The transport of sulphate and sulphite in rat liver mitochondria

1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO(2) or an exchange of the sulphite anion for hydroxyl ion(s).     

Effect of phosphate and uncouplers on substrate transport and oxidation by isolated corn mitochondria

A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O₂ uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K⁺ permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V_max, probably reflecting a diffusion-limited process.     

The transport of oxaloacetate in isolated mitochondria

The mechanism of mitochondrial oxaloacetate transport has been investigated by measuring the rate and the extent of exchange reactions between intramitochondrial anions and added oxaloacetate. The exchange between oxaloacetate and intramitochondrial oxoglutarate is insensitive to mersalyl at a concentration which completely inhibits the dicarboxylate carrier. Oxaloacetate causes efflux of intramitochondrial P_i, malonate, and malate. Mersalyl inhibits completely the oxaloacetate/P_i exchange, but only partially the oxaloacetate/malonate and the oxaloacetate/malate exchanges. The inhibition of the last two reactions decreases on increasing the time of incubation. Butylmalonate inhibits more than phenylsuccinate the exchange oxaloacetate_out/³²P_iin, whereas phenylsuccinate is a more effective inhibitor than butylmalonate of the oxaloacetate_out/[¹⁴C]oxoglutarate_in exchange. The apparent K_m values ranged from 0.6 to 1.2 mm for the oxaloacetate/oxoglutarate exchange and from 6.5 to 10 mm for the oxaloacetate/P_i exchange. The inhibition of oxoglutarate uptake by oxaloacetate is competitive. Oxaloacetate inhibits the malonate/P_i exchange competitively and it is a noncompetitive inhibitor of the $\text{P}{}_{\mathrm{i}}\text{P}{}_{\mathrm{i}}$ exchange. It is concluded that oxaloacetate may be transported across the mitochondrial membrane by the oxoglutarate carrier and, much less effectively, by the dicarboxylate carrier. The implications of these findings are discussed.     

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent K_m for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.     

Transport of dicarboxylic acids in castor bean mitochondria

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K_m values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.

The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [¹⁴C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [¹⁴C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4°C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [¹⁴C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K_m of 2.5 millimolar and V_max of 250 nanomoles per minute per milligram protein at 4°C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

     

Synthesis of malate from phosphoenolpyruvate by rabbit liver mitochondria: implications for lipogenesis

(1) Rabbit liver mitochondria can convert exogenous phosphoenolpyruvate to malate. (2) Malate production is dependent on phosphoenolpyruvate and HCO3- and is stimulated by CN- or malonate alone and especially in combination. (3) Malate production is inhibited 70% by 3-mercaptopicolinate, a specific inhibitor of phosphoenolpyruvate carboxykinase, and 50-60% by 1,2,3-benzenetricarboxylate, an inhibitor of the tricarboxylate transporter. (4) Rat liver mitochondria incubated with phosphoenolpyruvate under identical conditions do not produce malate. (5) Malate production from phosphoenolpyruvate is stimulated by exogenous GDP or IDP but not by ADP. (6) Data support the conclusion that malate is being produced from oxalacetate generated by reversal of mitochondrial phosphoenolpyruvate carboxykinase. A possible role for this enzyme in hepatic lipogenesis is suggested.     

Purification and characterization of the reconstitutively active citrate carrier from maize mitochondria.

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, alpha-ketoglutarate, oxaloacetate, and alpha-ketoadipate and was inhibited by pyridoxal 5'-P, phenylisothiocyanate, mersalyl, and p-hydroxymercuribenzoate (but not N-ethylmaleimide), 1,2, 3-benzentricarboxylate, benzylmalonate, and butylmalonate. The activation energy of the citrate/citrate exchange was 66.5 kJ/mol between 10 degrees C and 35 degrees 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 micromol min-1 mg-1 protein at 25 degrees C.     

The transport of inorganic phosphate by the mitochondrial dicarboxylate carrier

1. N-Ethylmaleimide inhibited the influx and efflux of P(i) in rat liver mitochondria. 2. The efflux was stimulated by either succinate or malate in the presence of N-ethylmaleimide, and this stimulation was reversed by 2-n-butylmalonate. 2-Oxoglutarate and citrate, even in the presence of low concentrations of malate, were relatively ineffective in stimulating efflux of P(i) under these conditions, as was glutamate. 3. By using radioactively labelled P(i) and dicarboxylate ions an exchange was demonstrated, the stoicheiometry of which was 1.3+/-0.5 dicarboxylate ions:1 P(i) (n=10). 4. An exchange between unlabelled and labelled P(i) in the presence of N-ethylmaleimide was found which was sensitive to 2-n-butylmalonate. 5. It is concluded that the mitochondrial dicarboxylate carrier can transport phosphate by an exchange diffusion with certain penetrant dicarboxylic acids or with phosphate itself. The exchange mechanism is sensitive to 2-n-butylmalonate but is unaffected by N-ethylmaleimide; the action of mersalyl in this context is commented on.     

Anion transporters in plant mitochondria

The swelling of potato (Solanum tuberosum L.) mitochondria in isosmotic ammonium salts of phosphate, chloride, malate, succinate, and citrate was investigated by measuring light scattering. Potato mitochondria swell spontaneously in ammonium phosphate, and this swelling can be inhibited in N-ethylmaleimide. They swell in ammonium malate or succinate only after the addition of inorganic phosphate and in ammonium citrate only after the addition of both phosphate and a dicarboxylic acid. Pentylmalonate inhibits swelling in ammonium citrate solutions by competing for dicarboxylate entry. The results indicate that potato mitochondria possess a phosphate-hydroxyl carrier, a dicarboxylate carrier, and a tricarboxylate carrier.     

Isolation and reconstitution of the n-butylmalonate-sensitive dicarboxylate transporter from rat liver mitochondria

The mitochondrial dicarboxylate carrier has been substantially purified from rat liver mitoplasts by extraction with Triton X-114 in the presence of cardiolipin followed by chromatography on hydroxylapatite. Upon incorporation of the hydroxylapatite eluate into phospholipid vesicles, an n-butylmalonate-sensitive malonate/malate exchange has been demonstrated. This exchange activity is enhanced 226-fold relative to the starting material (i.e. detergent-extracted mitoplasts). Silver-stained sodium dodecyl sulfate-polyacrylamide gradient gels verify the high purity of this fraction relative to the starting material. Nonetheless, the banding pattern indicates that several protein species are still present. As isolated, the dicarboxylate transporter is rather unstable but can be stabilized either by the addition of 10% ethylene glycol and subsequent storage at -20 degrees C or by incorporation into phospholipid vesicles in the presence of malate followed by freezing in liquid nitrogen. Such proteoliposomes catalyze a [14C]malonate uptake which is characterized by a first order rate constant of 1.02 min-1 and a t 1/2 of 41 s. This uptake can be inhibited by dicarboxylates (e.g. succinate, malate, unlabeled malonate) but not by either alpha-ketoglutarate or by tricarboxylates (e.g. citrate, threo-Ds-isocitrate). Furthermore, the reconstituted malonate transport is dependent on internal malate and can be inhibited by n-butylmalonate, mersalyl, p-chloromercuribenzoate, and Pi, but not by N-ethylmaleimide. It is concluded that this highly purified fraction contains a reconstitutively active dicarboxylate transporter which, based on its substrate specificity and inhibitor sensitivity, appears to be identical to the native dicarboxylate transport system found in intact rat liver mitochondria.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

Phosphate-induced efflux of adenine nucleotides from rat-heart mitochondria: evaluation of the roles of the phosphate/hydroxyl exchanger and the dicarboxylate carrier

Upon the addition of inorganic phosphate, isolated rat-heart mitochondria released endogenous adenine nucleotides. To elucidate the mechanism of this phosphate-induced efflux, we evaluated the relative roles of three inner mitochondrial membrane carriers: the adenine nucleotide translocase, the phosphate/hydroxyl exchanger, and the dicarboxylate carrier. Atractyloside (a specific inhibitor of the adenine nucleotide translocase) prevented this efflux, but did not inhibit mitochondrial swelling. Inhibitors of the phosphate/hydroxyl exchanger (200 microM n-ethylmaleimide and 10 microM mersalyl) did not inhibit phosphate-induced efflux. 200 microM mersalyl (which inhibited both the phosphate/hydroxyl exchanger and the dicarboxylate carrier) inhibited the rate of efflux approx. 65% Phenylsuccinate and 2-n-butylmalonate (inhibitors of the dicarboxylate carrier) partially inhibited phosphate-induced efflux and adenine nucleotide translocase activity. Mersalyl (200 microM) had no effect on adenine nucleotide translocase activity. Partial inhibition of the adenine nucleotide translocase by phenylsuccinate and butylmalonate could not explain the extent of inhibition of phosphate-efflux by these agents. Moreover, the rates of adenine nucleotide efflux in the presence of phenylsuccinate, butylmalonate, or mersalyl correlated well with the ability of these agents to inhibit succinate-supported respiration. We conclude that phosphate-induced efflux of adenine nucleotides from rat heart mitochondria occurs over the adenine nucleotide translocase, and that the site of action of the phosphate is not the phosphate/hydroxyl exchanger, but is likely the dicarboxylate carrier.     

Enzymatic synthesis of the 2',5'-A4 tetramer analog, 2',5'-ppp3'dA(p3'dA)3, by rabbit reticulocyte lysates: binding and activation of the 2',5'-A eta dependent nuclease, hydrolysis of mRNA, and inhibition of protein synthesis

The exchange between external [¹⁴C] malonate and internal malate or phosphate was reconstituted in liposomes prepared by incorporation of a Triton-extract of mitochondrial rat-liver inner membranes. The conditions of transport were investigated and the kinetic parameters of malonate-malate and malonate-phosphate exchanges were determined. The exchange was sensitive to butylmalonate and to organomercurials. Sulfate and phosphate decreased the rate of malonatemalate exchange and phosphate inhibition was found to be competitive. This report demonstrates the possibility to reconstitute a functional dicarboxylate transport and to use the system for further purification and for studies at the molecular level.     

Effect of inhibitors on ammonia-, 2-oxoglutarate-, and oxaloacetate-dependent o(2) evolution in illuminated chloroplasts

The evolution of O₂ in spinach chloroplasts in the presence of oxaloacetate (OAA) was inhibited by a wide range of dicarboxylates. In contrast, (ammonia, 2-oxoglutarate)-dependent O₂ evolution was stimulated by malate, succinate, fumarate, glutarate, maleiate, and l-tartrate although OAA has little effect. This increase in O₂ evolution was accompanied by a similar increase in ¹⁴C incorporation from [5-¹⁴C]oxoglutarate into amino acids which was sensitive to azaserine inhibition. Glutamate and aspartate inhibited (ammonia, 2-oxoglutarate)-dependent O₂ evolution, but this inhibition was relieved by the addition of succinate, malate, or fumarate. OAA-dependent O₂ evolution also was inhibited by glutamate and aspartate, but succinate, malate, or fumarate had little effect on this inhibition. Phthalonate and n-butyl malonate inhibited (ammonia, 2-oxoglutarate)-dependent O₂ evolution competitively with respect to 2-oxoglutarate and uncompetitively with respect to malate. Both these inhibitors inhibited OAA-dependent O₂ evolution competitively. This evidence suggests that different mechanisms might be involved in the transport of OAA, 2-oxoglutarate, and malate into the chloroplasts.     

Reactivity of the -SH groups of the mitochondrial phosphate carrier under native, solubilized and reconstituted conditions

The isolated and liposome-reconstituted mitochondrial phosphate carrier exhibits a sigmoidal inhibition curve by mersalyl, similar to that found with intact mitochondria. In contrast a hyperbolic inhibition curve is found (a) by titration of the soluble carrier with mersalyl before reconstitution in liposomes and (b) by titration of the reconstituted carrier with mersalyl after successively pretreatment of the mitochondria with low, non-inhibitory concentrations of mersalyl, excess N-ethylmaleimide and dithiothreitol. The inhibition of the reconstituted, but not of the soluble, phosphate carrier by mersalyl can be reversed by dithiothreitol. Cupric di(1,10-phenanthroline) inhibits the soluble but not the reconstituted phosphate carrier. The inhibited phosphate carrier can be reactivated by dithiothreitol in the soluble state but not after reconstitution in liposomes. The data support the previously suggested model of the phosphate carrier, assuming a dimer of two identical subunits for the active unit.     

The inhibition of mitochondrial dicarboxylate transport by inorganic phosphate, some phosphate esters and some phosphonate compounds

1. P(i) competitively inhibited succinate oxidation by intact uncoupled mitochondria in the presence of sufficient N-ethylmaleimide to block the phosphate carrier, with a K(i) of 2.5mm. 2. Of a large number of phosphate esters and phosphonate compounds, phenyl phosphate and phenylphosphonate were found to inhibit competitively uncoupled succinate oxidation by intact but not broken mitochondria. By comparison, benzoate was a relatively weak competitive inhibitor of succinate oxidation by intact mitochondria but a relatively potent inhibitor of succinate dehydrogenase. 3. Phenyl phosphate and phenylphosphonate were non-penetrant, and inhibited P(i)-dependent swelling of mitochondria suspended in isosmolar ammonium malate in a manner non-competitive with P(i). The inhibitors did not affect mitochondrial swelling when tested with P(i) alone. 4. It is concluded that: (i) phenyl phosphate and phenylphosphonate behaved as non-penetrant analogues of P(i), since their inhibitory properties were in strict contrast with those of benzoate; (ii) phenyl phosphate and phenylphosphonate interacted with the dicarboxylate carrier but not with the phosphate carrier; (iii) P(i) was effective as a competitive inhibitor of succinate oxidation because of its being either an alternative substrate for the dicarboxylate carrier or competitive with succinate for the intramitochondrial cations as proposed by Harris & Manger (1968).     

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP⁺ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP⁺ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H₂O₂ exposure, implying that Yhm2p has an antioxidant function.     

The mechanism of phosphate permeation in purified bean mitochondria

The permeability properties and mechanism of Pi transport wereinvestigated in purified bean mitochondria.

1. Purified bean mitochondria are impermeable to small moleculesand ions. However, Pi, arsenate, acetate and formate can enterthe osmotically active space of bean mitochondria.
2. Nigericinor the association of valinomycin and FCCP cause mitochondrialswelling in isoosmotic potassium phosphate.
3. The SH-blockingreagents mersalyl, pHMB and NEM inhibit variousmitochondrialfunctions dependent on the translocation of Piand arsenateacross the membrane. These include the respirationstimulatedby ADP, Ca²⁺+Pi, and K⁺+valinomycin +Pi; the swellingin ammoniumphosphate medium and, in the presence of nigericin,in potassiumphosphate medium; the energy-linked yalinomycin-inducedswellingand the subsequent CICCP-induced shrinking. The uncoupler-stimulatedrespiration, as well as the other processes when acetate issubstituted for Pi, are not influenced by SH reagents.
4. Mersalyland pHMB cause complete inhibition at about 20 nmoles/mgprotein,whereas, NEM is effective at about 1 µmole/mgprotein.The inhibition by mersalyl and pHMB, but not that byNEM, issigmoidal and reversed by 2-mercaptoethanol. Non-inhibitoryamounts of mersalyl protect the Pi transport from irreversibleinhibition by NEM.
5. We concluded that a carrier-mediated transportsystem for Piis present in bean mitochondria, and that someof its propertiesare similar to the Pi carrier of animal mitochondria.

(Received June 5, 1975; )     

The Phosphate Transporter from Pea Mitochondria (Isolation and Characterization in Proteolipid Vesicles)

The phosphate transporter from mitochondria will exchange matrix phosphate for cytosolic phosphate and facilitate either phosphate/proton symport or phosphate/hydroxyl ion antiport. The phosphate transported into the matrix by this carrier is either used for ATP synthesis or exchanges back out to the cytosol on the dicarboxylate transporter, permitting entry of malate and succinate into the matrix. The phosphate transporter was solubilized from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes with Triton X-114, purified approximately 500-fold by hydroxylapatite chromatography, and reconstituted into azolectin vesicles that were preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as the exchange of preloaded phosphate for external [32P]phosphate. Phosphate/phosphate exchange occurred for over 40 min at room temperature with an apparent K0.5 of 1.6 mM and a maximum velocity of over 700 nmol (mg protein)-1 min-1. Diethyl pyrocarbonate was used as an inhibitor-stop reagent. Transport was inhibited by p-hydroxyphenylglyoxal, p-hydroxymercuribenzoate, pyridoxal 5-phosphate, and dansyl chloride but was insensitive to sulfate, nitrate, and N-ethylmaleimide, the standard inhibitor for the mammalian phosphate transporter. Phosphate/hydroxyl exchange was stimulated when the proton gradient was collapsed with carbonyl cyanide m-chlorophenylhydrazone, but phosphate/phosphate exchange was unaffected by the uncoupler.     

The reduction of diamide by rat liver mitochondria and the role of glutathione.

Diamide is reduced by mitochondria utilizing endogenous substrates with Vmax. 20nmol/min per mg of protein and Km 75micrometer. The reaction is inhibited by: (a) thiol-blocking reagents (N-ethylmaleimide, p-hydroxymercuribenzoate, mersalyl and 2,6-dichlorophenol-indophenol);(b) respiratory inhibitors (arsenicals, malonate and antimycin, but not cyanide or oligomycin; inhibition by antimycin is reversed by ATP); (c) uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol and valinomycin with K+; inhibition by the first of these uncouplers is not reversed by cyanide); (d) reagents affecting energy conservation (Ca2+, increasing pH, phosphate; phosphate inhibition is augmented by catalytic ADP or ATP and augmentation is abolished by respiratory inhibitors). Concentrations of mitochondrial glutathione are high when diamide reduction is uninhibited, but low after adding one of the above inhibitors such that the reduction rate is roughly proportional to the glutathione concentration. Endogenous ATP concentrations are lower in the presence of diamide than without, but the difference is abolished by respiratory inhibitors. With oligomycin added, however, ATP concentrations are higher in the presence of diamide and this positive increment is decreased by antimycin, N-ethylmaleimide and malonate. In the presence of diamide and an uncoupler, the mitochondrial glutathione content does not fall if various reducible substrates are present, although the inhibition of diamide reduction is not relieved. Some of these substrates prevent the fall in reduced glutathione concentration found with diamide and phosphate. They also relieve the inhibition of diamide reduction and the relief is sensitive to butylmalonate. The inhibition of diamide reduction by N-ethylmaleimide, mersalyl or p-hydroxymercuribenzoate is not relieved by reducible substrates, but the latter mitigate the fall in the concentration of glutathione. Inhibitors of carriers of tricarboxylic acid-cycle intermediates also inhibit reduction of diamide. The reduced glutathione concentration remains high when they are added singly, but falls when two of them are combined. It is proposed that diamide may enter the matrix as a protonated adduct formed with the thiol groups of mitochondrial carriers and then be reduced in the matrix by glutathione, which is regenerated via NADH, energy-dependent transhydrogenase and NADP+-specific glutathione reductase. Some of the high-energy equivalents required for the transhydrogeneration may be generated by the substrate phosphorylation step of the tricarboxylic acid cycle.