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).     

Sulphate transport by H+ symport and by the dicarboxylate carrier in mitochondria.

1. Swelling of mitochondria was induced in non-respiring mitochondria by 30 mM or more Na2SO4 or by respiration in the presence of K2SO4. Respiration-drive swelling resulted in loss of respiratory control. Sulphate, when present at 10 mM concentration, promoted the release of accumulated Ca2+. 2. Swelling was prevented by N-ethylmaleimide and formaldehyde, known inhibitors of the phosphate carrier. Sulphate-induced swelling was more sensitive to the inhibitors than was phosphate-induced swelling. At lower concentration of sulphate, 5 mM, an alkalinisation of the medium was observed in addition of sulphate, indicating H+-sulphate symport. There was competition between sulphate and phosphate for transport by this mechanism. It is suggested that sulphate may be transported, though at a comparatively slow rate, by the phosphate carrier. 3. Uptake of sulphate was stimulated when preceded by energy-dependent accumulation of Ba2+, especially when acetate was also present, indicating precipitation of BaSO4 in the matrix. Using this system the influx of sulphate was studied at lower concentrations, 10 mM or less. the contributions of the H+ symporter (sensitive to N-ethylmaleimide) and the dicarboxylate carrier (sensitive to butylmalonate) could then be studied. The dicarboxylate carrier had a lower Km and was mainly responsible for sulphate transport at lower concentration range. At 10 mM-sulphate the transport rates by the two systems appeared to be similar; at still higher concentrations the H+ symporter may become more important.     

Characterisation of phosphate binding to mitochondrial and bacterial membrane-bound ATP synthase by studies of inhibition with 4-chloro-7-nitrobenzofurazan

The effect of phosphate on the inhibition by 4-chloro-7-nitrobenzofurazan of the ATPase activity of the proton-translocating ATP synthase in heart submitochondrial particles was investigated. Binding of phosphate protected strongly against the inhibition. A dissociation constant of 0.2 mM was determined for the enzyme X Pi complex and shown to be independent of pH in the range 7.0-8.0. The protective effect of phosphate was mimicked by arsenate but not by sulphate or malonate. Similar results were obtained for the enzyme from Paracoccus denitrificans. 2,4-Dinitrophenol enhanced phosphate binding to the mitochondrial enzyme since the protective effect of phosphate was increased. The data are compatible with protection arising from binding of phosphate to a catalytic site.     

The sodium gradient induces conformational changes in the renal phosphate carrier

Phosphate transport across brush border membranes from kidney cortex is very sensitive to inhibition by phenylglyoxal, an arginine modifier. Sodium-dependent phosphate influx into brush border membrane vesicles was inhibited by 60%. In contrast, phenylglyoxal had no effect on passive influx or on sodium-dependent efflux of phosphate. Preincubation of the vesicles with sodium prior to the addition of phenylglyoxal demonstrated a strong protective effect of intravesicular sodium (73% protection). Phosphate also protected the transporter from inhibition, but from the extravesicular side only (63%). Substitution of phosphate by sulfate offered no protection at all, indicating the specificity of protection. Addition of both substrates (sodium and phosphate) offered an additional protection from the extravesicular side compared to that offered by phosphate alone (92 versus 55%). There was no additional protection when both substrates were added to the intravesicular side. Phosphate influx measured in the presence of sodium but in the absence of a sodium gradient was totally unaffected by phenylglyoxal modification. There was no inhibition on phosphate influx measured in equilibrium exchange conditions. We propose a model for the phosphate carrier in which the sodium gradient induces a conformational change and an arginine residue is essential for the coupled flux of sodium and phosphate.     

Phosphate transport by capillaries of the blood-brain barrier.

Capillaries were isolated from bovine brain cortex and used for phosphate transport studies. The influx of phosphate through capillary membranes was studied by incubation with [32Pi]phosphate followed by a rapid filtration technique. Phosphate uptake by brain capillaries was mediated by a saturable high-affinity system which is independent of the sodium concentration in the incubation medium. The apparent half-saturation constant (Km) and maximal influx (Vmax) were estimated to 160 microM and 0.37 nmol/mg protein/30 s. Transport was inhibited by the phosphate analogues arsenate and phosphonoformic acid with apparent inhibition constants of 5 and 11 mM, respectively. The metabolic inhibitors cyanide and ouabain had no effect on the transport activity. Competition experiments showed that phosphate uptake was inhibited up to 41% by various anions (pyruvate, acetate, citrate, glutamate, and sulfate). In addition, phosphate uptake was significantly decreased by two selective inhibitors of anionic exchangers, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Chloride was not a substrate of the phosphate carrier as the replacement of external chloride, by nitrate, thiocyanate, or gluconate, did not increase phosphate transport. Aminohippuric acid and N'-methylnicotinamide, two specific substrates of anionic and cationic drug exchangers, did not compete with the phosphate carrier of cerebral capillaries. However, trans-stimulation with bicarbonate increased phosphate transport by 28%, and this stimulation was inhibited by 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that the carrier of the cerebral capillaries could exchange phosphate with bicarbonate.     

Carrier-mediated transport of metabolites in purified bean mitochondria

1. The mechanism of transport of Krebs cycle intermediates, phosphateand sulfurcontaining compounds across the membrane of purifiedbean mitochondria was investigated by directly measuring dieexchange between intramitochondrial labelled substrates andexternal anions and by testing die inhibitor sensitivity ofdiese transport processes.
2. The exchange between intramitochondrialphosphate and externalphosphate or sulfite is insensitive toN-ediylmaleimide or butylmalonatewhen either is added alone,but is completely inhibited by N-ethylmaleimideplus butylmalonateor by mersalyl. Internal phosphate is exchangedwith malate,succinate, oxaloacetate, sulfate and thiosulfate;these reactionsare inhibited by butylmalonate but not affectedby N-ethylmaleimide.
3. Internal sulfate is exchanged with malate, malonate, succinate,phosphate and sulfite in a butylmalonate- and mersalyl-sensitivereaction. Also the exchanges of malonate with phosphate, sulfateand sulfite are inhibited by butylmalonate and mersalyl. Onthe other hand, the exchange between intra- and extramitochondrialmalonate is completely inhibited only by the combination ofbutylmalonate and 1,2,3-benzenetricarboxylate.
4. Citrate isexchanged with some di- and tricarboxylates and phosphoenolpyruvate(but not with phosphate, sulfate, oxoglutarate, trans-aconitateand benzenetricarboxylates). These exchanges are inhibited by1,2,3-benzenetricarboxylate, but not by 1,2,4-benzenetricarboxylateor 1,3,5-pentanetricarboxylate.
5. Oxoglutarate is exchangedwith succinate, malate, malonate andoxaloacetate (but not withphosphate, citrate or phosphoenolpyruvate)in a mersalyl-insensitive,butylmalonate- and phenylsuccinate-sensitivereaction.
6. Weconcluded that bean mitochondria contain the following transportsystems: a phosphate carrier inhibited by N-ethylmaleimide ormersalyl, a dicarboxylate carrier inhibited by butylmalonateor mersalyl, a citrate carrier inhibited by 1,2,3-benzenetricarboxylateand an oxoglutarate carrier inhibited by phenylsuccinate orbutylmalonate but insensitive to mersalyl.

(Received June 23, 1976; )     

Kinetic discrimination of two substrate binding sites of the reconstituted dicarboxylate carrier from rat liver mitochondria

The kinetic interaction of various substrates and inhibitors with the dicarboxylate carrier from rat liver mitochondria was investigated using the isolated and reconstituted carrier protein. Due to their inhibitory interrelation the ligands could be divided into two classes: dicarboxylates, sulphate, sulphite and butylmalonate on the one hand and phosphate, thiosulphate and arsenate on the other. The mutual inhibition of substrates or inhibitors taken from one single class was found to be competitive, whereas the kinetic interaction of ligands when taken from the two different classes could be described as purely non-competitive. The half-saturation transport constants Km and the corresponding inhibition constants Ki of one single ligand, either used as substrate or as inhibitor, respectively, were found to be very similar. These kinetic data strongly support the presence of two different binding sites at the dicarboxylate carrier for the two different classes of substrates considering the external side of the reconstituted protein. When these two sites were saturated simultaneously with malate and phosphate, the turnover of the carrier was considerably reduced, hence indicating that a non-catalytic ternary complex is formed by the two substrates and the carrier molecule.     

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.     

Kinetics of the reconstituted dicarboxylate carrier from rat liver mitochondria

The dicarboxylate carrier from rat liver mitochondria was purified by the Amberlite/hydroxyapatite procedure and reconstituted in egg yolk phospholipid vesicles by removing the detergent with Amberlite. The efficiency of reconstitution was optimized with respect to the ratio of detergent/phospholipid, the concentration of phospholipid and the number of Amberlite column passages. In the reconstituted system the incorporated dicarboxylate carrier catalyzed a first-order reaction of malate/phosphate exchange. V of the reconstituted malate/phosphate exchange was determined to be 6000 mumol/min per g protein at 25 degrees C. This value was independent of the type of substrate present at the external or internal space of the liposomes (malate, phosphate or malonate). The half-saturation constant was 0.49 mM for malate, 0.54 mM for malonate and 1.41 mM for phosphate. The activation energy of the exchange reaction was determined to be 95.8 kJ/mol. The transport was independent of the external pH in the range between pH 6 and 8.     

Depression of Nitrate and Ammonium Transport in Barley Plants with Diminished Sulphate Status. Evidence of Co-regulation of Nitrogen and Sulphate Intake

When barley plants were grown in a solution with nitrate asthe sole N-source but deprived of sulphate (–Splants)for 1 to 5 d, the capacity for sulphate transport by the rootsincreased very markedly; subsequent measurement of influx using³⁵S-labelled showed increases of > 10-fold compared to plants continuously supplied with sulphate (+S plants).There were only small effects on plant growth over a 5 d periodand yet the influx of , labelled with the short-lived tracer ¹³N, was diminished by approximately 30%.By contrast, the influx of phosphate was little affected bysulphate-deprivation. When a sulphate supply was restored to– S plants, the sulphate influx was quickly repressedover the subsequent 24 h and the nitrate influx was restoredto >90% of the value in +S plants. When plants were grown in a solution with a mixed nitrate andammonium supply and deprived of sulphate for 1 d or 5 d thedepression of nitrate influx was more strongly marked (up to55% depression). The influx of ammonium was also depressed after5 d of sulphate-deprivation, but not at 1 d, nor where the concentrationof ammonium in the uptake solution was lowered to 20 mmol m^–3or less. Additional measurements with ¹⁵N-labelled nitrate and ammoniumover longer periods were used to determine net uptake. Net uptakeof nitrate was depressed to a similar extent to efflux, butnet ammonium uptake was depressed only in unbuffered uptakesolution where the pH decreased to pH 4.9 during the uptakeperiod. The ¹⁵N-tracer experiments showed that the translocationof label to the shoot, from both nitrate and ammonium, was depressedto a greater extent than net uptake in –S plants. Thedepression of nitrate influx, caused by 5 d of sulphate deprivation,could be relieved almost completely by providing plants with1.0 mol m^–3 L-methionine during the day prior to influxmeasurement. This treatment substantially decreased sulphateand potassium (⁸⁶Rb-labelled) influx in both +S and –Splants, but greatly increased total S-status of the plants.This methionine treatment had no effect on ammonium influx ornet uptake in – S plants but increased influx significantlyin +S ones. When plants were grown with sulphate but deprived of nitratefor 4 d there was a marked depression of the sulphate influx(by 48–65%) but a smaller effect on phosphate influx (21–37%of +N). The results are discussed in relation to the effects of sulphate-deprivationon growth rate and the root: shoot weight ratio. It is concludedthat the effects on influx and net uptake of nitrogen are moresevere than could be accounted for by these factors. The decreasedtranslocation of either nitrate, or the products of nitrateand ammonium assimilation from the roots, is suggested as areason for the depression of influx. The restoration of nitrateinflux and net uptake by methionine suggests that, for thision at least, a shortage of S-amino acids within the plant maylead to the accumulation of inhibitory concentrations of non-Samino acids in the transport pool. Key words: ¹³N, sulphate, nitrate, ammonium, ion-uptake, barley     

Mechanism of active shrinkage in mitochondria. I. Coupling between weak electrolyte fluxes.

1. A passive penetration of (NH4)2 HPO4 or of K2HPO4+nigericin occurs in respiratory-inhibited liver mitochondria. Addition of succinate at the end of the passive swelling initiates a shrinkage phase which leads to restoration of the initial mitochondrial volume. The rate and time of onset of the active shrinkage depend on the degree of stretching of the mitochondrial membrane. The rate of active shrinkage increases proportionally to the concentration of nigericin while it is strongly inhibited by valinomycin. 2. A number of SH inhibitors such as N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuriphenylsulphonate, dithiobisnitrobenzoate, exert a marked enhancing effect on the rate of shrinkage. The enhancing effect parallels titration of the phosphate carrier and inhibition of the passive phosphate efflux. In contrast, mersalyl is a powerful inhibitor of the rate of active shrinkage. The inhibition parallels that on phosphate passive efflux and requires higher mersalyl concentrations in respect to inhibition of phosphate influx. 3. The active shrinkage is discussed in terms of (a) a mechanoenzyme, (b) an electrogenic proton pump and (c) a proton-driven Pi pump.     

Control of Net Uptake of Nutrients by Regulation of Influx in Barley Plants Recovering from Nutrient Deficiency

Rates of influx and net uptake of nitrate, phosphate and sulphatewere measured in intact barley plants, and concurrent effluxwas obtained by difference. Net uptake of these anions variedwidely depending on the nutrient status of the plants, and thedifferences in net uptake could be accounted for almost entirelyby changes in influx. Efflux played only a minor role in regulatingnet uptake of nitrate, phosphate or sulphate during recoveryfrom N-, P-, or S-deficiency. Nitrate influx and short-term ammonium absorption by N-deficientbarley plants were closely correlated, and varied in parallelwith rates of net uptake of nitrate or ammonium by similar plants.Again, it would seem that net uptake of ammonium is controlledpredominantly by changes in the rate of influx.Copyright 1993,1999 Academic Press Hordeum vulgare, barley, nutrient absorption, influx, nitrate, phosphate, sulphate, ammonium     

Sulphate influx in wheat and barley roots becomes more sensitive to specific protein-binding reagents when plants are sulphate-deficient

When young wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) plants were deprived of an external sulphate supply (-S plants), the capacity of their roots to absorb sulphate, but not phosphate or potassium, increased rapidly (derepression) so that after 3–5 d it was more than tenfold that of sulphate-sufficient plants (+S plants). This increased capacity was lost rapidly (repression) over a 24-h period when the sulphate supply was restored. There was little effect on the uptake of L-methionine during de-repression of the sulphate-transport system, but S input from methionine during a 24-h pretreatment repressed sulphate influx in both+S and-S plants.Sulphate influx of both+S and-S plants was inhibited by pretreating roots for 1 h with 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) at concentrations > 0.1 mol · m^-3. This inhibition was substantially reversed by washing for 1 h in DIDS-free medium before measuring influx. Longer-term pretreatment of roots with 0.1 mol·m^-3 DIDS delayed de-repression of the sulphatetransport system in-S plants but had no influence on+S plants in 3 d.The sulphydryl-binding reagent, n-ethylmaleimide, was a very potent inhibitor of sulphate influx in-S roots, but was much less inhibitory in +S roots. Its effects were essentially irreversible and were proportionately the same at all sulphate concentrations within the range of operation of the high-affinity sulphate-transport system. Inhibition of influx was 85–96% by 300 s pretreatment by 0.3 mol·m^-3 n-ethylmaleimide. No protection of the transport system could be observed by including up to 50 mol·m^-3 sulphate in the n-ethylmaleimide pre-treatment solution. A similar differential sensitivity of-S and+S plants was seen with p-chloromercuriphenyl sulphonic acid.The arginyl-binding reagent, phenylglyoxal, supplied to roots at 0.25 or 1 mol·m^-3 strongly inhibited influx in-S wheat plants (by up to 95%) but reduced influx by only one-half in+S plants. The inhibition of sulphate influx in-S plants was much greater than that of phosphate influx and could not be prevented by relatively high (100 mol·m^-3 sulphate concentrations accompanying phenylglyoxal treatment. Effects of phenylglyoxal pretreatment were unchanged for at least 30 min after its removal from the solution but thereafter the capacity for sulphate influx was restored. The amount of new carrier appearing in-S roots was far greater than in+S roots over a 24-h period.The results indicate that, in the de-repressed state, the sulphate transporter is more sensitive to reagents binding sulphydryl and arginyl residues. This suggests a number of strategies for identifying the proteins involved in sulphate transport.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenyl sulphonic acid     

Mechanism of active shrinkage in mitochondria I. Coupling between weak electrolyte fluxes

1. A passive penetration of (NH₄)₂ HPO₄ or of K₂HPO₄+nigericin occurs in respiratory-inhibited liver mitochondria. Addition of succinate at the end of the passive swelling initiates a shrinkage phase which leads to restoration of the initial mitochondrial volume. The rate and time of onset of the active shrinkage depend on the degree of stretching of the mitochondrial membrane. The rate of active shrinkage increases proportionally to the concentration of nigericin while it is strongly inhibited by valinomycin.2. A number of SH inhibitors such as N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuriphenylsulphonate, dithiobisnitrobenzoate, exert a marked enhancing effect on the rate of shrinkage. The enhancing effect parallels titration of the phosphate carrier and inhibition of the passive phosphate influx. The above SH inhibitors do not inhibit passive phosphate efflux. In contrast, mersalyl is a powerful inhibitor of the rate of active shrinkage. The inhibition parallels that on phosphate passive efflux and requires higher mersalyl concentrations in respect to inhibition of phosphate influx.3. The active shrinkage is discussed in terms of (a) a mechanoenzyme, (b) an electrogenic proton pump and (c) a proton-driven P_i pump.     

The mitochondrial phosphate carrier reconstituted in liposomes is inhibited by doxorubicin

The phosphate carrier has been isolated from beef heart mitochondria in the presence of cardiolipin and reconstituted in asolectin vesicles. It has been found that 100 microM doxorubicin and 100 microM Br-daunomycin inhibit the unidirectional phosphate uptake in the reconstituted liposomes to the same extent as N-ethylmaleimide. The inhibition by Br-daunomycin is not due to covalent interaction with the carrier. The specific interaction between doxorubicin and cardiolipin is responsible for the inhibition of the phosphate carrier. Br-daunomycin interacts with 3 mitochondrial proteins of apparent Mr approximately 45 000, approximately 35 000 and approximately 30 000.     

pH gradient as an additional driving force in the renal re-absorption of phosphate.

The effects of the Na+ gradient and pH on phosphate uptake were studied in brush-border membrane vesicles isolated from rat kidney cortex. The initial rates of Na(+)-dependent phosphate uptake were measured at pH 6.5, 7.5 and 8.5 in the presence of sodium gluconate. At a constant total phosphate concentration, the transport values at pH 7.5 and 8.5 were similar, but at pH 6.5 the influx was 31% of that at pH 7.5. However, when the concentration of bivalent phosphate was kept constant at all three pH values, the effect of pH was less pronounced; at pH 6.5, phosphate influx was 73% of that measured at pH 7.5. The Na(+)-dependent phosphate uptake was also influenced by a transmembrane pH difference; an outwardly directed H+ gradient stimulated the uptake by 48%, whereas an inwardly directed H+ gradient inhibited the uptake by 15%. Phosphate on the trans (intravesicular) side stimulated the Na(+)-gradient-dependent phosphate transport by 59%, 93% and 49%, and the Na(+)-gradient-independent phosphate transport by 240%, 280% and 244%, at pH 6.5, 7.5 and 8.5 respectively. However, in both cases, at pH 6.5 the maximal stimulation was seen only when the concentration of bivalent trans phosphate was the same as at pH 7.5. In the absence of a Na+ gradient, but in the presence of Na+, an outwardly directed H+ gradient provided the driving force for the transient hyperaccumulation of phosphate. The rate of uptake was dependent on the magnitude of the H+ gradient. These results indicate that: (1) the bivalent form of phosphate is the form of phosphate recognized by the carrier on both sides of the membrane; (2) protons are both activators and allosteric modulators of the phosphate carrier; (3) the combined action of both the Na+ (out/in) and H+ (in/out) gradients on the phosphate carrier contribute to regulate efficiently the re-absorption of phosphate.     

Contrasting responses of sulphate and phosphate transport in barley (Hordeum vulgare L.) roots to protein-modifying reagents and inhibition of protein synthesis

The uptake of sulphate into roots of barley seedlings is highly sensitive to phenylglyoxal (PhG), an arginine-binding reagent. Uptake was inhibited by >80% by a 1-h pre-treatment of roots with 0.45 mol · m^–3 PhG. Inhibition was maximal in pre-treatment solutions buffered between pH 4.5 and 6.5. Phosphate uptake, measured simultaneously by double-labelling uptake solutions with ³²P and ³⁵S, was less susceptible to inhibition by PhG, particularly at pH <6.5, and was completely insensitive to the less permeant reagent p-hydroxyphenylglyoxal (OH-PhG) administered at 1 mol · m^–3 at pH at 5.0 or 8.2; sulphate uptake was inhibited in -S plants by 90% by OH-PhG-treatment. Root respiration in young root segments was unaffected by OH-PhG pre-treatment for 1 h and inhibited by only 17% after 90 min pre-treatment. The uptake of both ions was inhibited by the dithiol-specific reagent, phenylarsine oxide even after short exposures (0.5–5.0 min). Sulphate uptake was more severely inhibited than that of phosphate, but in both cases inhibition could be substantially reversed by 5 min washing of treated roots by 5 mol · m^–3 dithioerythritol. After longer pre-treatment (50 min) with phenylarsine oxide, inhibition of the ion fluxes was not relieved by washing with dithioerythritol. Inhibition of sulphate influx by PhG was completely reversed by washing the roots for 24 h with culture solution lacking the inhibitor. The reversal was dependent on protein synthesis; less than 20% recovery was seen in the presence of 50 mmol · m^–3 cycloheximide. Sulphate uptake declined rapidly when -S roots were treated with cycloheximide. In the same roots the phosphate influx was little affected, small significant inhibitions being seen only after 4 h of treatment. Respiration was depressed by only 20% in apical and by 31% in basal root segments by cycloheximide pre-treatment for 2 h. Similar rates of collapse of the sulphate uptake and insensitivity of phosphate uptake were seen when protein synthesis was inhibited by azetidine carboxylic acid, p-fluorophenylalanine and puromycin. Considering the effects of all of the protein-synthesis inhibitors together leads to the conclusion that the sulphate transporter itself, or some essential sub-component of the uptake system, turns over rapidly with a half-time of about 2.5 h. The turnover of the phosphate transporter is evidently much slower. The results are discussed in relation to strategies for identifying the transport proteins and to the regulation of transporter activity during nutrient stress.Abbreviations CAP chloramphenicol - CHM cycloheximide - DTE dithioerythritol - OH-PhG p-hydroxyphenylglyoxal - PhAsO phenylarsine - PhG phenylglyoxal Paper dedicated to the memory of the late Ken Treharne who did much to encourage this collaboration.D.T.C. gratefully acknowledges a fellowship provided by Le Ministére des Etrangers during his stay in Montpellier.     

Interaction of phenylisothiocyanates with the mitochondrial phosphate carrier. I. Covalent modification and inhibition of phosphate transport

The effects of phenylisothiocyanate (PITC) and of the polar analogue p-sulfophenylisothiocyanate (p-sulfoPITC) on the phosphate carrier of bovine heart mitochondria have been investigated. Incubation of mitochondria with the two phenylisothiocyanates leads to inhibition of the phosphate carrier protein. The inhibition of phosphate transport by PITC is unaffected by the addition of dithioerythritol (DTE) or by variation of the pH. The inhibition by p-sulfoPITC is in part removed by DTE; the remaining inactivation of the phosphate carrier, which can be attributed to the reaction with NH2 groups, is temperature and pH-dependent. Inhibition of phosphate transport by both p-sulfoPITC and PITC depends on the time of incubation and the concentration of the inhibitor. Preincubation with mersalyl protects the carrier protein against the inactivation by p-sulfoPITC but not against PITC. Other SH reagents tested do not show any protective effect. It can thus be concluded that two types of lysine residues are essential for the activity of the phosphate carrier. Lysine(s) of the former type are located at the surface of the membrane and are topologically related to the functional SH groups of the protein. Lysine residue(s) of the latter type are buried in the hydrophobic phase of the membrane.     

Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter

Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).