Net efflux of citrate in Penicillium simplicissimum is mediated by a transport protein

Penicillium simplicissimum excreted citrate, isocitrate, and succinate when grown in a strongly buffered medium [1 M Mes (pH 6) or 1 M Hepes (pH 7.3)]. Growth in a weakly buffered medium did not lead to citrate excretion despite a similar intracellular citrate concentration. When nongrowing, citrate-excreting hyphae were aerated in a glucose solution, the following steady-state intracellular concentrations of organic acids were measured: succinate (25 mM); citrate, isocitrate, malate, and fumarate (all less than 5 mM). After 2 h of incubation, the extracellular concentrations of these acids were [μmol (g dry wt.)^–1]: isocitrate [100], citrate [60], succinate [30], and malate, fumarate, and α-ketoglutarate [<5]. The excretion of citrate was due neither to an unspecific change in the permeability of the plasma membrane nor to simple diffusion of undissociated citric acid. The involvement of a transport protein in citrate excretion was indicated because N-ethylmaleimide and sodium azide inhibited citrate excretion strongly despite an unchanged outward-directed citrate gradient. Arguments are given why efflux via a citrate uptake carrier is not considered probable. These results indicate that citrate is excreted by P. simplicissimum via a transport protein that probably specifically mediates the efflux of citrate. Received: 28 July 1997 / Accepted: 19 November 1997     

Active enzyme sedimentation of pig heart fumarase

Active band sedimentation studies of pig heart fumarase indicate that the enzyme is predominantly tetrameric at enzyme concentrations between 0.0125 and 0.25 mg/ml and at a fumarate concentration of 2.5 mM. At enzyme concentrations of 0.25--1.0 mg/ml and fumarate concentrations known to activate and inhibit the enzyme, the sedimentation band of fumarase becomes disperse and indicates the presence of polymers greater than tetramers.     

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape.

Na+ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na+ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the 31P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg2+/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.     

Cyclic 2,3-diphosphoglycerate metabolism in Methanobacterium thermoautotrophicum (strain ΔH): characterization of the synthetase reaction

The levels of cyclic 2,3-diphosphoglycerate (cDPG) in methanogenic bacteria are governed by the antagonistic activities of cDPG synthetase and cDPG hydrolase. In this paper we focus on the synthetase from Methanobacterium thermoautotrophicum. The cytoplasmic 150 kDa enzyme catalyzed cDPG synthesis from 2,3-diphosphoglycerate (apparent K_m=21 mM), Mg²⁺ (K_m=3.1 mM) and ATP (K_m=1–2 mM). In batch-fed cultures, the enzyme was constitutively present (6–6.5 nmol per min per mg protein) during the different growth phases. In continuous cultures, activity decreased in response to phosphate limitation. The synthetase reaction proceeded with maximal rate at pH 6 and at 65° C and was specifically dependent on high (>0.3M) K⁺ concentrations. The reaction conditions remarkably contrasted to those of cDPG degradation catalyzed by the previously described membrane-bound cDPG hydrolase.Abbreviations cDPG Cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-Diphosphoglycerate - 2-PG 2-Phosphoglycerate - 3-PG 3-Phosphoglycerate     

Kinetic regulation of yeast NAD-specific isocitrate dehydrogenase by citrate

The present results suggest that the enzyme modifier citrate and the substrate isocitrate are bound at different sites on yeast NAD-specific isocitrate dehydrogenase and that citrate diminishes the binding of the positive effector 5'-AMP, thereby causing a decreased rate of enzyme catalysis. This interpretation differs from the earlier proposal that citrate can replace isocitrate at an activator site on the enzyme and can cause inhibition by binding at its catalytic site [Atkinson et al. (1965) J. Biol. Chem. 240, 2682]. The present proposal is supported by the following observations: At constant subsaturating levels of isocitrate, NAD+, and Mg2+ without AMP, up to 10 mM citrate was an activator and not an inhibitor. Citrate decreased velocity for AMP-activated enzyme; however, with increasing citrate the specific activity with AMP asymptotically approached but did not decrease below the level of the enzyme maximally activated by citrate in the absence of AMP. When added singly, AMP decreased S0.5 for isocitrate without changing the Hill number (n), whereas citrate lowered n without changing S0.5 for isocitrate. The difference in action of these modifiers indicated that they were bound at separate sites on the enzyme. The binding of citrate appeared to cause a conformational change in the protein that lowered the enzyme's affinity for AMP. This was consistent with the findings that citrate (or the citrate agonist fluorocitrate) (i) resulted in an increase in S0.5 for isocitrate with the AMP-activated enzyme and (ii) decreased binding of the positive effector analogue TNP-AMP as measured by fluorescence change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in allosteric properties of phosphofructokinase bound to erythrocyte membranes.

Human and rabbit erythrocyte membranes prepared by hypotonic hemolysis contained 5 to 15% of the phosphofructokinase in the erythrocytes. The membrane-bound phosphofructokinase can be eluted by a saline wash. Human erythrocyte and rabbit muscle phosphofructokinase bind to the saline-washed membranes. This binding is specific for the inner surface of the membrane. The amount of phosphofructokinase bound is dependent on pH; at pH 7, 6 times more enzyme is bound than at pH 7.5. Unlike free phosphofructokinase, the membrane-bound phosphofructokinase is not inhibited by ATP or 2,3-diphosphoglycerate, and its fructose-6-P saturation curve is nonsigmoidal.     

Affinity labeling of the allosteric ADP activation site of NAD-dependent isocitrate dehydrogenase by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate

Pig heart NAD-dependent isocitrate dehydrogenase is allosterically activated by ADP which reduces the Km of isocitrate. The new ADP analogue 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate (BDB-TADP) reacts irreversibly with the enzyme at pH 6.1 and 25 degrees C, causing a rapid loss of the ability of ADP to increase the initial velocity of assays conducted at low isocitrate concentrations and a slower inactivation measured using saturating isocitrate concentrations. The rate constant for loss of ADP activation exhibits a nonlinear dependence on BDB-TADP concentration; in the presence of 0.2 mM MnSO4, KI for the reversible enzyme-reagent complex is 0.069 mM with kmax at saturating reagent concentrations equal to 0.031 min-1. For reaction at the site causing overall inactivation, KI for the initial reversible enzyme-reagent complex is estimated to be 0.018 mM with kmax = 0.0083 min-1 in the presence of 0.2 mM MnSO4. Total protection against both reactions is provided by 1 mM ADP plus 0.2 mM MnSO4 or by 0.1 mM ADP plus 0.2 mM MnSO4 plus 0.2 mM isocitrate, but not by NAD, ATP, or ADP plus EDTA. The BDB-TADP thus appears to modify two distinct metal-dependent ADP-binding sites. Incubation of isocitrate dehydrogenase with 0.14 mM BDB-[beta-32P]TADP at pH 6.1 in the presence of 0.2 mM MnSO4 results in incorporation of 0.81 mol of reagent/mol of average subunit when the ADP activation is completely lost and the enzyme is 68% inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 0.5 mol of BDB-TADP/mol of average enzyme subunit causes complete loss of ADP activation, while reaction with another 0.5 mol of BDB-TADP would lead to total inactivation. The enzyme is composed of three distinct subunits in the approximate ratio 2 alpha:1 beta:1 gamma. The distribution of BDB-[beta-32P]TADP incorporated into modified enzyme is 63:30:7% for alpha:beta:gamma throughout the course of the reaction. These results indicate the 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate functions as an affinity label of two types of potential metal-dependent ADP sites of NAD-dependent isocitrate dehydrogenase and that these allosteric sites are present on two (alpha and beta) of the enzyme's three types of subunits.     

Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB

Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and approximately 54° C. The K _m values for fumarate and l-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe²⁺ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe-4S] cluster. Under reducing conditions, only a trace amount of a [4Fe-4S] cluster was detected. Addition of fumarate resulted in a significant increase of this [4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%). Received: 15 September 1995 / Accepted: 13 November 1995     

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape

Na⁺ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na⁺ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the ³¹P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg²⁺/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.     

Purification and properties of the NAD(P)-dependent malic enzyme from human placental mitochondria

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 mumol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn2+ and Co2+ were by far more effective activators than Mg2+ and Ni2+, whereas the reaction did not proceed in the presence of Ca2+. The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for L-malate and NAD were 2.82 and 0.33 mM; and in the presence of fumarate 1.18 and 0.22 mM, respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.     

Regulation of aconitate hydratase activity from rat kidney cortex by bicarbonate.

1. The increase in pH value and bicarbonate concentration stimulated citrate synthesis from pyruvate and malate, inhibiting simultaneously conversion of isocitrate to citrate. 2. Bicarbonate inhibited competitively the activity of aconitate hydratase, probably binding with the two active sites of the enzyme. The Ki values for the cytoplasmic and mitochondrial enzyme were, respectively, 27 and 38 mM. The pH optimum for both forms of the enzyme in Tris-HCl buffer was in the range 7.8-8.6, and in bicarbonate buffer varied from 7.2 to 8.0, depending on the form of the enzyme and the substrate used. 3. Only free, completely dissociated citrate anion acts as a substrate for aconitate hydratase. 4. The role of aconitate hydratase as a factor controlling the rate of citrate metabolism in kidney in metabolic alkalosis is discussed.     

Expression and characterization of fumarase (FUMR) from Rhizopus oryzae

Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30°C and pH 7.2. The enzyme was stable below 45°C and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.     

Organic phosphate binding to hemoglobin in intact human erythrocytes determined by 31P nuclear magnetic resonance spectroscopy.

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to estimate the percent of 2,3-diphosphoglycerate and ATP bound to hemoglobin in intact human erythrocytes at 37 degrees C. Binding was assessed by comparing the chemical shifts (delta) of 2,3-diphosphoglycerate and of ATP observed in intact cells with the delta values of these organic phosphates determined in model solutions closely simulating intracellular conditions, in which percent binding was directly evaluated by membrane ultrafiltration. The results showed that the percent of bound 2,3-diphosphoglycerate in intact cells varied with pH, the state of oxygenation, and 2,3-diphosphoglycerate concentration. The values ranged from 33% in cells incubated with glucose in air at an intracellular pH of 7.2 to 100% in cells incubated with inosine in N2 at a pH of 6.75. At the same 2,3-diphosphoglycerate concentration, a greater percentage of the compound appeared to be bound in erythrocytes than in the closely simulated model system. ATP was not significantly bound to hemoglobin under any condition examined, but appeared to be strongly complexed to Mg2+ inside the erythrocyte. The binding percentages for both 2,3-diphosphoglycerate and ATP in intact cells estimated by 31P NMR spectroscopy were lower than those calculated by others from individual association constants determined for the binding of different ligands to hemoglobin.     

Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes

Summary Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.     

The mitochondrial malic enzymes. I. Submitochondrial localization and purification and properties of the NAD(P)+-dependent enzyme from adrenal cortex.

Rat and calf adrenal cortex homogenates were found to contain three different malic enzymes. Two were strictly NADP+-dependent and were localized, one each, in the cytosol and the mitochondrial fractions, respectively. These two enzymes appear to be identical to those described by Simpson and Estabrook (Simpson, E. R., and Estabrook, R. W. (1969) Arch. Biochem. Biophys. 129, 384-395). The third was NAD(P)+-linked and was present in the mitochondrial fraction only. All three malic enzymes separated as distinct bands during electrophoresis on 5 percent polyacrylamide slab gels at pH 9.0. Marker enzymes and the mitochondrial malic enzymes migrated together in intact mitochondria during sucrose density gradient centrifugations despite changes in the equilibrium position of the mitochondria promoted by energy-dependent calcium phosphate accumulation. In adrenal cortex mitochondria subfractionated by the method of Sottocasa et al. (SOTTOCASA, G.L., KUYLENSTIERNA, B., ERNSTER, L., and BERGSTAND, A. (1967) J. Cell Biol. 32, 415-438), both malic enzymes were associated with the inner membrane-matrix space. Sonication solubilized the two malic enzymes along with the matrix space marker enzymes. The NAD(P)+-dependent malic enzyme was purified 100-fold from calf adrenal cortex mitochondria. The final preparation was free of malic dehydrogenase, fumarase, the strictly NADP+-linked malic enzyme and adenylate kinase. Either Mn24 orMg2+ was required for activity and 1 mol of pyruvate was formed for each mole of NAD+ and NADP+ reduced. The pH optima with NAD+ and NADP+ were 6.5 tp 7.0 and 6.0 to 6.5, respectively. Michaelis-Menten kinetics were observed on the alkaline side. Fumarate, succinate, and isocitrate were positive and ATP and ADP were negative modulators of the regulatory enzyme. The modulators did not influence the stoichiometry and they were not metabolized during the reaction. Under Vmax conditions the ratios for the rate of NAD+:NADP+ reduction were 1.76 and 1.15 at pH 7.4 and 6.0, respectively. The apparent Michaelis constants also differed depending on the pH and the coenzyme. At pH 7.4 (in the presence of 5 mM fumarate) and at pH 6.0 (no fumarate) the Km values for (-)-malate, NAD+, and Mn2+ were 1.7, 0.16, and 0.15 mM, and 0.31, 0.06, and 0.09 mM, respectively. At pH 7.4 (5MM fumarate) and pH 6.0 (no fumarate), the Km values for (-)-malate, NADP+, and Mn2+ were 6.5, 0.62, and 0.59 mM, and 0.68. 0.12, and 0.31 mM, respectively. The apparent Ki values for ATP with NAD+ and NADP+ as coenzyme were 0.42 and 0.27 mM, respectively.     

Tricarboxylic acid cycle enzymes of a pseudophyllid cestode Penetrocephalus ganapatii

Studies on the tricarboxylic acid cycle (TCA cycle) enzymes of Penetrocephalus ganapatii reveal that the TCA cycle is only partially operative, as some of the enzymes at the start of the cycle viz. citrate synthase, aconitase and isocitrate dehydrogenase are found to be low in their activities. The high activities of malate dehydrogenase and fumarase, showing affinity towards a reverse direction, indicate that the TCA cycle operates in the reverse direction resulting in the formation of fumarate. The low succinate dehydrogenase/fumarate reductase ratio suggests that ATP generation may occur at site I of the respiratory chain during the reduction of fumarate into succinate.     

Enzyme inactivation by a cellular neutral protease: enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation.

A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.     

Inhibition by alloxan of mitochondrial aconitase and other enzymes associated with the citric acid cycle

Considerable variations were found in the in vitro effect of alloxan on mouse liver enzymes associated with the citric acid cycle. The following approximative alloxan concentrations induced 50% inhibition of enzyme activity: 10(-6)M for aconitase, 10(-4)M for NAD-linked isocitrate dehydrogenase, glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase and fumarase, and 10(-3)M for citrate synthase and NADP-linked isocitrate dehydrogenase. Pyruvate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were not inhibited by 10(-3)M alloxan. The inhibition of aconitase was competitive both when using mouse liver and purified porcine heart enzyme. The Ki values for the purified enzyme in the presence of 5 microM alloxan were 0.22 microM with citrate, 4.0 microM with cis-aconitate and 0.62 microM with isocitrate as substrate. The high sensitivity of aconitase for inhibition by alloxan probably plays a prominent role for the toxic effects of alloxan.     

Evidence for a critical glutamyl and an aspartyl residue in the function of pig heart diphosphopyridine nucleotide dependent isocitrate dehydrogenase.

The pH dependence of the maximum velocity of the reaction catalyzed by diphosphopyridine nucleotide (DPN) dependent isocitrate dehydrogenase indicates the requirement for the basic form of an ionizable group in the enzyme-substrate complex with a pK of 6.6. This pK is unaltered from 10 to 33 degrees C, suggesting the ionization of a carboxyl rather than an imidazolium ion. The enzyme is inactivated upon incubation with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide in the presence of glycinamide or glycine ethyl ester. This inactivation is dependent on pH and the rate constant (k) increases as the pH is decreased in the range 7.3 to 6.25. A plot of 1/(H+) vs. 1/k suggests that the enzyme is inactivated as a result of the modification of a single ionizable group in this pH range. The coenzyme DPN and substrate alpha-ketoglutarate do not affect the rate of inactivation. In contrast, manganous ion (2 mM) and isocitrate (60 mM) produce a sevenfold decrease in the rate constant. The allosteric activator ADP (1 mM) does not itself influence the rate of inactivation; however, it reduces the concentration of Mn2+ (1 mM) and isocitrate (20 mM) required to produce the same decrease in the inactivation constant. These observations imply that the modification occurs at the substrate-binding site. Experiments employing [1-14C]glycine ethyl ester show a net incorporation of 2 mol of glycine ethyl ester per subunit (40 000), concomitant with the complete inactivation of the enzyme. The radioactive modified enzyme, after removal of excess reagent by dialysis, was exhaustively digested with proteolytic enzymes. High voltage electrophoretic analyses of the hydrolysate at pH 6.4 and 3.5 yield two major radioactive spots with approximately equal intensity, which correspond to gamma-glutamylglycine and beta-aspartylglycine, the ultimate products of reaction with glutamic and aspartic acids, respectively. Modification in the presence of manganous ion and isocitrate results in significant reduction in the incorporation of radioactivity into the two dipeptides. These results suggest that carbodiimide attacks one glutamyl and one aspartyl residue per subunit of the enzyme and that the integrity of these residues is crucial for the enzymatic activity.     

Participation of extracellular fumarase in the utilization of malate in cultured carrot cells

Carrot suspension cells were found to be unable to transport malate directly into the cell but utilized it as a single carbon source in a unique manner -they converted malate extracellularly to fumarate and subsequently used it instead. The uptake of fumarate proved to be inducible and sensitive to pH and protonophore. Immuno-blot experiments using an antibody raised against Arabidopsis fumarase showed that fumarase polypeptide appeared in the medium. Fumarase was not detected in medium when fumarate or glucose was used as a carbon source. The activity of fumarase, which catalyzes the reversible hydration reactions, was induced both in the medium (malate into fumarate, releasing protons) and in the cells (fumarate into malate, requiring protons) and resulted in an increase in the pH gradient across the plasma membrane. The reason for the participation of fumarase in the utilization of malate is discussed.