Identification and characterisation of a new class of highly specific and potent inhibitors of the mitochondrial pyruvate carrier

Two novel thiazolidine compounds, GW604714X and GW450863X, were found to be potent inhibitors of mitochondrial respiration supported by pyruvate but not other substrates. Direct measurement of pyruvate transport into rat liver and yeast mitochondria confirmed that these agents inhibited the mitochondrial pyruvate carrier (MPC) with K(i) values <0.1 muM. Inhibitor titrations of pyruvate-dependent respiration by heart mitochondria gave values (+/-S.E.) for the concentration of inhibitor binding sites (pmol per mg protein) and their K(i) (nM) of 56.0+/-0.9 and 0.057+/-0.010 nM for the more hydrophobic GW604714X; for GW450863X the values were 59.9+/-4.6 and 0.60+/-0.12 nM. [(3)H]-methoxy-GW450863X binding was also used to determine the MPC content of the heart, kidney, liver and brain mitochondria giving values of 56, 40, 26 and 20 pmol per mg protein respectively. Binding to yeast mitochondria was <10% of that in rat liver mitochondria, consistent with the slow rate of pyruvate transport into yeast mitochondria. [(3)H]-methoxy-GW450863X binding was inhibited by GW604714X and by the established MPC inhibitor, UK5099. The absorbance spectra of GW450863X and GW604714X were markedly changed by the addition of beta-mercaptoethanol suggesting that the novel inhibitors, like alpha-cyanocinnamate, possess an activated double bond that attacks a critical cysteine residue on the MPC. However, no labelled protein was detected following SDS-PAGE suggesting that the covalent modification is reversible. GW604714X and GW450863X inhibited l-lactate transport by the plasma membrane monocarboxylate transporter MCT1, but at concentrations more than four orders of magnitude greater than the MPC.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Decreased activity of the pyruvate translocator and changes in the lipid composition in heart mitochondria from hypothyroid rats

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 concentration of the mitochondrial pyruvate carrier in rat liver and heart mitochondria determined with alpha-cyano-beta-(1-phenylindol-3-yl)acrylate.

alpha-Cyano-beta-(1-phenylindol-3-yl)acrylate inhibited pyruvate transport into both liver and heart mitochondria approximately linearly with respect to its concentration until 65% inhibition was achieved. The extent of inhibition was dependent on the mitochondrial protein concentration. By extrapolation of plots of inhibition versus inhibitor concentration to total inhibition, or by mathematical analysis of the plots, the concentration of pyruvate transporter molecules per mg of protein was calculated to be approximately 100 pmol/mg for both heart and liver mitochondria, and the Ki about 7 nM. The data also suggest that pyruvate transport is rate-limiting for pyruvate oxidation by heart mitochondria in State 3, but not by liver mitochondria.     

A reexamination of the role of the cytosolic alanine aminotransferase in hepatic gluconeogenesis

The relative importance of the mitochondrial and cytosolic alanine aminotransferase isozymes for providing pyruvate from alanine for further metabolism in the mitochondrial compartment was examined in the isolated perfused rat liver. The experimental rationale employed depends upon the supposition that gluconeogenesis from alanine and the decarboxylation of infused [1-14C]alanine should be diminished by pyruvate transport inhibitors (e.g., alpha-cyanocinnamate) in proportion to the contribution of the cytosolic alanine aminotransferase for generating pyruvate. alpha-Cyanocinnamate inhibited the endogenous rate of glucose production in perfused livers derived from 24-h-fasted rats. The rate of [1-14C]alanine decarboxylation at low (1 mM) and high (10 mM) perfusate alanine concentrations was inhibited by 9.5 and 42%, respectively, in the presence of alpha-cyanocinnamate. In livers from fasted animals perfused with either 1 or 10 mM alanine, alpha-cyanocinnamate caused a substantial increase in the rates of both lactate and pyruvate production. Elevating the hepatic ketogenic rate during infusion of acetate in livers, perfused with alanine, stimulated both the rates of alanine decarboxylation and glucose production; the extent of stimulation of these two metabolic parameters was determined to be a function of the alanine concentration in the perfusate. The stimulation of the rate of alanine decarboxylation during acetate-induced ketogenesis was reversed by co-infusion of alpha-cyanocinnamate with simultaneous increases in the rates of lactate and pyruvate production. The results indicate that during rapid ketogenesis, cytosolic transamination of alanine contributes at least 19% (at 1 mM alanine) and 55% (at 10 mM alanine) of the pyruvate for gluconeogenesis.     

Measurement of the intramitochondrial volume in hepatocytes without cell disruption and its elevation by hormones and valinomycin.

Methods have been developed to measure the lysophospholipid content and matrix volume of liver cell mitochondria in situ in order to test the hypothesis that these parameters may be important in the hormonal control of mitochondrial function [Armston, Halestrap & Scott (1982) Biochim. Biophys. Acta 681, 429-439]. No change in the labelling of mitochondrial lysophospholipids with [32P]Pi was detected after treatment of liver cells with glucagon, phenylephrine or vasopressin. Incorporation of [32P]Pi into mitochondrial phosphatidylinositol was enhanced by phenylephrine and vasopressin. Mitochondrial volumes were measured using rapid disruption of cells by sonication into 3H2O and [14C]sucrose or without cell disruption using 3H2O and [14C]mannitol. In control cells the two methods gave values of 1.09 and 0.40 microliters/mg of mitochondrial protein respectively, which represent 19 and 7% respectively of the total cell volume measured with 3H2O and inulin [14C]carboxylic acid. Both methods showed that glucagon, phenylephrine and 1 nm-valinomycin produced significant increases (13% and 26% using sucrose and mannitol respectively) in mitochondrial volume. The increase was coincident with the stimulation of gluconeogenesis from L-lactate and pyruvate and of mitochondrial respiratory chain activity. The effects of glucagon and phenylephrine were additive on both mitochondrial volume and respiratory chain activity, but not on gluconeogenesis. Liver cells exposed to gluconeogenic hormones or low concentrations of valinomycin showed a decrease in light scattering at 520 nM correlating with the change in mitochondrial volume but without a change in whole-cell volume. The time course and hormone sensitivity of this response were similar to those for the hormonal stimulation of gluconeogenesis. The light-scattering response to glucagon, phenylephrine and vasopressin, but not to valinomycin, were greatly reduced or abolished in Ca2+-free media.     

The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.

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.     

A re-evaluation of the role of mitochondrial pyruvate transport in the hormonal control of rat liver mitochondrial pyruvate metabolism.

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.     

Pyruvate transport by thermogenic-tissue mitochondria.

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.     

Interaction of (3H) bongkrekic acid with the mitochondrial adenine nucleotide translocator.

Chemical labeling by 3H and biosynthetic labeling by 14C of bongkrekic acid (BA) are described. In the rat liver cell, mitochondria are the only subcellular particles to bind [3H]BA with high affinity. The high affinity sites for BA in mitochondria are located in the inner membrane. High affinity binding sites for BA are only displayed at pH below 7; they amount to 0.15-0.20 nmol/mg of protein in rat liver mitochondria and to 1.1-1.3 nmol/mg of protein in rat heart mitochondria. These values are similar to those found for the high affinity atractyloside binding sites and for the carboxyatractyloside binding sites. The kinetic parameters for BA binding to rat heart mitochondria at 20 degrees C are Kd = 10-40 X 10(-9) M, k+1 = 0.7 X 10(5) M-1 s-1, k-1 = 1.4 X 10(-3) M s-1. Binding assays carried out with rat heart mitochondria, under equilibrium conditions, showed that the amount of BA bound to high affinity sites increases with temperature and reaches the maximum value of 1.1-1.3 nmol/mg of protein at 32-35 degrees C. At lower temperatures, and under equilibrium conditions, a significant fraction of high affinity sites remains masked and is not titrated by BA; these masked BA sites are revealed by addition of micromolar concentrations of ADP or by energization of the mitochondria. Carboxyatractyloside added to rat heart mitochondria preloaded with [3H]BA is able to displace part of the bound [3H]BA. Displacement of the bound BA is enhanced by simultaneous additions of carboxyatractyloside plus ADP, or by energization of the mitochondria. The synergistic effect of carboxyatractyloside and ADP on displacement of bound [3H]BA is also observed in isolated inner membrane vesicles from rat liver mitochondria. When BA is preincubated with rat heart mitochondria before addition of [14C]ADP for assay of ADP transport, the inhibition of ADP transport is a mixed-type inhibition. When BA is preincubated with the mitochondria together with a very small concentration of ADP (less than 0.5 muM), the inhibition of [14C]ADP transport is markedly increased (up to ten times) and it becomes typically uncompetitive, which suggests the formation of a ternary complex, carrier-ADP-BA. The transition from a mixed-type inhibition, with high Ki value, to an uncompetitive type of inhibition, with low Ki value, upon addition of ADP, is explained by an ADP-induced conformational change of the ADP translocator.     

Carnitine-acylcarnitine translocase. Inhibition by alpha-cyano-4-hydroxycinnamate and evidence for separate identity from the pyruvate transporting system of mitochondria

Some of the known inhibitors of pyruvate transport inhibited the activity of carnitine-acylcarnitine translocase. Their order of effectiveness with millimolar concentration required for 50% inhibition given in parentheses, was: Compound UK-5099 (alpha-cyano-beta-(1-phenylindol-3-yl)acrylate) (0.1); alpha-cyano-4-hydroxycinnamate (0.17); alpha-cyano-3-hydroxycinnamate (1); alpha-cyanocinnamate (1); alpha-fluorocinnamate (7); transcinnamate (10); p-hydroxycinnamate (10); phenylpyruvate (22); p-hydroxyphenylpyruvate (25). Kinetically, the alpha-cyano-4-hydroxycinnamate inhibition was mixed and the p-hydroxyphenylpyruvate inhibition was noncompetitive with respect to external (-)-carnitine. The alpha-cyano-4-hydroxycinnamate inhibition was reversible and resulted from its ability to act as a thiol reagent. In general, alpha-cyanocinnamate and its derivatives inhibit carnitine transport at concentrations 100 to 5000 times as high as those known to pyruvate transport. At millimolar concentrations, alpha-cyano-4-hydroxycinnamate inhibited the mitochondrial transport of molecules other than carnitine as well as the activity of carnitine acyltransferases. Pyruvate and carnitine did not complete for transport into and out of mitochondria. These results establish that transmitochondrial transport mechanisms for carnitine and pyruvate involve different carriers.     

Transport of D-beta-hydroxybutyrate across rat liver mitochondrial membranes

1. D-beta-hydroxybutyrate, a major ketone body, is produced or converted in mitochondria from various animal tissues. 2. It is an easy permeate anion of the inner mitochondrial membrane. However, its translocation is not a passive diffusion process since it is inhibited by pyruvate transport inhibitors like alpha-cyanocinnamate and derivatives. 3. This carrier mediated process is associated with proton movements. Besides, dicarboxylate anions strongly inhibit the penetration into mitochondria. 4. This is in agreement with the existence of a second transport process related to the dicarboxylate carrier.     

Effect of phosphate and ionophores on (14C)-NEM incorporation in mitochondrial membranes and relationships with phosphate carrier system.

Phosphate transport in mitochondria was investigated with respect to its inhibition by NEM. The reactivity of the Pi carrier SH groups was influenced by phosphate or ionophores during preincubation before the addition of NEM. Furthermore in order to obtain some mitochondrial protein fractions where the typical effects of phosphate and ionophores on [14C]-NEM fixations were observed, mitochondria were submitted to hypotonic treatment and sonication. The following results were obtained: 1. -- Phosphate and grisorixin (a new ionophore of the nigericin group) decreased the inhibition of phosphate transport by NEM. The same effect was observed for [14C]-NEM incorporation. 2. -- Valinomycin increased [14C]-NEM incorporation. The valinomycin effect was abolished by phosphate. ClCCP alone affected [14C]-NEM incorporation slightly. Valinomycin plus ClCCP decreased NEM inhibition of phosphate transport and [14C]-NEM incorporation like grisorixin. 3. -- The variability of SH group reactivity can be interpreted by a control of SH group accessibility by transmembrane delta pH as previously suggested. 4. -- Typical effects of phosphate or ionophores were observed in whole pig heart and rat liver mitochondria. These effects were enhanced in the same supernatant protein fraction resulting from sonication in pig heart mitochondria : phosphate decreased [14C]-NEM incorporation by 1,50 nmoles/mg protein, grisorixin by 0.95 nmoles, whereas valinomycin increased it by 0.75 nmoles. For rat liver mitochondria the phosphate effect and the valinomycin increased it by 0.75 nmoles. For rat liver mitochondria the phosphate effect valinomycin effect on [14C]-NEM incorporation were observed in the subparticular fraction obtained after sonification.     

Multiple biotin-containing proteins in 3T3-L1 cells.

Extracts of 3T3-L1 cells prepared after labelling the monolayer cultures with [3H]biotin contained numerous protein bands that were detected by fluorography of dried SDS/polyacrylamide electrophoresis gels. All labelled proteins in the extracts could be removed by avidin affinity chromatography. The biotin-containing subunits of acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, with molecular masses of approx. 220, 120, 75 and 72 kDa respectively, were detected together with minor bands at 100, 85 and 37 kDa that did not appear to be partial degradation products. Additional labelled bands increased in amount during incubation of cell extracts or did not occur in extracts prepared with trichloroacetic acid, 9.5 M-urea or proteolytic inhibitors, and were tentatively classified as partial degradation products. The unknown bands were not removed by incubation of cell monolayers for 24 h, a treatment that gave degradation rate constants of 0.47 day-1 for acetyl-CoA carboxylase and 0.28 day-1 for pyruvate carboxylase. Upon two-dimensional electrophoresis, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase had isoelectric points of 6.4, 7.2 and 6.4 respectively. Several additional discrete spots with isoelectric points below 6.2 were also present. All the unknown biotin-containing proteins banded with intact mitochondria during density-gradient centrifugation. We conclude that several unknown biotin-containing proteins are present in the mitochondria of 3T3-L1 cells, whereas others are partial breakdown products of mitochondrial proteolysis.     

Accumulation of pyruvate by isolated rat liver mitochondria.

1. Various methods to measure the rate of accumulation of [3-14C]pyruvate in the sucrose-impermeable space of isolated rat liver mitochondria are tested and compared with respect to their ability to distinguish between carrier-linked pyruvate transport and non-carrier-linked processes (adsorption and diffusion). 2. Evidence is presented that the cinnamic acid derivatives commonly used as specific inhibitors of the pyruvate carrier (i) do not completely abolish all carrier-mediated pyruvate transport; (ii) inhibit pyruvate adsorption, and (iii) at higher concentrations lead to a removal of previously accumulated pyruvate from the mitochondria. It is concluded that procedures which avoid the use of transport inhibitors allow more reliable estimates of carrier-linked pyruvate transport. 3. It is proposed to measure pyruvate adsorption as the accumulation of pyruvate in the presence of an uncoupler. Using this procedure, it could be shown that, with 1 mM pyruvate, adsorption represents only a small part of the total pyruvate accumulation, the main part being carrier-linked transport driven by the pH gradient across the mitochondrial inner membrane.     

Transport of pyruvate and lactate in yeast mitochondria.

Evidence for the existence of mediated transport of pyruvate and lactate in isolated mitochondria of Saccharomyces cerevisiae is presented. 1. The mitochondrial oxidation of pyruvate is specifically inhibited by the monocarboxylic oxoacids alpha-ketoisocaproate and by alpha-cyano-3-hydroxycinnamate, while pyruvate and malate dehydrogenases activities are not inhibited. 2. The stimulation of the mitochondrial oxidations of succinate, alpha-ketoglutarate and citrate by pyruvate are also inhibited by alpha-cyano-3-hydroxycinnamate. 3. The [14C]pyruvate uptake by yeast mitochondria follows saturation kinetics and is completely inhibited by alpha-cyano-3-hydroxycinnamate. 4. Large amplitude passive swellings of mitochondria of the wild type and of cytoplasmic rho- and rho-n mutants are induced by isoosmotic ammonium pyruvate and lactate. These pH-dependent swellings are inhibited by alpha-cyano-3-hydroxycinnamate suggesting that the carrier system is not coded by mitochondrial DNA.     

Heart Mitochondrial TTP Synthesis and the Compartmentalization of TMP

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [³H]thymidine or [³H]TMP as precursors of [³H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [³H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [³H]TMP produced far less [³H]TTP than the amount observed with [³H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [³H]TTP from [³H]TMP was effectively blocked, demonstrating that synthesis of [³H]TTP from [³H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [³H]TMP to [³H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [³H]thymidine was readily converted to [³H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.     

Inhibition of pyruvate carboxylase degradation and total protein breakdown by lysosomotropic agents in 3T3-L1 cells.

1. Exposure to [3H]biotin during the differentiation of 3T3-L1 cells to adipocytes selectively labelled pyruvate carboxylase (EC 6.4.1.1). A subsequent incubation of labelled cells permitted the measurement of the degradation rate constant of this mitochondrial enzyme. 2. In medium without serum, pyruvate carboxylase was degraded with a half-life of 64 h, considerably longer than that found for average cell protein. The long half-life is commensurate with the enzyme being catabolized when whole mitochondria are destroyed. 3. The breakdown of pyruvate carboxylase was inhibited to a greater extent than the breakdown of total cell protein by insulin, NH4Cl and inhibitors of lysosomal proteinases, suggesting that the enzyme is degraded by the autophagic lysosomal system of the cell. 4. The above evidence implies that whole mitochondria are degraded in lysosomes, a conclusion that agrees with earlier electron-microscopic evidence showing mitochondria within autophagic vacuoles. 5. A second degradative pathway must be invoked to account for the breakdown of mitochondrial proteins of short half-life.     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.