Search

One of the globally common cancers is colorectal cancer (CRC). At present, a surgical approach remains a good option for CRC patients; however, 20% of surgically treated CRC patients experience metastasis. Currently, even the first-line used drug, oxaliplatin, remains inadequate for treating metastatic CRC, and its side effect of neurotoxicity is a major problem when treating CRC. The Gene Omnibus GSE42387 database contains gene expression profiles of parental and oxaliplatin-resistant LoVo cell lines. Differentially expressed genes (DEGs) between parental and oxaliplatin-resistance LoVo cells, protein-protein interactions (PPIs), and a pathway analysis were determined to identify overall biological changes by an online DAVID bioinformatics analysis. The ability of DEGs to predict overall survival (OS) and disease-free survival (DFS) was validated by the SPSS 22.0, using liver metastasis CRC patient samples of GSE41258. The bioinformatics web tools of the GEPIA, the Human Protein Atlas, WebGestalt, and TIMER platforms were used. In total, 218 DEGs were identified, among which 105 were downregulated and 113 were upregulated. After mapping the PPI networks and pathways, 60 DEGs were identified as hub genes (with high degrees). Six genes (TGFB1, CD36, THBS1, FABP1, PCK1, and IRS1) were involved with malaria, PPAR signaling, and the adipocytokine signaling pathway. High expressions of CD36 and PCK1 were associated with the poor survival of CRC patients in the GSE41258 database. We predicted specific micro (mi)RNAs that targeted the 3 ′ untranslated region (UTR) of PCK1 by using miRWalk. It was found that three miRNAs, viz., miR-7-5p, miR-20a-3p, and miR-636, may be upstream targets of those genes. High expression levels of miR-7-5p, miR-20a-3p, and miR-636 were associated with poor OS of CRC patients, and the small-molecule compound, mersalyl, is a promising drug for treating oxaliplatin-resistant CRC. In conclusion, miR-7-5p miR-20a-3p, and miR-636 targeted the PCK1 biomarker in the PPAR signaling pathway, which is involved in oxaliplatin-resistant CRC. Meanwhile, mersalyl was identified as a potential drug for overcoming oxaliplatin resistance in CRC. Our findings may provide novel directions and strategies for CRC therapies.

The effects of both Tl+ and thiol reagents were studied on the content of the inner membrane free SH-groups, detected with Ellman reagent, and the inner membrane potential as well as swelling and respiration of succinate-energized rat liver mitochondria in medium containing TlNO3 and KNO3. These effects resulted in a rise in swelling and a decrease in the content, the potential, and mitochondrial respiration in 3 and 2,4-dinitrophenol-uncoupled states. A maximal effect was seen when phenylarsine oxide reacting with thiol groups recessed into the hydrophobic regions of the membrane. Compared with phenylarsine oxide, the effective concentrations of other reagents were approximately one order of magnitude higher in experiments with mersalyl and 4,4′-diisothiocyanostilbene-2,2′-disulfonate, and two orders of magnitude higher in experiments with tert-butyl hydroperoxide and diamide. The above effects of Tl+ and the thiol reagents became even more pronounced with calcium overload of mitochondria. However, the effects were suppressed by inhibitors of the mitochondrial permeability transition pore (cyclosporine A, ADP, and n-ethylmaleimide). These findings suggest that opening of the pore induced by Tl+ in the inner membrane can be dependent on the conformation state of the adenine nucleotide translocase, which depends on the activity of its thiol groups.

In this study, the mechanism of non-specific membrane permeability (yPTP) in the Endomyces magnusii yeast mitochondria under oxidative stress due to blocking the key antioxidant enzymes has been investigated. We used monitoring the membrane potential at the cellular (potential-dependent staining) and mitochondrial levels and mitochondria ultra-structural images with transmission electron microscopy (TEM) to demonstrate the mitochondrial permeability transition induction due to the pore opening. Analysis of the yPTP opening upon respiring different substrates showed that NAD(P)H completely blocked the development of the yPTP. The yPTP opening was inhibited by 5–20 mM Pi, 5 mM Mg2+, adenine nucleotides (AN), 5 mM GSH, the inhibitor of the Pi transporter (PiC), 100 μM mersalyl, the blockers of the adenine nucleotide transporter (ANT) carboxyatractyloside (CATR), and bongkrekic acid (BA). We concluded that the non-specific membrane permeability pore opens in the E. magnusii mitochondria under oxidative stress, and the ANT and PiC are involved in its formation. The crucial role of the Ca2+ ions in the process has not been confirmed. We showed that the Ca2+ ions affected the yPTP both with and without the Ca2+ ionophore ETH129 application insignificantly. This phenomenon in the E. magnusii yeast unites both mitochondrial unselective channel (ScMUC) features in the Saccharomyces cerevisiae mitochondria and the classical membrane pore in the mammalian ones (mPTP).

The effect of α,ω-hexadecanedioic acid (HDA) as an inducer of Ca2+-dependent permeability of the inner membrane (pore opening) of isolated rat liver mitochondria was studied in the absence and presence of pore blocker cyclosporin A (CsA) and one of its effective modulator – inorganic phosphate (Pi). It was shown that the addition of HDA at a concentration of 30 µM to Ca2+-loaded mitochondria induces swelling of the organelles, rapid Ca2+ release from the matrix, and almost total drop in Δψ, which indicates the induction of the Ca2+-dependent permeability of the inner membrane. It was found that 1 µM CsA or 1 mM Pi added separately do not affect these effects of HDA. At the same time, in the presence of both CsA and Pi, HDA added to Ca2+-loaded mitochondria does not induce their swelling, Ca2+ release from the matrix, and a drop in Δψ. It is found that unlike HDA, the induction of Ca2+-dependent lipid pore by palmitic acid is not blocked by the combined action of CsA and Pi. On the basis of the obtained data Pi is considered as a blocker of the HDA-induced Ca2+-dependent pore in the presence of CsA. In this case, Pi can not be replaced by a similar permeable anion vanadate. It was established that this effect of Pi was eliminated if mitochondria were incubated with SH-reagents mersalyl (10 nmol/mg protein) and n-ethylmaleimide (200 nmol/mg protein), which are known as Pi-carrier inhibitors. We conclude that the mechanisms of the effects of HDA and palmitic acid as inducers of the Ca2+-dependent permeability of liver mitochondria differ significantly. The Ca2+-dependent effect of HDA can be considered as the formation of a pore sensitive to the combined action of CsA and Pi, while the Ca2+-dependent effect of palmitic acid is the formation of a lipid pore. Possible causes of the blocking action of Pi on the HDA-induced Ca2+-dependent mitochondrial pore are discussed.

The effect of the lipophilic penetrating cation dequalinium on rat liver mitochondria was studied. It was found that dequalinium dose-dependently inhibits the respiration rate of rat liver mitochondria in ADP-stimulated (V3) and DNP-stimulated (uncoupled) states. This can be due to the fact that dequalinium is a potent inhibitor of complex III of the mitochondrial respiratory chain. It was shown that dequalinium induces a high-amplitude swelling of rat liver mitochondria. The dequalinium-induced swelling of the organelles depends on the presence of inorganic phosphate in the incubation medium: in the absence of phosphate or in the presence of the phosphate carrier inhibitor mersalyl in the phosphate-containing medium, no swelling of the mitochondria was observed. At low concentrations of dequalinium (≤10 μM), this swelling is inhibited by cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. At the same time, at high concentrations of dequalinium (>10 μM), cyclosporin A becomes ineffective. It was found that in the presence of dequalinium the rate of the H2O2 production increased in rat liver mitochondria. Possible mechanisms of toxic effect of dequalinium chloride are discussed.

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 resulted in the Tl+-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca2+ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat liver mitochondria.

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO

The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients ( C vi JATP ) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C vi JATP were for system A: Complex I - 0.19, Complex III – 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) – 0.11, ATP synthase – 0.01, Pi carrier - 0.20, and the sum of C vi JATP was 0.75. In the presence of 10 mM creatine (system B) the C vi JATP were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C vi JATP were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C vi JATP was 1.33 and 3.84, respectively. Thus, C vi JATP were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin βII which restricts permeability of the mitochondrial outer membrane.

In liver mitochondria, the phosphate carrier is involved in protonophoric uncoupling effect of fatty acids together with ADP/ATP and aspartate/glutamate antiporters (Samartsev et al. 2003. Biochemistry (Moscow). 68, 618–629). Liver mitochondria depleted of endogenous oxidation substrates (exhausted mitochondria) have been used in the present work. In these mitochondria, like in the intact liver mitochondria, the specific inhibitor of ADP/ATP antiporter (carboxyatractylate) and the substrate of aspartate/glutamate antiporter (aspartate) suppress the uncoupling activity of palmitic acid. It is shown that in exhausted mitochondria the substrate of phosphate carrier (inorganic phosphate) and its nonspecific inhibitor mersalyl partially suppress palmitic acid-induced uncoupling due to decrease in the component of uncoupling activity sensitive to carboxyatractylate and aspartate. In the presence of inorganic phosphate or mersalyl, carboxyatractylate and aspartate added separately subsequent to palmitic acid do not suppress its uncoupling activity. They are effective only when added jointly. In the presence of thiourea or pyruvate, such effects of inorganic phosphate and mersalyl are not observed. It is supposed that in the presence of inorganic phosphate or mersalyl and under the condition of oxidation of critical SH-groups in mitochondria, the phosphate carrier, ADP/ATP antiporter, and aspartate/glutamate antiporter are involved in uncoupling function together with the general fatty acid pool as an uncoupling complex. The role of phosphate carrier in this complex may consist in facilitation of lateral transfer of the fatty acid molecules from one antiporter to another.

The effect of HgCl(2), methylmercury and mersalyl on the glutamine/amino acid (ASCT2) transporter reconstituted in liposomes has been studied. Mercuric compounds externally added to the proteoliposomes, inhibited the glutamine/glutamine antiport catalyzed by the reconstituted transporter. Similar effects were observed by pre-treating the proteoliposomes with the mercurials and then removing unreacted compounds before the transport assay. The inhibition was reversed by DTE, cysteine and N-acetyl-cysteine but not by S-carboxymethyl-cysteine. The data demonstrated that the inhibition was due to covalent reaction of mercuric compounds with Cys residue(s) of the transporter. The IC(50) of the transporter for HgCl(2), methylmercury and mersalyl, were 1.4+/-0.10, 2.4+/-0.16 or 3.1+/-0.19 microM, respectively. Kinetic studies of the inhibition showed that the reagents behaved as non-competitive inhibitor. The presence of glutamine or Na(+) during the incubation of the mercuric compounds with the proteoliposomes did not exerted any protective effect on the inhibition. None of the compounds was transported by the reconstituted transporter. A metal binding motif CXXC has been predicted as possible site of interaction of the mercuric compounds with the transporter on the basis of the homology structural model of ASCT2 obtained using the glutamate transporter homologue from Pyrococcus horikoshii as template.

To date, the existence of the plant inner membrane anion channel (PIMAC) has been shown only in potato mitochondria, but its physiological role remains unclear. In this study, by means of swelling experiments in K(+) and ammonium salts, we characterize a PIMAC-like anion-conducting pathway in mitochondria from durum wheat (DWM), a monocotyledonous species phylogenetically far from potato. DWM were investigated since they possess a very active potassium channel (PmitoK(ATP)), so implying a very active matching anion uniport pathway and, possibly, a coordinated function. As in potato mitochondria, the electrophoretic uptake of chloride and succinate was inhibited by matrix [H(+)], propranolol, and tributyltin, and was insensitive to Mg(2+), N,N'-dicyclohexylcarbodiimide (DCCD) and mercurials, thus showing PIMAC's existence in DWM. PIMAC actively transports dicarboxylates, oxodicarboxylates, tricarboxylates and Pi. Interestingly, a novel mechanism of swelling in ammonium salts of isolated plant mitochondria is reported, based on electrophoretic anion uptake via PIMAC and ammonium uniport via PmitoK(ATP). PIMAC is inhibited by physiological compounds, such as ATP and free fatty acids, by high electrical membrane potential (Delta Psi), but not by acyl-CoAs or reactive oxygen species. PIMAC was found to cooperate with dicarboxylate carrier by allowing succinate uptake that triggers succinate/malate exchange in isolated DWM. Similar results were obtained using mitochondria from the dicotyledonous species topinambur, so suggesting generalization of results. We propose that PIMAC is normally inactive in vivo due to ATP and Delta Psi inhibition, but activation may occur in mitochondria de-energized by PmitoK(ATP) (or other dissipative systems) to replace or integrate the operation of classical anion carriers.

In this work we examined the effect of low concentrations of Cu(2+) on the opening of the mitochondrial non-specific pore. The purpose was addressed to further contribute to the knowledge of the mechanisms that regulate the open/closed cycles of the permeability transition pore. Membrane leakage was established by measuring matrix Ca(2+) efflux and mitochondrial swelling. The experimental results indicate that Cu(2+) at very low concentrations promoted the release of accumulated Ca(2+), as well as mitochondrial swelling, provided 1,10-phenanthroline has been added. Carboxyatractyloside and Cu(2+) exhibited additive effects on these parameters. After Cu(2+) titration of membrane thiols, it might be assumed that the blockage of 5.9nmol of SH/mg protein suffices to open the non-specific pore. Taking into account the reinforcing effect of carboxyatractyloside, the increasing ADP concentrations, and that N-ethylmaleimide inhibited the Cu(2+)-induced Ca(2+) efflux, it is proposed that the target site for Cu(2+) is located in the ADP/ATP carrier.

The purpose of this work was addressed to provide new information on the effect of thiol reagents on mitochondrial non-specific pore opening, and its response to cyclosporin A (CSA). To meet this proposal phenylarsine oxide (PHA) and mersalyl were employed as tools to induce permeability transition and CSA to inhibit it. PHA-induced mitochondrial dysfunction, characterized by Ca2+ efflux, swelling, and membrane de-energization, was inhibited by N-ethylmaleimide and CSA. Conversely, mersalyl failed to inhibit the inducing effect of phenylarsine oxide, it rather strengthened it. In addition, the effect of mersalyl was associated with cross-linking of membrane proteins. The content of membrane thiol groups accessible to react with PHA, mersalyl, and PHA plus mersalyl was determined. In all situations, permeability transition was accompanied by a significant decrease in the whole free membrane thiol content. Interestingly, it is also shown that mersalyl hinders the protective effect of cyclosporin A on PHA-induced matrix Ca2+ efflux.

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in different isoforms. Two of these have been characterized and are localized in different subcellular compartments. hFADS2 containing 490 amino acids shows a two domain organization: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and a resembling molybdopterin-binding (MPTb) domain. By a multialignment of hFADS2 with other MPTb containing proteins of various organisms from bacteria to plants, the critical residues for hydrolytic function were identified. A homology model of the MPTb domain of hFADS2 was built, using as template the solved structure of a T. acidophilum enzyme. The capacity of hFADS2 to catalyse FAD hydrolysis was revealed. The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. The recombinant PAPS reductase domain is not able to perform the same function. The mutant C440A catalyses the same hydrolytic function of WT with no essential requirement for mersalyl, thus indicating the involvement of C440 in the control of hydrolysis switch. The enzyme C440A is also able to catalyse hydrolysis of FAD bound to the PAPS reductase domain, which is quantitatively converted into FMN.

H 2 O 2 inactivation of particular GST isoforms has been reported, with no information regarding the overall effect of other ROS on cytosolic GST activity. The present work describes the inactivation of total cytosolic GST activity from liver rats by the oxygen radical-generating system Cu 2+ /ascorbate. We have previously shown that this system may change some enzymatic activities of thiol proteins through two mechanisms: ROS-induced oxidation and non-specific Cu 2+ binding to protein thiol groups. In the present study, we show that nanomolar Cu 2+ in the absence of ascorbate did not modify total cytosolic GST activity; the same concentrations of Cu 2+ in the presence of ascorbate, however, inhibited this activity. Micromolar Cu 2+ in either the absence or presence of ascorbate inhibited cytosolic GST activity. Kinetic studies show that GSH but no 1-chloro-2,4-dinitrobenzene prevent the inhibition on cytosolic GST induced by micromolar Cu 2+ either in the absence or presence of ascorbate. On the other hand, NEM and mersalyl acid, both thiol-alkylating agents, inhibited GST activity with differential reactivity in a dose-dependent manner. Taken together, these results suggest that an inhibitory Cu 2+ -binding effect is likely to be negligible on the overall inhibition of cytosolic GST activity observed by the Cu 2+ /ascorbate system. We discuss how modification of GST-thiol groups is related to the inhibition of cytosolic GST activity.

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

The ethylmaleimide-sensitive phosphate-transport system of heart mitochondria was isolated and the activity reconstituted in liposomes. The identification of the phosphate-carrier protein was complicated by several factors. 1 The phosphate-carrier fraction, isolated through different procedures in different laboratories, contains 4–5 protein components of very similar apparent molecular weight, as shown by gradient dodecylsulfate gel electrophoresis. 2 The amino acid composition of protein 1, 2 and 3 (apparent Mr= 34500, 34000 and 33000, respectively) is quite similar although not identical. 3 Including several protease inhibitors during isolation of the phosphate-carrier fraction does not influence the protein pattern. 4 Labeled N-ethylmaleimide binds only protein 1 and 3, labeled N,N′-dicyclohexylcarbodiimide bonds only to protein 1 and 2. 5 N,N′-Dicyclohexylcarbodiimide had no effect on the reconstituted phosphate-exchange activity after incubation with intact mitochondria or proteoliposomes. 6 In the presence of protecting mersalyl concentrations N-ethylmaleimide inhibits the phosphate transport in intact mitochondria. Under these conditions no label ethylmaleimide was bound to the phosphate-carrier fraction. 7 Comparison of the reconstituted transport activity with the protein pattern of fractions, isolated with or without cardiolipin, shows correlation of transport with the amount of protein 2 but not with that of protein 1, which binds ethylmaleimide. A model of the mitochondrial phosphate carrier is presented which suggests a proteolytic degradation of the phosphate-carrier protein during its isolation. The model explains the contradictory results of this investigation.

Recent studies have suggested that parts of the hepatic activities of diacylglycerol acyltransferase and acyl cholesterol acyltransferase are expressed in the lumen of the endoplasmic reticulum (ER). However the ER membrane is impermeable to the long-chain fatty acyl-CoA substrates of these enzymes. Liver microsomal vesicles that were shown to be at least 95% impermeable to palmitoyl-CoA were used to demonstrate the membrane transport of palmitoylcarnitine and free L-carnitine - processes that are necessary for an indirect route of provision of ER luminal fatty acyl-CoA through a luminal carnitine acyltransferase (CAT). Experimental conditions and precautions were established to permit measurement of the transport of [14C]palmitoylcarnitine into microsomes through the use of the luminal CAT and acyl-CoA:ethanol acyltransferase as a reporter system to detect formation of luminal [14C]palmitoyl-CoA. Rapid, unidirectional transport of free L-[3H]carnitine by microsomes was measured directly. This process, mediated either by a channel or a carrier, was inhibited by mersalyl but not by N-ethylmaleimide or sulfobetaine - properties that differentiate it from the mitochondrial inner membrane carnitine/acylcarnitine exchange carrier. These findings are relevant to the understanding of processes for the reassembly of triacylglycerols that lipidate very low density lipoprotein particles as part of a hepatic triacylglycerol lipolysis/re-esterification cycle.

Biochemists recognize that all enzymatic reactions are reversible to some degree and much of respiratory chain is reversible. Although they meet the statements that the electron transport relying on the external electron-transport chain of mitochondrial suface takes the electrons out from extramitochondrial NADH using NADH-cytochrome b5 reductase (abbr. to fp5 below) for the final reduction of oxygen, the present reporter observed earlier that this fp5 catalysis could be reversed by ferrocyanide, an artificial reductant. Thus this artificial reductant could provide the fp5, composing the external electron-transport chain, with the electrons for NAD reduction. Notwithstanding the present shortcoming that the immediate electron acceptor making contact with the ferrocyanide is unknown, it seemed likely that one of the oxidoreductases on the electron-transport chain composing outer membrane was involved in the contact. Some authors, e.g., Solomons, describe the capacity of alcohols to reduce nicotinamide adenine dinucleotide. The hydroxyl carbon of the reductant alcohol is regarded to be functional for the reduction. The bovine-heart mitochondria, catalyzing an efficient oxidation for the extramitochondrial NADH, were made to reduce NADP for NADPH production with the assistance of ethanol in this study. Showing this kind of influence by ethanol depending on mitochondrial surface would make one explain the cellular fatty acid production, an event derived from usual alcohol drinking. The capaciousness put on fp5 for relaying electrons from ethanol to NADP was also examined using mersalyl treatment to fp5.

The respiration rate of liver mitochondria in the course of succinate oxidation depends on temperature in the presence of palmitate more strongly than in its absence (in state 4). In the Arrhenius plot, the temperature dependence of the palmitate-induced stimulation of respiration has a bend at 22°C which is characterized by transition of the activation energy from 120 to 60 kJ/mol. However, a similar dependence of respiration in state 4 is linear over the whole temperature range and corresponds to the activation energy of 17 kJ/mol. Phosphate partially inhibits the uncoupling effect of palmitate. This effect of phosphate is increased on decrease in temperature. In the presence of phosphate the temperature dependence in the Arrhenius plot also has a bend at 22°C, and the activation energy increases from 128 to 208 kJ/mol in the range from 13 to 22°C and from 56 to 67 kJ/mol in the range from 22 to 37°C. Mersalyl (10 nmol/mg protein), an inhibitor of the phosphate carrier, similarly to phosphate, suppresses the uncoupling effect of laurate, and the effects of mersalyl and phosphate are not additive. The recoupling effects of phosphate and mersalyl seem to show involvement of the phosphate carrier in the uncoupling effect of fatty acids in liver mitochondria. Possible mechanisms of involvement of the phosphate carrier in the uncoupling effect of fatty acids are discussed.

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.

The adenine nucleotide carrier from Jerusalem artichoke (Helianthus Tuberosus L.) tubers mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 33 kDa. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5′-phosphate-sensitive ATP/ATP exchange. It was purified 75-fold with a recovery of 15% and a protein yield of 0.18% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ATP, ADP, and GTP and was inhibited by bongkrekate, phenylisothiocyanate, pyridoxal 5′-phosphate, mersalyl and p-hydroxymercuribenzoate (but not N-ethylmaleimide). Atractyloside and carboxyatractyloside (at concentrations normally inhibitory in animal and plant mitochondria) were without effect in Jerusalem artichoke tubers mitochondria. V max of the reconstituted ATP/ATP exchange was determined to be 0.53 μmol/min per mg protein at 25°C. The half-saturation constant K m and the corresponding inhibition constant K i were 20.4 μM for ATP and 45 μM for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30°C. The N-terminal amino acid partial sequence of the purified protein showed a partial homology with the ANT protein purified from mitochondria of maize shoots.

The polyamine spermine is transported into the mitochondrial matrix by an electrophoretic mechanism having as driving force the negative electrical membrane potential (ΔΨ). The presence of phosphate increases spermine uptake by reducing ΔpH and enhancing ΔΨ. The transport system is a specific uniporter constituted by a protein channel exhibiting two asymmetric energy barriers with the spermine binding site located in the energy well between the two barriers. Although spermine transport is electrophoretic in origin, its accumulation does not follow the Nernst equation for the presence of an efflux pathway. Spermine efflux may be induced by different agents, such as FCCP, antimycin A and mersalyl, able to completely or partially reduce the ΔΨ value and, consequently, suppress or weaken the force necessary to maintain spermine in the matrix. However this efflux may also take place in normal conditions when the electrophoretic accumulation of the polycationic polyamine induces a sufficient drop in ΔΨ able to trigger the efflux pathway. The release of the polyamine is most probably electroneutral in origin and can take place in exchange with protons or in symport with phosphate anion. The activity of both the uptake and efflux pathways induces a continuous cycling of spermine across the mitochondrial membrane, the rate of which may be prominent in imposing the concentrations of spermine in the inner and outer compartment. Thus, this event has a significant role on mitochondrial permeability transition modulation and consequently on the triggering of intrinsic apoptosis.

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH-group reagents (N-ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4-aminobutyrate (GABA) in a competitive way (Ki = 20.9 +/- 2.4 mM). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1-[2-([(diphenylmethylene)amino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711) acid decreased carnitine accumulation while betaine, taurine and beta-alanine had no effect. The GABA transporters expressed in Xenopus laevis oocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nalecz, D. Dymna, J. E. Mroczkowska, A. Broer, S. Broer, M. J. Nalecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.

Oxidative damage of mammalian mitochondria induced by Ca2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca2+ uniport, undergo inner membrane permeabilization when treated with Ca2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.

To further understand the process of Al-induced citrate secretion from soybean roots, the effect of protein synthesis inhibitor, anion channel blockers, and citrate carrier inhibitors on Al-induced citrate exudation was investigated in Al-resistant soybean cultivar PI 416937. Citrate exudation from roots increased with the increase of Al concentration from 10 to 50 μM and initiated after 4 h of Al exposure. Protein synthesis inhibitor, cycloheximide (CHM; 25 μM) completely inhibited Al-induced citrate secretion during 12-h exposure, suggesting that novel protein synthesis was necessary in Al-induced citrate efflux. Also both anion channel blocker anthracene-9-carboxylic acid (A-9-C) and citrate carrier inhibitor mersalyl acid (Mersalyl) significantly reduced citrate secretion, suggesting that both anion channels in plasma membrane and citrate carriers in mitochondria membrane were the rate limiting factors of Al dependent citrate release. However, Al-induced citrate secretion was insensitive to anion channel blockers phenylglyoxal (PG), 4,4′-diisothiocyanostibene-2,2′-disulfonat (DIDS) and citrate carrier inhibitor pyridoxal 5′-P (PP).

Ascorbate peroxidase (APX) has been purified from potato tuber ( Solanum tuberosum L.) mitochondria. The potato enzyme appeared to be localized inside mitochondria. The mitochondrial APX was purified to homogeneity and its physico-chemical and kinetic properties were compared with those of the cytosolic enzyme. The molecular mass of mAPX was 31 kDa, as estimated by SDS-PAGE, and was similar to that of the cytosolic enzyme, but its relative mobility in non-denaturing PAGE was different from the cytosolic APX. The K m values of mAPX for AsA and H 2 O 2 were 76.1 ± 23.1 and 80.3 ± 24.9 μM, respectively, and were higher than those of the cytosolic enzyme. Mitochondrial APX was sensitive to inhibition by sulfhydryl reagents, such as mersalyl and p-hydroxymercuribenzoate (p-HMB). A role for the mitochondrial ascorbate-ascorbate peroxidase system in the scavenging of toxic oxygen species inside potato tuber mitochondria is proposed.

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4 ± 1.3 µm and 35 ± 5 pmol·min−1·(mg protein)−1, respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4 ± 0.9 µm and 22.9 ± 1.4 pmol·min−1·(mg matrix protein)−1, respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.

Mitochondrial transporters, in particular uncoupling proteins and the ADP/ATP carrier, are known to mediate uniport of anionic fatty acids (FAs), allowing FA cycling which is completed by the passive movement of FAs across the membrane in their protonated form. This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furthermore, how this putative activity is related to the previously observed HgCl(2)-induced uniport mode. The yeast mitochondrial phosphate carrier was expressed in Escherichia coli and then reconstituted into lipid vesicles. The FA-induced H(+) uniport or Cl(-) uniport were monitored fluorometrically after HgCl(2) addition. These transport activities were further characterized by testing various inhibitors of the two different transport modes. The phosphate carrier was found to mediate FA cycling, which led to H(+) efflux in proteoliposomes. This activity was insensitive to ATP, mersalyl or N-ethylmaleimide and was inhibited by methylenediphosphonate and iminodi(methylenephosphonate), which are new inhibitors of mitochondrial phosphate transport. Also, the HgCl(2) induced Cl(-) uniport mediated by the reconstituted yeast PIC, was found to be inhibited by these reagents. Both methylenediphosphonate and iminodi(methylenephosphonate) blocked unidirectional Cl(-) uptake, whereas Cl(-) efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformic acid only. These results suggest that a hydrophobic domain, interacting with FAs, exists in the mitochondrial phosphate carrier, which is distinct from the phosphate transport pathway. This domain allows for FA anion uniport via the phosphate carrier and consequently, FA cycling that should lead to uncoupling in mitochondria. This might be considered as a side function of this carrier.

In the presence of K + , addition of ATP or ethanol to yeast mitochondria triggers the depletion of the transmembrane potential (ΔΨ) and this is prevented by millimolar concentrations of phosphate (PO 4 ). Different monovalent and polyvalent anions were tested for their protective effects on mitochondria from Saccharomyces cerevisiae . Only arsenate (AsO 4 ) and sulfate (SO 4 ) were as efficient as PO 4 to protect mitochondria against the K + mediated swelling, depletion of the ΔΨ, and decrease in the ratio of uncoupled state to state 4 respiration rates. Protection by PO 4 , SO 4 or AsO 4 was inhibited by mersalyl, suggesting that these anions interact with a site located in the matrix side. In addition, the effects of SO 4 and AsO 4 on the F 1 F 0 -ATPase were tested: both SO 4 and AsO 4 inhibited the synthesis of ATP following competitive kinetics against PO 4 and non-competitive kinetics against ADP. The mersalyl sensitive uptake of 32 PO 4 was not inhibited by SO 4 or AsO 4 , suggesting that the synthesis of ATP was inhibited at the F 1 F 0 -ATPase. The hydrolysis of ATP was not inhibited, only a stimulation was observed when AsO 4 or sulfite (SO 3 ) were added. It is suggested that the structure and charge similarities of PO 4 , AsO 4 and SO 4 result in undiscriminated binding to at least two sites located in the mitochondrial matrix: at one site, occupation by any of these three anions results in protection against uncoupling by K + ; at the second site, in the F 1 F 0 -ATPase, AsO 4 and SO 4 compete for binding against PO 4 leading to inhibition of the synthesis of ATP.

The purpose of this study was to investigate the effects exerted by thiol-modifying reagents on themitochondrial sulfonylurea receptor. The thiol-oxidizing agents (timerosal and 5, 5'-dithio-bis(2-nitrobenzoic acid)) were found to produce a large inhibition (70% to 80%) of specific binding of [(3)H]glibenclamide to the beef heart mitochondrial membrane. Similar effects were observed with membrane permeable (N-ethylmaleimide) and non-permeable (mersalyl) thiol modifying agents. Glibenclamide binding was also decreased by oxidizing agents (hydrogen peroxide) but not by reducing agents (reduced gluthatione, dithiothreitol and the 2,3-dihydroxy-1,4-dithiolbutane). The results suggest that intact thiol groups, facing the mitochondrial matrix, are essential for glibenclamide binding to the mitochondrial sulfonylurea receptor.

The citrate carrier from maize(Zea mays) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and hydroxyapatite/celite in the presence of cardiolipin. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31 kD. When reconstituted into liposomes, the citrate carrier catalyzed a pyridoxal 5′-P-sensitive citrate/citrate exchange. It was purified 224-fold with a recovery of 50% and a protein yield of 0.22% with respect to the mitochondrial extract. In the reconstituted system the purified citrate carrier catalyzed a first-order reaction of citrate/citrate (0.065 min−1) or citrate/malate exchange (0.075 min−1). Among the various substrates and inhibitors tested, the reconstituted protein transported citrate,cis-aconitate, isocitrate, l-malate, succinate, malonate, glutarate, α-ketoglutarate, oxaloacetate, and α-ketoadipate and was inhibited by pyridoxal 5′-P, phenylisothiocyanate, mersalyl, andp-hydroxymercuribenzoate (but notN-ethylmaleimide), 1,2,3-benzentricarboxylate, benzylmalonate, and butylmalonate. The activation energy of the citrate/citrate exchange was 66.5 kJ/mol between 10°C and 35°C; the half-saturation constant (Km) for citrate was 0.65 ± 0.05 mm and the maximal rate (Vmax) of the citrate/citrate exchange was 13.0 ± 1.0 μmol min−1 mg−1 protein at 25°C.

The phosphate carrier of the inner mitochondrial membrane has been purified from potato tuber in a nearly homogenous form and characterized after reconstitution into liposomes. The mitochondrial membrane was solubilised by Triton X-114 in the presence of cardiolipin and the soluble fraction was chromatographied on hydroxylapatite. The flow-through fractions were collected and analyzed by SDS-gel electrophoresis. In some fractions, a single protein band of Mr=35 kDa was found. This protein was reconstituted into phosphatidylcholine vesicles and its capability to transport phosphate was measured by phosphate–phosphate exchange. A maximum velocity of 28.1 μmol min−1. (mg of protein)−1 was detected. Phosphate transport was sensitive to mersalyl and N-ethylmaleimide. On the other hand, no proton transport was apparent in these proteoliposomes, unlike to what was seen with similar hydroxylapatite-purified fractions obtained after solubilization of the membrane by decyl-pentaoxyethylene ether, which contained several polypeptides. Thus the phosphate carrier is distinct from the uncoupling protein (UCP) family and its behavior shows that not all anion carriers are able to act as UCPs.

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.

Glutamine transport into rat brain mitochondria (synaptic and non-synaptic) was monitored by the uptake of [3H]glutamine as well as by mitochondrial swelling. The uptake is inversely correlated to medium osmolarity, temperature-dependent, saturable and inhibited by mersalyl, and glutamine is upconcentrated in the mitochondria. These results indicate that glutamine is transported into an osmotically active space by a protein catalyzed mechanism. The uptake is slightly higher in synaptic mitochondria than in non-synaptic ones. It is inhibited both by rotenone and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the latter at pH 6.5, showing that the transport is activated by an electrochemical proton gradient. The K+/H+ ionophore nigericin also inhibits the uptake at pH 6.5 in the presence of external K+, which indicates that glutamine, at least in part, is taken up by a proton symport transporter. In addition, glutamine uptake as measured by the swelling technique revealed an additional glutamine transport activity with at least 10 times higher Km value. This uptake is inhibited by valinomycin in the presence of K+ and is thus also activated by the membrane potential. Otherwise, the two methods show similar results. These data indicate that glutamine transport in brain mitochondria cannot be described by merely a simple electroneutral uniport mechanism, but are consistent with the uptake of both the anionic and the zwitterionic glutamine.

The effect of pyridoxal 5'-phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by pyridoxal 5'-phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal 5'-phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [3H]pyridoxal 5'-phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by pyridoxal 5'-phosphate. These results indicate that pyridoxal 5'-phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal glycine residue) which is essential for substrate translocation and may be localized at or near the substrate-binding site.

Contact allergy to thimerosal (TH) has not been considered a marker for mercury allergy, since there is a low degree of cross-sensitivity to inorganic as well as to organic mercury salts. 40 subjects, who previously gave a positive patch test reaction only to thimerosal 0.1% pet. (Hermal), when simultaneously repatch-tested to solutions containing TH, mersalyl acid, p-amino-phenylmercuric acid, mercuric acetate and thiosalicylic acid, respectively, gave positive reactions only to TH. 36 out of 40 subjects were divided into 2 groups of 18 subjects and simultaneously repatch-tested to solutions containing TH, methylmercury chloride (MeHgCl), thiosalicylic acid, and, ethylmercury chloride (EtHgCl), respectively. EtHgCl was tested in the 1st group at 0.031% and in the 2nd group at 0.015%. The results showed that all subjects gave concomitant positive reactions to TH, EtHgCl and MeHgCl. EtHgCl 0.031% gave a higher number of reactions than EtHgCl 0.015%, underlining the rôle of the solvent in these reactions. Patch test results in 300 consecutive patients to a standard series, to which MeHgCl was added, showed that MeHgCl and TH were never able to give isolated positive reactions, and that the concomitant positive reactions occurred in only 3.6% of subjects. In conclusion, our data seem to suggest that the positive reactions to TH found in our patients were due to EtHgCl, and that the structural similarities with MeHgCl were so close that the skin reacted against each as if they were identical.

Blood–brain barrier transport of aluminum citrate was assessed in rats by microdialysis of the jugular vein as well as the right and left frontal cortices. Previous studies (Allen et al., 1995. Evidence for energy-dependent transport of aluminum out of brain extracellular fluid. Toxicology 92, 193–202; Ackley and Yokel, 1997. Aluminum citrate is transported from brain into blood via the monocarboxylic acid transporter located at the blood–brain barrier. Toxicology 120, 89–97), and the current study, demonstrated that the steady-state brain-to-blood ratio of the unbound extracellular aluminum immediately surrounding the microdialysis probe is less than 1, suggesting the presence of a process other than diffusion across the blood–brain barrier. It was speculated that a monocarboxylate transporter at the blood–brain barrier was maintaining this ratio at less than 1 (Ackley and Yokel, 1997). Monocarboxylate transporters are proton co-transporters. Decreasing extracellular pH (increasing proton availability) increases monocarboxylate transport. After alkalinizing the dialysate perfusing a brain microdialysis probe (to pH=10.2), the steady-state aluminum brain-to-blood ratio increased from 0.35 to 0.80. The addition of the proton ionophore, p -(trifluoromethoxy)phenylhydrazone (FCCP) (1 mM), to brain dialysate increased this ratio from 0.21 to 0.61. These increased ratios suggest that a proton-dependent process is removing Al from brain extracellular fluid. The monocarboxylate transporter is the only known proton-dependent transporter at the blood–brain barrier. There are two known isoforms of this transporter in the rodent, MCT1 and MCT2. Organomercurial thiol reagents, such as mersalyl acid, inhibit MCT1 but not MCT2. Mersalyl acid (50 mM) addition to brain dialysate increased the steady-state aluminum brain-to-blood ratio from 0.19 to 0.87, suggesting that MCT1 is at least partially mediating the efflux of aluminum from brain extracellular fluid.

Yeast mitochondria (Saccharomyces cerevisiae) contain a permeability transition pore which is regulated differently than the pore in mammalian mitochondria. In a mannitol medium containing 10 mM Pi and ethanol (oxidizable substrate), yeast mitochondria accumulate large amounts of Ca2+ (>400 nmol/mg of protein) upon the addition of an electrophoretic Ca2+ ionophore (ETH129). Pore opening does not occur following Ca2+ uptake, even though ruthenium red-inhibited rat liver mitochondria undergo rapid pore opening under analogous conditions. However, a pore does arise in yeast mitochondria when Ca2+ and Pi are not present, as monitored by swelling, ultrastructure, and matrix solute release. Pore opening is slow unless a respiratory substrate is provided (ethanol or NADH) but also occurs rapidly in response to ATP (2 mM) when oligomycin is present. Pi and ADP inhibit pore opening (EC50 approximately 1 and 4 mM, respectively), however, cyclosporin A (7 microg/ml), oligomycin (20 microg/ml), or carboxyatractyloside (25 microM) have no effect. The pore arising during respiration is also inhibited by nigericin or uncoupler, indicating that an acidic matrix pH antagonizes the process. Pi also inhibits pore opening by lowering the matrix pH (Pi/OH- antiport). However, inhibition of the ATP-induced pore by Pi is seen in the presence of mersalyl, suggesting a second mechanism of action. Since pore induction by ATP is not sensitive to carboxyatractyloside, ATP appears to act at an external site and Pi may antagonize the interaction. Isoosmotic polyethylene glycol-induced contraction of yeast mitochondria swollen during respiration, or in the presence of ATP, is 50% effective at a solute size of 1.0-1.1 kDa. This suggests that the same pore is induced in both cases and is comparable in size with the permeability transition pore of heart and liver mitochondria.

The matrix free magnesium ion concentration, [Mg2+]m, estimated using the fluorescent probe furaptra, averaged 0.67 mM in 15 preparations of beef heart mitochondria containing an average of 21 nmol total Mg2+ per mg protein. [Mg2+]m was compared with total Mg2+ during respiration-dependent uptake and efflux of Mg2+ and during osmotic swelling. In the absence of external Pi these mitochondria contain about 32 nmol/mg non-diffusible Mg-binding sites with an apparent Kd of 0.34 mM. [Mg2+]m depends on both the size of the total Mg2+ pool and the ability of matrix anions to provide Mg-ligands. Pi interacts strongly with Mg2+ to decrease [Mg2+]m and, in the absence of external Mg2+, promotes respiration-dependent Mg2+ efflux and a decrease in [Mg2+]m to very low levels. The uptake of Pi by respiring mitochondria converts delta pH to membrane potential (delta psi) and provides additional Mg-binding sites. This permits large accumulations of Mg2+ and Pi with little change in [Mg2+]m. Nigericin also converts delta pH to delta psi in respiring mitochondria and induces a large and rapid increase in both total Mg2+ and [Mg2+]m. Mersalyl increases the permeability of the mitochondrial membrane to cations and this also induces a marked increase in both total Mg2+ and [Mg2+]m. These results suggest that mitochondria take up Mg2+ by electrophoretic flux through membrane leak pathways, rather than via a specific Mg2+ transporter. Mitochondria swollen by respiration dependent uptake of potassium phosphate show decreased [Mg2+]m, whereas those swollen to the same extent in potassium acetate do not. This suggests that [Mg2+]m is well-buffered during osmotic volume changes unless there is also a change in ligand availability.

The adenine nucleotide carrier from maize (Zea mays L. cv B 73) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. Sodium dodecyl sulfate-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 32 kD. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5[prime]-phosphate-sensitive ATP/ATP exchange. It was purified 168-fold with a recovery of 60% and a protein yield of 0.25% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ADP, ATP, GDP, and GTP, and was inhibited by atractyloside, bongkrekate, phenylisothiocianate, pyridoxal 5[prime]-phosphate, and mersalyl (but not N-ethylmaleimide). Maximum initial velocity of the reconstituted ATP/ATP exchange was determined to be 2.2 [mu]mol min-1 mg-1 protein at 25[deg]C. The half-saturation constants and the corresponding inhibition constants were 17 [mu]M for ATP, 26 [mu]M for ADP, 59 [mu]M for GTP, and 125 [mu]M for GDP. The activation energy of the ATP/ATP exchange was 48 kilojoule/mol between 0 and 15[deg]C, and 22 kilojoule/mol between 15 and 35[deg]C. Partial amino acid sequences showed that the purified protein was the product of the ANT-G1 gene sequenced previously (B. Bathgate, A. Baker, C.J. Leaver [1989] Eur J Biochem 183: 303–310).

The effect of arginine-specific reagents on the function of the purified and reconstituted oxoglutarate carrier protein of the inner mitochondrial membrane has been investigated. The α-dicarbonyl reagents 2,3-butanedione, 2,3-pentanedione, 2,3- and 3,4-hexanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, and phenylglyoxal derivatives caused a concentration-dependent inhibition of oxoglutarate transport with an IC 50 of 0.05 m M for 2,3-hexanedione, 0.08 m M for 4-hydroxy-3-nitrophenylglyoxal, and 0.17 m M for 2,3-pentanedione. The inhibition increased with pH from 6.0 to 8.0, indicating that the p K of the reacting group(s) is rather high. Mersalyl and pyridoxal 5′-phosphate (or 4,4′-dinitrostilbene-2,2′-disulfonate), which are known to react specifically and reversibly with cysteine residues and lysine residues, respectively, were unable to protect the oxoglutarate carrier against inhibition by α-dicarbonyl reagents. Other diketone compounds, which do not react with arginine residues, had no significant effect on the oxoglutarate transport activity. Oxoglutarate and L -malate effectively protected the oxoglutarate carrier against inactivation caused by arginine-specific reagents; other dicarboxylates, which are not substrates of the carrier, had no protective effect. A 50% substrate protection was observed at half-saturation of the external binding site. These results indicate that the arginine-specific reagents used in this investigation interact with the oxoglutarate carrier at the level of an arginine residue(s), which is essential for binding and/or translocation of substrates and which may be localized in, or near, the substrate-binding site.