scholarly journals Regulation of pyruvate metabolism via pyruvate carboxylase in rat brain mitochondria

1973 ◽  
Vol 132 (2) ◽  
pp. 185-192 ◽  
Author(s):  
Mulchand S. Patel ◽  
Shirley M. Tilghman
Keyword(s):  
Rat Brain ◽  
Pyruvate Carboxylase ◽  
Inorganic Phosphate ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Brain Mitochondria ◽  
Tricarboxylic Acid ◽  
Pyruvate Metabolism ◽  
Rat Brain Mitochondria ◽  
Tricarboxylic Acid Cycle Intermediates

1. The fixation of CO2 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 H14CO3- 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 H14CO3- 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 Km 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.

scholarly journals The regulation and glutamate metabolism by tricarboxylic acid-cycle activity in rat brain mitochondria

1978 ◽  
Vol 172 (1) ◽  
pp. 155-162 ◽  
Author(s):  
Steven C. Dennis ◽  
John B. Clark
Keyword(s):  
Rat Brain ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Brain Mitochondria ◽  
Inhibitory Effect ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
Rat Brain Mitochondria ◽  
Fixed Concentration ◽  
K Concentration

1. The interrelationship of metabolism of pyruvate or 3-hydroxybutyrate and glutamate transamination in rat brain mitochondria was studied. 2. If brain mitochondria are incubated in the presence of equimolar concentrations of pyruvate and glutamate and the K+ concentration is increased from 1 to 20mm, the rate of pyruvate utilization is increased 3-fold, but the rate of production of aspartate and 2-oxoglutarate is decreased by half. 3. Brain mitochondria incubated in the presence of a fixed concentration of glutamate (0.87 or 8.7mm) but different concentrations of pyruvate (0 to 1mm) produce aspartate at rates that decrease as the pyruvate concentration is increased. At 1mm-pyruvate, the rate of aspartate production is decreased to 40% of that when zero pyruvate was present. 4. Brain mitochondria incubated in the presence of glutamate and malate alone produce 2-oxoglutarate at rates stoicheiometric with the rate of aspartate production. Both the 2-oxoglutarate and aspartate accumulate extramitochondrially. 5. Externally added 2-oxoglutarate has little inhibitory effect (Ki approx. 31mm) on the production of aspartate from glutamate by rat brain mitochondria. 6. It is concluded that the inhibitory effect of increased C2 flux into the tricarboxylic acid cycle on glutamate transamination is caused by competition for oxaloacetate between the transaminase and citrate synthase. 7. Evidence is provided from a reconstituted malate–aspartate (or Borst) cycle with brain mitochondria that increased C2 flux into the tricarboxylic acid cycle from pyruvate may inhibit the reoxidation of exogenous NADH. These results are discussed in the light of the relationship between glycolysis and reoxidation of cytosolic NADH by the Borst cycle and the requirement of the brain for a continuous supply of energy.

Determination of gluconeogenesis in vivo with 14C-labeled substrates

1985 ◽  
Vol 248 (4) ◽  
pp. R391-R399 ◽  
Author(s):  
J. Katz
Keyword(s):  
Pyruvate Carboxylase ◽  
Tricarboxylic Acid Cycle ◽  
Specific Radioactivity ◽  
Tricarboxylic Acid ◽  
Pyruvate Metabolism ◽  
Acetyl Coenzyme A ◽  
Glucose Carbon ◽  
Labeling Pattern

A mitochondrial model of gluconeogenesis and the tricarboxylic acid cycle, where pyruvate is metabolized via pyruvate carboxylase and pyruvate dehydrogenase, and pyruvate kinase is examined. The effect of the rate of tricarboxylic acid flux and the rates of the three reactions of pyruvate metabolism on the labeling patterns from [14C]pyruvate and [24C]acetate are analyzed. Expressions describing the specific radioactivities and 14C distribution in glucose as a function of these rates are derived. Specific radioactivities and isotopic patterns depend markedly on the ratio of the rates of pyruvate carboxylation and decarboxylation to the rate of citrate synthesis, but the effect of phosphoenolpyruvate hydrolysis is minor. The effects of these rates on 1) specific radioactivity of phosphoenolpyruvate, 2) labeling pattern in glucose, and 3) contribution of pyruvate, acetyl-coenzyme A, and CO2 to glucose carbon are illustrated. To determine the contribution of lactate or alanine to gluconeogenesis, experiments with two compounds labeled in different carbons are required. Methods in current use to correct for the dilution of 14C in gluconeogenesis from [14C]pyruvate are shown to be erroneous. The experimental design and techniques to determine gluconeogenesis from 14C-labeled precursors are presented and illustrated with numerical examples.

scholarly journals The role of ADP in the modulation of the calcium-efflux pathway in rat brain mitochondria

1985 ◽  
Vol 225 (1) ◽  
pp. 41-49 ◽  
Author(s):  
J Vitorica ◽  
J Satrústegui
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Tricarboxylic Acid Cycle ◽  
Brain Mitochondria ◽  
Ruthenium Red ◽  
Calcium Efflux ◽  
Rat Brain Mitochondria ◽  
Pi Complex ◽  
Efflux Pathway

The role of ADP in the regulation of Ca2+ efflux in rat brain mitochondria was investigated. ADP was shown to inhibit Ruthenium-Red-insensitive H+- and Na+-dependent Ca2+-efflux rates if Pi was present, but had no effect in the absence of Pi. The primary effect of ADP is an inhibition of Pi efflux, and therefore it allows the formation of a matrix Ca2+-Pi complex at concentrations above 0.2 mM-Pi and 25 nmol of Ca2+/mg of protein, which maintains a constant free matrix Ca2+ concentration. ADP inhibition of Pi and Ca2+ efflux is nucleotide-specific, since in the presence of oligomycin and an inhibitor of adenylate kinase ATP does not substitute for ADP, is dependent on the amount of ADP present, and requires ADP concentrations in excess of the concentrations of translocase binding sites. Brain mitochondria incubated with 0.2 mM-Pi and ADP showed Ca2+-efflux rates dependent on Ca2+ loads at Ca2+ concentrations below those required for the formation of a Pi-Ca2+ complex, and behaved as perfect cytosolic buffers exclusively at high Ca2+ loads. The possible role of brain mitochondrial Ca2+ in the regulation of the tricarboxylic acid-cycle enzymes and in buffering cytosolic Ca2+ is discussed.

Characterization of two ‘mitochondrial’ particulates from rat brain

1960 ◽  
Vol 198 (2) ◽  
pp. 467-470 ◽  
Author(s):  
Dennis R. Dahl ◽  
Roberta J. Jacobs ◽  
Frederick E. Samson
Keyword(s):  
Rat Brain ◽  
Tricarboxylic Acid Cycle ◽  
Chemical Properties ◽  
Oxidative Capacity ◽  
Brain Mitochondria ◽  
P Fraction ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Physical And Chemical ◽  
The Brain

Two particulate fractions were isolated from a preparation of rat brain mitochondria and some of the physical and chemical properties were studied. The distinguishing physical difference was the color; the heavier, more densely packed particles (P) were dark, while the lighter, loosely packed particles (W) were almost white. The amount of (P) and (W) are approximately equal. P has a higher concentration of protein than W but the nucleic acid (RNA and DNA) concentration is about the same. The per cent water of W is slightly greater than P. The P fraction was capable of oxidative-phosphorylation with several tricarboxylic acid cycle intermediates but W had no oxidative capacity. P appears to undergo the typical ‘swelling’ of mitochondria whereas W does not. P and W both increase in amount with the neonatal maturation of the brain.

scholarly journals Development of mitochondrial energy metabolism in rat brain

1977 ◽  
Vol 164 (2) ◽  
pp. 339-348 ◽  
Author(s):  
John M. Land ◽  
Robert F. G. Booth ◽  
Ruud Berger ◽  
John B. Clark
Keyword(s):  
Rat Brain ◽  
Pyruvate Dehydrogenase ◽  
Citrate Synthase ◽  
Aerobic Glycolysis ◽  
Biological Significance ◽  
Brain Mitochondria ◽  
Developmental Pattern ◽  
Mitochondrial Energy Metabolism ◽  
Rat Brain Mitochondria ◽  
Total Brain

1. The development of pyruvate dehydrogenase and citrate synthase activity in rat brain mitochondria was studied. Whereas the citrate synthase activity starts to increase at about 8 days after birth, that of pyruvate dehydrogenase starts to increase at about 15 days. Measurements of the active proportion of pyruvate dehydrogenase during development were also made. 2. The ability of rat brain mitochondria to oxidize pyruvate follows a similar developmental pattern to that of the pyruvate dehydrogenase. However, the ability to oxidize 3-hydroxybutyrate shows a different developmental pattern (maximal at 20 days and declining by half in the adult), which is compatible with the developmental pattern of the ketone-body-utilizing enzymes. 3. The developmental pattern of both the soluble and the mitochondrially bound hexokinase of rat brain was studied. The total brain hexokinase activity increases markedly at about 15 days, which is mainly due to an increase in activity of the mitochondrially bound form, and reaches the adult situation (approx. 70% being mitochondrial) at about 30 days after birth. 4. The release of the mitochondrially bound hexokinase under different conditions by glucose 6-phosphate was studied. There was insignificant release of the bound hexokinase in media containing high KCl concentrations by glucose 6-phosphate, but in sucrose media half-maximal release of hexokinase was achieved by 70μm-glucose 6-phosphate 5. The production of glucose 6-phosphate by brain mitochondria in the presence of Mg2++glucose was demonstrated, together with the inhibition of this by atractyloside. 6. The results are discussed with respect to the possible biological significance of the similar developmental patterns of pyruvate dehydrogenase and the mitochondrially bound kinases, particularly hexokinase, in the brain. It is suggested that this association may be a mechanism for maintaining an efficient and active aerobic glycolysis which is necessary for full neural expression.

scholarly journals A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast,Drosophila, and Humans

Science ◽  
2012 ◽  
Vol 337 (6090) ◽  
pp. 96-100 ◽  
Author(s):  
Daniel K. Bricker ◽  
Eric B. Taylor ◽  
John C. Schell ◽  
Thomas Orsak ◽  
Audrey Boutron ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Inner Mitochondrial Membrane ◽  
Pyruvate Metabolism ◽  
Genetic Studies ◽  
Mitochondrial Pyruvate Carrier ◽  
Families With Children ◽  
Tricarboxylic Acid Cycle Intermediates

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast,Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the inner mitochondrial membrane. Yeast andDrosophilamutants lackingMPC1display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, and silencing ofMPC1orMPC2in mammalian cells impairs pyruvate oxidation. A point mutation inMPC1provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped toMPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.

Inhibition of CO2 fixation in group D streptococci

1969 ◽  
Vol 15 (1) ◽  
pp. 57-60 ◽  
Author(s):  
Victor F. Lachica ◽  
Paul A. Hartman
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Pyruvate Carboxylase ◽  
Co2 Fixation ◽  
Tricarboxylic Acid Cycle ◽  
Inhibitory Effect ◽  
Tricarboxylic Acid ◽  
Acetyl Coa ◽  
Acid Cycle ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Group D

The stimulatory effect of acetyl-CoA and the inhibitory effect by L-aspartate and some intermediates of the tricarboxylic acid cycle in the assimilation of CO2 by crude extracts of group D streptococci suggest that the pyruvate carboxylase of Streptococcus faecium and the phosphoenolpyruvate carboxylase of S. bovis are allosteric enzymes. This implies that these enzymes are sites for the control of the amount of aspartate and of the tricarboxylic acid cycle intermediates synthesized.

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

1981 ◽  
Vol 200 (1) ◽  
pp. 27-33 ◽  
Author(s):  
M Forissier ◽  
G Baverel
Keyword(s):  
Glutamine Synthetase ◽  
Guinea Pig ◽  
Pyruvate Carboxylase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Kidney Cortex ◽  
Acid Cycle ◽  
Ammonia Release ◽  
Tricarboxylic Acid Cycle Intermediates

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.

scholarly journals Comparative development of the pyruvate dehydrogenase complex and citrate synthase in rat brain mitochondria

1986 ◽  
Vol 238 (3) ◽  
pp. 729-736 ◽  
Author(s):  
G D A Malloch ◽  
L A Munday ◽  
M S Olson ◽  
J B Clark
Keyword(s):  
Enzyme Activity ◽  
Rat Brain ◽  
Pyruvate Dehydrogenase ◽  
Citrate Synthase ◽  
Post Partum ◽  
Pyruvate Dehydrogenase Complex ◽  
Brain Mitochondria ◽  
Rat Brain Mitochondria ◽  
Developing Rat ◽  
Dehydrogenase Complex

The enzyme activity of the pyruvate dehydrogenase complex (PDHC) was measured in mitochondria prepared from developing rat brain, before and after steady-state dephosphorylation of the E1 alpha subunit. A marked increase in dephosphorylated (fully activated) PDHC activity occurred between days 10 and 15 post partum, which represented approx. 60% of the difference in fully activated PDHC activity measured in foetal and adult rat brain mitochondria. There was no detectable change in the active proportion of the enzyme during mitochondrial preparation nor any qualitative alteration in the detectable catalytic and regulatory components of the complex, which might account for developmental changes in PDHC activity. The PDHC protein content of developing rat brain mitochondria and homogenates was measured by an enzyme-linked immunoadsorbent assay. The development of PDHC protein in both fractions agreed closely with the development of the PDHC activity. The results suggest that the developmental increase in PDHC activity is due to increased synthesis of PDHC protein, which is partly a consequence of an increase in mitochondrial numbers. However, the marked increase in PDHC activity measured between days 10 and 15 post partum is mainly due to an increase in the amount of PDHC per mitochondrion. The development of citrate synthase enzyme activity and protein was measured in rat brain homogenates and mitochondria. As only a small increase in citrate synthase activity and protein was detected in mitochondria between days 10 and 15 post partum, the marked increase in PDHC protein and enzyme activity may represent specific PDHC synthesis. As several indicators of acquired neurological competence become apparent during this period, it is proposed that preferential synthesis of PDHC may be crucial to this process. The results are discussed with respect to the possible roles played by PDHC in changes of respiratory-substrate utilization and the acquisition of neurological competence occurring during the development of the brain of a non-precocial species such as the rat.

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