Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.37 (malate dehydrogenase)
4,591 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter-Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients. An isolation method was developed using Triton X-100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub-optimal Triton X-100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane. The mitochondria and peroxisomes could not be clearly separated using conventional sucrose-Ficoll density gradients. Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes ("heavy-labelling"). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact. The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen-peroxide-producing oxidases were detected. The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 muM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine.
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PMID:Mitochondria and peroxisomes from the cellular slime mould Dictyostelium discoideum. Isolation techniques and urate oxidase association with peroxisomes. 24 46

Multiple genes for thioredoxins (TRX) have been demonstrated in Dictyostelium discoideum. We expressed the cDNA for one of these genes (DdTrx1) in E. coli and purified the protein to homogeneity. The interaction with classic substrates as well as TRX reductases was analysed. It reacted with every tested substrate: insulin, NADP-dependent malate dehydrogenase and fructose-1,6-bisphosphatase. With a S0.5 of 20 microM, the reactivity with the fructose-1,6-bisphosphatase is the highest ever found for a heterologous TRX. DdTRX1 itself is accepted as a substrate by the chloroplast ferredoxin-dependent TRX reductase, as well as by the E. coli NADPH-dependent TRX reductase. Thus, the Dictyostelium TRX is functionally promiscuous. Its reactivity with insulin, chloroplast NADP-dependent malate dehydrogenase and ferredoxin-dependent TRX reductase resemble those of other TRX. It is, however, clearly different in its good interaction with chloroplast fructose-1,6-bisphosphatase and in its poor interaction with E. coli NADP-dependent TRX reductase.
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PMID:Biochemical characterization of thioredoxin 1 from Dictyostelium discoideum. 133 May 54

The examination of model robustness in the previous paper (Shiraishi, F., and Savageau, M. A. (1992) J. Biol. Chem. 267, 22919-22925 led to the suggestion that the current model for the tricarboxylic acid cycle in Dictyostelium discoideum is ill-determined with respect to one or more of the features reflecting pyruvate metabolism. This conclusion is further supported here by results of steady state and dynamic analyses. The tricarboxylic acid cycle, according to the current model, is poised on a knife's edge with its behavior rigidly determined; any alteration of the system's components leads to nonviable behavior, as exemplified by explosive accumulation of pyruvate and loss of steady state in response to a minute change in the level of malate dehydrogenase. With the additional results in this paper, we are able to refine the diagnosis of the problem and suggest three different areas of the current model that might profitably be re-examined by experiment. These include the kinetics of the reactions at the malate branch point, the turnover times for the alanine, glutamate, and aspartate pools in vivo, and the dynamic mass balances for the cofactor NAD. We also suggest a minimal modification in the current model that could alleviate or circumvent some of these problems.
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PMID:The tricarboxylic acid cycle in Dictyostelium discoideum. III. Analysis of steady state and dynamic behavior. 142 43

Thioredoxins are low molecular weight proteins which serve as hydrogen donors in a wide variety of redox reactions via reversible formation of a disulfide bridge between two neighboring cysteins. We present data demonstrating that in Dictyostelium discoideum thioredoxins constitute a highly conserved multigene family. We have isolated cDNA clones coding for three different Dictyostelium thioredoxins which show 80% mutual identity. Analysis of genomic Southern blots suggests the presence of additional genes. Except for the active site (Trp-Cys-Gly-Pro-Cys), there are only a few amino acid identities with thioredoxins from other organisms. Identity scores do not exceed 43%, the value found with the human lymphocyte protein. DdTRX1 was expressed in Escherichia coli, purified, and shown to have thioredoxin activity, as judged by its capacity to activate the NADP-malate dehydrogenase. Due to its life cycle, during which individual amoebae form a multicellular fruiting body, Dictyostelium is used to study developmental processes such as cell-type differentiation and regulation of gene expression. Transcript levels of Dictyostelium thioredoxins were regulated during the developmental cycle. Low levels of mRNAs could be detected during growth. After the onset of development, where essentially no cell divisions take place, message levels increased with maximal expression during aggregation. In later multicellular stages, RNA levels declined again. The same expression pattern could be seen for all cloned thioredoxins. Protein levels paralleled this time course with a delay of several hours as judged by Western blot and activity measurements.
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PMID:Thioredoxins from Dictyostelium discoideum are a developmentally regulated multigene family. 157 20

A steady-state computer model of the tricarboxylic cycle in Dictyostelium discoideum was analyzed using metabolic control theory. The steady state had variations of less than 0.04% over the last half of the simulation for both metabolite concentrations and fluxes. Metabolite and flux control coefficients were determined by varying enzymatic activities within 2% of their initial values and simulating the responses of metabolite concentrations and fluxes to these changes. Under these conditions, summation properties were met for most metabolite and all flux control coefficients. Maximum flux control coefficients were found for succinate dehydrogenase (0.35), malic enzyme (0.24), and malate dehydrogenase (-0.18). Comparable control was found for the reaction supplying pyruvate (0.14) and for the sum of the input amino acids (0.43), which serve as an energy source for D. discoideum. The time-dependent processes by which a new steady state was established were examined after increasing malic enzyme or malate dehydrogenase activities. This provided a method for an analysis of the mechanisms by which the observed control coefficients were generated. In addition, the effects of increasing the stimuli within 5-20% of the original enzyme activity were examined. Under these conditions, more typical of experimental stimuli and measurable responses, the metabolic model failed to return to steady state, and thus summation properties were not met. Whether "true" steady states ever occur or whether metabolic control theory can be applied in vivo is discussed.
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PMID:Systems analysis of the tricarboxylic acid cycle in Dictyostelium discoideum. II. Control analysis. 173 67

Malate dehydrogenase (MDH; EC 1.1.1.37) from Dictyostelium discoideum was purified and characterized MDH activity from whole cells was purified 275-fold. The mitochondrial and cytoplasmic MDH present co-purified through three ion exchange and affinity chromatography steps. The isoenzymes were barely separable by either disc gel electrophoresis or isoelectric focusing. The purified preparation containing both isoenzymes had a single pH optimum (9.3-9.5) and an apparent molecular weight of 70000. It exhibited linear kinetics and responded to known inhibitors of MDH, i.e. thyroxine and hydroxymalonate. Michaelis and dissociation constants obtained with this preparation were similar to those obtained with a 10-fold purified mitochondrial MDH.
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PMID:Kinetic characterization of mitochondrial malate dehydrogenase from Dictyostelium discoideum. 714 59

The MADS box transcription factor SrfA is required for spore differentiation in Dictyostelium discoideum. srfA null strains form rounded spores that do not resist adverse environmental conditions. Five genes whose expression is dependent on SrfA have been isolated by differential hybridization. One of these genes, sigC, is identical to phg1b, previously characterized in mutants with altered adhesive properties and found to encode a nine-transmembrane-domain protein. This gene is transcribed into two mRNAs as the result of alternative splicing of two internal exons. The slower-migrating mRNA codes for a shorter protein that lacks the first transmembrane fragment and is not expressed in srfA null strains. The other four genes (sigA, sigB, sigD, and 45D) are expressed only during late developmental stages. In situ hybridization experiments showed that expression of sigA, sigB, and sigD is restricted to the sorus of developing structures. sigA codes for a homologue of malate dehydrogenase that converts pyruvate to malate to replenish the tricarboxylic acid cycle. sigB encodes a protein with significant similarity to the GP63 metalloproteinase of Leishmania, leishmanolysin. The sequence of SigD is highly similar to that of several spore coat proteins of D. discoideum, and it may play a role in that structure. The gene 45D codes for an RNA-binding protein homologue whose expression is also dependent on the GATA transcription factor stalky (StkA). The expression of sigB is also dependent on both SrfA and StkA. The expression of 45D, but not of sigA, sigB, sigC, and sigD, can be induced in srfA null cells by constitutive protein kinase A activation. Strains in which either sigA, sigB, or sigD is disrupted were isolated and found to form spores that are not detectably different from those of wild-type strains.
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PMID:Dictyostelium discoideum developmentally regulated genes whose expression is dependent on MADS box transcription factor SrfA. 1466 66

A method for the isolation and purification of plasma membranes of Dictyostelium discoideum by equilibrium centrifugation on sucrose followed by Renografin continuous density gradients has been developed and monitored both with electron microscopy and a number of enzyme assays. On electron microscopy, the final plasma membrane fractions are judged to be freethe basis of of nuclei, rough endoplasmic reticulum, lysosomes and peroxisomes. Some profiles of the mitochondrial inner membranes are found within the plasma membrane fractions, but this contamination has been estimated to be only 5%. On the basis on enzyme assays, the plasma membrane fractions contain all the 5'-nucleotidase activity in the final gradients and are free of catalase, acid phosphatase and malate dehydrogenase activity (markers for peroxisomes, lysosomes, soluble enzymes and the matrix of mitochondria). Their content of glucose-6-phosphatase is reduced by more than 70%. The large majority of RNA and DNA have been removed from the preparation.
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PMID:The involvement of the plasma membrane in the development of Dictyostelium discoideum. I. Purification of the plasma membrane. 1625 Mar 37

We have reported that the differentiation-inducing factors (DIFs) DIF-1 and DIF-3, morphogens secreted from Dictyostelium discoideum, inhibit proliferation of several cancer cells via suppression of the Wnt/beta-catenin signaling pathway. However, the target molecules of DIFs involved in the anti-proliferative effects are still unknown. In the present study, DIF-1-tethered resins were synthesized to explore the target molecules of DIFs, and mitochondrial malate dehydrogenase (mMDH) was identified as one of the target molecules. In the in vitro assay, DIF-1 and other analogs including 2-MIDIF-1, DIF-3, and 6-MIDIF-3 were found to be capable of binding to mMDH but not to cytoplasmic MDH. However, only DIF-1 and 2-MIDIF-1 inhibited the enzymatic activity of mMDH. The effects of DIF analogs on ATP content and cell proliferation were then analyzed using HeLa cells. DIF-1 and 2-MIDIF-1 were found to lower the ATP content and both chemicals inhibited HeLa cell proliferation, suggesting that inhibition of mMDH activity affected cell energy production, probably leading to the inhibition of proliferation. These results suggest that the inhibition of mMDH activity by DIF-1 and 2-MIDIF-1 could be one of the mechanisms to induce anti-proliferative effects, independent of the inhibition of the Wnt/beta-catenin signaling pathway.
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PMID:Dictyostelium differentiation-inducing factor-1 binds to mitochondrial malate dehydrogenase and inhibits its activity. 2017 10

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