The effect of 2-oxoglutarate or 3-hydroxybutyrate on pyruvate dehydrogenase complex in isolated cerebro-cortical mitochondria

The oxidation of pyruvate is mediated by the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) whose catalytic activity is influenced by phosphorylation and by product inhibition. 2-Oxoglutarate and 3-hydroxybutyrate are readily utilized by brain mitochondria and inhibit pyruvate oxidation. To further elucidate the regulatory behavior of brain PDHC, the effects of 2-oxoglutarate and 3-hydroxyburyrate on the flux of PDHC (as determined by [1-¹⁴C]pyruvate decarboxylation) and the activation (phosphorylation) state of PDHC were determined in isolated, non-synaptic cerebro-cortical mitochondria in the presence or absence of added adenine nucleotides (ADP or ATP). [1-¹⁴C]Pyruvate decarboxylation by these mitochondria is consistently depressed by either 3-hydroxybutyrate or 2-oxoglutarate in the presence of ADP when mitochondrial respiration is stimulated. In the presence of exogenous ADP, 3-hydroxybutyrate inhibits pyruvate oxidation mainly through the phosphorylation of PDHC, since the reduction of the PDHC flux parallels the depression of PDHC activation state under these conditions. On the other hand, in addition to the phosphorylation of PDHC, 2-oxoglutarate may also regulate pyruvate oxidation by product inhibition of PDHC in the presence of 0.5 mM pyruvate plus ADP or 5 mM pyruvate alone. This conclusion is based upon the observation that 2-oxoglutarate inhibits [1-¹⁴C]pyruvate decarboxylation to a much greater extent than that predicted from the PDHC activation state (i.e. catalytic capacity) alone. In conjunction with the results from our previous study (Lai, J. C. K. and Sheu, K.-F. R. (1985) J. Neurochem. 45, 1861–1868), the data of the present study are consistent with the notion that the relative importance of the various mechanisms that regulate brain and peripheral tissue PDHCs shows interesting differences.     

Effects of Dichloroacetate on Brain Tissue Pyruvate Dehydrogenase

The activation of the pyruvate dehydrogenase complex (PDHC) by dichloroacetate (DCA) was studied in brain tissue. Chronic administration of DCA to rats caused no significant change of PDHC activation in brain. DCA brain concentrations were comparable to those of other tissues in which activation is known to occur. No effect of DCA on PDHC could be demonstrated from isolated brain mitochondria, whereas DCA reversed the deactivation of PDHC by ATP, alpha-ketoglutarate plus malate, and succinate in liver mitochondria. This study suggests that the regulation of PDHC activation in neural tissue differs from that in other tissues.     

Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.     

Pyruvate dehydrogenase complex is inhibited in calcium-loaded cerebrocortical mitochondria

An impairment of mitochondrial functions as a result of Ca-loading may be one of the significant events that lead to neuronal death after an ischemic insult. To assess the metabolic consequences of excess Ca on brain mitochondria, pyruvate oxidation was studied in isolated cerebrocortical mitochondria loaded with Ca in vitro. The flux of pyruvate dehydrogenase complex (PDHC) ([1-¹⁴C]pyruvate decarboxylation) was inhibited as the mitochondria accumulated excess Ca under the conditions tested: the inhibition in state 3 (i.e., in the presence of added ADP) was greater than in state 4 (i.e., in the absence of added adenine nucleotides). In state 4, the inhibition of the PDHC flux was accompanied by a similar reduction of the in situ activity of PDHC, indicating a change in PDHC phosphorylation. In state 3, the inhibition of the PDHC flux was greater than the corresponding decrease of the in situ PDHC activity. Thus, mechanisms other than the phosphorylation of PDHC might also contribute to the inhibition of pyruvate oxidation. Measurement of PDHC enzymatic activity in vitro indicated that PDHC, similar to -ketoglutarate dehydrogenase complex, was inhibited by millimolar levels of Ca. This observation suggests that PDHC may also be inhibited non-covalently in Ca-loaded mitochondria in a manner similar to that of -ketoglutarate dehydrogenase complex.     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: activity, properties, and subcellular localization

The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.     

Studies on the Pyruvate Dehydrogenase Complex in Brain with the Arylamine Acetyltransferase-Coupled Assay

Abstract: A spectrophotometric assay for the brain pyruvate dehydrogenase complex (PDHC) with arylamine acetyltransferase (ArAT; EC 2.3.1.5) to follow the production of acetyl-CoA has been standardized. Activity was proportional to time and protein. It depended completely on added pyruvate, CoA, NAD, and MgCI₂, and partially on thiamine pyrophosphate, Triton X-100, and a sulfhydryl compound. The activities are the highest in the literature for brain PDHC (50 nmol/min/mg protein) and equal the maximum recorded rates of pyruvate flux for brain in vivo . Activities as low as 0.6 nmol/min could be measured. Use of ArAT of different purities (1–2-fold and 11–%-fold) allowed convenient measurement of total PDHC (ArAT-I) and of the active form of PDHC (ArAT-II). The proportion of PDHC in the active form was 50% in mouse brain, 30% in rat brain, and 10% in mouse liver. Total PDHC activity was unchanged postmortem during storage of mouse brain in situ at +4°C or at -20°C for 3 days or at +20°C for 24 h. The relative specific activity of PDHC in cytoplasmic or synaptoplasmic fractions was less than that of two other mitochondrial enzymes, fumarase (EC 4.2.1.2) and monoamine oxidase (EC 1.4.3.4), which argues strongly against the hypothesis of a cytoplasmic PDHC in cholinergic nerve endings.     

Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..     

Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl(2) during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H(+) ions, and inhibited by ATP, NADH and Ca(2+). 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a ;classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.     

Plant Desiccation and Protein Synthesis. IV. RNA Synthesis, Stability, and Recruitment of RNA into Protein Synthesis during Desiccation and Rehydration of the Desiccation-Tolerant Moss, Tortula ruralis

We have studied the effects of ATP and ADP on the oxidation of malate by coupled and uncoupled mitochondria prepared from etiolated hypocotyls of mung bean (Vigna radiata L.).

In coupled mitochondria, ATP (1 millimolar) increased pyruvate production and decreased oxaloacetate formation without altering the rate of oxygen consumption. ATP also significantly decreased oxaloacetate production and increased pyruvate production in mitochondria that were uncoupled by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone plus oligomycin.

In coupled mitochondria, ADP (1 millimolar) increased the production of both pyruvate and oxaloacetate concomitantly with the acceleration of oxygen uptake to the state 3 rate. The effects of ADP were largely eliminated in uncoupled mitochondria. These results indicate that, whereas the ADP stimulation of oxaloacetate and pyruvate production in the coupled mitochondria is brought about primarily as the result of the accelerated rates of electron transport and NADH oxidation by the respiratory chain in state 3, ATP has significant regulatory effects independent of those that might be exerted by control of electron transport.

     

Mitochondria from the hepatopancreas of the marine clam Mercenaria mercenaria: substrate preferences and salt and pH effects on the oxidation of palmitoyl-L-carnitine and succinate

A method is presented for the isolation of mitochondria with good respiratory control from the hepatopancreas of the marine clam Mercenaria mercenaria. Palmitoyl-L-carnitine is the preferred substrate of the mitochondria of the hepatopancreas based on state 3 rates of oxidation (in the presence of ADP). Rates of oxidation of pyruvate and glutamate were about one-half that of the lipid substrate in state 3. alpha-Glycerophosphate was oxidized at a rate about one-third that of palmitoyl-L-carnitine. All Krebs cycle intermediates were oxidized to some extent. Proline was not oxidized at detectable levels. The optimal range of KCl concentrations for the oxidation of palmitoyl-L-carnitine is between 250 and 500 mM whereas the optimal range of KCl concentration for the oxidation of succinate is between 200 and 350 mM. The optimal range of pH for the oxidation of succinate and for the oxidation of palmitoyl-L-carnitine lies between pH 6.5 and 7.5 based on the respiratory control ratio.     

Immunochemical Characterization of Pyruvate Dehydrogenase Complex in Rat Brain

Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme-linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the alpha peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the alpha E1, beta E1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyruvate metabolism in castor-bean mitochondria.

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.     

Regulation of citric acid cycle by calcium

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.     

Pyruvate and acetate metabolism in termite mitochondria

Intact mitochondria have been successfully prepared from body tissues from the termites Nasutitermes walkeri and Coptotermes formosanus. This is the first report of the successful isolation of mitochondria from termites (Isoptera: Termitidae). Using an oxygen electrode, oxygen consumption by the mitochondria during the oxidation of various respiratory substrates was determined and their properties measured in terms of respiratory control index and ADP/O. ADP/O was as expected for substrates such as pyruvate, acetylcarnitine and acetyl-CoA and carnitine. Pyruvate and acetate were the major respiratory substrates in both species. The total activity of the pyruvate dehydrogenase complex (PDHc) in the mitochondria from N. walkeri and C. formosanus was determined to be 72.87+/-8.98 and 8.29+/-0.42 nmol/termite/h, respectively. Mitochondria isolated in the presence of inhibitors of PDHc interconversion were used to determine that about 60% of the PDHc was maintained in the active form in both N. walkeri and C. formosanus. The sufficient PDHc activity and high rate of pyruvate oxidation in mitochondria from N. walkeri suggest that pyruvate is rapidly metabolised, whereas the low mitochondrial PDHc activity of C. formosanus suggests that in this species more pyruvate is produced than can be oxidised in the termite tissues.     

The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The steady-state concentrations of citrate, isocitrate 2-oxoglutarate and malate in flight muscle and isolated mitochondria.

1. Blowfly (Phormia regina) flight-muscle mitochondria were allowed to oxidize pyruvate under a variety of experimental conditions, and determinations of the citrate, isocitrate, 2-oxoglutarate and malate contents of both the mitochondria and the incubation medium were made. For each intermediate a substantial portion of the total was present within the mitochondria. 2. Activation of respiration by either ADP or uncoupling agent resulted in a decreased content of citrate and isocitrate and an increased content of 2-oxoglutarate and malate when the substrate was pyruvate, APT and HCO3 minus. Such a decrease in citrate content was obscured when the substrate was pyruvate and proline owing to a large rise in the total content of tricarboxylate-cycle intermediates in the presence of proline and ADP. 3. An experiment involving oligomycin and uncoupling agent demonstrated that the ATP/ADP ratio is the main determinant of flux through the tricarboxylate cycle, with the redox state of nicotinamide nucleotide being of lesser importance. 4. Addition of ADP and Ca-2+ to activate the oxidation of both glycerol 3-phosphate and pyruvate, simulating conditions on initiation of flight, gave a decrease in citrate and isocitrate and an increase in 2-oxoglutarate and malate content. 5. There was a good correlation between these results with isolated flight-muscle mitochondria and the changes found in fly thoraces after 30s and 2 mihorax. 6. It is concluded that NAD-isocitrate dehydrogenase (EC 1.1.1.41) controls the rate of pyruvate oxidation in both resting fly flight muscle in vivo and isolated mitochondria in state 4 (nomenclature of Change & Williams, 1955).     

The decrease of mitochondrial substrate uptake caused by trialkyltin and trialkyl-lead compounds in chloride media and its relevance to inhibition of oxidative phosphorylation.

1. In a 100 mM-KCl medium (pH 6.8) containing ATP, triethyltin (1 muM) causes a decrease in the uptake of pyruvate, malate, citrate or beta-hydroxybutyrate by rat liver mitochondria, but no decrease is observed in a 100 mM-KNO3 medium. This response is not modified by the presence of rotenone in the incubation medium. 2. In the KCl medium at least 1 muM-triethyltin is required to cause maximum inhibition of pyruvate uptake. 3. Trimethyltin, tributyltin and the trialkyl-lead analogues at 1 muM, to varying degrees, also cause a decrease in pyruvate uptake by mitochondria only in the KCl medium. 4. Triethyltin stimulates resting respiration of mitochondria with all the substrates tested in the KCl medium but not in the KNO3 medium, yet this stimulation of O2 uptake occurs under conditions when substrate uptake is decreased. 5. In contrast, both O2 uptake during state 3 respiration and ATP synthesis when linked to the oxidation of pyruvate, malate or citrate are strongly inhibited by 1 muM-triethyltin in a KCl medium, but O2 uptake and ATP synthesis during the oxidation of beta-hydroxybutyrate are only slightly affected. In a KNO3 medium O2 uptake and ATP synthesis linked to the oxidation of all substrates are only slightly affected. 6. The relevance of the decrease in substrate uptake by mitochondria caused by triethyltin in a KCl medium to the greater sensitivity of various mitochondrial functions observed in vitro is discussed. It is concluded that decrease of matrix substrate content is probably not the major cause of the greater sensitivity of oxidative phosphorylation to triethyltin in a KCl medium observed previously.     

Distribution of control of oxidative phosphorylation in mitochondria oxidizing NAD-linked substrates.

The flux control distribution of the net rate of state 3 respiration was determined in heart and kidney mitochondria incubated with low concentrations of pyruvate (0.5 mM) or 2-oxoglutarate (1 mM), and in conditions that led to activation of NAD-linked dehydrogenases, i.e., high substrate or Ca2+ concentrations. Control of flux was exerted by the ATP/ADP carrier (flux control coefficient, ci = 0.37) and Site 1 of the respiratory chain (ci = 0.28) when dehydrogenase activity was low. Control of the process shifted to the ATP synthase (ci = 0.32) and the Pi carrier (Ci = 0.27) when dehydrogenases were activated by high pyruvate and high Ca2+. The changes in the control exerted by the ATP/ADP carrier and the ATP synthase were not due to changes in the transmembrane potential, nor to a modification of intramitochondrial ATP/ADP ratios. Applying the summation theorem of the control analysis, it was found that at low Ca2+ and pyruvate concentrations the dehydrogenases shared the control of state 3 respiration with other steps. The NAD-linked dehydrogenases did not exert any significant control at high Ca2+ or high pyruvate concentrations.     

Energy Metabolism in Rat Brain: Inhibition of Pyruvate Decarboxylation by 3-Hydroxybutyrate in Neonatal Mitochondria

Abstract: The effect of 3-hydroxybutyrate on pyruvate decarboxylation by neonatal rat brain mitochondria and synaptosomes was investigated. The rate of [1 -¹⁴C]pyruvate decarboxylation (1 mm final concentration) by brain synaptosomes derived from 8-day-old rats was inhibited by 10% in the presence of 2 mm -d ,l -3-hydroxybutyrate and by more than 20% in the presence of 20 mm -d ,l -3-hydroxybutyrate. The presence of 2 mm -l ,d -3-hydroxybutyrate did not affect the rate of [1-¹⁴T]pyruvate decarboxylation (1 mm final concentration) by brain mitochondria; however, at a concentration of 20 mm -d ,l -3-hydroxybutyrate, a marked inhibition was seen in preparations from both 8-day-old (35% inhibition) and 21-day-old (24% inhibition) but not in those from adult rats. Although the presence of 100 mm -K⁺ in the incubation medium stimulated the rate of pyruvate decarboxylation by approximately 50% compared with the rate in the presence of 1 mm -K⁺, the presence of 20 mm -d ,l -3-hydroxybutyrate still caused a marked inhibition in both media (1 and 100 mm -K⁺). The presence of 20 mm -d ,l -3-hydroxybutyrate during the incubation caused an approximately 20% decrease in the level of the active form of the pyruvate dehydrogenase complex in brain mitochondria from 8-day-old rats. The concentrations of ATP, ADP, NAD⁺, NADH, acetyl CoA, and CoA were measured in brain mitochondria from 8-day-old rats incubated in the presence of 1 mm -pyruvate alone or 1 mm -pyruvate plus 20 mm -d ,l -3-hydroxybutyrate. Neither the ATP/ADP nor the NADH/NAD⁺ ratio showed significant changes. The acetyl CoA/CoA ratio was significantly increased by more than twofold in the presence of 3-hydroxybutyrate. The possible mechanisms and physiological significance of 3-hydroxybutyrate inhibition of pyruvate decarboxylation in neonatal rat brain mitochondria are discussed.     

Regulation of fatty acid oxidation in rat brain mitochondria: inhibition of high rates of palmitate oxidation by ADP

Regulation of oxidation of [1-14C]palmitate in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin prepared by use of a Ficoll/sucrose density gradient. The mitochondrial preparation contained considerable Mg2+-ATPase activity, but was virtually free of contamination with nonmitochondrial fractions. Palmitate oxidation was inhibited by increasing the concentration of ATP in the assay system to near-physiological levels (2 mM), and the inhibition at 2 or 4 mM ATP was analyzed by comparing it with palmitate oxidation at near-maximal rates with low levels of ATP (0.5 or 1 mM). Inhibition was increased by the addition of ADP or by increasing the concentration of Mg2+ in the assay system, whereas inhibition was decreased by decreasing the concentration of mitochondrial protein or L-carnitine in the assay system. Increasing CoA concentration also had a deinhibitory effect. With 0.5 or 1 mM ATP, however, neither inhibition by added ADP nor protein concentration-dependent inhibition was observed, and the rate of oxidation was saturated with increasing concentrations of Mg2+, L-carnitine, or CoA. These results indicated that ADP was involved in the inhibition of high rates of palmitate oxidation in the presence of sufficient ATP and L-carnitine. The inhibitory effect of increasing the concentration of mitochondrial protein could be explained by the enhanced amounts of ADP present in the preparation; similarly, increased concentrations of Mg2+ would provide higher levels of ADP by stimulating the Mg2+-ATPase reaction. We discuss the possibility that the transport of ADP across the inner membrane of brain mitochondria is coupled to the inhibition of palmitate oxidation.