Forms Of Malate Dehydrogenase Research Articles

Cloning and characterization of the Bambusa oldhamii BoMDH-encoded malate dehydrogenase

Malate dehydrogenase (MDH), which is ubiquitously occurred in nature, catalyzes the interconversion of malate and oxaloacetate. Higher plants contain multiple forms of MDH that differ in coenzyme specificity, subcellular localization and physiological function. A putative Bambusa oldhamii BoMDH cDNA was screened with the specific probe from the bamboo cDNA library. Sequence alignment shows that there's a high homology between the deduced amino acid sequence of BoMDH and MDH protein in Oryza sativa glyoxysome (92%). A 57 kDa fusion protein was expressed by IPTG induction in Escherichia coli BL21 (DE3), and an obvious MDH activity was detected in the recombinant protein. The molecular mass of recombinant BoMDH was estimated to be 120 kDa, and the subunit form was 57 kDa by denatured SDS-PAGE, indicating that BoMDH presents as a homodimer. The optimum temperature and pH for BoMDH activity were 40 °C and 9.5, respectively. The Km values of BoMDH for malate and NAD+ were 5.2 mM and 0.52 mM. The kcat/Km values of BoMDH for malate and NAD+ were 163 min−1 mM−1 and 3060 min−1 mM−1.

The existence of a Cryptic Allosteric Site on Plasmodium falciparum Malate Dehydrogenase

Approximately one million people die from malaria each year, and thus this disease is a prevalent and pressing matter. Plasmodium falciparum, the protozoan parasite responsible for malaria has been subject to intensive study with the goal of developing specific drugs for malaria treatment. Plasmodium falciparum has a distinctive Malate Dehydrogenase (MDH), being tetrameric compared to the canonical dimeric forms of MDH, yet shares the same catalytic and substrate binding site motives as human mitochondrial and cytosolic forms. To investigate the possible existence of a cryptic allosteric site in the P falciparum enzyme, we have used sequence and structural bioinformatics and identified several short sequences,(G175‐L176, Q211‐M216) each containing highly conserved (in Plasmodium and related species) amino acids that are different in the Plasmodium falciparum enzyme than the human mitochondrial or cytosolic forms. To investigate the possibility that these regions form a cryptic allosteric site in Plasmodium falciparum we expressed, purified, using NiNTA Affinity chromatography, and characterized all three forms (Plasmodium falciparum, human cytosolic and human mitochondrial malate dehydrogenases). In terms of kinetic parameters the Plasmodium falciparum enzyme resembled the mammalian mitochondrial isoform, exhibiting similar substrate inhibition with oxaloacetate and weaker affinity for NADH than the mammalian cytosolic isoform. To determine the potential that the Plasmodium falciparum enzyme had a cryptic allosteric site we designed two mutations, one in each of the two significant sequence differences between the human isoforms and the Plasmodium falciparum enzyme (.D176N and R214E ). Each mutation was constructed using Quikchange mutagenesis, transformed into XL Gold cells and expressed. The purified mutant proteins were characterized using enzyme kinetics and size exclusion chromatography and their overall secondary structures compared to the wildtype enzyme using circular dichroism spectroscopy. Stability was compared using CD thermal melts at 222nm and a Fluorescence based Thermal Shift assay. While both mutations showed altered specific activities compared to wildtype, the D176N mutant showed a 90% lower activity than wildtype Plasmodium falciparum malate dehydrogenase. Both mutations significantly lowered affinity for the cofactor NADH, >20fold decrease in affinity. Taken together these results suggest that Plasmodium falciparum malate dehydrogenase may have a highly conserved cryptic allosteric site distinct from any such site in the mammalian isoforms. This Cryptic Allosteric Site could be exploited to develop inhibitors of Plasmodium falciparum malate dehydrogenase that would not impact the human isoforms.Support or Funding InformationThis work was supported by NSF Grants 1726932 and 0448905.

Abstract 4381: Investigating the reversible MDH1 catalytic reaction in squamous non-small cell lung cancer

Abstract Non-small-cell lung cancer (NSCLC) is a leading cause of death in men and women worldwide. NSCLC relies on glycolysis to support cell proliferation and to synthesize proteins, nucleic acids and lipids. Malate dehydrogenase (MDH) is an enzyme critical for replenishing intermediates in the tricarboxylic acid (TCA) cycle and for helping to drive glycolysis through its reversible coenzyme system NAD+/NADH by interconverting oxaloacetate and malate. The cytosolic form of MDH (MDH1) has been reported to be amplified in squamous cell NSCLC, which has high glycolytic activity. Elucidation of the role of MDH1 in altering cellular metabolism to drive tumorigenic progression has yet to be determined. Thus, our interest lies in identifying the chemical and cellular characteristics required to drive the reversible MDH1 catalytic reaction in squamous cell NSCLC. Previously, we have shown that metabolic dehydrogenases are regulated by microenvironmental changes in pH and oxidation levels, and we are currently extending this work to MDH1. We are also evaluating various cell lines to gain a better understanding of how the MDH1 reaction can be altered to decrease the tumorigenic capacity of squamous cell NSCLC. Experimental knowledge obtained has the potential to increase our understanding of the MDH1 reaction and educate the design of future anti-tumor therapeutics. Citation Format: Joi L. Weeks, Grace Wells, Sati Alexander, Christian Metallo, Christal D. Sohl. Investigating the reversible MDH1 catalytic reaction in squamous non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4381.

Mechanism of malate dehydrogenase isoform formation in Sphaerotilus natans D-507 under different cultivation conditions

Electrophoretically homogenous preparations of malate dehydrogenase (MDH) isoforms of the bacteria Sphaerotilus natans D-507 with specific activity 7.46 U/mg and 5.74 U/mg with respect to protein concentration have been obtained. The dimeric isoform of the enzyme was shown to function under organotrophic growth conditions, whereas the tetrameric isoform was induced under mixotrophic cultivation conditions. PCR-analysis revealed a single gene encoding the malate dehydrogenase molecule. The topography of the MDH isoform surface was studied by atomic-force microscopy, and a 3D-structure of the enzyme was obtained. Spectraphotometric analysis data allowed us to suggest that stabilization of the tetrameric form of MDH is due to additional bounds implicated in the quaternary structure formation.

Crystallization and preliminary X-ray diffraction studies of tetrameric malate dehydrogenase from the novel Antarctic psychrophile Flavobacterium frigidimaris KUC-1.

Flavobacterium frigidimaris KUC-1 is a novel psychrotolerant bacterium isolated from Antarctic seawater. Malate dehydrogenase (MDH) is an essential metabolic enzyme in the citric acid cycle and has been cloned, overexpressed and purified from F. frigidimaris KUC-1. In contrast to the already known dimeric form of MDH from the psychrophile Aquaspirillium arcticum, F. frigidimaris MDH exists as a tetramer. It was crystallized at 288 K by the hanging-drop vapour-diffusion method using ammonium sulfate as the precipitating agent. The crystal diffracted to a maximum resolution of 1.80 A. It contains one tetrameric molecule in the asymmetric unit.

Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat

Malate dehydrogenase (MDH), which is ubiquitous in nature, catalyzes the interconversion of oxaloacetate and malate. Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function. Cytosolic NAD-dependent MDH (cyMDH) is one class of MDH that has not been extensively characterized in plants. Here we report the cloning of a cDNA from wheat by RT-PCR and cDNA library screening, which is designated as TaMDH. Sequence analysis indicated that TaMDH exhibits a highly similarity to other plant cyMDHs. Immunological analysis confirmed that TaMDH encoded a cytosolic NAD-dependent MDH. The secondary and three-dimensional structures of TaMDH were analyzed by molecular modeling. DNA gel-blot analyses demonstrated that TaMDH gene exists as two copies in the wheat genome. RNA and protein gel-blot hybridization indicated that both TaMDH mRNA and protein were constitutively expressed in vegetative tissues of wheat, with slightly lower levels in roots than in leaves and stems. In silico analysis indicated that TaMDH was also expressed in various reproductive tissues and tissues under many different stress conditions. Kinetic analysis of bacterially expressed and purified protein confirmed that TaMDH catalyzed a reaction driven towards malate synthesis, which is consistent with other cyMDHs. Evolutionary analysis showed that this class of genes evolved from a very ancestral gene. The cyMDH represents an ancestral form of MDH, which is highly conserved in plants, animals and bacteria. This implies that cyMDHs are housekeeping genes and may have very essential functions in plant metabolism.

Gene Modifies Electrophoretic Properties of Malate Dehydrogenase in Norway Spruce (Picea abies (L.) Karst.)

The mobility of two malate dehydrogenase (MDH) isozymes (encoded by Mdh-2 and Mdh-3) was found to be altered by a modifier gene designated Mm(2,3) in an electrophoretic analysis of megagametophytes of Picea abies. This modifier gene exists in two allelic variants. The dominant allele Mmd(2,3)-N gives rise to the common electrophoretic phenotype whereas the recessive allele Mmd(2,3)-n produces faster migrating forms of both MDH-2 and MDH-3. In diploid tissues, genotypes heterozygous at Mmd(2,3) cannot be differentiated from homozygotes carrying dominant alleles. Certain allozymes encoded by Mdh2 and Mdh3 have very similar relative migration rates under the given electrophoretic conditions. If diploid tissue is analyzed, the enzyme products cannot be traced back to specific alleles. Therefore, haploid megagametophytes have to be used to identify properly MDH genotypes. It is suggested to analyze at least 13 megagametophytes per tree to ensure a misclassification smaller than the 5% level. Linkage of Mmd(2,3) to Aat3, Lap1, -2, Mdh3, Pepca, 6Pgd2, -3, Pgi2, Pgm1, -2, and Skdh1 was tested, revealing independent segregation. One locus (Mdh1) presumably codes for the cytoplasmatic form of MDH.

Purification and physicochemical properties of malate dehydrogenase from bacteria of the genus Beggiatoa.

Homogeneous malate dehydrogenase (MDH) with a specific activity of 20-24 units per mg protein was purified from the sulfur bacterium Beggiatoa leptomitiformis strain D-402 grown organotrophically and lithotrophically and from the organotrophic bacterium Beggiatoa alba. MDHs from the B. leptomitiformis strain D-402 grown under organotrophic conditions and from B. alba are homodimers with the subunit molecular weight of 40 kD. Tetrameric MDH is formed in B. leptomitiformis strain D-402 grown under lithotrophic conditions. The dimeric and tetrameric forms of MDH from B. leptomitiformis D-402 display some differences in kinetic properties.

Multiple soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes)

A recent locus duplication hypothesis for sMDH-B* was proposed to explain the complex electrophoretic pattern of six bands detected for the soluble form of malate dehydrogenase (MDH, EC 1.1.1.37) in 84% of the Geophagus brasiliensis (Cichlidae, Perciformes) analyzed (AB1B2 individuals). Klebe's serial dilutions were carried out in skeletal muscle extracts. B1 and B2 subunits had the same visual end-points, reflecting a nondivergent pattern for these B-duplicated genes. Since there is no evidence of polyploidy in the Cichlidae family, MDH-B* loci must have evolved from regional gene duplication. Tissue specificities, thermostability and kinetic tests resulted in similar responses from both B-isoforms, in both sMDH phenotypes, suggesting that these more recently duplicated loci underwent the same regulatory gene action. Similar results obtained with the two sMDH phenotypes did not show any indication of a six-banded specimen adaptive advantage in subtropical regions.

Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule-enhanced MDH.

Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate reversible malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C4 metabolism, pH balance, stomatal and pulvinal movement, respiration, beta-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh- mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.

Studies on associations of glycolytic and glutaminolytic enzymes in MCF-7 cells: role of P36.

Isoelectric focusing of MCF-7 cell extracts revealed an association of the glycolytic enzymes glyceraldehyde 3-phosphate-dehydrogenase, phosphoglycerate kinase, enolase, and pyruvate kinase. This complex between the glycolytic enzymes is sensitive to RNase. p36 could not be detected within this association of glycolytic enzymes; however an association of p36 with a specific form of malate dehydrogenase was found. In MCF-7 cells three forms of malate dehydrogenase can be detected by isoelectric focusing: the mitochondrial form with an isoelectric point between 8.9 and 9.5, the cytosolic form with pl 5.0, and a p36-associated form with pl 7.8. The mitochondrial form comprises the mature mitochondrial isoenzyme (pl 9.5) and its precursor form (pl 8.9). Refocusing of the pl 7.8 form of malate dehydrogenase also gave rise to the mitochondrial isoenzyme. Thus, the pl 7.8 form of malate dehydrogenase is actually the mitochondrial isoenzyme retained in the cytosol by the association with p36. Addition of fructose 1,6-bisphosphate to the initial focusing column induced a quantitative shift of the pl 7.8 form of malate dehydrogenase to the mitochondrial forms (pl 8.9 and 9.5). In MCF-7 cells p36 is not phosphorylated in tyrosine. Kinetic measurements revealed that the pl 7.8 form of malate dehydrogenase has the lowest affinity for NADH. Compared to both mitochondrial forms the cytosolic isoenzyme has a high capacity when measured in the NAD --> NADH direction (malate --> oxaloacetate direction). The association of p36 with the mitochondrial isoenzyme may favor the flow of hydrogen from the cytosol into the mitochondria. Inhibition of cell proliferation by AMP which leads to an inhibition of glycolysis has no effect on complex formation by glycolytic and glutaminolytic enzymes in MCF-7 cells. AMP treatment leads to an activation of malate dehydrogenase, which correlates with the increase of pyruvate and the decrease of lactate levels, but has no effect on the distribution of the various malate dehydrogenase forms.

Malate Dehydrogenase Forms a Complex with and Activates Phosphoenolpyruvate Carboxylase from Crassulacean Acid Metabolism Plants

Summary Gel chromatography and kinetic techniques give evidence for the association of malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) extracted from leaves of the Crassulacean Acid Metabolism plant Kalanchoe blossfeldiana . PEPC and one form of MDH co-eluted in the same peak from Ultrogel AcA 54, Ultrogel AcA 34 or Sephacryl 200. The complex was disrupted by gradient acrylamide gel electrophoresis and reconstituted in vitro by recovering the purified PEPC from the gel in the presence of MDH. This effect of MDH was not obtained with other proteins or enzymes at the same protein concentration. The molecular masses observed for the postulated complex are consistent with the association of one MDH dimer with one PEPC tetrameric form, or under higher ionic strength, one PEPC dimer. The PEPC-MDH interaction was shown to stimulate PEPC activity several times, or to depress it when MDH was in excess, which supports specificity of the effect. The inhibitory action of malate on PEPC activity was affected by the PEPC-MDH association.

Raman spectroscopic studies of NAD coenzymes bound to malate dehydrogenases by difference techniques

We report here on the Raman spectra of NADH, 3-acetylpyridine adenine dinucleotide, APAD+, and a fragment of these molecules, adenosine 5'-diphosphate ribose (ADPR) bound to the mitochondrial (mMDH) and cytoplasmic (or soluble, sMDH) forms of malate dehydrogenase. We observe changes in the Raman spectrum of the adenosine moiety of these cofactors upon binding to mMDH, indicating that the binding site is hydrophobic. On the other hand, there is little change in the spectrum of the adenosine moiety when it binds to sMDH. Such observations are in clear contrast with those results obtained in LDH and LADH, where there are significant changes in the spectrum of the adenosine moiety when it binds to these two proteins. A strong hydrogen bond is postulated to exist between amide carbonyl group of NAD+ and the enzyme in the binary complexes with both mMDH and sMDH on the basis of a sizable decrease in the frequency of the carbonyl double bond. The interaction energy for formation of a hydrogen bond is the same as found previously for LDH, and we estimate that it is 2.8 kcal/mol more favorable in the binary complex than in water. A hydrogen bond is also detected between the amide-NH2 group of NADH and sMDH that is stronger than that formed in water and is of the same size as found in LDH. Surprisingly, the hydrogen bond to the -NH2 group in mMDH is the same as that found for water.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of a null allele for MDH isozymes in the guppy (Poecilia reticulata), with special reference to sex-linked inheritance.

Malate dehydrogenase (MDH) was examined by starch gel electrophoresis for strain differences in the guppy, Poecilia reticulata. The electrophoretic patterns revealed duplicate gene loci encoding a mitochondrial form of MDH and the existence of a null allele at one of the duplicate loci. Different phenotypic frequencies at Mdh-4 were observed among 9 strains and between males and females within each strain. The progeny tests for Mdh-4 variants in the inter-strain crosses indicated a mode of partial sex-linked inheritance.

Immunocytochemical localization of transferrin and mitochondrial malate dehydrogenase in the developing nervous system of the rat.

Transferrin accumulates within neurons of the developing nervous system of humans, sheep, pigs and chickens. To assess the relationship of this accumulation with the ontogeny of oxidative metabolism, we studied the immunocytochemical localization of transferrin (Tf) and the mitochondrial form of malate dehydrogenase (mMDH) in developing neural tissues by the peroxidase-antiperoxidase method. Rabbit anti-rat Tf was obtained commercially and gave a single band of reaction product (MW = 80 kd) on Western blots. Antibodies to porcine heart mMDH were elicited in a rabbit. Western blot analysis showed that this anti-porcine mMDH antibody reacted with the mMDH from porcine, rat or avian tissue but not with the cytosolic MDH from pigs. Tf was first detected in rat brain neurons at about the 18th embryonic day and reached a peak at about the 6th postnatal day. All neurons were immunoreactive with large neurons throughout the brain showing a strong reaction for Tf. From this time onward, the level in brain neurons gradually decreased until adulthood. However, Tf immunoreactivity still remained strongly evident in capillary endothelial cells. The localization of Tf within rat spinal cord neurons peaked as early as the 1st postnatal day and remained elevated to the 6th postnatal day. By contrast, reactivity for Tf within dorsal root ganglia neurons was intense as early as the 18th embryonic day and diminished only gradually. Mitochondrial MDH, a marker for oxidative metabolism, appeared to reach a peak after the crest of intraneuronal Tf had been observed. For example, brain and spinal cord MDH immunoreactivity increased with intense staining in the cell bodies and fibers of neurons from the 6th to the 13th postnatal day; immunoreactivity gradually diminished into adulthood. The gradient of reactivity was low in some areas of the brain but more intense in areas containing large neuronal cell bodies such as the red nucleus. This occurred after the peak of intraneuronal Tf at day 6 and suggested a precursor-product relationship. By contrast, immunoreactivity for neuron-specific enolase, a glycolytic enzyme, showed a developmental pattern that differed from either Tf or MDH in that reactivity appeared later in development and was less intense. These data suggest that as cerebral metabolic rates begin to increase as early as 5-6 days after birth in the rat, an increase in mMDH occurs coincident with the onset of oxidative metabolism. Furthermore, this rise in intraneuronal mMDH follows the peak of intraneuronal Tf and suggests that Tf supplies the iron required for the synthesis of other mitochondrial ferroproteins.

Isoenzymes from Hulls and Seeds of Developing Pea Fruits

We have surveyed the distribution of multiple forms of thirteen enzyme activities in the pod wall (hull), seedcoats, cotyledons and embryonic axes from developing pods of the garden pea ( Pisum sativum L.). Many profiles reveal forms that appear to be confined to specific organs. Three forms of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) are confined to the embryonic axis; one form of phosphoglucoisomerase (EC 5.3.9.1) and two forms of glutamate dehydrogenase (EC 1.4.1.3) are confined to the embryo; one form of phosphoglucomutase (EC 2.7.5.1) and two forms of malate dehydrogenase (EC 1.1.1.37) are confined to the hull. For most activities, there are more isoenzymes in the reproductive organs than have hitherto been identified from leaves. This information should stimulate further studies on the properties, subcellular locations and functions of multiple enzyme forms, and assist the development of genetic linkage maps, which have so far depended substantially on leaf isoenzymes.

Electrophoretic study of some enzymatic genes inMusca domesiicaL.

Abstract Cellogel electrophoresis has been used to study isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), malic enzyme (ME), and alcohol dehydrogenase (ADH), in the housefly. Two forms of MDH (MDH, and MDH2) have been separated. These show properties similar to those ascribed to supernatant and mitochondrial MDH enzymes in other organisms. In the housefly zymograms of the mitochondrial fraction show MDH2 activity only. Allelic variants of MDH1 differing in thermostability and resistance to p‐hydroxymercuribenzoate have been revealed by qualitative analysis. MDH2 is invariant. These findings indicate that the two forms are isozymes under independent genetic control. Other enzymes examined here exhibit only one isozyme, which shows genetic polymorphism in some strains. Using the variants and visible markers, the linkage groups of the structural loci of the enzymes have been identified: for some of them the genetic map position has been determined.

Enzymatic Changes in Post-meiotic Anther Development in Petunia hybrida. I. Anther Ontogeny and Isozyme Analyses

Summary The relationship of the occurrence of isozymes of esterase, peroxidase, alcohol dehydrogenase (ADH) and malate dehydrogenase (MDH) with nine stages of the post-meiotic anther development of Petunia hybrida was examined. The esterases were primarily found in stages i to iii when tapetum degeneration was taking place, and later a single band was observed during pollen maturation. Peroxidases were also observed during the earlier stages and their occurrence was related to the differentiation of the endothecium. The activity of ADH was not observed until stage iv and it increased through to anther maturation. The occurrence of ADH was considered to be due to the anaerobic conditions of the maturing pollen and the dessication of anther tissue at later stages of development. Different forms of MDH were present at all nine stages of development and were suggested to be involved with the differentiation of various tissues.

Genetics of the malate dehydrogenase isozymes of the isopod porcellio scaber

AbstractElectrophoretic analysis of malate dehydrogenase (MDH) in extracts of the isopod Porcellio scaber Latr. indicates two zones of activity, MDH‐1 and MDH‐2, corresponding to the cytoplasmic and mitochondrial forms respectively. Electrophoretic variants of both forms have been isolated from natural populations. The locus encoding MDH‐1 has at least three alleles in appreciable frequency; the MDH‐2 locus has two. Breeding studies indicate that variants of both forms of MDH have Mendelian inheritance patterns, and that the two proteins are encoded by independently assorting autosomal genes. Neither MDH gene is apparently linked to the autosomal gene which encodes phosphoglucose isomerase.

Gemeinsame Präparation von drei Dehydrogenasen und zwei Kinasen in einem Arbeitsgang aus Schweineherz / The Preparation of Three Dehydrogenases and Two Kinases from Pig Heart by a Single Procedure

A single procedure for the preparation of lactate dehydrogenase (EC 1.1.1.27), the mitochondrial and cytoplasmic forms of malate dehydrogenase (EC 1.1.1.37), adenylate kinase (EC 2.7.4.3) and pyruvate kinase (EC 2.7.1.40) from pig heart is described. The five enzymes are obtained in preparative amounts in homogenous form with specific activities equal to or higher than those previously reported. Some molecular properties of pig heart pyruvate kinase are determined.