Glutamate metabolism and transport in rat brain mitochondria.
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ABSTRACT:
SUBMITTER: Dennis SC
PROVIDER: S-EPMC1163752 | biostudies-other | 1976 May
REPOSITORIES: biostudies-other
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Project description:1. Rat brain mitochondria did not swell in iso-osmotic solutions of ammonium or potassium (plus valinomycin) glutamate or aspartate, with or without addition of uncouplers. 2. Glutamate was able to reduce intramitochondrial NAD(P)(+); aspartate was able to cause partial re-oxidation. 3. These effects were inhibited by threo-hydroxy-aspartate in whole but not in lysed mitochondria. 4. The existence of a ;malate-aspartate shuttle' for the oxidation of extramitochondrial NADH was demonstrated. This shuttle requires the net exchange of glutamate for aspartate across the mitochondrial membrane. 5. Extramitochondrial glutamate did not inhibit intramitochondrial glutaminase under conditions in which the inhibition in lysed mitochondria was virtually complete. 6. The glutaminase activity of these mitochondria was not energy-dependent. 7. We conclude that these mitochondria do not possess a glutamate-hydroxyl antiporter similar to that of liver mitochondria nor a glutamate-glutamine antiporter similar to that of pig kidney mitochondria, but that they do possess a glutamate-aspartate antiporter.
| S-EPMC1167992 | biostudies-other
Project description:1. The pathway of glutamate metabolism in non-synaptic rat brain mitochondria was investigated by measuring glutamate, aspartate and ammonia concentrations and oxygen uptakes in mitochondria metabolizing glutamate or glutamine under various conditions. 2. Brain mitochondria metabolizing 10mm-glutamate in the absence of malate produce aspartate at 15nmol/min per mg of protein, but no detectable ammonia. If amino-oxyacetate is added, the aspartate production is decreased by 80% and ammonia production is now observed at a rate of 6.3nmol/min per mg of protein. 3. Brain mitochondria metabolizing glutamate at various concentrations (0-10mm) in the presence of 2.5mm-malate produce aspartate at rates that are almost stoicheiometric with glutamate disappearance, with no detectable ammonia production. In the presence of amino-oxyacetate, although the rate of aspartate production is decreased by 75%, ammonia production is only just detectable (0.3nmol/min per mg of protein). 4. Brain mitochondria metabolizing 10mm-glutamine and 2.5mm-malate in States 3 and 4 were studied by using glutamine as a source of intramitochondrial glutamate without the involvement of mitochondrial translocases. The ammonia production due to the oxidative deamination of glutamate produced from the glutamine was estimated as 1nmol/min per mg of protein in State 3 and 3nmol/min per mg of protein in State 4. 5. Brain mitochondria metabolizing 10mm-glutamine in the presence of 1mm-amino-oxyacetate under State-3 conditions in the presence or absence of 2.5mm-malate showed no detectable aspartate production. In both cases, however, over the first 5min, ammonia production from the oxidative deamination of glutamate was 21-27nmol/min per mg of protein, but then decreased to approx. 1-1.5nmol/min per mg. 6. It is concluded that the oxidative deamination of glutamate by glutamate dehydrogenase is not a major route of metabolism of glutamate from either exogenous or endogenous (glutamine) sources in rat brain mitochondria.
| S-EPMC1183800 | biostudies-other
Project description:1. The apparent Michaelis constants of the glutamate dehydrogenase (EC 1.4.1.3), the glutamate-oxaloacetate transaminase (EC 2.6.1.1) and the glutaminase (EC 3.5.1.2) of rat brain mitochondria derived from non-synaptic (M) and synaptic (SM2) sources were studied. 2. The kinetics of oxygen uptake of both populations of mitochondria in the presence of a fixed concentration of malate and various concentrations of glutamate or glutamine were investigated. 3. In both mitochondrial populations, glutamate-supported respiration in the presence of 2.5 mM-malate appears to be biphasic, one system (B) having an apparent Km for glutamate of 0.25 +/- 0.04 mM (n=7) and the other (A) of 1.64 +/- 0.5 mM (n=7) [when corrected for low-Km process, Km=2.4 +/- 0.75 mM (n=7)]. Aspartate production in these experiments followed kinetics of a single process with an apparent Km for glutamate of 1.8-2 mM, approximating to the high-Km process. 4. Oxygen-uptake measurement with both mitochondrial populations in the presence of malate and various glutamate concentrations in which amino-oxyacetate was present showed kinetics approximating only to the low-Km process (apparent Km for glutamate approximately 0.2 mM). Similar experiments in the presence of glutamate alone showed kinetics approximating only to the high-Km process (apparent Km for glutamate approximately 1-1.3 mM). 5. Oxygen uptake supported by glutamine (0-3 mM) and malate (2.5 mM) by the free (M) mitochondrial population, however, showed single-phase kinetics with an apparent Km for glutamine of 0.28 mM. 6. Aspartate and 2-oxoglutarate accumulation was measured in 'free' nonsynaptic (M) brain mitochondria oxidizing various concentrations of glutamate at a fixed malate concentration. Over a 30-fold increase in glutamate concentration, the flux through the glutamate-oxaloacetate transaminase increased 7--8-fold, whereas the flux through 2-oxoglutarate dehydrogenase increased about 2.5-fold. 7. The biphasic kinetics of glutamate-supported respiration by brain mitochondria in the presence of malate are interpreted as reflecting this change in the relative fluxes through transamination and 2-oxoglutarate metabolism.
| S-EPMC1164853 | biostudies-other
Project description: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 (K(i) approx. 31mm) on the production of aspartate from glutamate by rat brain mitochondria. 6. It is concluded that the inhibitory effect of increased C(2) 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 C(2) 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.
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Project description:1. The kinetics of glutamate transport into mitochondria were determined by using Bromocresol Purple to terminate the transport process. 2. Glutamate transport was found to have a V(max.) of 9.1nmol/min per mg of protein at pH6.9 and 20 degrees C; the K(m) for glutamate was 4mm. 3. The rate of glutamate deamination in intact mitochondria was tenfold slower than in disrupted mitochondria. 4. These results suggest that glutamate deamination may be controlled by the rate of glutamate transport. Possible consequences of these findings are discussed.
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Project description:Glutamate released from synaptic vesicles mediates excitatory neurotransmission by stimulating glutamate receptors. Glutamate transporters maintain low synaptic glutamate levels critical for this process, a role primarily attributed to astrocytes. Recently, vesicular release of glutamate from unmyelinated axons in the rat corpus callosum has been shown to elicit AMPA receptor-mediated currents in glial progenitor cells. Glutamate transporters are the only mechanism of glutamate clearance, yet very little is known about the role of glutamate transporters in normal development of oligodendrocytes (OLs) or in excitotoxic injury to OLs. We found that OLs in culture are capable of sodium-dependent glutamate uptake with a K(m) of 10 +/- 2 microm and a V(max) of 2.6, 5.0, and 3.8 nmol x min(-1) x mg(-1) for preoligodendrocytes, immature, and mature OLs, respectively. Surprisingly, EAAC1, thought to be exclusively a neuronal transporter, contributes more to [(3)H]l-glutamate uptake in OLs than GLT1 or GLAST. These data suggest that glutamate transporters on oligodendrocytes may serve a critical role in maintaining glutamate homeostasis at a time when unmyelinated callosal axons are engaging in glutamatergic signaling with glial progenitors. Furthermore, GLT1 was significantly increased in cultured mature OLs contrary to in vivo data in which we have shown that, although GLT1 is present on developing OLs when unmyelinated axons are prevalent in the developing rat corpus callosum, after myelination, GLT1 is not expressed on mature OLs. The absence of GLT1 in mature OLs in the rat corpus callosum and its presence in mature rat cultured OLs may indicate that a signaling process in vivo is not activated in vitro.
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