mM For Malate Research Articles

Kinetic Properties of NADP+-Dependent Decarboxylating Malate Dehydrogenase from Corn Leaves

One isoform of NADP+-dependent decarboxylating malate dehydrogenase (EC 1.1.1.40) was found in the leaf mesophyll of corn. The enzyme was purified in four stages: homogenization, fractionation with ammonium sulfate, gel filtration on Sephadex G-25, and ion exchange on Sephacel. The specific activity of the purified, electrophoretically homogeneous preparation was 92 E/mg of protein, while its yield was 15%, and the purification degree was 109. The relative electrophoretic mobility of the decarboxylating malate dehydrogenase was 0.1. The optimal pH for enzyme functioning during the catalysis of forward and reverse reactions was 8.0. The Michaelis constants were determined for the direct and reverse reactions: 5.5 mM for malate and 1.3 mM for pyruvate. Moreover, the affinity for coenzymes varied significantly. Thus, KM was equal to 4.5 mM for NADP+ and 0.9 mM for NADP.

Role of the malic enzyme in metabolism of the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The bacteria utilizing methane as a growth substrate (methanotrophs) are important constituents of the biosphere. Methanotrophs mitigate the emission of anthropogenic and natural greenhouse gas methane to the environment and are the promising agents for future biotechnologies. Many aspects of CH4 bioconversion by methanotrophs require further clarification. This study was aimed at characterizing the biochemical properties of the malic enzyme (Mae) from the halotolerant obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z. The His6-tagged Mae was obtained by heterologous expression in Escherichia coli BL21 (DE3) and purified by affinity metal chelating chromatography. As determined by gel filtration and non-denaturating gradient gel electrophoresis, the molecular mass of the native enzyme is 260 kDa. The homotetrameric Mae (65x4 kDa) catalyzed an irreversible NAD+-dependent reaction of L-malate decarboxylation into pyruvate with a specific activity of 32 ± 2 units mg-1 and Km value of 5.5 ± 0.8 mM for malate and 57 ± 5 μM for NAD+. The disruption of the mae gene by insertion mutagenesis resulted in a 20-fold increase in intracellular malate level in the mutant compared to the wild type strain. Based on both enzyme and mutant properties, we conclude that the malic enzyme is involved in the control of intracellular L-malate level in Mtm. alcaliphilum 20Z. Genomic analysis has revealed that Maes present in methanotrophs fall into two different clades in the amino acid-based phylogenetic tree, but no correlation of the division with taxonomic affiliations of the host bacteria was observed.

Purification and characterization of malate dehydrogenase from sheep liver (Ovis aries): Application in AST assay diagnostic kit

Malate dehydrogenase enzyme is a major component in aspartate aminotransferase (AST) diagnostic kit that used in diagnosis and monitoring of liver diseases. This study aimed at purification and characterization of MDH enzyme from sheep liver for direct application in preparation of AST kit. Two malate dehydrogenase isoenzymes were resolved from sheep liver by chromatography on DEAE-cellulose column, one major sheep liver MDH (SLMDH) and another minor one. SLMDH was extracted and purified by ammonium sulfate fractionation and chromatographical separation on anion exchanger and gel filtration resins. The purified sheep liver MDH specific activity is 6.7 units / mg protein representing 11.6 purification folds and 27.7 % yield. The molecular mass of SLMDH intact protein is 68 ± 1.6 kDa. SLMDH turned out to be homogeneous and consists of two identical subunits of 34 ± 1.2 kDa each. The SLMDH isoelectric point (pI) value is estimated at pH 6.2 and displayed its optimum activity at pH 9.6 and has Km value of 1.4 mM for NAD and 11 mM for malate. FeCl2, NiCl2 and ZnCl2 inhibited the SLMDH activity. The purified SLMDH is applied in the preparation of AST diagnostic kit that found sensitive and comparable with a commercially available one.

Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant.

NAD(+)-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene. The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions. The Km values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction). When malic enzyme was induced under appropriate culture conditions in a strain of E. coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism of glucose was restored. Succinic acid was the major fermentation product formed. When this fermentation was performed in the presence of hydrogen, the yield of succinic acid increased. The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks.

Effect of aspartate and glutamate on the oxoglutarate carrier investigated in rat heart mitochondria and inverted submitochondrial vesicles

Interaction of glutamate and aspartate with the oxoglutarate carrier was investigated in rat heart mitochondria or inverted submitochondrial particles. With mitochondria, glutamate and aspartate had no effect on the initial rate of oxoglutarate or malate uptake. With inverted submitochondrial vesicles, binding experiments indicated that aspartate bound to the oxoglutarate carrier on its matricial face and increased the affinity of the substrate binding site for malate but did not change the affinity for oxoglutarate. Glutamate had no effect on both substrate bindings. The dissociation constants of the binary substrate-carrier complexes on the matricial side were determined (1.28 ± 0.15 mM for oxoglutarate and 2.22 ± 0.26 mM for malate). These values, compared with those obtained previously on the cytosolic side of intact mitochondria, confirmed the asymmetry of the carrier in the native membrane (higher affinities on the cytosolic face). It is concluded that (1) aspartate and glutamate are not cytosolic effectors of the oxoglutarate carrier, (2) matricial aspartate is a positive effector of the binding of malate on the matricial side of the oxoglutarate carrier, and (3) such a characteristic may play a role in the regulation of the oxoglutarate carrier. Thus, it may be emphazised that (1) this observation is the first clear evidence of a well-defined ‘sophisticated regulation’ (allosteric) of a mitochondrial metabolite carrier, and (2) this regulation of the oxoglutarate carrier may have important consequences on the efficiency of reducing equivalent import in the matrix space by the malate-aspartate shuttle.

Role of malic acid in solubilizing excess berberine accumulating in vacuoles of Coptis japonica

The capability of cultured cells of Coptis japonica for accumulating a supersaturated solution of berberine (more than 70 mM) in vacuoles was investigated in relation to the solubility of various berberine salts. The results of experiments showed that the solubility of berberine is dependent on its paired anion, varying from 3 mM for nitrate to 640 mM for malate, suggesting that a high concentration of malic acid 168 mM found in the vacuole greatly contributes to the formation of a highly water-soluble salt of berberine. The production of berberine in cultured cells was accompanied by a steady increase of malic acid during the entire culture period. The present study has demonstrated an important role of a specific organic acid in solubilizing an excess of quarternary alkaloid in the vacuole.

Kinetic characterization of the reconstituted tricarboxylate carrier from rat liver mitochondria

The tricarboxylate carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted in phospholipid vesicles by removing the detergent using hydrophobic chromatography on Amberlite. Optimal transport activity was obtained by using a Triton X-114/phospholipid ratio of 0.8, 6% cardiolipin and 24 passages through a single Amberlite column. In the reconstituted system the incorporated tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The activation energy of the exchange reaction was 70.1 kJ/mol. The rate of the exchange had a pH optimum between 7 and 8. The half-saturation constant was 0.13 mM for citrate and 0.76 mM for malate. All these properties were similar to those described for the tricarboxylate transport system in intact mitochondria. In proteoliposomes the maximum exchange rate at 25°C reached 2000 μmol/min per g protein. This value was independent of the type of substrate present at the external or internal space of the liposomes (citrate or malate).

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.

Malate dehydrogenase from thermophilic Humicola lanuginosa and Mucor pusillus: purification and comparative properties of the enzymes with differing thermostabilities

Malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) was purified from the thermophiles Humicola lanuginosa and Mucor pusillus. The H. lanuginosa enzyme was homogeneous on sodium dodecyl sulphate – polyacrylamide gels, while the M. pusillus enzyme was more than 95% pure. The two enzymes appeared to be composed of two subunits of equal size, each of 36 000 daltons (H. lanuginosa) or 33 000 daltons (M. pusillus). The native enzymes revealed molecular weights of 68 000 as determined by gel filtration. The isoelectric points of malate dehydrogenase from H. lanuginosa and M. pusillus were 3.9 and 4.2, respectively. The reduction of oxaloacetate by the H. lanuginosa enzyme was optimum at pH 8.5–9 with apparent Km's of 0.12 mM for oxaloacetate and 0.027 mM for NADH. On the other hand, M. pusillus enzyme snowed a pH optimum of 7.8–8.5 with apparent Km's of 0.075 mM for oxaloacetate and 0.1 mM for NADH. The L-malate oxidation reaction was catalyzed optimally at pH 10 by the H. lanuginosa enzyme with apparent Km's of 5.8 mM for malate and 0.1 mM for NAD, while the M. pusillus enzyme catalyzed it optimally between pH 9.5 and 10 with apparent Km's of 4.44 mM for malate and 0.16 mM for NAD. The optimum temperature for reduction of oxaloacetate was 50 °C for both the enzymes. The H. lanuginosa enzyme was resistant to heat inactivation at 40 °C, but lost 60% of its activity after 15 min at 50 °C. Mucor pusillus enzyme, on the other hand, retained 90% activity at 60 °C after 10 min. The two enzymes were protected from heat inactivation by monovalent cations (viz Na+, K+, and NH4+), as well as citrate, which may possibly involve conformational changes.

Purification and properties of NADP-dependent malate dehydrogenase from pea leaves

A high-yield purification procedure for NADP-dependent malate dehydrogenase from pea leaves is described, involving acid precipitation, ammonium sulphate fractionation, and two subsequent chromatography steps on Procion HE 3B-Sepharose CL-4B and on Phenyl-Sepharose CL-4B. The final gel filtration step on Ultrogel AcA 44 yields a homogeneous preparation of high specific activity. Subunit molecular weight was determined to be 38 500. Both inactive and activated preparations of the native enzyme eluted from a gel filtration column, indicative of molecular weights of 40 000, 87 000 and 163 000, thus suggesting the enzyme to be present in monomeric, dimeric and tetrameric forms. At neutral pH, the enzyme was found to be tetrametric. With either high salt or high pH or both, the dimeric enzyme was eluted. The isoelectric point was determined to be at pH 4.35. Apparent K m values were 39 μM for NADH, 48 μM for oxaloacetate, 14.5 mM for malate and 63 μM for NADP.

Some properties of mitochondrial NAD + linked malic enzyme and malate dehydrogenase from the flight muscles of Leptinotarsa decemlineata

Malic enzyme and malate dehydrogenase were purified to homogeneity from the flight muscles of the Colorado potato beetle, Leptinotarsa decemlineata, by gel filtration on Sephadex G-200 and affinity chromatography on 5-AMP Sepharose. Malic enzyme utilizes NAD + preferentially and is activated by low concentrations of manganese ions. The molecular weights of the native enzyme and its subunits were 220,000 and 49,000 as determined by gel filtration and SDS polyacrylamide gel electrophoresis respectively. In the direction of pyruvate formation, the enzyme showed apparent K m values of 0.09 mM for malate and 0.10 mM for NAD + and was inhibited by oxaloacetate, succinate, pyruvate, ATP, ADP and AMP. Under alkaline pH condition, purified malate dehydrogenase utilises malate and oxaloacetate at approximately the same rate. The enzyme showed a molecular weight of about 61,000 composing two subunits of 30,000. In the direction of oxaloacetate formation, the enzyme showed apparent K m values of 1.5 mM for malate and 0.22 mM for NAD + and was inhibited by succinate, pyruvate and the adenine nucleotides. The physiological significance of the biochemical properties of both enzymes is discussed.

Kinetic study of the tricarboxylate carrier in rat liver mitochondria.

The kinetics of citrate uptake by malate‐loaded mitochondria were measured using the inhibitor stop method and analysed for possible carrier mechanisms. The citrate exchange is found to follow a first order reaction with a constant k of 1.18 min−1 and the corresponding rate of citrate uptake of 13.4 μmol × min−1× g protein−1 (at 0.5 mM citrate and 9 °C). The half time is 34 sec. The temperature dependence of citrate exchange was measured in the range between 0 and 14 °C. An activation energy of 20.1 kcal and a Q10 of 3.6 can be calculated from the Arrhenius plot. The concentration dependence of citrate exchange reveals hyperbolic saturation characteristics. The Km and V values for the rate of citrate uptake are 0.12 ± 0.01 mM and 22.5 ± 1.8 μmol citrate × min−1× g protein−1 respectively at 9 °C in 11 experiments. The rate of citrate uptake has a pH optimum of about 7. The inhibition by higher pH is competitive with citrate. At pH lower than 7, V is decreased, indicating that the citrate transporting system is inactivated by acid pH. The rate of citrate exchange is inhibited in a competitive manner by cis‐aconitate, threo‐Ds‐isocitrate, 1,2,3‐propanetricarboxylate, 1,2,3‐benzenetricarboxylate, citrate and propylcitrate. Other tricarboxylates, however, such as trans‐aconitate and 1,3,5‐pentanetricarboxylate have no effect on citrate exchange, even when added in large excess. Succinate, malonate, oxaloacetate and oxomalonate inhibit the rate of citrate uptake. In contrast, glutarate, adipate, pimelate, 2‐oxoglutarate, aspartate and glutamate do not. Maleate, but not the trans isomer fumarate, also inhibits citrate uptake. The inhibition of citrate uptake is greater with malate than with succinate. The rate of citrate uptake is also inhibited by the nonpenetrant anions phenylsuccinate, butylmalonate, benzylmalonate and pentylmalonate, previously thought to be specific inhibitors of the dicarboxylate carrier. The inhibition of the rate of citrate uptake by dicarboxylates and their analogues is found to be competitive. The affinity for dicarboxylates is lower than for tricarboxylates (K1 is 0.7 mM for malate). Phosphoenolpyruvate strongly inhibits the rate of citrate uptake, while P1 and pyruvate have only a slight effect. The inhibition by phosphoenolpyruvate is shown to be competitive (K1 is 0.11 mM). It is concluded that the tricarboxylate carrier has a single binding site for tricarboxylates, phosphoenolpyruvate and dicarboxylates. The implications of these findings are discussed.