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At least 19 recordsLinked to original sources
PubMed1999

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.

Acyltransferases↗
PubMed2003

Malate and malate-channel antibodies inhibit electrogenic and ATP-dependent citrate transport across the tonoplast of citrus juice cells.

Citrus juice cells accumulate high levels of citric acid in their vacuoles when compared to other organic ions including malate. Uptake of citrate into tonoplast vesicles from Citrus juice cells was investigated in the presence of malate, and after incubation with antibodies raised against the vacuolar malate-specific channel of Kalanchoë diagremontiana leaves. Antibodies against the vacuolar malate channel immunoreacted with a protein of similar size in tonoplast extracts from three Citrus varieties differing in citric acid content. Malate channel antibodies inhibited both delta MicroH(+)-dependent and delta MicroH(+)-independent ATP-dependent citrate transport, indicating common domains in both transport systems and to the malate-specific channel of Kalanchoë diagremontiana leaves. Malate strongly inhibited electrogenic citrate transport, whereas ATP-dependent citrate uptake was less affected. Kinetic analysis of citrate transport in the presence of malate confirmed the existence of two citrate transport mechanisms and indicated that both citrate and malate share a common transport channel across the tonoplast of Citrus juice cells.

Adenosine Triphosphate↗
PubMed1987

Amino acid sequence similarity between malate dehydrogenases (NAD) and pea chloroplast malate dehydrogenase (NADP).

Purified pea chloroplast malate dehydrogenase (NADP) was reduced, S-pyridylethylated with 4-vinyl-pyridine and cleaved with trypsin. The resulting peptides were separated by reversed-phase high-performance liquid chromatography. Several of these peptides were subjected to automated Edman degradation. The sequences obtained were compared to the published primary structures of malate dehydrogenase from the thermophilic bacterium Thermus flavus and with the sequence of heart mitochondrial and cytoplasmic malate dehydrogenase (NAD). Most peptides from choroplast malate dehydrogenase (NADP) showed high homology with sequences of the other malate dehydrogenases, especially with those of the bacterial enzyme. One of the sequenced peptides contains the active-site histidine residue which is conserved in all malate dehydrogenases. Our results suggest a common evolutionary origin for all malate dehydrogenases despite their different coenzyme specificities and regulatory properties. The sequenced peptides which revealed no homology were either located at the amino-terminal or at the carboxy-terminal region of chloroplast malate dehydrogenase (NADP). These novel sequences are most likely plant-specific extensions of an ancestral malate dehydrogenase and may be responsible for the unique light-dependent activation of the chloroplast enzyme.

Amino Acid Sequence↗
PubMed1992

Electrogenic malate uptake and improved growth energetics of the malolactic bacterium Leuconostoc oenos grown on glucose-malate mixtures.

Growth of the malolactic bacterium Leuconostoc oenos was improved with respect to both the specific growth rate and the biomass yield during the fermentation of glucose-malate mixtures as compared with those in media lacking malate. Such a finding indicates that the malolactic reaction contributed to the energy budget of the bacterium, suggesting that growth is energy limited in the absence of malate. An energetic yield (YATP) of 9.5 g of biomass.mol ATP-1 was found during growth on glucose with an ATP production by substrate-level phosphorylation of 1.2 mol of ATP.mol of glucose-1. During the period of mixed-substrate catabolism, an apparent YATP of 17.7 was observed, indicating a mixotrophy-associated ATP production of 2.2 mol of ATP.mol of glucose-1, or more correctly an energy gain of 0.28 mol of ATP.mol of malate-1, representing proton translocation flux from the cytoplasm to the exterior of 0.56 or 0.84 H+.mol of malate-1(depending on the H+/ATP stoichiometry). The growth-stimulating effect of malate was attributed to chemiosmotic transport mechanisms rather than proton consumption by the malolactic enzyme. Lactate efflux was by electroneutral lactate -/H+ symport having a constant stoichiometry, while malate uptake was predominantly by a malate -/H+ symport, though a low-affinity malate- uniport was also implicated. The measured electrical component (delta psi) of the proton motive force was altered, passing from -30 to -60 mV because of this translocation of dissociated organic acids when malolactic fermentation occurred.

Adenosine Triphosphate↗
PubMed1978

Malate dehydrogenase. Kinetic studies of substrate activation of supernatant enzyme by L-malate.

At pH 8.0 in 0.05 M Tris-acetate buffer at 25 degrees C, homogeneous supernatant malate dehydrogenase exhibits substrate activation by L-malate. The turnover number, Michaelis constant for L-malate, and Michaelis constant for NAD are: 0.46 X 10(4) min(-1), 0.036 mM, and 0.14 mM, respectively, for nonactivated enzyme and 1.1 X 10(4) min(-1), 0.2mM, and 0.047 mM for the same series of constants in activated enzyme. Nonactivating behavior is observed at concentrations between 0.02 and 0.15 mM L-malate and activating behavior is observed between 0.15 and 0.5 mM L-malate. L-Malate activation is compared with similar activation of mitochondrial malate dehydrogenase. While it is not possible to exclude unequivocally all mechanisms, the data seem to be consistent with the occurrence of a fundamentally ordered bi bi mechanism, possibly involving activation through the allosteric binding of L-malate. It is concluded that the data are consistent with a form of the "reciprocating compulsory order mechanism" in which nonactivated enzyme reflects catalysis by one subunit and activated catalysis expresses the coordinated activity of two subunits. The allosteric interaction and the "reciprocating mechanism/ are not mutually exclusive.

Animals↗
PubMed1981

Demonstration of physical interactions between consecutive enzymes of the citric acid cycle and of the aspartate-malate shuttle. A study involving fumarase, malate dehydrogenase, citrate synthesis and aspartate aminotransferase.

By means of covalently immobilized fumarase and mitochondrial or cytoplasmic malate dehydrogenase we were able to detect physical interactions between different enzymes of the citric acid cycle (fumarase with malate dehydrogenase, malate dehydrogenase with citrate synthase and fumarase with citrate synthase) and between the enzymes of both mitochondrial and cytoplasmic halves of the aspartate-malate shuttle (aspartate amino-transferase and malate dehydrogenase). The interactions between fumarase and malate dehydrogenase were also investigated by immobilizing one enzyme indirectly through antibodies bound to Sepharose-protein A. Our results are consistent with a model in which maximally four molecules of malate dehydrogenase are bound to one fumarase molecule. This complex is able to bind either citrate synthase or aspartate aminotransferase. We propose that these enzymes bind alternatively, in order to allow the cell to perform citric acid cycle or shuttle reactions, according to its needs. The physiological meaning and implications on the regulation of metabolism of the existence of a large citric acid cycle/malate-aspartate shuttle multienzyme complex are discussed.

Animals↗
PubMed1996

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.

Animals↗
PubMed1990

Factors affecting L-malate activation of mitochondrial malate dehydrogenase from chicken liver.

Mitochondrial malate dehydrogenase is activated by high concentrations of L-malate. In this paper, several factors affecting this activation on chicken liver enzyme have been investigated. The results obtained show clearly that this phenomenon is an intrinsic property of this enzyme since it does not depend on pH or ionic strength of the reaction medium. However, L-malate activation decreases when NAD+ concentration diminishes (5mM----0.2 mM) in such a way that when NAD+ concentration is 0.2 mM, L-malate does not activate mitochondrial malate dehydrogenase. On the other hand, several activators of this enzymatic system, such as citrate or phosphate, also produce the elimination of this activation by L-malate; in this case, the phenomenon seems be due to a competitive binding to a regulatory site of the different metabolites implied.

Animals↗
PubMed1994

Molecular characterization of Escherichia coli malate synthase G. Differentiation with the malate synthase A isoenzyme.

Two genes encoding the enzymes malate synthase G and glycolate oxidase, have been linked to locus glc (64.5 min), responsible for glycolate utilization in Escherichia coli. The gene encoding malate synthase G, for which we propose the notation glcB, has been cloned, sequenced and found to correspond to a 2262-nucleotide open-reading frame, which can encode a 723-amino-acid polypeptide, clearly different from the isoenzyme malate synthase A, which has 533 amino acids. Northern-blot experiments indicate that glcB was expressed as an apparently monocistronic transcript, inducible by glycolate. Malate synthase G was purified to near homogeneity. The molecular mass determined by gel filtration yielded a value of 82 kDa for the purified enzyme and the same value as for the crude extract enzyme, indicating a monomeric structure. Despite the lower sequence similarity between malate synthase G and the other reported malate synthases, three out of nine consensus boxes defined in most of these enzymes are conserved in addition to a cysteine residue that has been reported to be important for the catalytic mechanisms.

Amino Acid Sequence↗
PubMed1997

Specificity and direction of depolymerization of beta-poly(L-malate) catalysed by polymalatase from Physarum polycephalum--fluorescence labeling at the carboxy-terminus of beta-poly(L-malate).

Beta-poly(L-malate), a major constituent of nuclei in plasmodia of Physarum polycephalum, is enzymatically degraded to L-malate after secretion into the culture medium. This depolymerization is specifically catalysed by an endogenous polymalatase. The mode of action and the specificity criteria have been investigated by employing various chemical derivatives of beta-poly(L-malate), including substitution at the hydroxy-terminus and carboxy-terminus of the polymer, esterification of the pending alpha-carboxylate, and beta-poly(DL-malate). The results of the investigation were summarized in a specificity model that involved recognition of the hydroxy-terminus and of the alpha-carboxylate as substituents of the asymmetric carbon in the malic acid unit. Depolymerization proceeded from the hydroxy-terminus towards the carboxy-terminus, thereby degrading the polymer to L-malate. When the terminal beta-carboxylate had been amidated with the fluorescent N-(1-naphthyl)ethylenediamine, degradation was normal but was arrested at the level of the terminal beta-carboxy-substituted dimer. It should be possible to employ polymalatase as a tool for the detection of branching and other modifications of beta-poly(L-malate).

Animals↗
PubMed2003

The Bacillus subtilis YufLM two-component system regulates the expression of the malate transporters MaeN (YufR) and YflS, and is essential for utilization of malate in minimal medium.

The Gram-positive bacterium Bacillus subtilis has a complete set of enzymes for the tricarboxylic acid (TCA) cycle and can grow aerobically using most of the TCA cycle intermediates (malate, fumarate, succinate and citrate) as a sole carbon source. The B. subtilis genome sequence contains three paralogous two-component regulatory systems, CitST, DctSR and YufLM. CitST and DctSR activate the expression of a transporter of the Mg(2+)-citrate complex (CitM) and a fumarate and succinate transporter (DctP), respectively. These findings prompted an investigation of whether the YufL sensor and its cognate regulator, YufM, play a role in malate uptake. This paper reports that the YufM regulator shows in vitro binding to the promoter region of two malate transporter genes, maeN and yflS, and is responsible for inducing their expression in vivo. It was also found that inactivation of the yufM or maeN genes resulted in bacteria that could not grow in a minimal salts medium containing malate as a sole carbon source, indicating that the induction of the MaeN transporter by the YufM regulator is essential for the utilization of malate as a carbon source. Inactivation of the yufL gene resulted in the constitutive expression of MaeN. This expression was suppressed by reintroduction of the kinase domain of YufL, indicating that the YufL sensor is required for proper signal detection and signalling specificity. The authors propose that a phosphatase activity of YufL plays an important role in the YufLM two-component regulatory system. The studies reported here have revealed that members of a set of paralogous two-component regulatory systems in B. subtilis, CitST, DctSR and YufLM, are involved in a related function--uptake (and metabolism) of the TCA cycle intermediates--but with distinct substrate specificities.

Bacillus subtilis↗
PubMed2000

The role of vacuolar malate-transport capacity in crassulacean acid metabolism and nitrate nutrition. Higher malate-transport capacity in ice plant after crassulacean acid metabolism-induction and in tobacco under nitrate nutrition.

Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H(+)-transport by H(+)-pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, Deltamu(H+), across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, J(mal)(rel), of apparent ATP-dependent malate-stimulated H(+)-transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. J(mal)(rel) was high in the obligate crassulacean acid metabolism (CAM) species Kalanchoë daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO(3)(-) nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher J(mal)(rel). It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO(3)(-) nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins.

Adenosine Triphosphate↗
PubMed1995

Glyoxysomal malate dehydrogenase and malate synthase from soybean cotyledons (Glycine max L.): enzyme association, antibody production and cDNA cloning.

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD+ oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (Mr) of 670,000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M(r) 510,000 and of gMDH as a dimer of M(r) 73,000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X6)HL motif also found in plant and mammalian thiolases.

Amino Acid Sequence↗
PubMed1982

The use of a potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Uptake of L-malate and D-malate by luminal-membrane vesicles.

The mechanisms of uptake of dicarboxylic acids by rabbit renal luminal-membrane vesicles were studied by the use of filtration and spectrophotometric techniques as described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J.208, 359-368]. Addition of l- or d-malate to dye-membrane-vesicle suspensions in the presence of Na(+) gradients (extravesicular>intravesicular) resulted in spectral curves indicative of depolarization events. The renal uptake of dicarboxylic acids was dependent on the type of Na(+)-salt anion present and could be correlated with the ability of the anions to penetrate biological membranes (i.e. Cl(-)>SO(4) (2-)>gluconate). Identical results were obtained by a filtration technique with Sartorius membrane filters. The results indicate that the dicarboxylic acids are taken up by the membrane vesicles in an electrically positive form (i.e. Na(+)/substrate coupling ratio 3:1) by an Na(+)-dependent transport system. This proposal was further supported by spectrophotometric experiments with various ionophores such as valinomycin, gramicidin and nigericin. The absorbance changes associated with simultaneous addition of l- and d-malate and spectrophotometric competition studies revealed that the two isomers are taken up by a common transport system. Spectral changes of the dye induced by addition of increasing concentrations of l- or d-malate indicated that the transport system favours the unphysiological d-form rather than the l-form of malate. Furthermore, it was observed that the affinity of both isomers for the transport system was dependent on the concentration of Na(+) in the medium.

Animals↗
PubMed1999

The NAD-linked aromatic alpha-hydroxy acid dehydrogenase from Trypanosoma cruzi. A new member of the cytosolic malate dehydrogenases group without malate dehydrogenase activity.

Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.

Alcohol Oxidoreductases↗
PubMed1975

Malate dehydrogenase, anticooperative NADH, and L-malate binding in ternary complexes with Supernatant pig heart enzyme.

Supernatant malate dehydrogenase from pig heart, a dimeric protein containing two very similar or identical subunits, shows negatively cooperative (anticooperative) interactions between NADH binding sites in the presence, but not in the absence, of 0.1 M L-malate. This behavior is observed consitently whether the technique used employs protein fluorescence quenching, NADH fluorescence enhancement, or ultrafiltration dialysis. Fluorescence titration shows that L-malate is also anticooperatively bound in the presence of saturating concentrations of NADH. The data are consistent with an "induced asymmetry" model in which conformational change accompanies the formation of the ternary complex. Two of the three chromatographically resolvable forms of the enzyme have been tested and found to have anticooperative behavior.

Animals↗
PubMed1999

Structural basis of substrate specificity in malate dehydrogenases: crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase, alpha-ketomalonate and tetrahydoNAD.

The structural basis for the extreme discrimination achieved by malate dehydrogenases between a variety of closely related substrates encountered within the cell has been difficult to assess because of the lack of an appropriate catalytically competent structure of the enzyme. Here, we have determined the crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase with the alternative substrate alpha-ketomalonate and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide. Both subunits of the dimeric porcine heart, and from the prokaryotes Escherichia coli and Thermus flavus. However, large changes are noted around the active site, where a mobile loop now closes to bring key residues into contact with the substrate. This observation substantiates a postulated mechanism in which the enzyme achieves high levels of substrate discrimination through charge balancing in the active site. As the activated cofactor/substrate complex has a net negative charge, a positive counter-charge is provided by a conserved arginine in the active site loop. The enzyme must, however, also discriminate against smaller substrates, such as pyruvate. The structure shows in the closed (loop down) catalytically competent complex two arginine residues (91 and 97) are driven into close proximity. Without the complimentary, negative charge of the substrate side-chain of oxaloacetate or alpha-ketomalonate, charge repulsion would resist formation production of this catalytically productive conformation, hence minimising the effectiveness of pyruvate as a substrate. By this mechanism, malate dehydrogenase uses charge balancing to achieve fivefold orders of magnitude in discrimination between potential substrates.

Amino Acid Sequence↗