Dependent Malic Enzyme Research Articles

Estimation of TCA cycle flux, aminotransferase flux, and anaplerosis in heart: validation with syntactic model

Weiss et al. (Circ. Res. 70: 392-408, 1992) proposed a model of the citric acid cycle (CAC) in myocytes and a system of 17 differential equations that can be used to describe the changes over time in enrichment of carbons C-2 and C-4 of glutamate under conditions of metabolic steady state. They also proposed an empirical measure (KT) of flux through the CAC, which has been shown to be correlated to O2 consumption in rat hearts perfused with acetate or a mixture of glucose and acetate. We report a new method for estimation of the absolute rate of the flux through the CAC in heart (vTCA), without the numerical solution of differential equations. Unlike KT, our estimate is equal to the rate of flux catalyzed by the alpha-ketoglutarate dehydrogenase complex (vTCA), not merely correlated with it. We also estimate the rate of flux catalyzed by aspartate aminotransferase (vTA) and by NADP(+)-dependent malic enzyme (an anaplerotic reaction). The formula for vTCA during administration of [2-13C]acetate is as follows: vTCA = M[(C-2ssLC-4)/[C-4ss(LC-4-LC-2)]], where C-2ss and C-4ss represent steady-state fractional enrichment, LC-2 and LC-4 represent dominant rate constants of C-2 and C-4 of glutamate, respectively, and M is the sum of concentrations of aspartate, glutamate, and intermediates of the CAC. The assumptions underlying our formula are as follows: 1) metabolic steady state is maintained, 2) exchange of molecules between cytosolic and mitochondrial compartments is rapid, 3) 13C enters pools of the CAC only from acetyl CoA via citrate synthase, 4) [citrate]/[glutamate] < 1 + (vTCA/vTA), and 5) (m-[glutamate])/M < C-2ss/C-4ss.

Cloning, sequencing and functional expression of a cDNA encoding a NADP-dependent malic enzyme from human liver

This work reports the structure of a cDNA (ME) encoding a human malic enzyme (ME) (malate NADP oxidoreductase, EC 1.1.1.40) elucidated by joining several overlapping fragments amplified by PCR from human hepatic cDNA or from cDNA libraries. The full-length cDNA has an open reading frame (ORF) of 1719 bp that encodes a 572-aminoacid protein of 64 113 Da, similar to the native monomeric, cytosolic, NADP-dependent ME isolated from human liver. The comparison of the structure of this cDNA with that of the human mitochondrial NAD(P)-dependent ME (EC 1.1.1.39) shows a homology of 63%, suggesting that these two forms originated from the same gene. The expression of the cDNA in Escherichia coli as a translational fusion (glutathione S-transferase::ME) protein yielded a product of the predicted mass. The recombinant protein shows NADP-dependent malate oxidoreductase activity and is virtually inactive with NAD. It also shows other distinct features of the native cytosolic NADP-dependent ME, like Mn 2+ dependence, similar substrate ( K m = 117 μM) and cofactor affinity ( K m = 2 μM) constants, and a lack of allosteric regulation. In human proliferative cells, the NADP-dependent ME activity is poorly expressed and barely inducible by thyroid hormones.

Purification, cDNA cloning and heterologous expression of the human mitochondrial NADP+-dependent malic enzyme

Mitochondrial NADP(+)-dependent malic enzyme (ME; EC 1.1.1.39) has been purified to homogeneity and characterized kinetically from bovine heart. Partial amino acid sequence information allowed amplification of a specific bovine cDNA, which was used to isolate a full-length human cDNA of this isoform of ME. The cDNA is 1930 bp long and codes for a protein of 604 amino acids. Comparison of the amino acid sequence of this isoform with published sequences of other human ME isoforms shows stretches of homology interrupted by larger regions with significant differences. The human protein has been expressed in Escherichia coli, and the recombinant human protein has the same kinetic properties as the corresponding protein purified from bovine heart. Northern blot analysis showed a strong tissue-specific transcription with a predominantly high expression-rate in organs with a low division-rate.

Characterization of a bean (Phaseolus vulgaris L.) malic-enzyme gene.

We have isolated a genomic clone encoding a plant NADP(+)-dependent malic enzyme (NADP-ME). This clone, isolated from bean (Phaseolus vulgaris L.), covers the entire gene (exons, introns) and 5'-flanking regions. DNA sequencing defines 20 exons spanning approximately 4.5 kb, which range over 48-235 bp in size. All 19 introns are fairly small (79-391). The first intron resides in the 5'-untranslated leader sequence. Introns 10, 11 and 16 are located at positions identical to a rat malic-enzyme gene. In the promoter region, a TATA box (TATATATA) is easily recognized 41 bp upstream of a single transcription-initiation site. Two potential cis-acting elements with homology to elements from plant genes, activated by UV light and fungal elicitors, were identified at positions -153 and -312, respectively. Southern-blot analysis suggests a single gene copy, but also other distantly related genes, in the bean genome. The deduced NADP-ME protein of 589 amino acids exhibits features consistent with a cytoplasmic location. We describe the organization of the NADP-ME protein into functional domains located on separate exons. The evolution of malic-enzyme genes coding for isoforms in different cellular compartments of plants and animals is discussed.

Characterization of cytosolic malic enzyme in human tumor cells

Cytosolic NADP(+)-dependent malic enzyme (ME) from human tumor cells was characterized in detail and compared to ME from normal human tissues produced in recombinant E. coli. Kinetic properties, size as seen in SDS gels, and HPLC elution profiles of tryptic digests of human 'normal cell' ME and NADP(+)-ME from tumor cells were identical. Thus, NADP(+)-ME found in tumor cells does not constitute a tumor-specific isoform as suggested by other studies but is identical to the 'housekeeping protein' predominantly expressed in human liver and white adipose tissue.

Detection of an NAD+-dependent malic enzyme locus in the atlantic salmonSalmo salar and other salmonid fish

Electrophoretic studies of malate oxidoreductases routinely assess variation in two enzymes, malate dehydrogenase (EC 1.1.1.37) and malic enzyme (NADP+) (EC 1.1.1.40). By modification of the standard isozyme staining conditions for these enzymes, we have resolved a new NAD(+)-preferring, MgCl2-requiring malic enzyme which is indicated to be EC 1.1.1.39. The enzyme was detected in 10 salmonid fish species of the genera Salmo, Salvelinus, and Onchorynchus. Phenotypic variation indicates that the novel enzyme is tetrameric and coded by a single locus. Inheritance in S. salar follows a single-locus model and the phenotypes are unlinked to polymorphisms for sMDH-3,4* and mMEP-2*, two malate oxidoreductase loci previously shown to be variable in this species.

Cloning and Expression of Pigeon Liver Cytosolic NADP+-Dependent Malic Enzyme cDNA and Some of Its Abortive Mutants

A full-length 1927-base-pair cDNA of pigeon liver malic enzyme was obtained by utilizing the screening of the cDNA library and polymerase chain reaction techniques. The cDNA contained one open reading frame coding for 557 amino acid residues, flanked by 86 and 167 nucleotides at the 5′ and 3′ termini, respectively, and was successfully cloned and expressed in Escherichia coli cells. Functionally active recombinant malic enzyme was purified from the cells. This recombinant enzyme has a Km value for L-malate of 160 ± 30 μM, which is almost identical to that for the natural enzyme (150 ± 17 μM). The Km value for Mn2+ (4.2 ± 0.3 μM) is higher than that for the natural pigeon malic enzyme (1.4 ± 0.2 μM), while the Km value for NADP+ (3.8 ± 0.3 μM) is lower than that for the natural enzyme (10.8 ± 0.1 μm). The catalytic constant (kcat) for the recombinant enzyme is decreased by 3.6-fold, but the substrate inhibition constant for L-malate is increased by about 40-fold. Change in the quaternary structure of the recombinant enzyme was revealed in the pH perturbation examination. A truncated pigeon liver malic enzyme, lacking the first 13 amino acid residues, and a recombinant protein, mutated at F19S, N250S, and L353Q, showed no enzymatic activity. Both abortive recombinant mutant proteins were still able to bind with 2′,5′-ADP agarose; however, the fluorescence emission spectrum of the protein bound NADPH did not show a blue shift as the natural enzyme. In accordance with these observations, we suggest that the adenosine 2′,5′-bisphosphate binding domain of the NADP+ binding site in the βαβ motif may still be retained in these mutant proteins. However, the local hydrophobic environment for the binding of the nicotinamide moiety of the coenzyme molecule may be altered. Therefore, the lack of catalytic activity of the mutant proteins could be attributed to an improper orientation of the bound NADP+.

Plant mitochondrial NAD+-dependent malic enzyme. cDNA cloning, deduced primary structure of the 59- and 62-kDa subunits, import, gene complexity and expression analysis.

The 59- and 62-kDa subunits of the mitochondrial NAD+-dependent malic enzyme (EC 1.1.1.39) were purified from Solanum tuberosum L. (potato). NH2-terminal and internal amino acid sequence information was used to identify cDNAs encoding the two subunits. Comparison of the nucleotide sequences revealed that the subunits have 60% identity at the DNA level and 65% identity at the deduced amino acid level, implying that they are derived from a common ancestral gene. The plant NAD+-dependent malic enzymes belong to a family of related enzymes, including cytosolic and chloroplastic NADP+-dependent malic enzymes (EC 1.1.1.40) and bacterial NAD+-dependent malic enzymes (EC 1.1.1.38). The cDNAs were transcribed and translated in vitro and the resultant polypeptides imported into isolated mitochondria and shown to be processed. Southern blot analysis of potato genomic DNA revealed a simple pattern of hybridization for both subunits, indicating a simple gene structure or small number of genes encoding the two subunits. Northern blot analysis of RNA from a range of potato tissues has shown that the steady state levels for the two subunits are equivalent, suggesting that they are coordinately expressed.

NAD+ ‐dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD(+)-dependent and NADP(+)-dependent malic enzyme activities. The NAD+ malic enzyme exhibited more activity with NAD+ as cofactor, but also showed some activity with NADP+. The NADP+ malic enzyme only showed activity when NADP+ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NAD+ malic enzyme activity (dme-) but retained NADP+ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N2. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N2-fixation would normally begin. These results support the proposal that NAD+ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N2-fixing bacteroids which metabolize C4-dicarboxylic acids supplied by the plant.

Antagonism between cadmium chloride and divalent metal cations in the activation of malic enzyme

1. The effect of the divalent cation on the activation of the shrimp Crangon crangon abdomen muscle cytosol NADP-dependent malic enzyme (EC 1.1.1.40) and herring Clupea harengus testicular tissue mitochondrial NAD(P)-dependent malic enzyme (EC 1.1.1.39) was examined. 2. For cytosol the NADP-dependent malic enzyme K m for manganese is only slightly higher than that for cadmium and cobalt, while it is about 60-times lower than the value for magnesium. 3. For the mitochondrial NAD(P)-dependent malic enzyme in the NAD linked reaction K m for manganese is very similar to that for cobalt, while it is 2.7-fold and 41-fold lower than the value for cadmium and magnesium, respectively. In the NADP linked reaction, K m for manganese is only slightly higher than that for cobalt, while it is 1.6-fold and 36-fold lower than the value for cadmium and magnesium, respectively. 4. The cadmium ion shows strong inhibition of the malic enzyme activity. In cation combinations, cadmium causes inhibition of enzyme activity to the value as added alone only. These results demonstrate that malic enzyme is a molecule which is very sensitive to cadmium exposure.

Mitochondrial glutamine and glutamate metabolism in human placenta and its possible link with progesterone biosynthesis

Summary The effect of glutamine and glutamate on progesterone biosynthesis in humanplacental mitochondria has been investigated. Both amino acids stimulates this process, but glutamate to a greater extent. At pH 8.2 activity of placental mitochondrial glutaminase and progesterone biosynthesis in the presence of glutamine depend on inorganic phosphate concentration in the same manner. This suggests that glutaminase at first hydrolyses glutamine to glutamate and further metabolism of glutamate supports progesterone biosynthesis. Results of experiments in which progesterone biosynthesis has been measured at pH 7.2 and 8.2 indicate the possible involvement of NADP-dependent glutamate dehydrogenase, NAD(P)-dependent malic enzyme, and NADP-dependent isocitrate dehydrogenase in providing the reducing equivalents (NADPH) for progesterone biosynthesis from glutamine and glutamate. Data reported in this paper suggest that in human placental mitochondria metabolism of glutamine and glutamate may be closely correlated to progesterone biosynthesis.

Kinetic mechanism of the cytosolic malic enzyme from human breast cancer cell line

The kinetic mechanism of the cytosolic NADP +-dependent malic enzyme from cultured human breast cancer cell line was studied by steady-state kinetics. In the direction of oxidative decarboxylation, the initial-velocity and product-inhibition studies indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism with NADP + as the leading substrate followed by l-malate. The products are released in the order of CO 2, pyruvate, and NADPH. The enzyme is unstable at high salt concentration and elevated temperature. However, it is stable for at least 20 min under the assay conditions. Tartronate (2-hydroxymalonate) was found to be a noncompetitive inhibitor for the enzyme with respect to l-malate. The kinetic mechanism of the cytosolic tumor malic enzyme is similar to that for the pigeon liver cytosolic malic enzyme but different from those for the mitochondrial enzyme from various sources.

Purification and characterization of the cytosolic NADP+‐dependent malic enzyme from human breast cancer cell line

Cytosolic NADP(+)-dependent malic enzyme from a cultured human breast cancer cell line was purified to near homogeneity by two highly efficient chromatography systems: Pharmacia-LKB Q-Sepharose anion-exchange chromatography and adenosine-2',5'-bisphosphate-agarose affinity chromatography. The overall yield was 27%. The enzyme is presumably a tetramer composed of four probably identical subunits of Mr 65,000, which is similar to the enzyme from other sources. The pI and optimum reaction pH values for the tumor malic enzyme are 5.5 and 7.2, respectively. At pH 6.9, most of the enzyme exists as monomers. Activation energy for the enzyme-catalyzed oxidative-decarboxylation reaction is 57.4 kJ/mol. The enzyme is strictly NADP+ dependent, as NAD+ cannot support the oxidative-decarboxylation reaction. ATP at low concentration inhibits the enzyme activity. Fumarate at concentrations up to 5 mM does not affect the enzymatic reaction rate. Therefore the tumor cytosolic malic enzyme, unlike the mitochondrial malic enzyme, is not an allosteric regulatory enzyme.

Pyruvate carboxylation prevents the decline in contractile function of rat hearts oxidizing acetoacetate

Acetoacetate, when present as the only fuel for respiration in rat hearts, causes an impairment in contractile function that is reversible with the addition of substrates that can contribute to anaplerosis. To determine the importance of pyruvate carboxylation via NADP(+)-dependent malic enzyme on metabolism and function in hearts oxidizing acetoacetate, isolated working rat hearts were perfused with [1-14C]pyruvate and acetoacetate. While the cardiac power output after 60 min of perfusion in hearts utilizing acetoacetate alone had fallen to 44% of the initial value, the addition of pyruvate resulted in a stable performance with no fall in the work output. When hydroxymalonate, an inhibitor of NADP(+)-dependent malic enzyme and malate dehydrogenase, was added to the two substrates, function at 60 min was similar to the value for hearts oxidizing acetoacetate alone. Measurements of the specific activities of malate, aspartate, and citrate confirm inhibition of both pyruvate carboxylation and malate oxidation. The findings are consistent with a mechanism in which the enrichment of malate by pyruvate improves function by increasing the production of reducing equivalents by the malate dehydrogenase and the isocitrate dehydrogenase reactions increase flux through the span of the tricarboxylic acid cycle from malate to 2-oxoglutarate. The present study demonstrates the physiological importance of anaplerotic pathways in maintaining contractile function in the heart.

Analogues of NADP+ as inhibitors and coenzymes for NADP+ malic enzyme from maize leaves

Structural analogues of the NADP(+) were studied as potential coenzymes and inhibitors for NADP(+) dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N(6)-etheno-nicotinamide adenine dinucleotide phosphate (∈ NADP(+)), 3-acetylpyridine-adenine dinucleotide phosphate (APADP(+)), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP(+)) and β-nicotinamide adenine dinucleotide 2': 3'-cyclic monophosphate (2'3'NADPc(+)) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in Vmax for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP(+)), 3-aminopyridine-adenine dinucleotide phosphate (AADP(+)), adenosine 2'-monophosphate (2'AMP) and adenosine 2': 3'-cyclic monophosphate (2'3'AMPc) were competitive inhibitors with respect to NADP(+), while β-nicotinamide adenine dinucleotide 3'-phosphate (3'NADP(+)), NAD(+), adenosine 3'-monophosphate (3'AMP), adenosine 2': 5'-cyclic monophosphate (2'5'AMPc), 5'AMP, 5'ADP, 5'ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2'-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C4 atom of the pyridine ring is the major factor that governs the coenzyme activity.

Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli.

Mitochondrial NAD(+)-dependent malic enzyme (EC 1.1.1.40) is expressed in rapidly proliferating cells and tumor cells, where it is probably linked to the conversion of amino acid carbon to pyruvate. In this paper, we report the cDNA cloning, amino acid sequence, and expression in Escherichia coli of functional human NAD(+)-dependent mitochondrial malic enzyme. The cDNA is 1,923 base pairs long and contains an open reading frame coding for a 584-amino acid protein. The molecular mass is 65.4 kDa for the unprocessed precursor protein. Comparison of the amino acid sequence of the human protein with the published NADP(+)-dependent mammalian cytosolic or plant chloroplast malic enzymes reveals highly conserved regions interrupted with long stretches of amino acids without significant homology. Expression of the processed protein in E. coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells.

Control and function of the transamination pathways of glutamine oxidation in tumour cells

Parallel investigations of the transamination pathways of glutamine oxidation in Ehrlich ascites carcinoma (EAC) and AS 30D hepatoma revealed that hepatoma cells, unlike EAC, produce very little aspartate. This cannot be explained by differences in the activity of glutamine-metabolizing enzymes. Also, the mitochondria from the hepatoma respired at a similar rate to EAC mitochondria with glutamine as sole substrate producing substantial amounts of aspartate. Unlike their isolated mitochondria, intact hepatoma cells showed a very low rate of glutamine oxidation. Compared with EAC, the rate of L-[U-14C]glutamine consumption by AS 30D hepatoma cells was much lower, with insignificant production of 14C-labelled aspartate and CO2. This suggested that the glutamine-transporting system in the hepatoma cell plasma membrane had a very low activity. Isolated hepatoma mitochondria produced 3 times more pyruvate from malate than did EAC mitochondria, indicating a higher activity of NAD(P)-dependent malic enzyme. We postulate that an active malic enzyme may suppress the synthesis of aspartate in hepatoma cells, but further evidence is needed to confirm this assumption.

Mitochondrial NAD(P)-dependent malic enzyme from herring testicular tissue: Purification, kinetic behaviour and regulatory properties

Mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1.39, L-malate: NAD(+) oxidoreductase (decarboxylating)] was purified from herring testicular tissue to a specific activity of 26.4 μmol NADH/min/mg protein. Herring testicular tissue is one of the most abundant sources of this enzyme. The purification procedure involved differential centrifugation of mitochondria and then chromatography on DEAE-Sephacel, Red Agarose and Sephacryl S-300. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of Mn(2+) and either NAD or NADP. Under Vmax conditions the ratios for the rate of NAD/NADP reduction was 1.8. A study of the reductive carboxulation reaction indicated that this enzyme reaction is reversible; at pH 7.0 the reverse reaction exhibited 22% of the activity of forward reaction. Some kinetic characteristics of the enzyme were determined. ATP was found to be a competitive inhibitor with respect to malate. Fumarate reversed ATP inhibition. Regulation of NAD(P)-dependent malic enzyme from herring testicular tissue mitochondria could respond to changing levels of mitochondrial ATP and fumarate in vivo.

Purification of tumor mitochondrial malic enzyme by specific ligand affinity chromatography

A two-step chromatographic procedure, based on a specific ligand-binding approach, for the purification of tumor NAD(P) +-dependent malic enzyme is described. The enzyme was purified to near homogeneity by extraction from mitochondria, negative cellulose phosphate chromatography, ammonium sulfate precipitation, and application of specific elution from a malate-agarose column. The rationale for the use of the affinity column is also described.

Regulation of coenzyme utilization by mitochondrial NAD(P)-dependent malic enzyme

1. 1. Skeletal muscle mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1. 39, l-malate: NAD + oxidoreductase (decarboxylating)] from herring could use both coenzymes, NAD and NADP, in a similar manner. 2. 2. The coenzyme preference of mitochondrial NAD(P)-dependent malic enzyme was probed using dual wavelength spectroscopy and pairing the natural coenzymes, NAD or NADP with their respective thionicotinamide analogues, s-NADP or s-NAD, that have absorbance maxima in reduced forms at 400 nm. 3. 3. s-NAD and s-NADP were found to be good alternate substrates for NAD(P)-dependent malic enzyme, the apparent K m values for the thioderivatives were similar to those of the corresponding natural coenzymes. 4. 4. ATP produced greater inhibition of the NAD or s-NAD linked reactions than of the NADP or s-NADP-linked reactions of skeletal muscle mitochondrial NAD(P)-dependent malic enzyme. 5. 5. At 5 mM malate concentration and in the presence of 2 mM ATP the NADP-linked reaction is favoured and the activity ratios, v ( s- nadp) / v ( nad) or v ( nadp) / v ( s- nad) are 6 and 26, respectively.