The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide-adenine dinucleotide phosphate-linked isoenzymes.
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ABSTRACT:
SUBMITTER: Self CH
PROVIDER: S-EPMC1174318 | biostudies-other | 1972 Nov
REPOSITORIES: biostudies-other
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Project description:Of the two NADP-linked isocitrate dehydrogenases in Acinetobacter lwoffi the higher-molecular-weight form, isoenzyme-II, is reversibly stimulated sixfold by low concentrations of glyoxylate or pyruvate. Kinetic results indicate that this stimulation of activity involves both an increase in V(max.) and a decrease in the apparent K(m) values for substrates, most markedly that for NADP(+). Other changes brought about by glyoxylate or pyruvate include a shift in the pH optimum for activity and an increased stability to inactivation by heat or urea. Mixtures of glyoxylate plus oxaloacetate, known to inhibit isocitrate dehydrogenases from other organisms, produce inhibition of both A. lowffi isoenzymes, and do not reflect the stimulatory specificity of glyoxylate for isoenzyme-II. Isoenzyme-II is also stimulated by AMP and ADP, but the activation by glyoxylate or pyruvate is shown to be quite independent of the adenylate activation. Differential desensitization of the enzyme by urea to the two types of activator further supports the view that the enzyme possesses two distinct allosteric regulatory sites. The metabolic significance of the activations is discussed.
| S-EPMC1177580 | biostudies-other
Project description:Pre-steady-state studies of the isocitrate dehydrogenase reaction show that the rate constant for the hydride-transfer step is above 990s-1, and that both subunits of the enzyme are simulataneously active. After the fast formation of NADPH in amounts equivalent to the enzyme subunit concentration, the rate of NADPH formation is equal to the steady-state rate if the enzyme has been preincubated with isocitrate and Mg2+. If the enzyme has been preincubated with NADP+ and Mg2+, in 0.05 M-triethanolamine chloride buffer, pH 7.0, with the addition of 0.1 M-NaCl, the amount of NADPH formed in the fast phase is only 60% of the enzyme subunit concentration, and the turnover rate is at first lower than the steady-state rate. In 0.05 M-triethanolamine chloride buffer, pH 7.0, if the enzyme is preincubated with NADP+ or NADPH, the turnover rate increases 3-fold to reach the steady-state rate after about 5 s. Preincubation of the enzyme with isocitrate and Mg2+ abolishes this lag phase, the steady-state rate being reached at once. It is suggested that the enzyme exists in at least two conformational forms with different activities, and that the lag phase represents the transition (k = 0.4s-1) from a form with low activity to the fully active enzyme, induced by the binding of isocitrate and Mg2+.
| S-EPMC1184022 | biostudies-other
Project description:Here we report a new robust nicotinamide dinucleotide phosphate cofactor analog (carba-NADP+ ) and its acceptance by many enzymes in the class of oxidoreductases. Replacing one ribose oxygen with a methylene group of the natural NADP+ was found to enhance stability dramatically. Decomposition experiments at moderate and high temperatures with the cofactors showed a drastic increase in half-life time at elevated temperatures since it significantly disfavors hydrolysis of the pyridinium-N-glycoside bond. Overall, more than 27 different oxidoreductases were successfully tested, and a thorough analytical characterization and comparison is given. The cofactor carba-NADP+ opens up the field of redox-biocatalysis under harsh conditions.
| S-EPMC8252718 | biostudies-literature
Project description:1. Rat liver and heart major isoenzymes of NADP-isocitrate dehydrogenase have each been purified about 100-fold by a combination of ammonium sulphate fractionation and chromatography on ion-exchange cellulose and their properties compared. 2. The properties were similar in respect of pH, inhibition by Hg(2+) and Michaelis constants for isocitrate and NADP. 3. Some of the properties of the isoenzymes were different. 4. The heart isoenzyme was activated about 210% by 0.8m-ammonium sulphate whereas the liver isoenzyme was unaffected. The heart isoenzyme showed greater sensitivity to inactivation by heat (30 degrees C for 30min), whereas the liver isoenzyme was more sensitive to inactivation by p-chloromercuribenzoate and by Cu(2+). 5. The Michaelis constants with 3-acetylpyridine-adenine dinucleotide phosphate showed a twofold difference between liver and heart isoenzyme. 6. The differential sensitivity to heat and its mainly non-cytoplasmic location may be an explanation of the failure of plasma isocitrate dehydrogenase activity to increase after a myocardial infarction.
| S-EPMC1174258 | biostudies-other
Project description:1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems associated with the use of immobilized derivatives for enzyme binding and mechanistic studies are briefly discussed.
| S-EPMC1165874 | biostudies-other
Project description:1. The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.
| S-EPMC1184021 | biostudies-other
Project description:Nicotinamide adenine dinucleotide (NAD+) is a key redox compound in all living cells responsible for energy transduction, genomic integrity, life-span extension, and neuromodulation. Here, we report a new function of NAD+ as a molecular photocatalyst in addition to the biological roles. Our spectroscopic and electrochemical analyses reveal light absorption and electronic properties of two π-conjugated systems of NAD+. Furthermore, NAD+ exhibits a robust photostability under UV-Vis-NIR irradiation. We demonstrate photocatalytic redox reactions driven by NAD+, such as O2 reduction, H2O oxidation, and the formation of metallic nanoparticles. Beyond the traditional role of NAD+ as a cofactor in redox biocatalysis, NAD+ executes direct photoactivation of oxidoreductases through the reduction of enzyme prosthetic groups. Consequently, the synergetic integration of biocatalysis and photocatalysis using NAD+ enables solar-to-chemical conversion with the highest-ever-recorded turnover frequency and total turnover number of 1263.4 hour-1 and 1692.3, respectively, for light-driven biocatalytic trans-hydrogenation.
| S-EPMC6641943 | biostudies-literature
Project description:Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate oxidase (NOX) is required for liver fibrosis. This study investigates the role of NOX in ROS production and the differential contribution of NOX from bone marrow (BM)-derived and non-BM-derived liver cells. Hepatic fibrosis was induced by bile duct ligation (BDL) for 21 days or by methionine-choline-deficient (MCD) diet for 10 weeks in wild-type (WT) mice and mice deficient in p47phox (p47phox knockout [KO]), a component of NOX. The p47phox KO chimeric mice were generated by the combination of liposomal clodronate injection, irradiation, and BM transplantation of p47phox KO BM into WT recipients and vice versa. Upon BDL, chimeric mice with p47phox KO BM-derived cells, including Kupffer cells, and WT endogenous liver cells showed a ?25% reduction of fibrosis, whereas chimeric mice with WT BM-derived cells and p47phox KO endogenous liver cells, including hepatic stellate cells, showed a ?60% reduction of fibrosis. In addition, p47phox KO compared to WT mice treated with an MCD diet showed no significant changes in steatosis and hepatocellular injury, but a ?50% reduction in fibrosis. Cultured WT and p47phox KO hepatocytes treated with free fatty acids had a similar increase in lipid accumulation. Free fatty acids promoted a 1.5-fold increase in ROS production both in p47phox KO and in WT hepatocytes.NOX in both BM-derived and non-BM-derived cells contributes to liver fibrosis. NOX does not play a role in experimental steatosis and the generation of ROS in hepatocytes, but exerts a key role in fibrosis.
| S-EPMC2947612 | biostudies-literature
Project description:Reperfusion after stroke leads to infiltration of inflammatory cells into the ischemic brain. Nicotinamide adenine dinucleotide phosphate oxidase (NOX2) is a major enzyme system that generates superoxide in immune cells. We studied the effect of NOX2 derived from the immune cells in the brain and in blood cells in experimental stroke.To establish whether NOX2 plays a role in brain ischemia, strokes were created in mice, then mice were treated with the NOX2 inhibitor apocynin or vehicle and compared to mice deficient in NOX2's gp91 subunit and their wild-type littermates. To determine whether NOX2 in circulating cells versus brain resident cells contribute to ischemic injury, bone marrow chimeras were generated by transplanting bone marrow from wild-type or NOX2-deficient mice into NOX2 or wild-type hosts, respectively.Apocynin and NOX2 deletion both significantly reduced infarct size, blood-brain barrier disruption, and hemorrhagic transformation of the infarcts, compared to untreated wild-type controls. This was associated with decreased matrix metalloproteinase 9 expression and reduced loss of tight junction proteins. NOX2-deficient mice receiving wild-type marrow had better outcomes compared to the wild-type mice receiving wild-type marrow. Interestingly, wild-type mice receiving NOX2-deficient marrow had even smaller infarct sizes and less hemorrhage than NOX2-deficient mice receiving wild-type marrow.This indicates that NOX2, whether present in circulating cells or brain resident cells, contributes to ischemic brain injury and hemorrhage. However, NOX2 from the circulating cells contributed more to the exacerbation of stroke than that from brain resident cells. These data suggest the importance of targeting the peripheral immune system for treatment of stroke.
| S-EPMC3205431 | biostudies-literature
Project description:Uterine fibroids are the most common benign tumor in women. The goal of this study was to investigate whether nicotinamide adenine dinucleotide phosphate oxidase (NOX), a major source of superoxide and subsequent oxidative stress, was differentially regulated in myometrium versus leiomyoma. Expression levels of NOXs1-5, dual oxidase (DUOX), DUOX2, NOX organizer (NOXO) 1, NOX activator 1, p47(phox), p67(phox), and p22(phox) were determined in cells treated with hypoxia by real-time reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry in tissues. Expression of NOX4 increased in fibroid compared to myometrial tissues and cells. The NOX2, DUOX1, and p67(phox) were higher while p22(phox) was lower in fibroid than that in myometrial cells. Hypoxia increased NOX4, DUOX1, and NOXO1 and decreased p22(phox) in myometrial and reduced DUOX1 in fibroid cells. The NOX1, NOX3, NOX5, and DUOX2 were undetectable. Fibroid cells are characterized by a unique NOX profile, which promotes a severe prooxidant state that may be responsible for their development. Targeting these subunits may be beneficial for future therapeutic interventions.
| S-EPMC5933080 | biostudies-literature