Glutamate transprot in rat kidney mitochondria

The quantitative characteristics of [U-14C]glutamate transport were determined in rotenone-inhibited energized rat kidney mitochondria at pH 7.0 and 28 degrees C. Glutamate efflux was observed to be first order with respect to matrix glutamate with a rate constant of 0.457 min-1. Uptake kinetic studies indicated that the Km of external glutamate was 1.4 mM and the Vmax 3.2 nmol/mg X min. These kinetic values were found to be unchanged at pH 6.6 or in mitochondria obtained from kidneys of chronically acidotic rats. Parallel studies of glutamate deamination were performed in which mitochondria were incubated in state 3, state 4, and with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, in the presence of malonate. The oxidative deamination of glutamate determined with 1 and 10 mM glutamate never exceeded the simultaneously measured rate of glutamate transport. No glutamate was detectable within the mitochondrial matrix under the conditions of these metabolic experiments. The studies indicate that the glutamate hydroxyl transporter is quite slow and rate limiting for the oxidative deamination of external glutamate in rat kidney mitochondria.     

Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings

Abiotic stresses, such as high salinity or drought, can cause proline accumulation in plants. Such an accumulation involves proline transport into mitochondria where proline catabolism occurs. By using durum wheat seedlings as a plant model system, we investigated how proline enters isolated coupled mitochondria. The occurrence of two separate translocators for proline, namely a carrier solely for proline and a proline/glutamate antiporter, is shown in a functional study in which we found the following: (1) Mitochondria undergo passive swelling in isotonic proline solutions in a stereospecific manner. (2) Externally added l-proline (Pro) generates a mitochondrial membrane potential (ΔΨ) with a rate depending on the transport of Pro across the mitochondrial inner membrane. (3) The dependence of the rate of generation of ΔΨ on increasing Pro concentrations exhibits hyperbolic kinetics. Proline transport is inhibited in a competitive manner by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent N-ethylmaleimide (NEM). (4) No accumulation of proline occurs inside the mitochondria as a result of the addition of proline externally, whereas the content of glutamate increases both in mitochondria and in the extramitochondrial phase. (5) Glutamate efflux from mitochondria occurs at a rate which depends on the mitochondrial transport, and it is inhibited in a non-competitive manner by NEM. The dependence of the rate of glutamate efflux on increasing proline concentration shows saturation kinetics. The physiological role of carrier-mediated transport in the regulation of proline catabolism, as well as the possible occurrence of a proline/glutamate shuttle in durum wheat seedlings mitochondria, are discussed.Catello Di Martino, Roberto Pizzuto these authors contributed equally to the paper     

Glycine uptake by cultured human Y79 retinoblastoma cells: effect of changes in phospholipid fatty acid unsaturation

Glycine uptake was investigated in cultured Y79 retinoblastoma cells containing different degrees of phospholipid fatty acid unsaturation. The modifications were produced by growing the retinoblastoma cells in medium supplemented with various unsaturated fatty acids. Glycine was taken up by the retinoblastoma cells through two kinetically distinguishable process. The high-affinity system is totally dependent upon extracellular Na+ and partially dependent upon Ca2+. Of the glycine taken up by retinoblastoma cells, 85-90% remains as free intracellular glycine and less than 30% is incorporated into cellular protein. When the cells are grown in a medium containing 10% fetal bovine serum as the only source of fatty acids, the phospholipids contained 23% polyunsaturated fatty acids. Under these conditions the high-affinity system has a K'm of 34.2 +/- 3.7 micrometers and a V'max of 91.2 +/- 16.2 pmol min-1 mg protein -1. The low-affinity system has a K'm of 2.7 +/- 0.4 mM and a V'max of 4.1 +/- 0.5 nmol min-1 mg protein-1. When the polyunsaturated fatty acid content of the phospholipids was increased by supplementing the medium with linolenic or docosahexaenoic acids (n-3 polyunsaturates) or linoleic or arachidonic acids (n-6 polyunsaturates), the K'm and V'max of the high-affinity glycine uptake system were increased three- to fourfold. By contrast, supplementing the medium with oleic acid, and n-9 monounsaturate, did not significantly alter the K'm or V'max for glycine uptake. The results with this model system suggest that one of the effects of the high polyunsaturated fatty acid content normally present in neural cell membranes may be a modulation of the high-affinity transport system so that it functions more efficiently in regulating glycine uptake.     

Ammonia formation in isolated rat liver mitochondria

Studies of isolated rat liver mitochondria were undertaken in order to evaluate the importance of glutamate transport, oxidation reduction state, and product inhibition on the rates of formation of ammonia from glutamate. Uptake and efflux of glutamate across the mitochondrial membrane were measured isotopically in the presence of rotenone. Efflux was stimulated by H+ in the mitochondrial matrix and was found to be first order with respect to matrix glutamate except when the matrix pH was unphysiologically low. The data suggest that the Km of matrix glutamate for efflux is decreased by H+. Matrix H+ also appeared to stimulate glutamate uptake, but the effect was to increase both the Km of medium glutamates and Vmax. Mitochondria were incubated at 15 and 28 degrees C with glutamate and malonate. Under these conditions, glutamate was metabolized only by the deamination pathway. Flux was evaluated by assay of ammonia formation. Oxidation reduction state was varied with ADP and uncoupling agents. Matrix alpha-ketoglutarate was varied either by the omission of malonate from the incubation media or by adding alpha-ketoglutarate to the external media. Influx and efflux of glutamate could be calculated from previously determined transport parameters. The difference between calculated influx and efflux was found to be equal to ammonia formation under all conditions. It was, therefore, possible to evaluate the relative contributions of oxidation reduction state, transport, and product inhibition as effectors of ammonia formation. The contribution of transport was relatively small while oxidation reduction state exerted a large influence. alpha-Ketoglutarate was found to be a potent competitive inhibitor of ammonia production and glutamate dehydrogenase. Inhibition of glutamate dehydrogenase by alpha-ketoglutarate was judged to be a potentially important modulator of metabolic fluxes.     

pH regulation of mitochondrial branch chain alpha-keto acid transport and oxidation in rat heart mitochondria

The kinetics of branched chain alpha-keto acid uptake and efflux were studied as a function of varied external and matrix pH. Matrix pH was determined by the distribution of 5,5'-dimethyloxazolidine-2,4-dione. When rat heart mitochondria were incubated under transport conditions at pH 7.0 with succinate as respiratory substrate, the matrix pH was significantly greater than 8.0. Matrix pH remained greater than or equal to 8.0 when the medium pH was varied from 6.3 to 8.3, and it was lowered below 8.0 by addition of 5 mM phosphate or uncoupler. No pH gradient was detectable when mitochondria were incubated in the presence of valinomycin and uncoupler. Efflux of alpha-ketoisocaproate or alpha-ketoisovalerate from rat heart mitochondria obeyed first order kinetics. Varying the external pH from 6.6 to 8.3 had no significant effect on efflux, and at an external pH of 7.0, the first order rate constant for efflux was not affected by decreasing the matrix pH. On the other hand, exchange was sensitive to changes in medium but not matrix pH. The K0.5 for external branched chain alpha-keto acid was lowered by changing the medium pH from 7.6 to 6.3. At medium pH values greater than or equal to 8.0 both K0.5 and Vmax were affected. Uptake was determined either by measuring initial rates or was calculated after measuring the first order approach to a final equilibrium value. Unlike efflux, uptake was sensitive to changes in both external and matrix pH. The rate of branched chain alpha-keto acid uptake was stimulated by decreasing the medium pH from 8.3 to 6.3 and by alkalinization of the mitochondrial matrix. The estimated external pK for proton binding was 6.9. The data indicate that the branched chain alpha-keto acid transporter is asymmetric, that is, binding sites for substrate on the inside and outside of the mitochondrial membrane are not identical. alpha-Ketoisocaproate oxidation was measured at 37 degrees C in isolated mitochondria over the pH range of 6.6 to 8.1. Changes in the rate of branched chain alpha-keto acid oxidation, particularly when ATP was added (increase delta pH), were found to parallel the pH effects observed on branched chain alpha-keto acid uptake. Therefore, transport, and by implication oxidation, can be regulated by pH changes within the physiological range. Furthermore, intracellular pH may affect the degree of compartmentation between the cytosolic and mitochondrial branched chain alpha-keto acid pools.     

Glutamine transport and metabolism by mitochondria from dog renal cortex. General properties and response to acidosis and alkalosis.

Mitochondria from dog renal cortex were incubated with L-[14Cglutamine. Glutamate metabolism was prevented by inhibitors so that glutamate accumulated either in the mitochondrial matrix space or in the medium. The formation and accumuation of glutamate formed from glutamine and the distribution of glutamine in the mitochondrial fluid spaces were studied. In the matrix space glutamate rapidly reaches levels over 5 times that of glutamine in the medium. A more gradual accumulation occurs in the medium as glutamate is transported out of the mitochondria. Addition of an energy source such as succinate to the medium accelerates glutamate formation. A Km of 0.6 mM appears to govern the reaction at low concentrations of glutamine; at about 4 mM an abrupt change kinetics occurs with a Km of 5 mM above that level. Both NH4+ and glutamate inhibit glutamine metabolism and phosphate stimulates it, but little effect glutamate or phosphate occurs at low levels of these substances. The pH optimum of the reaction is between 7.4 and 7.8. Mersalyl and p-chloromercuribenzoate strongly inhibit glutamate formation; N-ethylmaleimide and bromcresol green have weaker inhibitory actions, and borate increases the reaction rate. In the presence of mersalyl, glutamine is striclly confined to the outer space of mitochondria and none is detectable in the matrix space. Similarly at ) degrees glutamine is confined to the simultaneously determined sucrose or mannitol spaces...     

Quantitative characteristics of glutamate transport in rat liver mitochondria

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.     

Ethanolamine and choline transport in cultured bovine aortic endothelial cells

The transport of the polar head groups, ethanolamine and choline, was examined in cultured bovine aortic endothelial cells. Both ethanolamine and choline are taken up by high- and low-affinity systems. The K'm and V'max for the Na+-dependent, high-affinity ethanolamine and choline transport system are 3.0 and 3.0 microM and 5.4 and 7.3 pmol/mg protein/min, respectively. Ethanolamine and choline competitively influence one another's transport as the presence of 50 microM ethanolamine increases the K'm but not the V'max of choline uptake. Likewise, 50 microM choline increases the K'm but not the V'max of ethanolamine transport. The concentration of ethanolamine that inhibits maximal velocity of 5 microM choline by 50% is 9.7 microM, while 12 microM choline inhibits 5 microM ethanolamine maximal velocity by 50%. Uptake of both head groups is only partially Na+-dependent and is inhibited similarly by 2-methylethanolamine and 2,2-dimethylethanolamine at all concentrations examined. Hemicholinium-3, a classic inhibitor of high-affinity, Na+-dependent choline transport, reduces both ethanolamine and choline accumulation in a concentration-dependent fashion, but has a greater effect on choline transport at higher concentrations. The major portion of these data is consistent with our hypothesis that the uptake of physiological concentrations of ethanolamine and choline may occur through the same transport system. However, the results of the effect of hemicholinium-3 and the extent of Na+-dependency of choline and ethanolamine uptake could be interpreted as meaning that separate transport systems for choline and ethanolamine exist which cross react or that a single transport system exists which has separate active sites for the two compounds.     

Relationship of phosphoenolpyruvate transport, acyl coenzyme A inhibition of adenine nucleotide translocase and calcium ion efflux in guinea pig heart mitochondria.

The transport of phosphoenolpyruvate by the adenine nucleotide translocase system of heart mitochondria may be directly involved in the mechanism of phosphoenolpyruvate-induced calcium ion efflux. In contrast to liver mitochondria, the transport of phosphoenolpyruvate via the tricarboxylate carrier system is low or absent in heart mitochondria. The translocation of phosphoenolpyruvate which catalyzed adenine nucleotide and calcium efflux from heart mitochondria was inhibited by palmitoyl-CoA as well as atractylate and ATP. These results suggest that phosphoenolpyruvate, which is preferentially transported on the tricarboxylate carrier of liver mitochondria, is transported primarily via the adenine nucleotide translocase system in heart mitochondria. As a result of its inward transport, phosphoenolpyruvate is able to catalyze calcium ion as well as adenine nucleotide efflux from the mitochondrial matrix. Although not yet proven, either or both phosphoenolpyruvate and long chain acyl-CoA esters may act as natural physiological effectors in the regulation and distribution of intracellular calcium.     

Arginine transport in mitochondria of Neurospora crassa.

Transport of arginine into mitochondria of Neurospora crassa has been studied. Arginine transport was found to be saturable (Km = 6.5 mM) and to have a pH optimum of pH 7.5. Mitochondrial arginine transport appeared to be facilitated transport rather than active transport because: (i) the arginine concentration within the mitochondrial matrix after transport was similar to that of the reaction medium, and (ii) uncouplers and substrates of oxidative phosphorylation did not affect the transport rate. The basic amino acids ornithine, lysine, and D-arginine inhibited arginine transport. The arginine transport system could be irreversibly blocked by treating mitochondria with the reactive arginine derivative, N-nitrobenzyloxycarbonyl-arginyl diazomethane.     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase.

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH4+-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibted by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitos of respiration or an uncoupler but not in ther absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition. On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibted by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Sodium-Dependent Glutamate Uptake by an Alkaliphilic, Thermophilic Bacillus Strain, TA2.A1

A strain of Bacillus designated TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70 degrees C. Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was an obligate requirement for growth. Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (DeltapNa) across the cell membrane. N, N-Dicyclohexylcarbodiimide inhibited the growth of Bacillus strain TA2.A1, suggesting that an F1F0-ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of V-type ATPases, did not affect the growth of Bacillus strain TA2.A1. Glutamate transport by Bacillus strain TA2.A1 could be driven by an artificial membrane potential (DeltaPsi), but only when sodium was present. In the absence of sodium, the rate of DeltaPsi-driven glutamate uptake was fourfold lower. No glutamate transport was observed in the presence of DeltapNa alone (i.e., no DeltaPsi). Glutamate uptake was specifically inhibited by monensin, and the Km for sodium was 5.6 mM. The Hill plot had a slope of approximately 1, suggesting that sodium binding was noncooperative and that the glutamate transporter had a single binding site for sodium. Glutamate transport was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport. The rate of glutamate transport increased with increasing glutamate concentration; the Km for glutamate was 2.90 microM, and the Vmax was 0.7 nmol. min-1 mg of protein. Glutamate transport was specifically inhibited by glutamate analogues.     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH₄⁺-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate.

1. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue.

2. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibited by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate.

3. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitors of respiration or an uncoupler but not in their absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition.

On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibited by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Prominent glutamine oxidation activity in mitochondria of avian transplantable hepatoma induced by MC-29 virus

Well coupled mitochondria were isolated from transplantable chicken hepatoma induced by MC-29 virus. The mitochondrial phosphate-dependent and phosphate-independent glutaminase activities were increased compared with those from normal chicken liver. Glutamate dehydrogenase was undetectable in the tumor mitochondria. Oxypolarographic tests showed the following: glutamine oxidation was prominent in the tumor mitochondria and was mediated through an NAD-linked reaction, while mitochondria from the liver showed a feeble glutamine oxidation; glutamine oxidation by tumor mitochondria was inhibited either by aminooxyacetate, inhibitor of transaminases, or prior incubation of mitochondria with DON (6-diazo-5-oxonorleucine), which inhibited mitochondrial glutaminases. Bromofuroate, inhibitor of glutamate dehydrogenase, had little or no effect; and glutamate oxidation was also inhibited by aminooxyacetate, while it was not affected by DON. These findings clearly show a high glutamate oxidation activity in the hepatoma and indicate that the product of glutamine hydrolysis, glutamate, is catabolized via transamination in the mitochondria to supply ATP.     

A mechanism of sulfite neurotoxicity: direct inhibition of glutamate dehydrogenase

Exposure of Neuro-2a and PC12 cells to micromolar concentrations of sulfite caused an increase in reactive oxygen species and a decrease in ATP. Likewise, the biosynthesis of ATP in intact rat brain mitochondria from the oxidation of glutamate was inhibited by micromolar sulfite. Glutamate-driven respiration increased the mitochondrial membrane potential (MMP), and this was abolished by sulfite but the MMP generated by oxidation of malate and succinate was not affected. The increased rate of production of NADH from exogenous NAD+ and glutamate added to rat brain mitochondrial extracts was inhibited by sulfite, and mitochondria preincubated with sulfite failed to reduce NAD+. Glutamate dehydrogenase (GDH) in rat brain mitochondrial extract was inhibited dose-dependently by sulfite as was the activity of a purified enzyme. An increase in the Km (glutamate) and a decrease in Vmax resulting in an attenuation in Vmax/Km (glutamate) at 100 microm sulfite suggest a mixed type of inhibition. However, uncompetitive inhibition was noted with decreases in both Km (NAD+) and Vmax, whereas Vmax/Km (NAD+) remained relatively constant. We propose that GDH is one target of action of sulfite, leading to a decrease in alpha-ketoglutarate and a diminished flux through the tricarboxylic acid cycle accompanied by a decrease in NADH through the mitochondrial electron transport chain, a decreased MMP, and a decrease in ATP synthesis. Because glutamate is a major metabolite in the brain, inhibition of GDH by sulfite could contribute to the severe phenotype of sulfite oxidase deficiency in human infants.     

Carrier mediated GABA translocation into rat brain mitochondria

GABA added to rat brain mitochondria causes oxidation of intramitochondrial NAD(P)H as well as inducing glutamate efflux from the mitochondrial matrix. The rate of NAD(P)H oxidation shows saturation characteristics, depends on GABA transport across the mitochondrial membrane and is inhibited by non-penetrant compounds and by the metal-complexing agent bathophenanthroline. These results show the existence of a specific GABA carrier. Inhibition studies strongly suggest the existence of two separate binding sites, namely the GABA binding site and the dicarboxylates binding site, as well as suggest the presence of a metal ion (ions) at GABA binding site. The occurrence of a GABA/GLUTAMATE antiport is proposed which allows a cyclical route to account for GABA synthesis and degradation in brain.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Inhibition of Glutamine Transport in Rat Brain Mitochondria by Some Amino Acids and Tricarboxylic Acid Cycle Intermediates

Glutamine transport into rat brain synaptic and non-synaptic mitochondria has been monitored by the uptake of [³H]glutamine and by mitochondrial swelling. The concentration of glutamate in brain mitochondria is calculated to be high, 5–10 mM, indicating that phosphate activated glutaminase localized inside the mitochondria is likely to be dormant and the glutamine taken up not hydrolyzed. The uptake of [³H]glutamine is largely stereospecific. It is inhibited by glutamate, asparagine, aspartate, 2-oxoglutarate and succinate. Glutamate inhibits this uptake into synaptic and non-synaptic mitochondria by 95 and 85%, respectively. The inhibition by glutamate, asparagine, aspartate and succinate can be explained by binding to an inhibitory site whereas the inhibition by 2-oxoglutarate is counteracted by aminooxyacetic acid, which indicates that it is dependent on transamination. The glutamine-induced swelling, a measure of a very low affinity uptake, is inhibited by glutamate at a glutamine concentration of 100 mM, but this inhibition is abolished when the glutamine concentration is raised to 200 mM. This suggests that the very low affinity glutamine uptake is competitively inhibited by glutamate. Furthermore, glutamine-induced swelling is inhibited by 2-oxoglutarate, succinate and malate, similarly to that of the [³H]glutamine uptake. The properties of the mitochondrial glutamine transport are not identical with those of a recently purified renal glutamine carrier.     

Reversibility of energy-dependent Ca2+ accumulation in mitochondria

Ca2+ accumulation in energized rat liver mitochondria has been studied after the blockage of mitochondrial permeability transition pore (MPTP) by cyclosporin A. It is shown that Ca2+ transport is coupled to the countertransport of protons: from the matrix of mitochondria in the medium in the course of Ca2+ accumulation, and, on the contrary, from the medium to mitochondrial matrix after membrane depolarization. In standard incubation medium containing K+, Cl-, oxidation substrate (glutamate) and inorganic phosphate (H2PO4(-)) the observed stoichiometry of the exchange is 1Ca2+ : 1H+. In accordance with this exchange ratio, proton, as well as cation, transport follows the same first-order kinetics, which is characterized in both cases by very close values of reaction half-times and rate constants. It is shown that reversion of Ca2+ -uniporter, sensitive to ruthenium red, is necessary for Ca2+ - efflux from the matrix ofdeenergized mitochondria when MPTP is blocked by cyclosporin A. It is also shown that Ca2+ -uniporter reversion takes place only after membrane depolarization and permeabilization by protonophore CCCP. Calcium release from mitochondria in the presence of CCCP is accompanied by proton flow into the matrix. Both calcium and proton fluxes are sensitive to Ca2+ uniporter blocker, ruthenium red, which gives the evidence of the identity of Ca2+ -efflux and influx pathways. The data obtained lead to the conclusion that calcium-proton exchange is necessary for Ca2+ -uniporter reversion and the reversibility of energy-dependent Ca2+ -uptake in mitochondria.     

Regulation of human eIF4E by 4E-BP1: binding analysis using surface plasmon resonance

The mitochondria play a pivotal role in regulating glucose-induced insulin secretion in the pancreatic beta cell. We have recently demonstrated that glutamate derived from mitochondria participates directly in the stimulation of insulin exocytosis. In the present study, mitochondria isolated from the beta cell line INS-1E generated glutamate when incubated with the tricarboxylic acid cycle intermediate succinate. The generation of glutamate correlated with stimulated mitochondrial activity monitored as oxygen consumption and was inhibited by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Glutamate is formed by the mitochondrial enzyme glutamate dehydrogenase from alpha-ketoglutarate. Transient overexpression of glutamate dehydrogenase in INS-1E cells resulted in potentiation of glucose-stimulated hormone secretion without affecting basal release. These results further point to glutamate as an intracellular messenger playing a key role in the control of insulin exocytosis.