caused nonenzymatic decarboxylation of pyruvate and alpha-ketoglutarate. "Lipids" obtained from human or pigeon liver homogenates using isopropanol/hexane also had very high nonenzymatic decarboxylating activity on these two alpha-ketoacids; most of this activity could be traced to the hexane (Eastman) used in the extraction. Optimum pH of the reaction with dithiothreitol and mercaptoethanol was 7-8 and with the other chemicals around 10, but considerable activity was present at pH 7-8. Liver homogenates had a scavenger effect on the decarboxylating activity of Triton X-100 and of dithiothreitol. Dithiothreitol and mercaptoethanol at high concentrations (greater than 1 mM) also had a scavenger effect on the decarboxylating activity of the "lipids." Pretreatment of Triton X-100, dithiothreitol, retinol, and the "lipids" with catalase markedly decreased the decarboxylating activity, while treatment with boiled catalase failed to do so. The results suggest that these compounds contain oxidizing contaminants, perhaps peroxide derivatives. Powerful oxidizing impurities have been reported in Triton X-100 from various sources by Y. Ashani and G. N. Catravas (1980, Anal. Biochem 109, 55-62). Such peroxide derivatives may cause nonenzymatic decarboxylation of pyruvate and alpha-ketoglutarate, presumably by a mechanism similar to the well-known nonenzymatic decarboxylation of alpha-ketoacids by hydrogen peroxide. In the absence of catalase and/or other protective agents against reactive oxygen derivatives, these chemicals would interfere in the assays of pyruvate dehydrogenase, pyruvate dehydrogenase complex, and alpha-ketoglutarate dehydrogenase complex which depend on the release of 14CO2 from alpha[1-14C]ketoacids.     

Role of pyruvate and S-adenosylmethioine in activating the pyruvate formate-lyase of Escherichia coli

The pyruvate formate-lyase activity of extracts of Escherichia coli is stimulated and the dilution effect is abolished by the addition of pyruvate to the extract. The activity can be purified fourfold from pyruvate-supplemented extracts by isoelectric precipitation under anaerobic conditions. The activity of extracts not supplemented with pyruvate has been separated into two fractions by treatment with protamine sulfate-fraction PS, the soluble portion, and fraction N, an extract of the precipitate formed upon the addition of protamine sulfate. After treatment of these fractions with charcoal, pyruvate formate-lyase activity is stimulated by the addition of S-adenosylmethionine. When sodium pyruvate is added to the crude extract before the fractionation, fraction PS has full enzymatic activity and is not stimulated by fraction N or by S-adenosylmethionine. Incubation of the inactive fractions with pyruvate and S-adenosylmethionine in the absence of other substrates similarly results in a highly active preparation, not subject to the "dilution effect" obtained when the fractions are added separately to the assay. These observations suggest that the component in the protamine supernatant fraction is activated by the other fraction and that S-adenosylmethionine and pyruvate are required for the activation reaction. The activating factor present in the protamine precipitate fraction may be further purified by heating for 10 min at 100 C under H(2) atmosphere. The yield of this factor from crude extract is not affected by activation of the pyruvate formate-lyase of the extract, indicating that the factor acts catalytically. The requirement for pyruvate is only partially satisfied by alpha-ketobutyrate and not at all by other alpha-keto acids, acetyl phosphate, or adenosine triphosphate. The rate of activation is maximal at 0.01 m sodium pyruvate and 3 x 10(-4)mS-adenosylmethionine; it is linearly dependent on the amount of activating factor added. The rate of activation is the same when the activation reaction is initiated by addition of any of the four required components, indicating that no slow step of activation can be carried out by any three of the components. A similar pyruvate formate-lyase system was found in extracts of the methionine/B(12) autotroph 113-3, grown with methionine supplement, indicating that vitamin B(12) derivatives do not participate in the system.