The transport of L-cysteinesulfinate in rat liver mitochondria.

1. The mechanism of L-cysteinesulfinate permeation into rat liver mitochondria has been investigated. 2. Mitochondria do not swell in ammonium or potassium salts of L-cysteinesulfinate in all the conditions tested, including the presence of valinomycin and/or carbonylcyanide p-trifluoromethoxyphenylhydrazone. 3. The activation of malate oxidation by L-cysteinesulfinate is abolished by aminooxyacetate, an inhibitor of the intramitochondrial aspartate aminotransferase, it is not inhibited by high concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (in contrast to the oxidation of malate plus glutamate) and it is decreased on lowering the pH of the medium. 4. All the aspartate formed during the oxidation of malate plus L-cysteinesulfinate is exported into the extramitochondrial space. 5. Homocysteinesulfinate, cysteate and homocysteate, which are all good substrates of the mitochondrial aspartate aminotransferase, are unable to activate the oxidation of malate. Homocysteinesulfinate and homocysteate have no inhibitory effect on the L-cysteinesulfinate-induced respiration, whereas cysteate inhibits it competitively with respect to L-cysteinesulfinate. 6. In contrast to D-aspartate, D-cysteinesulfinate and D-glutamate, L-aspartate inhibits the oxidation of malate plus L-cysteinesulfinate in a competitive way with respect to L-cysteinesulfinate. Vice versa, L-cysteinesulfinate inhibits the influx of L-aspartate. 7. Externally added L-cysteinesulfinate elicits efflux of intramitochondrial L-aspartate or L-glutamate. The cysteinesulfinate analogues homocysteinesulfinate, cysteate and homocysteate and the D-stereoisomers of cysteinesulfinate, aspartate and glutamate do not cause a significant release of internal glutamate or aspartate, indicating a high degree of specificity of the exchange reactions. External L-cysteinesulfinate does not cause efflux of intramitochondrial Pi, malate, malonate, citrate, oxoglutarate, pyruvate or ADP. The L-cysteinesulfinate-aspartate and L-cysteinesulfinate-glutamate exchanges are inhibited by glisoxepide and by known substrates of the glutamate-aspartate carrier. 8. The exchange between external L-cysteinesulfinate and intramitochondrial glutamate is accompanied by translocation of protons across the mitochondrial membrane in the same direction as glutamate. The L-cysteinesulfinate-aspartate exchange, on the other hand, is not accompanied by H+ translocation. 9. The ratios delta H+/delta glutamate, delta L-cysteinesulfinate/delta glutamate and delta L-cysteinesulfinate/delta aspartate are close to unity. 10. It is concluded that L-cysteinesulfinate is transported by the glutamate-aspartate carrier of rat liver mitochondria. The present data suggest that the dissociated form of L-cysteinesulfinate exchanges with H+-compensated glutamate or with negatively charged aspartate.

Mechanism of inhibition of the dicarboxylate carrier of mitochondria by thiol reagents.

The nature of the inhibition of the dicarboxylate carrier by compounds reacting with SH groups has been investigated. (1) Mersalyl and p-hydroxymercuribenzoate increase the Km without changing the V of malonate/Pi exchange, when they are added simultaneously with the dicarboxylate. If, on the other hand, the mitochondria are preincubated with SH reagents prior to the addition of malonate, the mersalyl inhibition of malonate/Pi exchange becomes predominantly non-competitive with respect to malonate. (2) In the case of Pi/Pi exchange, catalyzed by the dicarboxylate carrier, the mersalyl inhibition is competitive with respect to Pi (as indicated by Lineweaver-Burk plots), even when mersalyl is added before the substrate. Dixon plots of the rate of Pi uptake against mersalyl concentration are, however, non-linear, suggesting that the inhibition is partially competitive. (3) Dicarboxylates and dicarboxylate analogous protect against SH reagent inhibition of both dicarboxylate and Pi uptake via the dicarboxylate carrier. The protectors are effective when added before, or together with the SH reagents, but do not reverse the inhibition once it has been established. Protection by substrate analogues progressively decreases, as the time of incubation with the SH reagent increases. (4) The presence of Pi does not protect against the SH reagent inhibition of the Pi uptake. (5) The rate of SH reagent inhibition of the dicarboxylate carrier is competively inhibited by dicarboxylates. (6) It is concluded that SH reagents bind at or near the dicarboxylate specific binding site and distant from the Pi binding site. As a result of this reaction these inhibitors prevent dicarboxylate binding directly and decrease the affinity for Pi by an indirect conformational change.

Photoaffinity labeling of the mitochondrial oxoglutarate carrier by azido-phthalonate.

The effect of azido-phthalonate, a photoreactive analogue of oxoglutarate, on the transport of oxoglutarate was investigated in proteoliposomes reconstituted with the purified oxoglutarate carrier. In the dark, azido-phthalonate inhibits the reconstituted oxoglutarate/oxoglutarate exchange in a competitive manner with a Ki of 0.38 mM. Upon photoirradiation, the inhibition of the oxoglutarate exchange by azido-phthalonate is not removed by passing the proteoliposomes through a Sephadex column. The light-induced inhibition of the oxoglutarate/oxoglutarate exchange activity by azido-phthalonate is time- and concentration-dependent. The kinetic analysis of transport inhibition by azido-phthalonate reveals that one molecule of this substrate analogue bound to the functional carrier molecule is responsible for complete inhibition of the carrier function. Azido-[3H]phthalonate binds to the oxoglutarate carrier covalently. Incubation of the proteoliposomes with oxoglutarate during photoirradiation in the presence of azido-phthalonate protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the carrier protein. It is concluded that azido-phthalonate can be used for photoaffinity labeling of the mitochondrial oxoglutarate carrier at the substrate-binding site.

Purification of the active mitochondrial tricarboxylate carrier by hydroxylapatite chromatography.

The mitochondrial tricarboxylate carrier has been extracted from rat liver mitochondria or SMP with Triton X-100, in the presence of 1,2,3-BTA and DPG, and partially purified by chromatography on HTP. The purified fraction, which also contains the ADP/ATP carrier and the phosphate carrier, after incorporation into liposomes catalyzes a 1,2,3-BTA-sensitive [14C]citrate/citrate exchange. The tissue and substrate specificity, the inhibitor sensitivity and the kinetic properties of citrate transport in liposomes are similar to those described for the citrate transport in mitochondria. The maximal rate of citrate exchange in the reconstituted system is 338 mumol X min-1 X g protein-1, at 30 degrees C and pH 7.0.

Inhibition of the mitochondrial tricarboxylate carrier by arginine-specific reagents.

The effect of arginine-specific reagents on the activity of the partially purified and reconstituted tricarboxylate carrier of the inner mitochondrial membrane has been studied. It has been found that 1,2-cyclohexanedione, 2,3-butanedione, phenylglyoxal and phenylglyoxal derivatives inhibit the reconstituted citrate/citrate exchange activity. The inhibitory potency of the phenylglyoxal derivatives increases with increasing hydrophilic character of the molecule. Citrate protects the tricarboxylate carrier against inactivation caused by the arginine-specific reagents. Other tricarboxylates, which are not substrates of the carrier, have no protective effect. The results indicate that at least one essential arginine residue is located at the substrate-binding site of the tricarboxylate carrier and that the vicinity of the essential arginine(s) has a hydrophilic character.

Characterization of sulphydryl groups of the mitochondrial phosphate translocator by a maleimide spin label.

A maleimide spin label strongly inhibits the phosphate/H+ symporter of rat liver mitochondria. While inducing half-maximal inhibition of transport, the spin label reacts preferentially with the SH groups of the carrier, which are at least of two types. One type of SH group is localized close to the surface of the membrane and its environment does not significantly influence the mobility of the probe. The second type of SH group is buried in the membrane, is not accessible to ascorbate or chromium oxalate and its environment greatly restricts the motion of the probe.

[Relationship between cysteinesulfinate and aspartate transport in mitochondria]. / Interrelazione fra il trasporto di cisteinsulfinato e aspartato nei mitocondri.

L-CSA and L-Aspartate inhibit each other's transport in the mitochondria in a competitive manner. The D-isomers have very little effect. It is proposed that the two amino acids are transported by a common translocator, presumable the glutamate-aspartate carrier.

Effect of anthracycline antibiotics on the reconstituted mitochondrial tricarboxylate carrier.

The effect of anthracycline antibiotics on the activity of the partially purified and reconstituted tricarboxylate carrier system of the rat liver mitochondria was studied. It was found that the citrate/citrate exchange activity is inhibited by Br-daunomycin and with less potency by doxorubicin, daunomycin, epirubicin and idarubicin. The inhibition of the citrate transport activity is concentration and time-dependent. Cardiolipin protects against the inhibition by Br-daunomycin and the reconstituted citrate transport activity depends upon the ratio of cardiolipin/Br-daunomycin.

[Transport of H+ in mitochondria induced by the uptake of sulfite, sulfate and thiosulfate]. / Trasporto di h+ nei mitocondri indotto dall'aggiunta di solfito, solfato e tiosolfato.

The addition of sulphite to rat-liver mitochondria (RLM) causes an uptake of H+ that is unaffected by NEM and butylmalonate. The uptake of H+ induced by sulphate or thiosulphate is abolished by NEM and butylmalonate in freshly isolated RLM, whereas it is inhibited only by butylmalonate in sulphite-pretreated mitochondria. The data suggest that sulphite is cotransported with H+, whereas the movement of H+ associated to the uptake of sulphate or thiosulphate by RLM is mediated by either phosphate or sulphite.

The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria catalyses a second transport mode: ornithine+/H+ exchange.

The mechanism of unidirectional transport of ornithine (i.e. in the absence of a counter-metabolite) has been investigated in proteoliposomes reconstituted with the ornithine carrier purified from rat liver mitochondria. The efflux of [(3)H]ornithine from proteoliposomes was stimulated by the addition of H(+) (but not of other cations) to the incubation medium. On keeping the pH in the compartment containing ornithine constant at 8.0, the flux of ornithine into or out of the proteoliposomes increased on decreasing the pH in the opposite compartment from 8.0 to 6.0. Ornithine influx was also stimulated when a higher H(+) concentration was generated inside the vesicles relative to the outside by the K(+)/H(+) exchanger nigericin in the presence of an outwardly directed K(+) gradient. A valinomycin-induced electrogenic flux of K(+) did not affect ornithine transport in the absence of a counter-metabolite. Furthermore, changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the flux of ornithine is accompanied by translocation of H(+) in the opposite direction. It is concluded that the mitochondrial ornithine carrier catalyses an electroneutral exchange of ornithine(+) for H(+), in addition to the well-known 1:1 exchange of metabolites. Lysine(+), but not citrulline, can also be exchanged for H(+) by the ornithine carrier. The ornithine(+)/H(+) transport mode of the exchanger is an essential step in the catabolism of excess arginine.

Inhibition of mitochondrial substrate anion translocators by a synthetic amphipathic polyanion.

A synthetic polyanion (a copolymer of methacrylate, maleate, and styrene in 1:2:3 proportion with an average molecular weight of 10,000 dalton) inhibits the tricarboxylate, oxoglutarate, dicarboxylate, and adenine nucleotide translocators of rat liver mitochondria. The activity versus inhibitor concentration curves are sigmoidal. The inhibition of the oxoglutarate and tricarboxylate translocators by the polyanion is competitive, while that of the adenine nucleotide translocator is of mixed-type. The K1 values of the polyanion are the following: for oxoglutarate translocator 4.0 microM, tricarboxylate translocator 1.2 microM, and adenine nucleotide translocator 1.3 microM with ADP and 0.9 microM with ATP. It is suggested that the polyanion acts primarily by increasing the negative charge of the inner membrane at the outer surface, and the sensitivity of the translocators toward the polyanion depends on the number of negative charges of their substrates.

Identification and purification of the reconstitutively active glutamine carrier from rat kidney mitochondria.

The glutamine carrier from rat kidney mitochondria, solubilized in dodecyl octaoxyethylene ether (C12E8) and partly purified on hydroxyapatite, was identified and completely purified by Celite chromatography. On SDS/PAGE, the purified glutamine carrier consisted of a single protein band with an apparent molecular mass of 41.5 kDa. When reconstituted into liposomes, the glutamine carrier catalysed both the unidirectional flux of glutamine and the glutamine/glutamine countertransport, which were completely inhibitable by a mixture of pyridoxal 5'-phosphate and N-ethylmaleimide. The carrier protein was purified 474-fold with a recovery of 58% and a protein yield of 0.12% with respect to the mitochondrial extract. The glutamine carrier-mediated transport is quite specific for l-glutamine. l-Asparagine is the only other amino acid that is efficiently transported by the reconstituted carrier protein. d-Glutamine, l-glutamate and l-aspartate are very poor substrates. The transport activity was inhibited by several thiol-group and amino-group reagents.

The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria: electrical nature and coupling of the exchange reaction with H+ translocation.

The mechanism and the electrical nature of ornithine/citrulline exchange has been investigated in proteoliposomes reconstituted with the ornithine/citrulline carrier purified from rat liver mitochondria. The stoichiometry of the exchanging substrates was close to 1:1. The exchange was not affected by inducing electrogenic flux of K+ with valinomycin. In contrast, the pH gradient generated by the K+/H+ exchanger nigericin in the presence of an outwardly directed K+ gradient stimulated the ornithineout/citrullinein exchange, but not the ornithine/ornithine homoexchange. Experiments in which either the internal or the external pH was varied, while keeping constant the pH in the other compartment, indicated that maximal exchange rates are found at pH 6 in the compartment containing citrulline and at pH 8 in the compartment containing ornithine. Changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the exchanges ornithineout/citrullinein and citrullineout/ornithinein are accompanied by translocation of H+ in the same direction as citrulline. It is concluded that the mitochondrial ornithine/citrulline carrier catalyses an electroneutral exchange of ornithine+ for citrulline plus an H+. A reasonable model is one in which ornithine binds to a deprotonated carrier and citrulline to a protonated carrier and both substrate-carrier complexes are neutral. The physiological implications of this transport process are discussed.

Reconstitution of the isolated phosphate-transport system of pig-heart mitochondria.

The phosphate carrier of pig heart mitochondria has been isolated and reconstituted in liposomes. The highest specific activity for [32P]phosphate exchange was obtained with hydroxyapatite eluate from mitochondria, extracted with Triton X-114 in the presence of cardiolipin. This fraction, which is free from the ADP ATP-carrier, had a specific activity of 30 mumol 32Pi x min-1 x mg protein -1. The following conditions were found to inactivate the phosphate carrier irreversibly in the solubilized state: high ionic strength, high detergent concentrations and a high pH. The decrease of the activity by high detergent concentrations can be largely prevented by cardiolipin, present in the extraction buffer, suggesting a specific removal of this lipid by the detergent. After reconstitution in liposomes, the phosphate carrier is rather stable. The Arrhenius plot of the temperature-dependence of the reconstituted phosphate exchange showed different slopes above and below 27 degrees C. Between 0 degrees C and 27 degrees C the EA was 64 kJ . mol-1, between 27 degrees C and 42 degrees C 44 kJ . mol-1. The exchange of Pi followed a first-order kinetic.

Inhibition of the reconstituted mitochondrial oxoglutarate carrier by arginine-specific reagents.

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 alpha-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 IC50 of 0.05 mM for 2,3-hexanedione, 0.08 mM for 4-hydroxy-3-nitrophenylglyoxal, and 0.17 mM for 2,3-pentanedione. The inhibition increased with pH from 6.0 to 8.0, indicating that the pK 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 alpha-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.

The mitochondrial oxoglutarate carrier: cysteine-scanning mutagenesis of transmembrane domain IV and sensitivity of Cys mutants to sulfhydryl reagents.

Using a functional mitochondrial oxoglutarate carrier mutant devoid of Cys residues (C-less carrier), each amino acid residue in transmembrane domain IV and flanking hydrophilic loops (from T179 to S205) was replaced individually with Cys. The great majority of the 27 mutants exhibited significant oxoglutarate transport in reconstituted liposomes as compared to the activity of the C-less carrier. In contrast, Cys substitution for G183, R190, Q198, and Y202, in either C-less or wild-type carriers, yielded molecules with complete loss of oxoglutarate transport activity. G183 and R190 could be partially replaced only by Ala and Lys, respectively, whereas Q198 and Y202 were irreplaceable with respect to oxoglutarate transport. Of the single-Cys mutants tested, only T187C, A191C, V194C, and N195C were strongly inactivated by N-ethylmaleimide and by low concentrations of methanethiosulfonate derivatives. Oxoglutarate protects Cys residues at positions 187, 191, and 194 against reaction with N-ethylmaleimide. These positions as well as the residues found to be essential for the carrier activity, except Y202 which is located in the extramembrane loop IV-V, reside on the same face of transmembrane helix IV, probably lining part of a water-accessible crevice or channel between helices of the oxoglutarate carrier.

Increment of sister chromatid exchange frequencies (SCE) due to epichlorohydrin (ECH) in vitro treatment in human lymphocytes.

Although several studies have examined the effects on health of exposure to epichlorohydrin (ECH) through normal industrial operations and production, there is still considerable interest in its potential harmful effects on humans. The aim of the present study was to evaluate ECH effects in vitro through controlled investigations by using sister chromatid exchange (SCE), micronucleus (MN), and chromosome aberrations (CA) as the test battery. Cultures for cytogenetic tests were set up from blood samples of four healthy non-smoking and three smoking males. The experiments were performed using four different concentrations: 10(-10) M, 10(-8) M, 10(-6) M, and 10(-4) M, of ECH in DMSO. Analysis of variance showed that concentrations of ECH had significant effects on SCE/cell frequencies in the lymphocyte cultures of all donors (F=100.25, P<0.001). We were unable to find any evidence of significant increases in CA and MN frequencies in ECH-treated lymphocyte cultures with respect to the controls.