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[21] Extramitochondrial l-malate dehydrogenase of beef heart. [EC 1.1.1.37 l-Malate: NAD oxidoreductase]
Abstract
The chapter describes the method for the preparation of crystalline beef heart muscle extramitochondrial malate dehydrogenase. All operations are carried out at 3-5°. All additions of solid ammonium sulfate are carried out at 0°; the pH of the mixture is maintained between 7.1 and 7.3 by a dropwise addition of approximately 2 N ammonium hydroxide. The extramitochondrial malate dehydrogenase contains significantly more lysine, arginine, tyrosine, methionine, aspartic acid, and tryptophan than does the mitochondrial enzyme, and less phenylalanine, glycine, proline, and threonine. The enzyme appears to be homogeneous as determined by ultracentrifugation and electrophoretic criteria. Extramitoehondrial malate dehydrogenase activity is measured spectrophotometrically by the decrease in absorption at 340 mμ due to NADH oxidation in presence of oxaloacetate.
... The tricarboxylic acid cycle (TCA cycle) was the most important metabolic pathway in microbes, in which acetyl CoA was oxidized to produce CO 2 and ATP to provide energy for cell growth. It was the most efficient way to convert carbohydrates into energy as the metabolism link of sugars, lipids, and proteins [19,20]. Moreover, the TCA cycle was also capable of generating amino acid precursors and the cofactor NAD(P)H for a variety of chemical reactions [21]. ...
4-Acetylantroquinol B (4-AAQB) is a ubiquinone that has been shown to have multiple anticancer activities and is only found in the rare medicinal fungus A. cinnamomea in Taiwan. The large-scale production and application of 4-AAQB is thus limited due to the high host specificity, long production cycle, and low 4-AAQB content of A. cinnamomea. Additionally, the lack of molecular genetic studies on A. cinnamomea has hindered the study of the synthetic pathway of 4-AAQB. In this work, transcriptomic analysis was conducted to understand the essential metabolic nodes in the synthesis of 4-AAQB by A. cinnamomea based on the differences using glucose and fructose as carbon sources, respectively. The results showed that the glyoxylate and TCA cycle, terpenoid synthesis pathway, and the quinone ring modification pathway were clarified as the most significant factors associated with 4-AAQB synthesis. The enzymes ACS, ACU7, ACUE, GPS, PPT, P450, GEDA, YAT1, CAT2, and METXA in these pathways were the essential metabolic nodes in the synthesis of 4-AAQB. When fructose was used as the substrate, the expressions of these enzymes were upregulated, and the synthesis of some important intermediate metabolites was enhanced, thus promoting the accumulation of 4-AAQB. Our work understood the mechanism of fructose promoting the synthesis of 4-AAQB and identified the essential metabolic nodes which could provide the theoretical basis for the development of fermentation strategies to produce 4-AAQB by A. cinnamomea.
... All enzyme assays were carried out at room temperature (26 o ± 2 o C). Published procedures were used to assay the enzymes: isocitrate dehydrogenase (ICDH) (Khouw & Mc Curdy, 1969), succinate dehydrogenase (Ells, 1959), malate dehydrogenase (Englard & Siegel, 1969). Assay for each enzyme was run in triplicate, and the values obtained were averaged. ...
Benzene, petroleum-ether, chloroform and ethyl acetate extracts of Saxifraga ligulata were tested against three Gram positive bacteria viz., Bacillus subtilis, B. megaterium and Staphylococcus aureus, and five Gram negative bacteria viz., Escherichia coli, Salmonella typhimurium, Pseudomonas fluorescence, Ps. aeruginosa and Pantoea ananetis. The minimum inhibitory concentration (MIC) values of each fraction against all the bacteria were tested. Petroleum ether was found most effective, followed by ethyl acetate fraction. Both the fractions were found effective to kill all the bacteria tested, but at different doses. Benzene fraction was effective at very low concentration but was unable to produce any effect on Gram positive Staphyloccoccus aureus and Gram negative Escherichia coli. Morphology of the bacteria was severely affected when they were treated with sub lethal concentration of solvent fractions as revealed by the study of scanning electron microscopy. Also, the petroleum ether extract showed very strong antifungal activity against three plant pathogenic fungal species which were Alternaria solani, Helminthosporium oryzae and Fusarium oxysporum at nearly 250 μg/ml. This extract strongly interferes with oxido-reductase enzymes of the TCA cycle.
... The reduction of cytochrome c is measured by the increase in absorbance at 550 nm for 2 min. (ε 550nm = 20 M -1 cm -1 ):Malate dehydrogenase(Englard et al., 1969) Malate dehydrogenase catalyses the last reaction of TCA cycle, namely the NADHdependent reduction of oxaloacetate to malate. The enzyme activity is determined by measuring the oxidation of NADH for 2 min at λ = 340 nm. ...
Iron-sulfur (Fe/S) clusters are inorganic cofactors of many proteins found in nearly all prokaryotic and eukaryotic organisms. Fe/S proteins play important roles in different cellular processes, such as electron transport, enzyme catalysis or gene regulation. Eukaryotes contain Fe/S proteins in mitochondria, chloroplasts, cytosol and nucleus. In S. cerevisiae 3 different machineries cooperate to synthesise Fe/S proteins. The mitochondrial ISC-assembly machinery is required for maturation of all cellular Fe/S proteins, whereas the mitochondrial ISC-export and the cytosolic CIA-machineries are specifically involved in the formation of cytosolic and nuclear Fe/S proteins. In the first part of this study Isd11 was identified as a novel component of the mitochondrial ISC-assembly machinery. Isd11 is an essential protein of 11 kDa localized in the mitochondrial matrix and conserved only in eukaryotes. Depletion of Isd11 using generegulated yeast strains resulted in impaired activities of mitochondrial (e.g., aconitase, complex II) and cytosolic (Leu1) Fe/S enzymes. Strong defects were also observed in the de novo maturation of several mitochondrial, cytosolic and nuclear Fe/S proteins indicating that Isd11 is required for the biogenesis of all cellular Fe/S proteins. Here, it was shown that cells defective in Isd11 show deregulation of iron homeostasis. All these data indicated that Isd11 represents a novel component of the ISC-assembly machinery. Isd11 forms a stable complex with the cysteine desulfurase Nfs1 and also can interact with the scaffold protein Isu1. Surprisingly, Isd11 is not required for Nfs1 activity in vitro. However, depletion of Isd11 resulted in a strong reduction of Fe/S cluster formation on Isu1 indicating a function in early steps of biogenesis in vivo. Although, Isd11 is not needed for Nfs1 desulfurase activity, it is likely that the Isd11-Nfs1 complex is the physiological cysteine desulfurase, as both proteins are required for the Fe/S cluster assembly on Isu1. The second part of the present study was focused on the Nar1 protein. Nar1 is a component of the newly identified CIA-machinery. Biogenesis of extra-mitochondrial Fe/S proteins requires the CIA-machinery, which besides Nar1 encompasses at least three other proteins, Cfd1, Nbp35 and Cia1. Nar1 is an Fe/S protein itself, most likely containing two magnetically coupled Fe/S clusters. Yeast Nar1 is a highly conserved protein with relation to Feonly hydrogenases and contains eight conserved cysteines, four of them at the N-terminus and the other four at the C-terminus. Therefore, it was important to know whether the conserved cysteine residues are involved in coordination of the two Fe/S clusters. Using site-directed mutagenesis, it was demonstrated that three of the four N-terminal cysteines (C59A, C62A and C65) are essential residues for yeast cell viability and Nar1 function in the maturation of cytosolic Fe/S proteins, such as Leu1 or Rli1. Mutation of three of the N-terminal cysteines resulted in a loss of Fe/S cluster association. Mutation of the forth N-terminal cysteine residue (C20A) showed no effect, yet the combined mutation of both C20 and C65 lead to a more severe phenotype. These results indicate that all four N-terminal cysteines are ligands of an Fe/S cluster. Moreover, the data suggest that the N-terminal Fe/S cluster is required for stable insertion of the second Fe/S cluster at the C-terminus. Surprisingly, single mutations of the C-terminal cysteines had no influence on the incorporation of the Nar1 Fe/S clusters in vivo. However, simultaneous exchange of two cysteine residues at the C-terminus resulted in the loss of the Fe/S cluster located at the C-terminus, whereas the N-terminal cluster was still bound. The data presented in this study clearly indicate that the N- and C-terminal cysteine residues coordinate two Fe/S clusters and that these clusters are essential for Nar1 function in the maturation of cytosolic and nuclear Fe/S proteins. Furthermore, the N-terminal Fe/S cluster was found to be more labile than the C-terminal one. An explanation for this observation was suggested by the structural model of Nar1 which was derived from the crystal structure of Fe-only hydrogenases. The calculated model shows that the N-terminal cluster is surface-exposed, whereas the C-terminal Fe/S cluster is buried inside the protein. Eisen-Schwefel (Fe/S) Cluster sind anorganische Kofaktoren zahlreicher prokaryotischer und eukaryotischer Proteine. Diese Fe/S Proteine übernehmen wichtige Aufgaben bei verschiedenen zellulären Prozessen, wie dem Elektronentransport, bei Enzymkatalysen oder bei der Genregulation. In Eukaryoten sind Fe/S Proteine in den Mitochondrien, den Chloroplasten, im Cytosol und im Zellkern lokalisiert. In der Hefe Saccharomyces cerevisiae wird die Reifung der Fe/S Proteine von mindestens drei komplexen Maschinerien übernommen. Eine davon ist die in der mitochondrialen Matrix lokalisierte „iron-sulfur cluster“ (ISC) Assemblierungsmaschinerie, die an der Reifung aller zellulären Fe/S Proteine beteiligt ist. Dagegen werden die mitochondriale ISC-Export- und die „cytosolic iron-sulfur protein assembly“ (CIA) Maschinerien spezifisch nur für die Reifung cytosolischer und nukleärer Fe/S Proteine benötigt. Im ersten Teil dieser Arbeit wurde Isd11 als eine neue Komponente der mitochondrialen ISC-Assemblierungsmaschinerie in Hefe identifiziert. Isd11 ist ein essentielles Protein mit einer molekularen Masse von 11 kDa, das in der mitochondrialen Matrix lokalisiert ist. Es ist in allen Eukaryoten, nicht aber in Prokaryoten konserviert. Die Depletion von Isd11 durch regulierte Genexpression in einer Hefemutante führte zur Beeinträchtigung der Aktivität von mitochondrialen (z.B. Aconitase, Komplex II) und cytosolischen (Leu1) Fe/S Enzymen. Es wurden auch starke Defekte in der de novo Synthese von mitochondrialen, cytosolischen und nukleären Fe/S Proteinen beobachtet. Diese Ergebnisse deuten darauf hin, dass Isd11 an der Reifung aller zellulären Fe/S Proteine beteiligt ist. In dieser Arbeit wurde gezeigt, dass Isd11-defiziente Zellen eine fehl regulierte zelluläre Eisenhomöostase aufweisen. Diese Ergebnisse weisen darauf hin, dass Isd11 eine neue Komponente der ISC-Assemblierungsmaschinerie ist. Isd11 bildet einen stabilen Komplex mit der Cystein-Desulfurase Nfs1 und interagiert, vermutlich indirekt über Nfs1, mit dem Gerüstprotein Isu1. Zwar wird Isd11 nicht für eine in vitro Aktivität von Nfs1 benötigt, doch führte die Depletion von Isd11 zu einer stark verminderten Synthese von Fe/S Clustern auf Isu1. Damit kommt Isd11 eine in vivo Funktion in der frühen Phase der Fe/S Proteinbiogenese zu. Obwohl Isd11 nicht für die Aktivität von Nfs1 erforderlich ist, stellt ihr Komplex die physiologische Cystein-Desulfurase dar, die zur Assemblierung eines Fe/S Clusters auf Isu1 benötigt wird. Im zweiten Teil der Arbeit lag der Schwerpunkt auf der molekularen und funktionellen Charakterisierung von Nar1, einem Protein der erst vor kurzem identifizierten CIA Maschinerie. Diese Maschinerie wird für die Biogenese von extra-mitochondrialen Fe/S Proteinen benötigt und umfasst neben Nar1 noch mindestens die drei Proteine Cfd1, Nbp35 und Cia1. Nar1 ist ein Fe/S Protein und enthält vermutlich zwei gekoppelte Fe/S Cluster. Nar1 der Hefe ist ein hoch konserviertes Protein, das Homologien zu bakteriellen Fe-Hydrogenasen aufweist und acht konservierte Cysteinreste enthält. Je vier davon sind am N- und C-Terminus lokalisiert. Für eine Charakterisierung von Nar1 war es wichtig zu untersuchen, ob die konservierten Cysteinreste für die Koordination der beiden Fe/S Cluster benötigt werden. Mit Hilfe einer gerichteten Mutagenese wurde gezeigt, dass drei der vier N-terminalen Cysteinreste (C59, C62 und C65) für das Überleben der Hefe essentiell waren. Ein Austausch der Cysteinreste zu Alanin- oder Serinresten führte zum reduzierten Einbau von radioaktivem Eisen-55 (55Fe) in die Fe/S-Proteine Leu1 und Rli1, sowie zum Verlust der Enzymaktivität von Leu1. Beides wies auf eine essentielle Rolle der entsprechenden Cysteinreste für die Funktion von Nar1 bei der Reifung extra-mitochondrialer Fe/S Proteine hin. Die Mutation von jeweils einem der drei N-terminalen Cysteinreste führte zu einem fast kompletten Verlust an gebundenem Fe/S Cluster. Eine Mutation des vierten N-terminalen Cysteins (C20A) alleine zeigte keinen Effekt, jedoch führte die kombinierte Mutation von C20 und C65 zu einem stärkeren Phänotyp der C65 Mutante. Diese Daten legen nahe, dass alle vier N-terminalen Cysteinreste koordinierende Liganden sind. Überdies weisen die Ergebnisse darauf hin, dass der N-terminale Fe/S Cluster für die Assemblierung des zweiten Fe/S-Zentrums benötigt wird. Interessanterweise hatten Mutationen einzelner Cysteinreste am C-Terminus in vivo keinen Einfluss auf den Einbau von Fe/S Clustern in Nar1. Wurden jedoch zwei C-terminale Cysteinreste gleichzeitig ausgetauscht, so führte dies zum Verlust des C-terminalen, nicht jedoch des N-terminalen Fe/S Clusters. Damit konnte diese Studie zum einen belegen, dass die N- und C-terminalen Cysteinreste jeweils einen Fe/S Cluster koordinieren, und zum anderen, dass beide Fe/S Cluster essentiell für die Funktion von Nar1 bei der Reifung extramitochondrialer Fe/S Proteine sind. Darüber hinaus war der N-terminale Fe/S Cluster labiler als derjenige am C-Terminus. Eine Erklärung für diese Beobachtung ergibt sich aus der modellierten Struktur von Nar1, die aus der Kristallstruktur von Fe-Hydrogenasen berechnet wurde. Im Strukturmodel von Nar1 ist der N-terminale Fe/S Cluster zur Proteinoberfläche hin exponiert, während der C-terminale Fe/S Cluster im Inneren des Proteins verborgen liegt.
... was assayed by measuring the decrease in absorbance at 340 nm due to NADH oxidation (e NAD + 56.3 mM 21 cm 21 ) with oxaloacetate. The assay contained 50 mM triethanolamine.HCl (pH 7.4), 5 mM EDTA, 0.12 mM oxaloacetate (pH 7.0), 0.15 mM NADH, and was started by addition of protein (Englard, 1969). Fumarate hydratase (EC 4.2.1.2) was assayed under aerobic conditions as described by Genda et al. (2006) in 100 mM phosphate buffer (pH 7.6), 50 mM malate and protein to start the reaction. ...
A mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml(-1). Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP(+) oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD(+) or FMN(+) as electron acceptor but is inactive with NAD(+), Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.
... However, when the low concentrations of both initial substrates (oxaloacetate and NADH) and the relatively high levels of products (malate, NAD') are considered, a different picture emerges. An approximate calculation based on values in the literature [44] shows that the enzyme is probably operating at well below its apparent K, values for NADH and oxaloacetate, and that the net rate of operation of the enzyme in vivo could be as low 3 -4 pmol x min-' x g-'. Therefore, when ethanol is oxidised, NADH accumulates to the point at which its rate of removal by malate dehydrogenase equals its rate of production by alcohol dehydrogenase. ...
1. Ethanol was oxidised more slowly by rats which were given an ethanol dose of 5.1 g/kg than by rats which were given an ethanol dose of 1.4 g/kg. 2. A positive correlation was found between [lactate]/[pyruvate] ratios and rates of ethanol oxidation. 3. Acetaldehyde concentrations varied widely between rats, but in some cases were high enough to influence rates of ethanol oxidation. 4. Liver alcohol dehydrogenase levels were just sufficient to account for ethanol oxidation rates observed in vivo. 5. Pre-administration of a large ethanol dose (6.5 g/kg) did not alter mean [lactate]/[pyruvate] ratios or ethanol oxidation rates during metabolism of test doses of 2.5 g/kg. 6. Injection of pyruvate did not increase rates of ethanol oxidation. 7. The results do not support suggestions that a high-Km ethanol oxidising system plays an important role in vivo, that increased rates of ethanol oxidation can be induced by large, acute ethanol doses or that the rate of NADH reoxidation controls rates of ethanol metabolism. 8. The results support other evidence which has indicated that the level of alcohol dehydrogenase is the major factor limiting rates of ethanol oxidation in vivo.
Sucrose is a vital ingredient in numerous food items consumed regularly. However, exposure to excessive sucrose for a prolonged period can promote health issues. The reproductive system has a delicate physiology that can be
targeted by various chemical stressors, including sucrose. Hence, the present in vivo study aims to unveil the impacts of High-Sucrose Diet (HSD) on the reproductive fitness of Drosophila melanogaster. In addition, the present work has also assessed the protective potential of a bioactive compound, rutin, against it. Here, first instar larvae were exposed to HSD (30 %) alone and in combination with rutin (100–300 µM) till their adult stage. HSD disturbed sex comb morphology in adult males, while fecundity and hatchability of eggs in females. Moreover, HSD triggered gonadal ROS production, oxidative stress, and modulated endogenous antioxidants such as SOD, catalase, and glutathione in both sexes. Nuclear fragmentation and tissue injuries, along with protein and lipid oxidation, were also apparent. Elevated levels of cytosolic Iron suggested an active Fenton reaction in adults. Further, HSD modulated the activities of reproductive and metabolic mediators, including
vitellogenin, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and angiotensin-converting enzymes
that are critical to maintain the overall reproductive fitness. Interestingly, co-treatment with rutin, mainly at
200 µM, mitigated these adverse effects and restored reproductive fitness. The protective potential of rutin might be attributed to its ability to normalize redox homeostasis, reduce oxidative stress, and optimize critical enzymes involved in reproductive physiology. These findings suggest that rutin has potential therapeutic implications for counteracting the reproductive hazards induced by HSD.
Peroxiredoxins (Prxs) play dual roles as both thiol-peroxidases and molecular chaperones. Peroxidase activity enables various intracellular functions, however, the physiological roles of Prxs as chaperones are not well established. To study the chaperoning function of Prx, we previously sought to identify heat-induced Prx-binding proteins as the clients of a Prx chaperone. By using His-tagged Prx I as a bait, we separated ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a heat-induced Prx I binding protein from rat brain crude extracts. Protein complex immunoprecipitation with HeLa cell lysates revealed that both Prx I and Prx II interact with UCH-L1. However, Prx II interacted considerably more favorably with UCH-L1 than Prx I. Prx II exhibited more effective molecular chaperone activity than Prx I when UCH-L1 was the client. Prx II interacted with UCH-L1 through its C-terminal region to protect UCH-L1 from thermal or oxidative inactivation. We found that chaperoning via interaction through C-terminal region (specific-client chaperoning) is more efficient than that involving oligomeric structural change (general-client chaperoning). Prx II binds either thermally or oxidatively unfolding early intermediates of specific clients and thereby shifted the equilibrium towards their native state. We conclude that this chaperoning mechanism provides a very effective and selective chaperoning activity.
The distribution of α-mannosidase (EC 3.2.1.24) in various tissues of Canavalia ensiformis has been studied and a new form of the enzyme has been identified in the leaf. The new enzyme is immunologically distinct from the well-characterized seed enzyme since the polyclonal rabbit antisera against the latter, failed to cross-react with it in immuno-difrusion, immunoprecipitation and immunoblot experiments. The native enzyme has an approximate Mr of 217 000 and a pH optimum near 4, and it is stimulated by Zn2+. In these respects, it is similar to the seed enzyme.
During starvation, muscle glycogen in Boleophthalmus boddaerti was utilized preferentially over liver glycogen. In the first 10 days of fasting, the ratio of the active‘a’form of glycogen phosphorylase to total phosphorylase present in the liver was small. During this period, the active‘I’form of glycogen synthetase increased in the same tissue. In the muscle, the phosphorylase‘a’activity declined during the first 7 days and increased thereafter while the total glycogen synthetase activity showed a drastic decline during the first 13 days of fasting.
The glycogen level in the liver and muscle of mudskippers starved for 21 days increased after refeeding. After 6 and 12 h refeeding, liver glycogen level was 8·5 ± 2·3 and 6·9 ± 4·5 mg·g wet wt ¹ , respectively, as compared to 5·8 ± l·6mg·g wet wt ¹ in unfed fish. Muscle glycogen level after 6 and 12 h refeeding was 0·96±0·76 and 0·82 ± 0·50 mg·g wet wt ¹ , respectively, as opposed to 0·21 ± 0·12 mg·g wet wt ¹ in the 21‐days fasted fish. At the same time, activities of glycogen phosphorylase in the muscle and liver increased while the active‘I’form of glycogen synthetase showed higher activity in the liver. Since glycogen was resynthesized upon refeeding, this eliminated the possibility that glycogen depletion during starvation was due to stress or physical exhaustion after handling by the investigator.
Throughout the experimental starvation period, the body weight of the mudskipper decreased, with a maximum of 12% weight loss after 21 days. Liver lipid reserves were utilized at the onset of fasting but were thereafter resynthesized. Muscle proteins were also metabolized as the fish were visibly thinner. However, no apparent change in protein content expressed as per gram wet weight was detected as the tissue hydration state was maintained constant. The increased degradation of liver and muscle reserves was coupled to an increase in the activities of key gluconeogenic enzymes in the liver (G6Pase, FDPase, PEPCK, MDH and PC). The increase in glucose synthesis was possibly necessary to counteract hypoglycemia brought about by starvation in B. boddaerti.
Onchidium tumidium, an intertidal pulmonate, has evolved to depend mainly on the formation of succinate, rather than lactate and opines, to survive in anoxia. For our study O. tumidum were collected from the mud flats of the mangrove swamp at Mandai, Singapore between 1988 and 1991. After 24 h of anoxic exposure, the lactate and succinate contents of the anoxic individuals were approximately 10 and 150 times, respectively, the corresponding values of the normoxic individuals. Alanine and acetate accumulations also occurred during anoxia, though to a much lesser extent. No propionate or octopine was detected. The depletion in aspartate content in O. tumidium could not account for the amount of succinate accumulated during anoxia. The succinate formed might have originated from glycogen involving the flow of carbon through the phosphoenolpyruvate (PEP) branch point of glycolysis. In support of such a hypothesis, results indicate that there was a decrease in the affinity of pyruvate kinase from O. tumidium exposed to 24h of anoxia to PEP to facilitate succinate formation through phosphoenolpyruvate carboxykinase (PEPCK). In comparison, the affinity of PEPCK from O. tumidium exposed to anoxia to PEP was apparently unaltered.
This is the first report on in vivo effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on mitochondrial respiration in mouse brain. C57/BL mice, injected with 40 mg/kg of MPTP subcutaneously, were sacrificed by cervical dislocation 5–6 h after the injection. Mitochondrial suspensions were prepared from whole brains. Mitochondrial respiration was studied polarographically. The state 3 respiration, i.e., the active respiration in the presence of tricarboxylic acid (TCA) cycle substrates and ADP with coupled phosphorylation of ADP to ATP, was significantly inhibited in mice treated with MPTP. This inhibition was prevented by pretreatment of mice with pargyline. Activity of mitochondrial NADH-ubiquinone oxidoreductase was also inhibited in mice treated with MPTP.
We report herein the complete coding sequence of a Taenia solium cytosolic malate dehydrogenase (TscMDH). The cDNA fragment, identified from the T. solium genome project database, encodes a protein of 332 amino acid residues with an estimated molecular weight of 36517Da. For recombinant expression, the full length coding sequence was cloned into pET23a. After successful expression and enzyme purification, isoelectrofocusing gel electrophoresis allowed to confirm the calculated pI value at 8.1, as deduced from the amino acid sequence. The recombinant protein (r-TscMDH) showed MDH activity of 409U/mg in the reduction of oxaloacetate, with neither lactate dehydrogenase activity nor NADPH selectivity. Optimum pH for enzyme activity was 7.6 for oxaloacetate reduction and 9.6 for malate oxidation. K(cat) values for oxaloacetate, malate, NAD, and NADH were 665, 47, 385, and 962s(-1), respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56Kb genomic contig assembly is also reported.
Malate dehydrogenase (l-malate: NAD oxidoreductase, EC 1.1.1.37) from the cytoplasm of Taenia solium cysticerci (cMDHTs) was purified 48-fold through a four-step procedure involving salt fractionation, ionic exchange, and dye affinity chromatography. cMDHTs had a native M
r of 64,000, while the corresponding value per subunit, obtained under denaturing conditions, was 32,000. The enzyme is partially positive, with an isoelectric point of 8.7, and had a specific activity of 2,615 U mg−1 in the reduction of oxaloacetate. The second to the 21st amino acids from cMDHTs N-terminal group were P G P L R V L I T G A A G Q I A Y N L S. This sequence is 100% identical to that of Echinococcus granulosus. Basic kinetic parameters were determined for this enzyme. The optimum pH for enzyme reaction was at 7.6 for oxaloacetate reduction and at 9.6 for malate oxidation. K
m values for oxaloacetate, malate, NAD, and NADH were 2.4, 215, 50, and 48 µM, respectively. V
max values for the substrates and cosubstrates as described above were 1,490, 87.8, 104, and 1,714 µmol min−1 mg−1. Several NAD analogs, structurally altered in either the pyridine or purine moiety, were observed to function as coenzymes in the reaction catalyzed by the purified malate dehydrogenase. cMDHTs activity was uncompetitive inhibited by arsenate for both the forward (Ki = 8.2 mM) and reverse (Ki = 77 mM) reactions. The mechanism of the cMDHTs reactivity was investigated kinetically by the product inhibition approach. The results of this study are qualitatively consistent with an Ordered Bi Bi reaction mechanism, in which only the coenzymes can react with the free enzyme.
Effects of 1-methyl-4-phenylpyridinium ion (MPP+) on cellular respiration were studied using mitochondria prepared from mouse brains. State 3 and state 4 respiration supported by glutamate plus malate or pyruvate plus malate were significantly inhibited by 0.05 mM MPP+. On the other hand, respirations supported by succinate or alpha-glycerophosphate were not inhibited at all. Activity of mitochondrial NADH-ubiquinone oxidoreductase was significantly inhibited by MPP+. This inhibition was markedly potentiated by preincubating mitochondria with MPP+ together with glutamate plus malate. The latter observation suggested accumulation of MPP+ within the mitochondria during preincubation. When mitochondria were pretreated with an uncoupling agent such as carbonylcyanide m-chlorophenylhydrazone (CCCP) or dinitrophenol, MPP+-induced inhibition of state 3 respiration or of activity of complex I could no longer be seen. A potassium ionophore, valinomycin, showed a similar effect. Adenosine triphosphate (ATP) synthesis was also inhibited by MPP+. Among the NAD+-linked dehydrogenases in the tricarboxylic acid cycle, alpha-ketoglutarate dehydrogenase complex was significantly inhibited by MPP+. This inhibition was reversible and competitive with NAD+. Energy crisis appears to be one of the most important mechanisms of neuronal degeneration in MPTP-induced parkinsonism. Biochemical mechanisms underlying MPP+-induced inhibition of mitochondrial respiration were discussed.
A successful method for the preparation of plant malate dehydrogenase (MDH) was developed. Three isoenzymes were isolated and crystallized from maize seed. Purification of these proteins involved a course of acetone fractionation, batch and column adsorption on hydroxylapatites, gel permeation chromatography, and ionexchange on DEAE-cellulose columns. In addition, final separation of one of the component isoenzymes was accomplished by continuous flow elution electrophoresis on acrylamide gels. By these techniques it was possible to prepare 5–10 mg of each isoenzyme at one time. Two of the proteins (designated M1-MDH and M2-MDH) are very similar with respect to their charge properties and association with mitochondrial fractions. The other isoenzyme (S-MDH) is associated with the supernatant or cytosol fraction. Antibodies prepared against one of the mitochondrial forms (M1-MDH) cross-reacts with the other form from the mitochondria (M2-MDH) and shows a reaction of identity on agar double diffusion tests. The antibodies against the mitochondrial malate dehydrogenase show no cross-reactivity with the supernatant protein. This preparation of malate dehydrogenase isoenzymes represents the first procedure for obtaining these proteins in a homogenous state from a plant, source, and it is the first purification and separation of multiple mitochondrial isoenzymes as separate entities.
The theory that the rate of ethanol oxidation is governed by rates of NADH reoxidation is based in part on the observation that the ratio of free cytosolic [NADH]/[NAD+] increases during ethanol metabolism. However, it has recently been suggested that the amount of alcohol dehydrogenase governs rates of ethanol metabolism, which then leaves the change in cytosolic redox state unexplained. In this paper the kinetic parameters for rat liver malate dehydrogenase, determined at 37 degrees C and pH 7.4, are used to provide an explanation for the change in cytosolic redox state that is compatible with rate control by alcohol dehydrogenase.
When leupeptin, a thiol protease inhibitor of microbial origin, was injected into rats, the activity of fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) in the liver decreased to about 60% of that in control rats. However, the concentration of aldolase protein in the liver extracts, measured with a specific antibody obtained with enzyme purified on a phosphocellulose column, remained unchanged. Injection of leupeptin also caused a marked increase in the activities of free lysosomal proteases, such as cathepsin B (EC 3.4.22.1), cathepsin L (EC 3.4.22.-), cathepsin D (EC 3.4.23.5) and lysosomal carboxypeptidase A in the cytosol fraction. A clear inverse relationship between aldolase and cathepsin B activities in the cytosol fraction was demonstrated. The possibility that the less active form of aldolase detected in the livers of leupeptin-treated rats was produced during homogenization was excluded by showing that the aldolase activity was not changed by addition of various protease inhibitors to the homogenization medium., When insulin was coinjected with leupeptin, increase in the activity of free cathepsin L and decrease of activity of aldolase produced by the injection of leupeptin was prevented. These findings indicate that modification of aldolase may be due to the action of a lysosomal protease(s). Enhanced sensitivity of lysosomes to osmotic shock was demonstrated in the livers of leupeptin-treated rats, suggesting that the lysosomal membrane is labilized by administration of leupeptin. Incubation of the purified aldolase with the lysosomal fraction produced the same changes in properties of aldolase as those observed in vivo on injection of leupeptin.
The two malic dehydrogenases from beef heart, identified as being supernatant and mitochondrial in origin, have been compared further with regard to kinetic behavior and additional striking differences have been observed.With respect to sulfhydryl groups of mitochondrial malic dehydrogenase and supernantant malic dehydrogenase the following have been established. First, mitochondrial malic dehydrogenase has twice the number of half-cystine residues contained in supernatant malic dehydrogenase (12 as opposed to 6). Secondly, all sulfhydryl groups of undenatured mitochondrial malic dehydrogenase can be titrated with p-mercuribenzoate, although reaction occurs slowly, while only half of the sulfhydryl groups of supernantant malic dehydrogenase can be titrated even in presence of excess reagent. Finally, the titration of mitochondrial malic dehydrogenase with p-mercuribenzoate results in loss of enzymic activity after addition of only three equivalents of reagent, whereas no loss of activity of supernatant-malic dehydrogenase occurs after half its sulfhydryl groups have been titrated.Significant differences in amino acid composition exist between mitochondrial malic dehydrogenase and supernatant malic dehydrogenase. The latter enzyme contains more lysine, arginine, tyrosine, methionine, aspartic acid and tryptophan than does the former. On the other hand, mitochondrial malic dehydrogenase contains more phenylalanine, glycine, proline and threonine.
Differential extraction of beef-heart muscle revealed the presence of two malic dehydrogenases. One, apparently derived from the supernatant fraction, is easily extractable and exhibits no inhibition of oxidation of DPNH by elevated concentrations of oxaloacetate. The other, presumably mitochondrial in origin, requires more drastic means of extraction and under similar defined conditions of assay is inhibited by elevated concentrations of oxaloacetate. A method of purifying the supernatant malic dehydrogenase is described. By a combination of ammonium sulfate fractionation, heat inactivation, chromatography on DEAE-cellulose, starch-block electrophoresis, and finally dialysis against ammonium sulfate solutions, a crystalline malic dehydrogenase has been prepared. Electrophoretic studies over a wide range of pH values revealed that the preparation was essentially homogeneous. The enzyme was also homogeneous in the ultracentrifuge, and had an s20,w0 of 5.1·10-13 sec and a D20,w0 of 9.1·10-7 cm2/sec. From these data, assuming a partial specific volume of 0.74 ml/g, a molecular weight of 52 000 was calculated. Some kinetic constants of the enzyme have been determined and its specificity examined.
Malic dehydrogenase has been purified from acetone-dried powders of thoroughly washed minces of whole beef heart.