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Androgen receptor (AR) signaling plays a central role in metabolic reprogramming for prostate cancer (PCa) growth and progression. Mitochondria are metabolic powerhouses of the cell and support several hallmarks of cancer. However, the molecular links between AR signaling and the mitochondria that support the metabolic demands of PCa cells are poorly understood. Here, we demonstrate increased levels of dynamin-related protein 1 (DRP1), a mitochondrial fission mediator, in androgen-sensitive and castration-resistant AR-driven PCa. AR signaling upregulates DRP1 to form the VDAC-MPC2 complex, increases pyruvate transport into mitochondria, and supports mitochondrial metabolism, including oxidative phosphorylation and lipogenesis. DRP1 inhibition activates the cellular metabolic stress response, which involves AMPK phosphorylation, induction of autophagy, and the ER unfolded protein response, and attenuates androgen-induced proliferation. Additionally, DRP1 expression facilitates PCa cell survival under diverse metabolic stress conditions, including hypoxia and oxidative stress. Moreover, we found that increased DRP1 expression was indicative of poor prognosis in patients with castration-resistant PCa. Collectively, our findings link androgen signaling-mediated mitochondrial dynamics to metabolic reprogramming; moreover, they have important implications for understanding PCa progression.

Fisetin is a flavonoid dietary ingredient found in the smoke tree (Cotinus coggyria) and in several fruits and vegetables. The effects of fisetin on glucose metabolism in the isolated perfused rat liver and some glucose-regulating enzymatic activities were investigated. Fisetin inhibited glucose, lactate, and pyruvate release from endogenous glycogen. Maximal inhibitions of glycogenolysis (49%) and glycolysis (59%) were obtained with the concentration of 200 microM. The glycogenolytic effects of glucagon and dinitrophenol were suppressed by fisetin 300 microM. No significant changes in the cellular contents of AMP, ADP, and ATP were found. Fisetin increased the cellular content of glucose 6-phosphate and inhibited the glucose 6-phosphatase activity. Gluconeogenesis from lactate and pyruvate or fructose was inhibited by fisetin 300 microM. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC(50) = 163.10 +/- 12.28 microM); no such effect was observed in freeze-thawing disrupted mitochondria. It was concluded that fisetin inhibits glucose release from the livers in both fed and fasted conditions. The inhibition of pyruvate transport into the mitochondria and the reduction of the cytosolic NADH-NAD(+) potential redox could be the causes of the gluconeogenesis inhibition. Fisetin could also prevent hyperglycemia by decreasing glycogen breakdown or blocking the glycogenolytic action of hormones.

The action of gatifloxacin, the broad-spectrum fluoroquinolone antibiotic commonly used in the therapy of various bacterial infections, was investigated in isolated rabbit hepatocytes and kidney-cortex tubules by measuring the activity of gluconeogenesis, a process that maintains whole body glucose homeostasis. The data show that in kidney-cortex tubules, application of gatifloxacin at up to 100 μM was followed by a marked accumulation of the drug in the intracellular milieu and a decrease in the rate of glucose formation from pyruvate by 20–50%. Gatifloxacin did not affect the rate of gluconeogenesis from either alanine + glycerol + octanoate or aspartate + glycerol + octanoate. At concentrations between 25 and 200 μM the drug decreased mitochondrial oxygen consumption by 20–45% with pyruvate + malate and ADP. As in the case of alpha-cyano-4-hydroxycinnamate, a well-established inhibitor of the mitochondrial pyruvate transporter, it diminished pyruvate uptake by both renal and hepatic mitochondria. The inhibitory action of gatifloxacin was less pronounced in hepatocytes where reduction in pyruvate-dependent glucose formation and mitochondrial respiration was by no more than 25%. The antibiotic did not influence mitochondrial oxygen consumption with glutamate + malate in either kidney-cortex or liver mitochondria. A differential substrate dependence of gatifloxacin action on gluconeogenesis and mitochondrial respiration combined with a decrease in pyruvate uptake by mitochondria suggest that the inhibitory action of this drug on gluconeogenesis might result from its impairment of pyruvate transport into mitochondria.

The characteristics of pyruvate transport across the plasma membrane in the bloodstream form of Trypanosoma brucei were studied using [14C]pyruvate in combination with the silicone-oil centrifugation technique. We present evidence for the existence of a facilitated diffusion carrier in the plasma membrane of T. brucei which specifically mediates the translocation of pyruvate. The uptake of pyruvate followed saturation kinetics (Km 1.96 +/- 0.28 mM; Cmax 36.61 +/- 1.15 nmol pyruvate/30 sec.mg protein), after correction of the data for a nonsaturable diffusion component. The uptake of pyruvate was competitively inhibited by a number of (oxo)monocarboxylic acids, including pyruvate analogs and metabolically related substances, but not by L-lactate. The transport exhibited the phenomenon of transacceleration, indicative for the involvement of a facilitated diffusion carrier. The carrier is highly specific for pyruvate and differs from other known monocarboxylate carriers present in the mitochondrial and/or plasma membrane of other eukaryotic cells in that it does not transport L-lactate.

The effect of aging on the activity of the pyruvate translocator and on the lipid composition in rat-heart mitochondria has been investigated. It has been found that the rate of pyruvate transport in mitochondria from aged rats (28 months old) is markedly reduced (38%) as compared with that obtained with mitochondria from young adults rats (4 months old). Kinetic analysis of the pyruvate transport shows that only the Vmax of this process is decreased, while there is no change in the Km values. The age-related decrement in the activity of the pyruvate carrier is not due to a decrease in the transmembrane delta pH value, neither does it depend on a decrease in the total number of the pyruvate carrier molecules, titrated with radioactive alpha-cyanocinnamate. The lower activity of the pyruvate translocator in mitochondria from aged rats is associated to a parallel decrement of the rate of pyruvate-dependent oxygen uptake. There is, however no appreciable difference in either the respiratory control ratios or in the ADP/O ratios between these two types of mitochondrion. The Arrhenius plot characteristics differ for pyruvate transport activity in mitochondria from aged rats as compared with young rats in that the break point of the biphasic plot is shifted to a higher temperature. The heart mitochondrial lipid composition is significantly altered in aged rats. The total cholesterol increases (43%), the phospholipids decrease (15%) and the cholesterol/phospholipid molar ratio increases (68%). Among phospholipids, cardiolipin shows the greatest alteration (28% decrease in aged rats). The lower activity of the pyruvate carrier in mitochondria from aged rats may be ascribed to changes in the lipid domain surrounding the carrier molecule in the membrane.

The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent Mr after the labelling points to the presence of at least two cysteine residues. Pyruvate uptake in the reconstituted system is inhibited by both permeable (eosin-5-maleimide at 1 mM concentration) and impermeable (mersalyl, p-chloromercuribenzoate) -SH group reagents. Phenylarsine oxide inhibits pyruvate transport only slightly (20%), but the inhibition is enhanced after preincubation with the substrate.

Jesus A. Marcos, Dolores de Arriaga, Felix Busto, and Joaquin Soler 1 1997. Functional Characteristics of Pyruvate Transport in Phycomyces blakesleeanus. Fungal Genetics Biology 25 , 204-215. A saturable and accumulative transport system for pyruvate has been detected in Phycomyces blakesleeanus NRRL 1555(−) mycelium. It was strongly inhibited by α-cyano-4-hydroxycinnamate. l -Lactate and acetate were competitive inhibitors of pyruvate transport. The initial pyruvate uptake velocity and accumulation ratio was dependent on the external pH. The V max of transport greatly decreased with increasing pH, whereas the affinity of the carrier for pyruvate was not affected. The pyruvate transport system mediated its homologous exchange, which was essentially pH independent, and efflux, which increased with increasing external pH. The uptake of pyruvate was energy dependent and was strongly inhibited by inhibitors of oxidative phosphorylation and of the formation of proton gradients. Glucose counteracted the inhibitory effect of the pyruvate transport produced by inhibitors of mitochondrial ATP synthesis. Our results are consistent with a pyruvate/proton cotransport in P. blakesleeanus probably driven by an electrochemical gradient of H + generated by a plasma membrane H + -ATPase.

Pyruvate oxidation was measured in cardiac mitochondria from euthyroid and hypothyroid rats fed diets enriched with either omega-6 or omega-3 fatty acids. Both State 4 and State 3 rates of pyruvate-dependent respiration were markedly reduced in hypothyroid mitochondria, regardless of diet consumed, compared to euthyroid controls. Respiratory control ratios and ADP/O ratios were the same under all treatments. While there was no significant effect of diet on respiration in euthyroid mitochondria, pyruvate oxidation was 28% higher in hypothyroid mitochondria from animals fed the omega-3 diet compared to those fed the omega-6 diet. Depressed respiration in the hypothyroid state was correlated with 18% more phosphatidylcholine in the inner mitochondrial membrane whereas phosphatidylethanolamine was 17% lower and cardiolipin 32% lower compared to controls. The total phospholipid fatty acid composition was not affected by the hypothyroid state. However, enhanced respiration in hypothyroid animals fed the omega-3 diet was associated with a 3-fold increase in monounsaturated fatty acids in the cardiolipin fraction, and a 12-fold increase in omega-3 fatty acids, primarily 22:5(omega-3) and 22:6(omega-3). The data suggest that membrane levels of cardiolipin and its omega-3 fatty acid content modulate pyruvate transport in hypothyroid mitochondria.

The pyruvate produced by glycolysis in the bloodstream form of the trypanosome is excreted into the host bloodstream by a facilitated diffusion carrier. The sensitivity of pyruvate transport for alpha-cyano-4-hydroxycinnamate and the compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate], which are known to be selective inhibitors of pyruvate (monocarboxylate) transporters present in mitochondria and the plasma membrane of eukaryotic cells, was examined. The trypanosomal pyruvate carrier was found to be rather insensitive to inhibition by alpha-cyano-4-hydroxycinnamate (Ki = 17 mM) but could be completely blocked by UK5099 (Ki = 49 microM). Inhibition of pyruvate transport resulted in the retention, and concomitant accumulation, of pyruvate within the trypanosomes, causing acidification of the cytosol and osmotic destabilization of the cells. Our results indicate that this physiological state has serious metabolic consequences and ultimately leads to cell death; thereby identifying the pyruvate carrier as a possible target for chemotherapeutic intervention.

Pyruvate transport and carboxylation have been determined in mitochondria from liver and kidney cortex isolated from Wistar rats with acidosis produced by three different treatments: fasting, exercise and ingestion of ammonium chloride. Fasting for 48 h or swimming for 2 h resulted in an increased rate of CO2 fixation by mitochondria from both organs incubated with pyruvate. This increase was accompanied by a rise in the rate of pyruvate transport in all cases except in mitochondria derived from the kidney of the fasted animals. Acute acidosis produced by the ingestion of ammonium chloride resulted in increases in pyruvate transport and carboxylation in kidney mitochondria, but a drop in pyruvate carboxylation was observed in mitochondria from the liver. The results are discussed in terms of the differential regulation of the mitochondria steps for gluconeogenesis from three carbon precursors in liver and kidney, taking into consideration the hormonal status of the animals and the prevailing available substrates in each condition.

In the presence of glucose and ample oxygen, insect form African trypanosomes release pyruvate more than 100-fold more slowly than do bloodstream forms. This rate decrease could not be accounted for simply by an increased mitochondrial pyruvate oxidation rate as inhibiting mitochondrial respiration increases pyruvate efflux to rates only 2-3% of that observed for bloodstream form trypanosomes. Alternatively, decreased pyruvate efflux from insect form trypanosomes could not be accounted for by decreased pyruvate transporter activity, which, surprisingly, was nearly as high in insect form trypanosomes as reported by us earlier for bloodstream forms (J.P. Barnard, B. Reynafarje, and P.L. Pedersen (1993) J. Biol. Chem. 268, 3654-3661). Rather, the low pyruvate efflux rate appears to be due primarily to reduced levels of the enzyme pyruvate kinase, which, in contrast to conclusions of an earlier study, is readily detected in insect form trypanosomes in the absence of added activators at an activity level about 4% of that found in bloodstream forms. Insect form pyruvate kinase seems to be located in the cytosol and exhibits kinetic profiles and constants nearly identical to those reported by us earlier for the bloodstream form enzyme (J.P. Barnard, and P.L. Pedersen (1988) Mol. Biochem. Parasitol. 31, 141-148). It is suggested that the reduced levels of pyruvate kinase, and hence the reduced pyruvate efflux rates, in insect form trypanosomes result from down regulation of the gene encoding the cytosolic enzyme.

Myocardial pyruvate oxidation is work- or calcium-load-related, but control of pyruvate dehydrogenase (PDH) by the specific mitochondrial pyruvate transporter has also been proposed. To test the transport hypothesis distribution of pyruvate across the cell membrane as well as rates of mitochondrial pyruvate net transport plus oxidation were examined in isolated perfused but stable and physiologically working guinea-pig hearts. 150 microM-1.2 mM alpha-cyanohydroxycinnamate proved to specifically block mitochondrial pyruvate uptake in these hearts. When perfusate glucose as cytosolic pyruvate precursor was supplied in combination with octanoate (0.2 or 0.5 mM) as diffusible alternative fatty acid substrate, alpha-cyanohydroxycinnamate produced up to 20- and 3-fold increases in pyruvate and lactate efflux, respectively. Cinnamates did not alter myocardial hemodynamics nor sarcolemmal pyruvate and lactate export. In contrast the tested concentrations of cinnamate produced reversible, dose-dependent decreases in 14CO2 production from [1-14C]pyruvate or [U-14C]glucose by inhibiting mitochondrial pyruvate uptake. Linear least-squares estimates of available cinnamate-sensitive total pyruvate transport potential yielded rates close to 110 mumol/min per g dry mass at S0.5 approximately 120 microM, which compared reasonably well with literature values from isolated cardiac mitochondria. This transport potential was severalfold larger than total extractable myocardial PDH activity of approximately 32 mumol/min per g dry mass at 37 degrees C. Even when cytosolic pyruvate levels were in the lower physiologic range of about 90 microM, pyruvate oxidation readily kept pace with mitochondrial respiration over a wide range of workload and inotropism. Furthermore, dichloroacetate, a selective activator of PDH, stimulated pyruvate oxidation without affecting myocardial O2 consumption, regardless of the metabolic or inotropic state of the hearts. Consequently, little or no regulatory function with regard to pyruvate oxidation could be assigned to the native mitochondrial pyruvate carrier of the working heart. Therefore, mitochondrial pyruvate-H+ symport was the normal, highly efficient (rather than controlling) mechanism for pyruvate entry into the mitochondria where PDH regulation controlled pyruvate oxidation.

1. The kinetics of transport of pyruvate (Km 0.20 mM), L-lactate (Km 2.2 mM) and D-lactate (Ki 10.2 mM) into rat cardiac myocytes were studied and compared with those for guinea-pig heart cells [Poole, Halestrap, Price and Levi (1989) Biochem. J. 264, 409-418] whose equivalent values were 0.07, 2.3 and 6.6 mM respectively. Maximal rates of transport were about 5-fold higher in the rat heart cells. 2. 4,4′-Dibenzamidostilbene-2,2′-disulphonate (DBDS), a powerful inhibitor of monocarboxylate transport into erythrocytes [Poole & Halestrap (1991) Biochem. J. 275, 307-312], was found to be a potent but apparently partial inhibitor of lactate and pyruvate transport, with an apparent Ki value at 0.5 mM L-lactate of about 16 microM in both species. Maximal inhibition was 50% and 80% in rat and guinea-pig cells respectively. 3. The maximal extent of inhibition and apparent Ki values were dependent on both the substrate transported and its concentration. Maximum inhibition was less and the Ki was greater at higher substrate concentrations. 4. A variety of other stilbene disulphonates were studied which showed different Ki values and maximal extents of inhibition. 5. Phloretin was a significantly less potent inhibitor of transport into both rat (Ki 25 microM) and guinea-pig (Ki 16 microM) heart cells than into rat erythrocytes (Ki 1.4 microM). In the rat but not the guinea-pig heart cells, inhibition appeared partial (maximal inhibition 84%). 6. We demonstrate that our results can be explained by the presence of two monocarboxylate carriers in heart cells, both with Km values for L-lactate of about 2 mM and inhibited by alpha-cyano-4-hydroxycinnamate, but with different affinities for other substrates and inhibitors. One carrier is sensitive to inhibition by stilbene disulphonates and has lower Km values for pyruvate (0.05-0.10 mM) and D-lactate (5 mM), whereas the other has higher Km values for pyruvate (0.30 mM) and D-lactate (25 mM), and is relatively insensitive to stilbene disulphonates. Rat heart cells possess more of the latter carrier and guinea-pig heart cells more of the former. 7. The significance of these results for the study of lactate transport in the perfused heart is discussed.

The pyruvate carrier, of molecular mass 34 kDa, was purified from mitochondria isolated from rat liver, rat brain, and bovine heart, by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate. Its activity after reconstitution in phosphatidylcholine vesicles was measured either as uptake of [1- 14 C]pyruvate or as exchange with different 2-oxoacids. All preparations exhibited similar apparent K m values for pyruvate, but somewhat different V max values. The ability to exchange different anions of physiological significance, including branched-chain 2-oxoacids, confirmed the known substrate specificity described for the pyruvate carrier in mitochondria. The sensitivity of pyruvate transport toward phenylglyoxal suggested an important role of arginyl residues in the transport activity, while a role of lysyl and histydyl residues was not confirmed.

Inhibition of endogenous long chain fatty acids oxidation by tetradecylglycidate (TDGA) impeded gluconeogenesis from lactate or from low concentrations of pyruvate (0.5 mM), β-hydroxybutyrate or even ethanol was capable of overcoming the inhibitory effect of TDGA in the absence of significant changes in the hepatic content of acetyl-CoA. At low (, This work has been supported in part by grants from Dirección General de Investigación Científica y Técnica (87065 and PL87-0280), and Fondo de Investigaciones Sanitarias (89/0467 and 89/0468). C. González-Manchón was recipient of a fellowship from Ministerio de Educación y Ciencia.

The mechanisms of lactate and pyruvate transport across the plasma membrane of rat skeletal muscle under various pH and ionic conditions were studied in skeletal muscle sarcolemmal (SL) membrane vesicles purified from 22 female Sprague-Dawley rats. Transport by SL vesicles was measured as uptake of L(+)-[U-14C] lactate and [U-14C] pyruvate. Lactate (La-) transport is pH-sensitive; stimulations to fivefold overshoot above equilibrium values were observed both directly by a proton gradient directed inward, and indirectly by a monensin- or nigericin-stimulated exchange of Na+ or K+ for H+ across the SL. Isotopic pyruvate could utilize the transporter, and demonstrated pH gradient-stimulated overshoot and cis-inhibition characteristics similar to those of lactate. Overshoot kinetics were also demonstrated by pH gradient formed by manipulation of external media at pH 5.9, 6.6, and 7.4 and intravesicular media at 6.6, 7.4, and 8.0, respectively. Carbonyl cyanide m-chlorophenylhydrazone, an H+ ionophore, was used as a "pH clamp" to return all stimulated uptake courses back to equilibrium values. Lactate uptake was depressed when internal pH was lower than external pH. These data strongly suggest that La- and H+ are either cotransported by the carrier, or transported as the undissociated HLa, and can account for the majority of the lactate uptake at pH 7.4. The mechanism does not require cotransport of either K+ or Na+. However, an inwardly directed Na+ gradient without ionophore in the absence of a pH gradient doubled La- transport; treatment with amiloride, an inhibitor of the Na+/H+ exchanger, abolished this stimulation, suggesting that this transporter may be an important coregulator of intracellular pH, and could disrupt 1:1 H+ and La- efflux stoichiometry in vivo. We conclude that the majority of La- crosses the skeletal muscle SL by a specific carrier-mediated process that is saturable at high La- concentrations, but flux is passively augmented at low intracellular pH by undissociated lactic acid. In addition, a Na+/H+ exchange mechanism was confirmed in skeletal muscle SL, does affect both lactate and proton flux, and is potentially an important coregulator of intracellular pH and thus, cellular metabolism.

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.

Phenylephrine effect on liver and kidney cortex mitochondrial pyruvate concentration was investigated. While in liver the α 1 -adrenergic agent produced a decrease in pyruvate content, a significant increase was observed in kidney, even in the presence of 0.5 mM α-cyano-4-hydroxy-cinnamate. These changes were not observed when pyruvate was formed by intramitochondrial transamination of alanine, suggesting a role for the pyruvate transport across mitochondrial membranes in the regulation of mitochondrial pyruvate metabolism in kidney cortex. This was corroborated measuring the phenylephrine effect on pyruvate carboxylation.

The sensitivity of glucose production from L-lactate by isolated liver cells from starved rats to inhibition by alpha-cyano-4-hydroxycinnamate was studied. A small percentage of the maximal rate of gluconeogenesis was insensitive to inhibition by alpha-cyano-4-hydroxycinnamate, and evidence is presented to show that this is due to pyruvate entry into the mitochondria as alanine. After subtraction of this rate, Dixon plots of the reciprocal of the rate of gluconeogenesis against inhibitor concentration were linear both in the absence and presence of glucagon, phenylephrine or valinomycin, each of which stimulated gluconeogenesis by 30-50%. Pyruvate kinase activity was decreased by glucagon, but not by phenylephrine or valinomycin. Inhibition of gluconeogenesis by quinolinate (inhibitor of phosphoenolpyruvate carboxykinase) or monochloroacetate (probably inhibiting pyruvate carboxylation) caused a significant deviation from linearity of the Dixon plot obtained with alpha-cyano-4-hydroxycinnamate. Amytal, however, inhibited gluconeogenesis without affecting the linearity of this plot. These data, coupled with a computer simulation study, suggest that pyruvate transport may control gluconeogenesis from L-lactate and that hormones may stimulate this process through an effect on the respiratory chain. An additional role for pyruvate kinase and pyruvate carboxylase is quite compatible with the data presented.

A mutant of Escherichia coli K-12 lacking pyruvate dehydrogenase and phosphoenolpyruvate synthase was used to study the transport of pyruvate by whole cells. Uptake of pyruvate was maximal in mid-log phase cells, with a Michaelis constant for transport of 20 microM. Pretreatment of the cells with respiratory chain poisons or uncouplers, except for arsenate, inhibited transport up to 95%. Lactate and alanine were competitive inhibitors, but at nonphysiological concentrations. The synthetic analogs 3-bromopyruvate and pyruvic acid methyl ester inhibited competitively. The uptake of pyruvate was also characterized in membrane vesicles from wild-type E. coli K-12. Transport required an artificial electron donor system, phenazine methosulfate and sodium ascorbate. Pyruvate was concentrated in vesicles 7- to 10-fold over the external concentration, with a Michaelis constant of 15 microM. Energy poisons, except arsenate, inhibited the transport of pyruvate. Synthetic analogs such as 3-bromopyruvate were competitive inhibitors of transport. Lactate initially appeared to be a competitive inhibitor of pyruvate transport in vesicles, but this was a result of oxidation of lactate to pyruvate. The results indicate that uptake of pyruvate in E. coli is via a specific active transport system.

1Pyruvate equilibrates, like other carboxylic acids, across the membrane of rat-liver mitochondria according to the transmembrane pH difference. 2Comparison of the rate of pyruvate, acetoacetate and acetate efflux from mitochondria as well as a study of the effect of mersalyl and N-ethylmaleimide on this process, show that the diffusion of pyruvic and acetoacetic acid across the mitochondrial membrane is carrier-mediated. The process might consist of pyruvate (acetoacetate) hydroxyl ion exchange-diffusion or of pyruvic acid (acetoacetic acid) uniport. On the contrary acetic acid appears to diffuse freely across the membrane. 3The initial rate of the net pyruvate uptake by rat liver mitochondria follows saturation kinetics. The increase of the external pH of the medium as well as the addition of acetoacetate inhibit competitively pyruvate uptake. Pyruvate uptake is inhibited by mersalyl and N-ethylmaleimide. 4Mitochondria1 [14C]pyruvate exchanges with unlabelled pyruvate and acetoacetate but not with other monocarboxylates, Pi and citric-acid cycle intermediates. 5The pyruvate-pyruvate and pyruvate-acetoacetate exchanges follow saturation kinetics and exhibit the same V but different Km values. Both the exchanges are inhibited by mersalyl, but not by N-ethylmaleimide. 6It is concluded that pyruvate translocation across the mitochondrial membrane is mediated by a specific transport system.

1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite direction of the activity of pyruvate dehydrogenase (EC. 1.2.4.1). 3. Changes of the transmembrane pH gradient and of the membrane potential, brought about by the pretreatments of the mitochondria, cannot account for the observed changes in the rate of pyruvate transport. 4. It is proposed that the pretreatment of the mitochondria directly modulates the activity of the mitochondrial pyruvate carrier. The possible regulatory role of such a modulation system is discussed.

The effect of alloxan on anion transport in isolated mouse liver mitochondria was studied with the swelling technique. Mitochondria pre-incubated with alloxan exhibited partial inhibition of the transport of malate, citrate, pyruvate and glutamate. 2n-butylmalonate caused complete inhibition of malate transport, and the translocation of citrate was completely blocked by 1,2,3-benzenetricarboxylate, while N-ethylmaleimide inhibited glutamate transport partially. The observation that alloxan inhibits different mitochondrial anion transport mechanisms emphasizes that the drug profoundly affects mitochondrial function.

A study of the transport of pyruvate in heart mitochondria from normal and hypothyroid rats has been carried out. Heart mitochondria from hypothyroid rats translocate pyruvate via the alpha-cyanocinnamate sensitive carrier much more slowly than do mitochondria from normal rats. Kinetic analysis of the pyruvate transport shows that the Vmax of this process is decreased while there is practically no change in the Km values. Neither a decrease in the transmembrane delta pH value nor a decrease in the total number of the pyruvate carrier molecules, titrated with labeled alpha-cyanocinnamate, account for the decreased rate of pyruvate transport. The lower activity of the pyruvate translocator in mitochondria from hypothyroid rats is associated with a parallel decrease of the rate of pyruvate supported oxygen uptake. There is, however, no difference in either the respiratory control ratios or in the ADP/O ratios between these two types of mitochondria. The heart mitochondrial lipid composition is significantly altered in hypothyroid rats. Cardiolipin, particularly, was found to decrease by around 36%. In addition the pattern of fatty acids was found to be altered in mitochondrial membranes from hypothyroid rats. It is suggested that the decreased activity of the pyruvate translocator in heart mitochondria from hypothyroid rats can be ascribed to changes in the lipid environment which surrounds the pyruvate carrier molecule in the mitochondrial membrane.

The effect of the mitochondrial pyruvate transport inhibitors, α-cyanocinnamate and α-cyano-4-hydroxycinnamate, on the regulation of the pyruvate dehydrogenase multienzyme complex was investigated in the isolated perfused rat heart. Metabolic flux through pyruvate dehydrogenase was monitored by measuring 14 CO 2 production from [1- 14 C]pyruvate infused into the heart. A stepwise increase in the concentration of the inhibitor in the influent perfusate effected a stepwise reduction of the flux through the enzyme complex at all pyruvate concentrations tested. However, the magnitude of the α-cyanocinnamate-insensitive flux through pyruvate dehydrogenase increased markedly as the infused pyruvate concentration was elevated. The inhibition of pyruvate decarboxylation in the heart was nearly completely reversed following cessation of the inhibitor infusion. α-Cyanocinnamate was nearly 10 times more potent than α-cyano-4-hydroxycinnamate as an inhibitor of the flux through pyruvate dehydrogenase. Maximally inhibiting levels of α-cyano-4-hydroxycinnamate caused an increase in the ratio of the active form of pyruvate dehydrogenase to the total extractable enzyme complex from a value of 0.5 at 1 m m infused pyruvate (in the absence of the inhibitor) to a value of near unity. This result indicated that the intramitochondrial pyruvate concentration was severely depleted by the infusion of the inhibitor and that the enzyme complex was interconverted to its active form under these conditions. Removal of the inhibitor from the perfusion medium again lowered the ratio of the active/total pyruvate dehydrogenase to near its original level of 0.5 and restored the original flux through the enzyme complex indicating that mitochondrial pyruvate transport has been restored. The results of this study indicate that α-cyanocinnamate and its derivatives are effective inhibitors of pyruvate transport in the perfused heart and that carrier-mediated pyruvate transport can be an important parameter in the regulation of the activation state and the metabolic flux through the pyruvate dehydrogenase multienzyme complex in the heart.

Time courses of L-lactate and pyruvate uptake into isolated rat hepatocytes were measured in a citrate-based medium to generate a pH gradient (alkaline inside), by using the silicone-oil-filtration technique at 0 degrees C to minimize metabolism. At low concentrations of lactate and pyruvate (0.5 mM), transport was inhibited by over 95% by 5 mM-alpha-cyano-4-hydroxycinnamate, whereas at higher concentrations (greater than 10 mM) a significant proportion of transport could not be inhibited. The rate of this non-inhibitable transport was linearly related to the substrate concentration, was less with pyruvate than with L-lactate, and appeared to be due to diffusion of undissociated acid. Uptake of D-lactate was not inhibited by alpha-cyano-4-hydroxycinnamate and occurred only by diffusion. Kinetic parameters for the carrier-mediated transport process were obtained after correction of the initial rates of uptake of lactate and pyruvate in the absence of 5 mM-alpha-cyano-4-hydroxycinnamate by that in the presence of inhibitor. Under the conditions used, the Km values for L-lactate and pyruvate were 2.4 and 0.6 mM respectively and the Ki for alpha-cyano-4-hydroxycinnamate as a competitive inhibitor was 0.11 mM. Km values for the transport of L-lactate and pyruvate into rat erythrocytes under similar conditions were 3.0 and 0.96 mM. The Vmax. of lactate and pyruvate transport into hepatocytes at 0 degrees C was 3 nmol/min per mg of protein. Carrier-mediated transport of 0.5 mM-L-lactate was inhibited by 0.2 mM-p-chloromercuribenzenesulphonate (greater than 90%), 0.5 mM-quercetin (80%), 0.6 mM-isobutylcarbonyl-lactyl anhydride (70%) and 0.5 mM-4,4′-di-isothiocyanostilbene-2,2′-disulphonate (50%). A similar pattern of inhibition of lactate transport is seen in erythrocytes. It is suggested that the same or a similar carrier protein exists in both tissues. The results also show that L-lactate transport into rat hepatocytes is very rapid at physiological temperatures and is unlikely to restrict the rate of its metabolism. Differences between our results and those of Fafournoux, Demigne & Remesy [(1985) J. Biol. Chem. 260, 292-299] are discussed.

The effect of phenylglyoxal, an arginine-specific reagent, on the translocation of pyruvate and on the binding of alpha-cyanocinnamate by rat-heart mitochondria has been studied. It has been found that both the uptake and the oxidation of pyruvate by mitochondria are inhibited by phenylglyoxal. The inhibitory potency increases with the increasing of the pH of the medium. Phenylglyoxal does not affect the transmembrane delta pH. Phenylglyoxal also inhibits the binding of alpha-cyanocinnamate to mitochondria. Substrates of the carrier, such as pyruvate itself and monochloroacetate, partially prevent the inhibition of alpha-cyanocinnamate binding by phenylglyoxal, whilst acetate has no effect in this respect. Phenylglyoxal affects only the affinity of the alpha-cyanocinnamate binding site(s), without changing their total number. The results obtained indicate that arginine residues are involved in the mechanism of pyruvate translocation and of alpha-cyanocinnamate binding in rat-heart mitochondria.

The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zerotrans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and Δψ=0. Under zerotrans condition and Δψ=0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (K t =88 μm) and a low affinity for sodium (K t =57.7mm) (site I), the second one with a low affinity for pyruvate (K t =6.1mm) and a high affinity for sodium (K t =23.9mm) (site II). The coupling factor [Na]/[pyruvate] stoichiometry were determined at 0.25mm and 8mm pyruvate and estimated at 1.8 for site I, and 3 when the first and the second sites transport simultaneously. Under chemical equilibrium (Δψ≅0) single isotopic labeling, transport kinetics of pyruvate carrier systems have shown a double interaction of pyruvate with the transporter; the sodium/pyruvate stoichiometry also expressed according to a Hill plot representation wasn=1.7. The direct method of measuring Na+/pyruvate stoichiometry from double labeling kinetics and isotopic exchange, for a time course, gives an=1.67. Studies of transport specificity, indicate that the absence of inhibition of lactate transport by citrate and the existence of competitive inhibition of lactate and citrate transports by pyruvate leads to the conclusion that the low pyruvate affinity site can be attributed to the citrate carrier (tricarboxylate) and the high pyruvate affinity site to the lactate carrier (monocarboxylate).

The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The K m ranged from 15.0 mM at μ = 0.05 to 3.7 mM at μ = 0.01 , while the V went from 0.611 · 10 -15 to 0.137 · 10 -15 mol · min -1 · ghost -1 . Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, α-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4′-isothiocyanate-stilbene-2,2′-disulfonic acid and o-phenanthroline/CuSO4 mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.

A comparative study of the transport of pyruvate in heart mitochondria from normal and triiodothyronine-treated rats has been carried out. It has been found that the rate of carrier-mediated (α-cyanocinnamate-sensitive) pyruvate uptake is significantly enhanced in mitochondria from triiodothyronine-treated rats as compared with mitochondria from control rats. The kinetic parameters of the pyruvate uptake indicate that only the V max of this process is enhanced whilst there is no change in the K m value. The enhanced rate of pyruvate uptake is not dependent on the increase of the transmembrane ΔpH value (both mitochondria from normal and triiodothyronine-treated rats exhibit the same ΔpH value) neither does it depend on the increase of the pyruvate carrier molecules (titration of these last with α-cyanocinnamate gives the same total number of binding sites). The pyruvate-dependent oxygen uptake is stimulated by 35–40% in mitochondria from hyperthyroid rats when compared with mitochondria from control rats. There is, however, no difference in either the respiratory control ratios or in the ADP O ratios between these two types of mitochondria. The heart mitochondrial phospholipid composition is altered significantly in hyperthyroid rats; in particular, negatively charged phospholipid such as cardiolipin and phosphatidylserine were found to increase by more than 50%. Minor alterations were found in the pattern of fatty acids with an increase of 20:4 18:2 ratio. It is suggested that the changes in the kinetic parameters of pyruvate transport in mitochondria from hyperthyroid rats involve hormone-mediated changes in the lipid composition of the mitochondrial membranes which in turn modulate the activity of the pyruvate carrier.

The pattern of oxidative metabolism of pyruvate may be assessed by comparing the steadystate 14 CO 2 production from four isotopes in identical samples. The assay requires measuring the ratios of steady-state 14 CO 2 production from two isotope pairs, [2- 14 C]pyruvate:[3- 14 C]pyruvate and [l- 14 C]acetate:[2- 14 C]acetate. These ratios are defined as the “pyruvate 14 CO 2 ratio” and the “acetate 14 CO 2 ratio”, respectively. If pyruvate is metabolized exclusively via pyruvate dehydrogenase (PDH), the two ratios will be identical. Alternatively, if any pyruvate enters the tricarboxylic acid (TCA) cycle via pyruvate carboxylation (PC), the pyruvate 14 CO 2 ratio will be less than the acetate 14 CO 2 ratio. If pyruvate enters the TCA cycle only through PC (with oxaloacetate and fumarate in equilibrium) the pyruvate 14 CO 2 ratio will approach a value of 1.0. An equation is presented for the quantitative evaluation of pyruvate oxidation by these two pathways. We have used this method to detect relative changes in the pattern of pyruvate metabolism in rat liver mitochondria produced by exposure to 1 m m octanoyl carnitine, a compound known to alter the PC:PDH activity ratio. The major advantages of the method are (i) that it provides a sensitive method for detecting pyruvate carboxylation at physiological pyruvate concentrations and (ii) that it provides a method for distinguishing between effects on pyruvate transport and effects on pyruvate oxidation.

1. 1. Pyruvate oxidation by coupled rat heart and brain mitochondira is inhibited by phenylpyruvate and α-ketoisocaproate but not by α-ketoisovalerate or α-keto-β-methyl valerate; none of these compounds inhibit pyruvate dehydrogenase. 2. 2. Transport of pyruvate but not acetate into rat liver and brain mitochondria is inhibited by both phenylpyruvate and α-ketoisocaproate. 3. 3. Phenylpyruvate inhibits the transport of pyruvate but not acetate into human red blood cells. 4. 5. It is suggested that inhibition of pyruvate transport by phenylpyruvate and α-ketoisocaproate may be involved in the pathology of phenylketonuria and maple syrup urine disease.

Glucagon treatment of rats allowed the isolation of liver mitochondria with enhanced rates of pyruvate metabolism measured in either sucrose or KCl media. No change in the activity of the pyruvate carrier itself was apparent, but under metabolizing conditions, use of the inhibitor of pyruvate transport, alpha-cyano-4-hydroxycinnamate, demonstrated that pyruvate transport limited the rate of pyruvate metabolism. The maximum rate of transport under metabolizing conditions was enhanced by glucagon treatment. Problems involved in measuring the transmembrane pH gradient under metabolizing conditions are discussed and a variety of techniques are used to estimate the matrix pH. From the distribution of methylamine, ammonia and D-lactate and the Ki for inhibition by alpha-cyano-4-hydroxycinnamate it is concluded that the matrix is more acid than the medium and that the pH of the matrix rises after glucagon treatment. The increase in matrix pH stimulates pyruvate transport. The membrane potential, ATP concentration and O2 uptake were also increased under metabolizing conditions in glucagon-treated mitochondria. These changes were correlated with a stimulation of the respiratory chain which can be observed in uncoupled mitochondria [Yamazaki (1975) J. Biol. Chem. 250, 7924–7930]. The mitochondrial Mg2+ content (mean +/- S.E.M.) was increased from 38.8 +/- 1.2 (n = 26) to 47.5 +/- 2.0 (n = 26) ng-atoms/mg by glucagon and the K+ content from 126.7 +/- 10.3 (n = 19) ng-atoms/mg. This may represent a change in membrane potential induced by glucagon in vivo. The physiological significance of these results in the control of gluconeogenesis is discussed.

The monocarboxylate (pyruvate) carrier from bovine heart mitochondria was extracted from submitochondrial particles with Triton X-114 in the presence of cardiolipin. By a single hydroxylapatite chromatography step a 125-fold purification of the carrier protein could be achieved. High pyruvate/pyruvate-exchange activity was recovered, when the protein was reconstituted into phospholipid vesicles. No transport activity was observed, when the isolation occurred in the absence of phospholipids. The 2-cyano-4-hydroxycinnamate sensitive pyruvate exchange reaction was strongly temperature sensitive and dependent on the amount of protein reconstituted. Other 2-ketoacids caused competitive inhibition of the pyruvate uptake. Inhibitors of other mitochondrial carries, however, had very low or no effect on the monocarboxylate exchange. The influence of different -SH group reagents on the measured pyruvate/pyruvate-exchange in the reconstituted system was similar to the one observed with intact mitochondria. It is concluded that the described procedures for extraction, purification and reconstitution of the mitochondrial monocarboxylate carrier conserved the functional properties of the protein.

The exposure of spermatids to glucose in the absence of pyruvate and lactate resulted in an extremely low energy charge. The adenosine 5'-triphosphate (ATP) level rapidly declined and the fructose 1,6-bisphosphate (FBP) and triose levels increased. These changes were prevented by the addition of pyruvate or lactate. The levels of ATP and FBP were inversely correlated. In cells exposed to glucose, FBP did not flow appreciably through the step of glyceraldehyde 3-phosphate dehydrogenase (GA3PDH). The lactate level did not change. However, when pyruvate or lactate was administered to cells exposed to glucose, the FBP level declined rapidly. This drop was accompanied by a commensurate increase in lactate. In these cells, pyruvate transport was suppressed, and the pyruvate taken up by these cells was mostly oxidized in the tricarboxylic acid (TCA) cycle without its being reduced to lactate. In this case, the ATP level increased, but to a level still lower than existed before exposure to glucose. Furthermore, when kinetic studies on the activity of 6-phosphofructokinase (PFK) were carried out, PFK appeared to be fully activated at intracellular levels of fructose 6-phosphate, ATP and adenosine 5'-monophosphate (AMP). These results indicate that the rate of glucose metabolism in glycolysis depends heavily on the energy charge. In cells exposed to glucose, the sugar does not flow appreciably through the glycolytic pathway due to inhibition of GA3PDH. Moreover, the ATP level cannot be recovered fully from the lowest level by the addition of pyruvate or lactate.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibitor of mitochondrial pyruvate transport alpha-cyano-beta-(1-phenylindol-3-yl)-acrylate was used to inhibit progressively pyruvate carboxylation by liver mitochondria from control and glucagon-treated rats. The data showed that, contrary to our previous conclusions [Halestrap (1978) Biochem. J. 172, 389-398], pyruvate transport could not regulate metabolism under these conditions. This was confirmed by measuring the intramitochondrial pyruvate concentration, which almost equilibrated with the extramitochondrial pyruvate concentration in control mitochondria, but was significantly decreased in mitochondria from glucagon-treated rats, where rates of pyruvate metabolism were elevated. Computer-simulation studies explain how this is compatible with linear Dixon plots of the inhibition of pyruvate metabolism by alpha-cyano-4-hydroxycinnamate. Parallel measurements of the mitochondrial membrane potential by using [3H]triphenylmethylphosphonium ions showed that it was elevated by about 3 mV after pretreatment of rats with both glucagon and phenylephrine. There was no significant change in the transmembrane pH gradient. It is shown that the increase in pyruvate metabolism can be explained by a stimulation of the respiratory chain, producing an elevation in the protonmotive force and a consequent rise in the intramitochondrial ATP/ADP ratio, which in turn increases pyruvate carboxylase activity. Mild inhibition of the respiratory chain with Amytal reversed the effects of hormone treatment on mitochondrial pyruvate metabolism and ATP concentrations, but not on citrulline synthesis. The significance of these observations for the hormonal regulation of gluconeogenesis from L-lactate in vivo is discussed.

Alternative oxidase activity (oxygen uptake in the presence of KCN, antimycin or myxothiazol) in mitochondria isolated from the roots of soybean seedlings was very slow, even with succinate as substrate. This activity was stimulated substantially (100–400%) by the addition of pyruvate, with half maximal stimulation occurring at 0.1 mM pyruvate. Mitochondria from soybean shoots displayed high alternative oxidase activity with succinate and malate as substrates but lower activity with exogenous NADH; addition of pyruvate stimulated the activity with NADH up to that seen with succinate. This stimulation of cyanide-insensitive NADH oxidation was seen also with mitochondria from other species. Hydroxypyruvate and oxoglutarate could substitute for pyruvate, although higher concentrations were required to achieve maximum stimulation. Pyruvate stimulation of cyanide-insensitive oxygen uptake was observed with exogenous quinols as substrates, with sub-mitochondrial particles, and in the presence of the pyruvate transport inhibitor, cyanohydroxycinnamic acid, but was not observed with detergent-solubilised mitochondria. It is suggested that pyruvate acts allosterically on the alternatlve oxidase to stimulate its activity. The implications of these findings for respiration in vivo are discussed.

The existence of a specific carrier for pyruvate in the inner membrane of rat liver mitochondria is now well established [l-4] . Based on indirect evidence it has been suggested that glucagon, adrenaline or dibutyryl cyclic AMP act on liver cells to stimulate the operation of the mitochondrial pyruvate carrier and that this effect is involved in the increase in the rate of gluconeogenesis which follows exposure to those agents [.5,6]. In an earlier paper [7] we obtained direct evidence for this suggestion. Glucagon was injected into rats and 30 min later the livers were removed and mitochondria were prepared. We observed increased maximum initial rates of pyruvate transport into these mitochondria compared to mitochondria from control rat livers. The increased mobility of the pyruvate carrier was not due to preaccumulation of counter ions pyruvate, malate or palmitoyl carnitine within the mitochondrial matrix but an increased hydroxyl anion concentration therein was not excluded. We now report experiments of similar design in which injection of adrenaline or production of diabetes by streptozotocin injection also led to increases in initial rates of mitochondrial pyruvate transport. We have also measured various other parameters of mitochondrial

Pyruvate transport into rat liver mitochondria is inhibited by a variety of thiol reagents. alpha-Cyanocinnamate and its derivates, potent and reversible inhibitors of pyruvate transport, react reversibly with mercaptoethanol and cysteine to form addition products. It is concluded that these inhibitors react with an essential thiol group on the pyruvate carrier.

A sudden pH decrease (pH jump) of the medium enhanced pyruvate uptake in the dark in mesophyll chloroplasts (MCp) of Zea mays and Sorghum bicolor, NADP-malic enzyme type C4 plants, while it was reported that a Na+ jump enhanced pyruvate uptake in MCp of P. miliaceum, a NAD-malic enzyme type [(1987) FEBS Lett. 219, 347]. The enhancement effect of the pH jump decayed completely in 5 min and the decay was accelerated by proton gradient-collapsing reagents. The results suggest that active pyruvate uptake into MCp of NADP-malic enzyme type C4 species is primarily driven by the proton gradient across the envelope.

1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.

The effect of doxorubicin on the transport of pyruvate in rat-heart mitochondria was studied. It was found that the rate of pyruvate transport is inhibited by doxorubicin, half maximal inhibition being obtained at concentration of 125 microM of the drug. The inhibition is not due to a change in the transmembrane delta pH nor does it depend on an interaction of doxorubicin with thyol groups of the pyruvate carrier. Doxorubicin also inhibits the pyruvate dependent oxygen uptake and the specific binding of alpha-cyanocinnamate to mitochondria. It is proposed that doxorubicin affects the pyruvate transport by interacting with cardiolipin molecules surrounding the pyruvate carrier in the mitochondrial membrane.

Data are presented in support of the transport of (-)-D-3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were Vmax 2.0 mumol.g-1.min-1, Km 29 mM, and KD 0.024 ml.g-1.min-1. The value for Vmax was the same as that for L-lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-oxo-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The Ki values for the 2-oxo acids were severalfold lower than the respective Km values. 2-Oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.

1. 1. A method has been developed for measuring the facilitated diffusion of pyruvate anions across the isolated human erythrocyte membrane. Ghosts were resealed in the presence of an excess of lactate dehydrogenase and NADH so that the influx of pyruvate could be followed by a continuous spectrophotometric recording of the oxidation of NADH (Rice, W.R. and Steck, T.L. (1976) Biochim. Biophys. Acta 433, 39–53). This approach has now been extended to sealed inside-out vesicles derived from the same membrane. 2. 2. Transport across inside-out and right-side-out membranes was similar in certain respects. In both cases, the influx of pyruvate was greatly stimulated by increasing the ionic strength in the external medium. Salts of transported anions (such as chloride) but not non-transported anions (such as malate or acetate) were inhibitory at high concentrations, presumably a competitive effect. The non-competitive inhibitors of anion flux, salicylate, 4-acetamido-4′-isothiocyanato-2,2′-stilbene disulfonate (SITS) and 1-fluoro-2,4-dinitrobenzene, reduced pyruvate transport from both sides of the membrane. 3. 3. Characteristic asymmetries in the transport of pyruvate were noted. Probenecid was found to act only at the cytoplasmic surface, although it inhibited flux in both directions. Mixtures of o- phenanthroline and CuSO 4 catalyzed the disulfide cross-linking of band 3, the purported anion transport protein, only at the membrane's cytoplasmic surface. This treatment stimulated pyruvate flux into resealed ghosts, but inhibited flux into inside-out vesicles. 4. 4. The binding of glyceraldehyde-3-phosphate dehydrogenase and aldolase to the cytoplasmic surface of the membrane did not significantly alter pyruvate flux in either direction, even though these enzymes are known to associate specifically with band 3. Proteolytic excision of the cytoplasmic domain of band 3, amounting to approximately 40% of its mass, also had no effect on pyruvate flux.

1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Exposure of mitochondrial to unlabelled N-phenylmaleimide in the presence of alpha-cyanocinnamate, followed by removal of alpha-cyanocinnamate and exposure to [3H]N-phenylmaleimide, produced specific labelling of the same protein. 4. Both labelling and kinetic experiments with inhibitors gave values for the approximate amount of carrier present in liver and heart mitochondria of 100 and 450 pmol/mg of mitochondrial protein respectively. 5. The turnover numbers for net pyruvate transport and pyruvate exchange at 0 degrees C were 6 and 200 min-1 respectively.

1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of pyruvate transport at higher temperatures to be made. The activation energy of mitochondrial pyruvate transport was found to be 113 kJ (27 kcal)/mol and by extrapolation the rate of transport of pyruvate at 37 degrees C to be 42 nmol/min per mg of protein. The possibility that pyruvate transport into mitochondria may be rate limiting and involved in the regulation of gluconegenesis is discussed. 6. The transport of various monocarboxylic acids into mitochondria was studied by monitoring proton influx. The transport of dichloroacetate, difluoroacetate and oxamate appeared to be largely dependent on the pyruvate carrier and could be inhibited by pyruvate-transport inhibitors. However, many other halogenated and 2-oxo acids which could exchange with pyruvate on the carrier entered freely even in the presence of inhibitor.