L-malate transport and proton symport in vesicles prepared from Pseudomonas putida

In vesicles from glucose-grown Pseudomonas putida, L-malate is transported by nonspecific physical diffusion. L-Malate also acts as an electron donor and generates a proton motive force (delta p) of 129 mV which is composed of a membrane potential (delta psi) of 60 mV and a delta pH of 69 mV. In contrast, vesicles from succinate-grown cells transport L-malate by a carrier-mediated system with a Km value of 14.3 mM and a Vmax of 313 nmol X mg protein-1 X min-1, generate no delta psi, delta pH, or delta p when L-malate is the electron donor, and produce an extravesicular alkaline pH during the transport of L-malate. A kinetic analysis of this L-malate-induced proton transport gives a Km value of 16 mM and a Vmax of 667 nmol H+ X mg protein-1 X min-1. This corresponds to a H+/L-malate ratio of 2.1. The failure to generate a delta p in these vesicles is considered, therefore, to be consistent with the induction in succinate-grown cells of an electrogenic proton symport L-malate transport system.     

Fermentation of fumarate and L-malate by Clostridium formicoaceticum.

The fermentation of fumarate and L-malate by Clostridium formicoaceticum was investigated. Growing and nongrowing cells degraded fumarate by dismutation to succinate, acetate, and CO2; on the other hand, only small amounts of succinate were detected when the organism was grown on L-malate. This dicarboxylic acid was mainly converted to acetate and CO2. The fermentation balances were modified if bicarbonate or formate were present in the medium. When C. formicoaceticum was grown in the presence of both dicarboxylic acids, fumarate was consumed before L-malate. The latter was mainly converted to acetate, whereas fumarate was fermented to acetate and succinate. Molar growth yields were determined to be 6 g of dry weight per mol of fumarate and 8 g of dry weight per mol of L-malate fermented.     

Synthetic substrates and inhibitors of beta-poly(L-malate)-hydrolase (polymalatase).

Polymalatase from Physarum polycephalum calalysed the hydrolysis of beta-poly[L-malate] and of the synthetic compounds beta-di(L-malate), beta-tetra(L-malate), beta-tetra(L-malate) beta-propylester, and L-malate beta-methylester. Cyclic beta-tri(L-malate), cyclic beta-tetra(L-malate), and D-malate beta-methylester were not cleaved, but were competitive inhibitors. The O-terminal acetate of beta-tetra(L-malate) was neither a substrate nor an inhibitor. L-Malate was liberated; the Km, Ki and Vmax values were measured. The appearance of comparable amounts of beta-tri(L-malate), and beta-di(L-malate) during the cleavage of beta-tetra(L-malate) indicated a distributive mechanism for small substrates. The accumulation of a series of oligomers, peaking with the 11-mer and 12-mer in the absence of higher intermediates, indicated that the depolymerization of beta-poly(L-malate) was processive. The results indicate that beta-poly(L-malate) is anchored at its OH-terminus by the highly specific binding of the penultimate malyl residue. The malyl moieties beyond 12 residues downstream from the OH-terminus extend into a diffuse second, electrostatic binding site. The catalytic site joins the first binding site, accounting for the cleavage of the polymer into malate residues. It is proposed that the enzyme does not dissociate from beta-poly(L-malate) during hydrolysis, when both sites are filled with the polymer. When only the first binding site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation.     

Effects of ferulic acid on L-malate oxidation in isolated soybean mitochondria

The effects of ferulic acid on L-malate oxidation in mitochondria isolated from soybean (Glycine max L.) seedlings were investigated. Oxygen uptake and the products of L-malate oxidation were measured under two conditions: pH 6.8 and 7.8. At acidic pH, the activity of the NAD+-linked malic enzyme (L-malate:NAD+oxidoreductase [decarboxylating] EC 1.1.1.39) was favoured, whereas at alkaline pH a predominance of the L-malate dehydrogenase activity (L-malate:NAD+oxidoreductase EC 1.1.1.37) was apparent. Ferulic acid inhibited basal and coupled respiration during L-malate oxidation either at acidic or alkaline pH, reducing also the amounts of pyruvate or oxaloacetate produced. The results suggest that the site of ferulic acid action is situated at some step that precedes the respiratory chain. An interference with the L-malate entry into the mitochondria could be an explanation for the effects of ferulic acid, but the possibility of a direct inhibition of both enzymes involved in L-malate oxidation cannot be ruled out. This revised version was published online in August 2006 with corrections to the Cover Date.     

Carbon-13 nuclear magnetic resonance analysis of [1-13C]glucose metabolism in Crithidia fasciculata. Evidence of CO2 fixation by phosphoenolpyruvate carboxykinase

The non-invasive technique of 13C nuclear magnetic resonance was applied to study glucose metabolism in vivo in the insect parasite Crithidia fasciculata. It was found that under anaerobic conditions [1-13C]glucose underwent a glycolytic pathway whose main metabolic products were identified as [2-13C]ethanol, [2-13C]succinate and [1,3-13C2]glycerol. These metabolites were excreted by C. fasciculata into the incubation medium, while in the cells [3-13C]phosphoenolpyruvate was also detected in addition to the aforementioned compounds. The C3 acid is apparently the acceptor of the primary CO2 fixation reaction, which leads in Trypanosomatids to the synthesis of succinate. By addition of sodium bicarbonate to the incubation mixture L-[3-13C]malate was detected among the excretion products, while the ethanol:succinate ratio of 2.0 in the absence of bicarbonate changed to a ratio of 0.6 in the presence of the latter. This was due to a shift of the balance between carboxylation of phosphoenolpyruvate, leading to succinate, and pyruvate decarboxylation leading to ethanol. The addition of 25% 2H2O to the incubation mixture led to the formation of [2-13C, 2-2H]ethanol derived from the prior incorporation of 2H+ into pyruvate in the reactions mediated by either pyruvate kinase or malic enzyme. However, no 2H+ incorporation into L-malate was detected, excluding the possibility that the latter was formed by carboxylation of pyruvate, and lending support to the idea that L-malate results from the carboxylation of phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxykinase. The formation of [2-13C, 2-2H]-succinate under the same conditions reflected the uptake of 2H+ during the reduction of fumarate. When the incubations were carried out in the presence of 100% 2H2O, several [1-13C, 1-2H]ethanol species were detected, as well as [2-13C, 2-2H]malate and [1,3-13C2, 1,3-2H2]glycerol. The former deuterated compounds reflect the existence of NAD2H species when the incubations were carried out in 100% 2H2O, while the incorporation of 2H+ into [1,3-13C2]glycerol must be attributed to the phosphoglucose-isomerase-mediated reaction during glycolysis.     

Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions.

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.     

Energization of mitochondrial inner membranes caused by l-malate

It was found that 0.06 μg antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c₁ completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.     

Effect of different physico-chemical conditions on malolactic fermentation of fourLactobacillus plantarum wild strains isolated from wines of northwestern Spain

Summary Four strains ofLactobacillus plantarum, were tested for malolactic fermentation under conditions of variations in temperature, pH and SO2, L-malate and ethanol levels. When the pH value was below 3.5, malolactic fermentation was lower and was more sensitive to temperature changes. Malolactic fermentation decreased when the SO2 and ethanol levels were increased. The effects of L-malate levels were not significant.     

Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy.

The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate. Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylcarbodiimide. Since substrate-level phosphorylation does not occur during malolactic fermentation, the generation of metabolic energy must originate from the uptake of L-malate and/or excretion of L-lactate. The initiation of malolactic fermentation is stimulated by the presence of L-lactate intracellularly, suggesting that L-malate is exchanged for L-lactate. Direct evidence for heterologous L-malate/L-lactate (and homologous L-malate/L-malate) antiport has been obtained with membrane vesicles of an L. lactis mutant deficient in malolactic enzyme. In membrane vesicles fused with liposomes, L-malate efflux and L-malate/L-lactate antiport are stimulated by a membrane potential (inside negative), indicating that net negative charge is moved to the outside in the efflux and antiport reaction. In membrane vesicles fused with liposomes in which cytochrome c oxidase was incorporated as a proton motive force-generating mechanism, transport of L-malate can be driven by a pH gradient alone, i.e., in the absence of L-lactate as countersubstrate. A membrane potential (inside negative) inhibits uptake of L-malate, indicating that L-malate is transported an an electronegative monoanionic species (or dianionic species together with a proton). The experiments described suggest that the generation of metabolic energy during malolactic fermentation arises from electrogenic malate/lactate antiport and electrogenic malate uptake (in combination with outward diffusion of lactic acid), together with proton consumption as result of decarboxylation of L-malate. The net energy gain would be equivalent to one proton translocated form the inside to the outside per L-malate metabolized.     

Role of the divalent metal ion in the NAD:malic enzyme reaction: an ESEEM determination of the ground state conformation of malate in the E:Mn:malate complex.

The conformation of L-malate bound at the active site of Ascaris suum malic enzyme has been investigated by electron spin echo envelope modulation spectroscopy. Dipolar interactions between Mn2+ bound to the enzyme active site and deuterium specifically placed at the 2-position, the 3R-position, and the 3S-position of L-malate were observed. The intensities of these interactions are related to the distance between each deuterium and Mn2+. Several models of possible Mn-malate complexes were constructed using molecular graphics techniques, and conformational searches were conducted to identify conformers of malate that meet the distance criteria defined by the spectroscopic measurements. These searches suggest that L-malate binds to the enzyme active site in the trans conformation, which would be expected to be the most stable conformer in solution, not in the gauche conformer, which would be more similar to the conformation required for oxidative decarboxylation of oxalacetate formed from L-malate at the active site of the enzyme.     

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

The steady state concentrations of coenzyme A-SH and coenzyme A thioester, citrate, and isocitrate during tricarboxylate cycle oxidations in rabbit heart mitochondria.

The steady state mitochondrial content of coenzyme A-SH (CoA), acetyl-CoA, succinyl-CoA, and long chain acyl-CoA has been determined during the oxidation of palmitoylcarnitine by rabbit heart mitochondria. Variation of the substrate concentration during ADP-stimulated (state 3) respiration varies the mitochondrial content of long chain acyl-CoA and the rate of O2 uptake, and permits the conclusion that the Km of beta oxidation for intramitochondrial long chain acyl-CoA is approximately 1 nmol/mg of mitochondrial protein. At near saturating concentrations of palmitoylcarnitine, plus L-malate, the addition of ADP causes a decrease in acetyl-CoA, an increase in CoA and succinyl-CoA, and no clear change in long chain acyl-CoA content. These changes reverse upon the depletion of ADP (state 3 leads to 4 transition). Similar changes in CoA, acetyl-CoA, and succinyl-CoA are seen during state 4 leads to 3 leads to 4 transitions with pyruvate plus L-malate and octanoate plus L-malate as substrates. These results suggest a limitation of flux by citrate synthase during the controlled oxidation of these three substrates. The ratio acetyl-CoA/succinyl-CoA was determined not only during state 3 and state 4 oxidation of palmitoylcarnitine plus L-malate and pyruvate plus L-malate, but also during intermediate respiratory states (state 3 1/2) generated by adding glucose and varying amounts of hexokinase. These intermediate states are characterized by a high succinyl-CoA content, relative to either state 3 or state 4, and a suboptimal flux through citrate synthase, estimated either by pyruvate disappearance or by O2 uptake.     

Is beta-poly(L-malate) synthesis catalysed by a combination of beta-L-malyl-AMP-ligase and beta-poly(L-malate) polymerase?

beta-Poly(L-malate) is supposed to function in the storage and transport of histones, DNA polymerases and other nuclear proteins in the giant syncytical cells (plasmodia) of myxomycetes. Here we report on the biosynthesis of [14C]beta-poly(L-malate) from injected L-[14C]malate in the plasmodium of Physarum polycephalum. The effects of KCN, arsenate, adenosine 5'-(alpha, beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, guanosine 5'-(beta, gamma-methylene)triphosphate, desulfo coenzyme A and phenylarsinoxid on beta-poly(L-malate) synthesis were studied after their coinjection with L-[14C]malate. The synthesis was not affected by KCN or desulfo coenzyme A, but was blocked by arsenate and adenosine 5'-(alpha,beta-methylene)triphosphate. The plasmodium lysate catalysed an L-malate-dependent ATP-[32P]pyrophosphate exchange, but was devoid of beta-poly(L-malate) synthetic activity under all experimental conditions tested. The results suggested an extramitochondrial synthesis of beta-poly(L-malate), involving the polymerization of beta-L-malyl-AMP. It is assumed that the lack of synthesis in the lysate is caused by the inactivation of beta-poly(L-malate) polymerase involving a cell injury kinase pathway. Because injected guanosine 5'-(beta, gamma-methylene)triphosphate blocks the synthesis, the injury signal is likely to be GTP dependent.     

Active oxidative decarboxylation of malate by mitochondria isolated from L-1210 ascites tumor cells

Mitochondria from L-1210 mouse ascites tumor show a very high rate of oxidation of L-malate in comparison with mitochondria from normal tissues. They were found to contain large amounts of malic enzyme (E.C.1.1.1.39) catalyzing oxidative decarboxylation of L-malate to pyruvate. Malic enzyme in extracts of tumor mitochondria requires Mn²⁺ or Mg²⁺, utilizes either NAD⁺ or NADP⁺ as electron acceptor, and shows positive cooperativity in binding of L-malate. These observations suggest that L-1210 tumor mitochondria actively convert excess tricarboxylate cycle intermediates and their precursors into pyruvate for further oxidation.     

Staining of argininosuccinate lyase activity in polyacrylamide gel.

A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to L-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of L-malate to give CO2 and pyruvate with concomitant reduction of NADP+ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer.     

Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.     

L-malate reverses oxidative stress and antioxidative defenses in liver and heart of aged rats

The intracellular levels of antioxidant and free radical scavenging enzymes are gradually altered during the aging process. An age-dependent increase of oxidative stress occurring throughout the lifetime is hypothesized to be the major cause of aging. The current study examined the effects of L-malate on oxidative stress and antioxidative defenses in the liver and heart of aged rats. Sprague-Dawley male rats were randomly divided into four groups, each group consisting of 6 animals. Group Ia and Group IIa were young and aged control rats. Group Ib and Group IIb were young and aged rats treated with L-malate (210 mg/kg body weight per day). L-malate was orally administrated via intragastric canula for 30 days, then the rats were sacrificed and the liver and heart were removed to determine the oxidant production, lipid peroxidation and antioxidative defenses of young and aged rats. Dietary L-malate reduced the accumulation of reactive oxygen species (ROS) and significantly decreased the level of lipid peroxidation in the liver and heart of the aged rats. Accordingly, L-malate was found to enhance the antioxidative defense system with an increased activity of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) and increased glutathione (GSH) levels in the liver of aged rats, a phenomenon not observed in the heart of aged rats. Our data indicate that oxidative stress was reversed and the antioxidative defense system was strengthened by dietary supplementation with L-malate.     

Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata.

Non-autotrophic ( Aut -) mutants of Rhodopseudomonas capsulata B10 were tested for their efficiency of nitrogenase-mediated H2 production. Three of these mutants ( IR3 , IR4 and IR5 ) showed an increase stoichiometry of H2 production, mediated by nitrogenase, from certain organic substrates. For example, in a medium containing 7 mM-L-glutamate as nitrogen source, strain IR4 produced 10-20% more H2 than did the wild type with DL-lactate or L-malate as major carbon source, 20-50% more H2 with DL-malate, and up to 70% more with D-malate. Strain IR4 was deficient in 'uptake' hydrogenase activity as measured by H2-dependent reduction of Methylene Blue or Benzyl Viologen. However, this observation did not explain the increased efficiency of H2 production, since H2 uptake (H2 recycling) was undetectable in cells of the wild type. Instead, increased H2 production by the mutant appeared to be due to an improved conversion of organic substrates to H2 and CO2, presumably due to an altered carbon metabolism. The metabolism of D-malate by different strains was studied. An NAD+-dependent D-malic enzyme was synthesized constitutively by the wild type, and showed a Km for D-malate of 3 mM. The activity of this enzyme was approx. 50% higher in strain IR4 than in the wild type, and the mutant also grew twice as fast as the wild type with D-malate as sole carbon source.     

Purification by affinity chromatography of a membrane dicarboxylate binding protein from Bacillus subtilis

Membrane vesicles of Bacillus subtilis W23 actively transport the C4 and C5 dicarboxylates of the tricarboxylate cycle by system(s) of relatively high affinity for their requisite substrates (Km 4-53 microM). Glutamate and succinate binding activities were readily solubilized from membrane vesicles by nonionic detergents, particularly by Lubrol WX. From this extract, glutamate binding activity was highly enriched by affinity chromatography on phloroglucinol-expanded Sepharose-6B to which L-aspartate was coupled via divinylsulfone. Another protein (41000 molecular weight), which bound both L-glutamate and L-malate, was purified from affinity columns to which either L-glutamate or L-malate had been coupled via bisdiglycidyl ether. This protein bound labelled L-malate as well as L-glutamate with affinities similar to those seen with membrane vesicles (Kd's 8 microM L-malate and 52 microM L-glutamate).