NADP-dependent Isocitrate Dehydrogenase Research Articles

Metabolic changes in the mouse kidney after subcutaneous injection of butyl 2‐chloroethyl sulfide

Subcutaneous exposure to vesicants such as butyl 2-chloroethyl sulfide (butyl mustard, BCS) produces local tissue injury (vesication) primarily by alkylation and cross-linking of the purine nucleotides and rapidly binding to proteins. We recently reported that administering BCS can cause other biochemical and morphological alterations, not only in tissues at the injection site but in other areas as well. In this study, we have examined the metabolic effects of BCS administration on the mouse kidney. At 1, 24, and 48 h after injection (5 microliters neat, sc), treated mice were terminated along with an untreated control group, and the kidneys were analyzed for metabolic changes. Glutathione (GSH) peroxidase (GPx) activity markedly increased, (+78 and +85%), but NADP-dependent isocitrate dehydrogenase activity decreased (-43 and -37%) at 1 and 24 h, respectively. Glucose-6-phosphate dehydrogenase (G6PD) remained unchanged at 1 and 24 h, but increased 20% (p less than .05) at 48 h after injection. Kidney glutathione S-transferase (GST) was increased at 24 h after injection. Both total and oxidized GSH levels were significantly lower than control values (approximately 30%) at all time points. Lipid peroxidation, as estimated by the thiobarbituric (TBA) acid-reactive products, was 45% lower (p less than .05) after 1 h. Kidney GPx, G6PD, and GT activities and kidney GSH levels were consistent with changes associated with oxidative stress or detoxication mechanism for BCS. The decrease in TBA-reactive products suggests that mouse kidney metabolic response to BCS injection was different from responses observed for other organs (eyes, brain, and lung).

Enzyme levels inTrichosporon cutaneumgrown on acetate, phenol or glucose

The soil yeast Trichosporon cutaneum was grown in continuous culture on phenol, acetate or glucose. The effect of carbon source and its level on the activity of key enzymes were investigated. Cells grown on acetate or phenol showed higher activity of citrate synthase, malate dehydrogenase, succinate dehydrogenase and NADP-dependent isocitrate dehydrogenase than glucose grown cells, and the activity of these enzymes depended on the level of the carbon source. Isocitrate lyase was derepressed by acetate, but was barely detectable in phenol or glucose grown cells, indicating the absence of the glyoxylate cycle. The activity of glucose-6-phosphate dehydrogenase was higher in phenol or glucose grown cells than in acetate grown cells, and its activity varied with the specific growth rate. Hexokinase was the only enzyme, the level of which was not affected by carbon source. Phenol 2-monooxygenase and catechol 1,2-dioxygenase, the first two enzymes of the 3-oxoadipate pathway, showed barely detectable activities in glucose or acetate grown cells. Assays of enzymes in crude extracts and in isolated mitochondria from batch grown cells showed that NADP-dependent isocitrate dehydrogenase was found only in the cytoplasm, while citrate synthase and malate dehydrogenase occurred in the cytoplasm as well as in the mitochondria, both in glucose and phenol groen cells. A model is proposed for the central metabolism of the two products of the 3-oxoadipate pathway, acetyl-CoA and succinate.

Multinuclear NMR studies of the divalent metal binding site of NADP-dependent isocitrate dehydrogenase from pig heart

The metal activator site of NADP-dependent isocitrate dehydrogenase from pig heart has been probed by using 113Cd and 25Mg NMR as well as manganese paramagnetic relaxation of nuclei in the fast-exchanging ligands alpha-ketoglutarate and adenosine 2'-monophosphate. Cadmium NMR shows that cadmium, bound to the enzyme in the presence of isocitrate, has a resonance at 9 ppm relative to cadmium perchlorate, while the free Cd-isocitrate complex has a resonance at -23 ppm. Comparison with model compounds and previously studied proteins indicates that cadmium is coordinated with six oxygen ligands. Measurements as a function of cadmium concentration give a dissociation constant of 66 microM and a dissociation rate constant of 1.5 X 10(4) s-1 at pH 7.0. 25Mg NMR demonstrates that the line width of the magnesium resonance is increased upon binding to isocitrate dehydrogenase. A further increase in line width is observed upon addition of isocitrate. Measurement of line widths as a function of temperature reveals that in the binary complex between magnesium and enzyme, exchange is the major contributor to broadening while in the ternary complex containing isocitrate, the intrinsic relaxation in the bound state is also important, suggesting an increase in the dissociation rate constant for magnesium from the ternary complex. Paramagnetic relaxation studies of nuclei of alpha-ketoglutarate, bicarbonate, and adenosine 2'-monophosphate locate the divalent metal within the active site. The results with adenosine 2'-monophosphate show that atoms in the adenosine moiety of the coenzyme are at least 8 A from the metal site.(ABSTRACT TRUNCATED AT 250 WORDS)

A highly stable NADP-dependent isocitrate dehydrogenase from Thermus thermophilic HB8: purification and general properties

NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermus thermophilus HB8, and shown to be a dimeric protein of molecular weight 115,000, with a pI of 5.5. The amino acid composition of the present enzyme was similar to that reported for other bacterial counterparts, except for a high Arg/Lys ratio and a low Cys content. Divalent cations, such as Mn2+ and Mg2+, were essential for activity. The optimal pH was 7.8 at 55 degrees C. The Km values for NADP and D-isocitrate were 6.3 and 8.8 microM, respectively, with a Vmax of 77.6 mumol/min per mg at 55 degrees C. NAD was able to replace NADP with low efficiency. Backward reaction at 40 degrees C indicated that the Km value for 2-oxoglutarate was 63 microM with a Vmax of 4% that of the forward reaction at that temperature. The enzyme was highly stable against high temperature and denaturing reagents.

Effects of food restriction and hyperoxia on rat survival and lung polyamine metabolism.

We fed Sprague-Dawley rats either freely or by restricting them to 20% of their usual diet for 21 days. In one experiment, we refed half of the food-restricted rats for 12 h, then exposed the three groups to air or 85% O2 for 5 days. The mortalities in 85% O2 were 100, 33, and 0% for the food-restricted, restricted-refed, and freely fed groups, respectively. In air lung polyamine contents and glucose 6-phosphate dehydrogenase and NADP-dependent isocitrate dehydrogenase activities were significantly lower with food restriction. After hyperoxia, lung polyamine and protein contents and enzyme activities were increased in the two surviving groups, but spermine and DNA contents of refed rats did not increase. In a second experiment, we exposed rats to 60% O2 and found that DNA synthesis of food-restricted rats was lower than the freely fed rats in air and remained low after hyperoxia. We conclude that food restriction increases the mortality from 85% O2 and is associated with lower DNA synthesis and polyamine content. We speculate that food-restricted animals may accumulate greater lung injury partly because of a compromised repair process.

Monoamine Oxidase and Other Mitochondrial Enzymes in Density Subpopulations of Human Platelets

Normal human platelets have been separated according to density on continuous Percoll gradients and the platelet distribution divided into five fractions containing approximately equal numbers of platelets. The mean volumes and protein contents of the platelets in each fraction were found to correlate positively with density while the protein concentration did not differ significantly between the fractions. Four mitochondrial enzymes (monoamine oxidase, glutamate dehydrogenase, cytochrome oxidase and NADP-dependent isocitrate dehydrogenase) were assayed and their activities per unit volume were found to increase in a very similar monotonic fashion with platelet density. When MAO and GDH were assayed on the same set of density fractions the correlation between the two activities was very high (r = 0.94-1.00, p less than 0.001) and a similar close correlation was found between MAO and ICDH. The results support the hypothesis that high density platelets either have a higher concentration of mitochondria or have larger mitochondria than low density platelets.

Ferric Reducing Activity in Roots of Fe-deficient Phaseolus vulgaris Source of Reducing Equivalents

Summary Fe deficiency activates a system in the roots of dicotyledons which can reduce extracellular ferric chelates (ferric reductase); this system is located in the plasma membranes of the epidermal cells. In order to identify the source of reducing equivalents for the reduction of ferric chelates by roots of bean ( Phaseolus vulgaris L. var. Prelude), properties of NADP + reducing enzymes were studied and incubations were performed with glucose labeled with 14 C at positions Cl and C6. NADP-dependent isocitrate dehydrogenase was the most active of the enzymes tested in root protein extracts and it could reduce NADP+ even at very high NADPH/NADP + ratios. It is concluded that citrate, which accumulates in roots upon Fe deficiency, via transformation into isocitrate by mitochondrial aconitase, drives cytosolic NADP in bean roots to a strongly reduced state through isocitrate dehydrogenase. Upon addition of reducible ferric salts, the NADP couple becomes more oxidized making it possible for the oxidative pentose phosphate pathway to contribute to NADP + reduction.

NADP-specific isocitrate dehydrogenase of Mycobacterium phlei ATCC 354: purification and characterization.

NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) from Mycobacterium phlei ATCC 354 was purified to homogeneity by ammonium sulphate fractionation, followed by DEAE cellulose and Sephadex G-200 chromatography. The pH optimum of the enzyme was 8.5. The Km values for isocitrate and NADP were 74 and 53 microM, respectively. Mn2+ was essential for enzyme activity. The enzyme lost all activity on incubation at 70 degrees C for 15 min; isocitrate and NADP protected against this thermal inactivation. p-Chloromercuribenzoate inhibited the enzyme; pre-incubation of enzyme with isocitrate + Mn2+ prevented this inhibition. The purified enzyme showed concerted inhibition by glyoxylate + oxaloacetate and was inhibited by oxalomalate.

Interaction between NAD-dependent isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex, and NADH:ubiquinone oxidoreductase.

Interaction between the alpha-ketoglutarate dehydrogenase complex and NAD+-dependent isocitrate dehydrogenase was detected with a variety of techniques including polyethylene glycol precipitation, ultracentrifugation, and centrifugal gel filtration on a Sepharose 6B column. The interaction was specific in that citrate synthase, cytosolic malate dehydrogenase, and NADP-dependent isocitrate dehydrogenase did not interact with alpha-ketoglutarate dehydrogenase complex. The interaction was not inhibited by either 0.1 M KCl or 0.4 M (NH4)2SO4, but was completely prevented by 5% glycerol. A new method for the preparation of NADH: ubiquinone oxidoreductase resulted in an enzyme having a protein subunit composition similar to that of classical complex I preparation. Evidence is given for the existence of ternary complexes containing NADH:ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-NAD-dependent isocitrate dehydrogenase and NADH: ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-succinate thiokinase. These data suggest that a part of the citric acid cycle may be located in the vicinity of NADH: ubiquinone oxidoreductase. These complexes may facilitate the transport of metabolites among these enzymes without their equilibrating with the whole compartment.

NADPH/NADP ratio could regulate the glyoxylate cycle in Tetrahymena pyriformis

1. 1. The glyoxylic acid cycle pathway could be regulated through the modulation of the isocitrate dehydrogenase-NADP activity. This enzyme is inhibited by NADPH. 2. 2. The effect on the glyoxylate cycle flux of variations in the rate of the NADPH-consuming pathways has been studied. 3. 3. Increase in the rate of NADPH-consuming activity by addition of H 2O 2 produces inhibition of the glyoxylate cycle and decrease in the NADPH/NADP ratio. 4. 4. These results suggest that the glyoxylate flux in Tetrahymena could be modulated by regulation of NADP-dependent isocitrate dehydrogenase by the NADPH/NADP ratio.

Electrophoretic polymorphism and sexual dimorphism in the freshwater and anadromous threespine sticklebacks (Gasterosteus aculeatus) of the Little Campbell River, British Columbia

We used single-family crosses to confirm the Mendelian interpretation of allozyme variation and to examine linkage relationships at five polymorphic loci in freshwater and anadromous threespine sticklebacks (Gasterosteus aculeatus) from the Little Campbell River, British Columbia. The electrophoretic pattern of a sixth locus, mitochondrial NADP-dependent isocitrate dehydrogenase (IDH), was found to be sexually dimorphic but otherwise invariant in sticklebacks. This raised the possibility of a sex-linked Idh locus. No differences in IDH quantity or substrate affinities (apparent Km values) were detected between male and female sticklebacks. The electrophoretic pattern of IDH expression was not changed in sticklebacks in which sexual development was altered by hormonal treatment.

A multilocus system for studying tissue and subcellular specialization. The pH and temperature dependence of the two major NADP-dependent isocitrate dehydrogenase isozymes of the fish Fundulus heteroclitus.

In the teleost fish Fundulus heteroclitus, there are three NADP-dependent isocitrate dehydrogenase isozymes. IDH-B2 is the only cytoplasmic isozyme, and IDH-C2 dominates the mitochondria of all tissues other than liver, where IDH-A2 is expressed. Since fish are ectotherms, their intracellular temperature and pH change directly with environmental temperature. In order to evaluate the influence of these environmental parameters on a model fish NADP-isocitrate dehydrogenase system, the major cytoplasmic (IDH-B2) and mitochondrial (IDH-C2) isozymes were kinetically evaluated as a function of pH and temperature. Whereas Vfmax and KmISOCm (where ISOC is isocitrate) were pH-independent, the Km for NADP was pH-dependent for both isozymes. The cytoplasmic isozyme (IDH-B2) had smaller KmNADP values between pH 7.0 and pH 8.0 than the mitochondrial form (IDH-C2). Vfmax and Km for substrate and coenzyme were temperature-dependent. Energy of activation for IDH-B2 and IDH-C2 was 10.6 and 12.8 kcal/mol, respectively. Both proteins had delta G not equal to values of about 15.8 kcal/mol, with significantly different distributions between delta H not equal to and delta S not equal to. The cytoplasmic isozyme (IDH-B2) appears to have a greater rate of catalysis than the mitochondrial enzyme (IDH-C2) at temperatures less than 30 degrees C. Moreover, the IDH-B2 isozyme had lower KmNADP values than the IDH-C2 isozyme at all temperatures, whereas the KmISOC values for the two isozymes were indistinguishable. Our data suggest that the two major NADP-dependent isocitrate dehydrogenase isozymes have unique physiological and metabolic functions that are adapted to the tissues and cellular compartments in which they are expressed.

GENETIC DIVERGENCE AND GEOGRAPHIC SPECIATION IN LAYIA (COMPOSITAE).

Electrophoretic variability was examined in six species of Layia (Compositae), native to California, which have previously been studied by Clausen, Keck, and Hiesey, and are regarded as a classic example of geographic speciation in plants. The study was carried out to test the hypothesis that the extent of divergence in structural genes coding enzymes is concordant with divergence in morphological characteristics, ecological traits, and reproductive isolation. Eleven enzymes specified by 17 loci were analyzed. The genetic identity values were consistent with those expected on the model that the species diverged gradually as they adapted to geographically separate habitats. Thus, the values between the three species complexes proposed by Clausen, Keck, and Hiesey (L. chrysanthemoides/L. fremontii; L. jonesii/L. leucopappa/L. munzii; L. platyglossa) were substantially lower than the values between species within the complexes. The results provide an important contrast to the very high genetic identities between species which originated rapidly from their progenitors. The electrophoretic results also provided evidence that the cytosolic isozyme of phosphoglucomutase and the cytosolic NADP-dependent isocitrate dehydrogenase in the six species are coded by duplicate genes.

A multilocus system for studying tissue and subcellular specialization. The three NADP-dependent isocitrate dehydrogenase isozymes of the fish Fundulus heteroclitus.

Three NADP-dependent isocitrate dehydrogenase isozymes in the teleost, Fundulus heteroclitus (L.), exhibit differences in tissue and subcellular distribution. These three proteins were purified and characterized as to native and subunit molecular weight, isoelectric pH, susceptibility to thermal denaturation, and certain kinetic parameters (Km and Vmax) for the oxidative decarboxylation of isocitrate at 25 degrees C and pH 7.4. The enzymes are dimers of 90 +/- 4 kDa with subunit molecular masses of 45 +/- 3 kDa. Isoelectric pH values were 7.00, 5.19, and 5.29 for IDH-A2, IDH-B2 and IDH-C2 (where IDH represents isocitrate dehydrogenase), respectively. While the monomer-dimer equilibrium is not influenced by substrates, the equilibrium appears to respond to buffer concentration and temperature. Enzyme activity is not affected upon dilution in the presence of buffer containing bovine serum albumin, however, its activity declines rapidly in the absence of bovine serum albumin. Thermal stability varies among the isozymes, and they do not denature by a simple first-order process. The presence of substrates, metal, and coenzymes independently provided enzyme stability, suggesting a random mechanism of substrate and cofactor binding. While IDH-A2 and IDH-B2 have identical KISOCm, IDH-B2 has a lower KNADPm. The most common mitochondrial isozyme (IDH-C2) has a greater KISOCm than either the less common mitochondrial isozyme (IDH-A2) or the cytoplasmic enzyme (IDH-B2). The KNADPm for IDH-C2 was the same as that of IDH-A2 but greater than that of IDH-B2. These Km differences are consistent with the cytoplasmic-mitochondrial shuttling of NADPH-reducing equivalents into the cytoplasm.

Isocitrate dehydrogenase of the thermoacidophilic archaebacterium Sulpholobus acidocaldarius

The thermoacidophilic archaebacterium, Sulpholobus acidocaldarius has been found to possess both NAD- and NADP-dependent isocitrate dehydrogenase activities. Evidence is presented to suggest that both enzymic activities are functions of the same protein: NAD + and NADP + compete with each other for the enzyme and do so with K i values equal to their K m values; thermal inactivation results in the loss of both activities at the same rate and copurification was observed on gel filtration, ion-exchange chromatography and polyacrylamide gel electrophoresis. The evolutionary significance of this unique isocitrate dehydrogenase is discussed.

Tricarboxylic Acid Cycle Activity in Relation to Itaconic Acid Biosynthesis by Aspergillus terreus

Cell-free extracts of an itaconic acid producing strain of Aspergillus terreus contained NADP-dependent isocitrate dehydrogenase activity but no NAD-dependent activity was detected. During the acid-producing phase the activity of the NADP-linked enzyme fell to one-eighth of its value during the growth phase, whereas citrate synthase activity increased. Intact mitochondria were isolated from growth and production phase mycelium. The preparation from the growth phase oxidized exogenous NADH and all tricarboxylic acid cycle intermediates tested. That from the production phase oxidized tricarboxylic acid cycle intermediates at a reduced rate but exogenous NADH oxidation remained high. [2-14C]Acetate was incorporated into itaconic acid and over 90 % of the incorporated radioactivity was located in the methylene carbon. [1-14C]-Acetate was a poorer precursor and did not preferentially label the methylene carbon. These results support the view that itaconic acid biosynthesis in A. terreus occurs by decarboxylation of aconitic acid, the latter produced by the normal reactions of the tricarboxylic acid cycle.

Anaerobic acetate oxidation to CO2 by Desulfobacter postgatei

The strict anaerobe Desulfobacter postgatei oxidizes acetate to CO2 with sulfate as electron acceptor. During growth at 28°C with a doubling time of 16 h the oxidation and assimilation rate of acetate were 280 nmol and 20 nmol per min and mg protein, respectively. In cell extracts all the enzymes of the citric acid cycle were found (numbers in brackets=specific activities in nmol per min and mg protein at 28°C): Citrate (si)-synthase (250); aconitase (200); NADP-dependent isocitrate dehydrogenase (8500); 2-oxoglutarate: ferredoxin oxidoreductase (300); succinyl-CoA: acetate CoA transferase (160); membrane bound succinate dehydrogenase (3500); and membrane bound malate dehydrogenase with 2,3-dimethyl-1,4-naphthoquinone as artificial electron acceptor (54). The following enzymes catalyzing the synthesis of oxaloacetate from acetate and CO2 were also present: Acetyl-CoA synthetase (10); ferredoxin dependent pyruvate synthase (30); phosphoenolpyruvate synthetase (10); and phosphoenolpyruvate carboxylase (24). The key enzymes of the glyoxylate cycle were not detected. The order of magnitude of the observed enzyme activities was sufficient to account for an oxidation of acetate via the citric acid cycle and for a synthesis of oxaloacetate from acetate and CO2 as anaplerotic reaction.

Production of NADPH and acetyl CoA for lipid biosynthesis in the pyloric caeca of the sea star, Asterias rubens

1. 1. Enzyme systems for the translocation of acetyl CoA and for the production of NADPH in lipid biosynthesis were studied in the pyloric caeca of the sea star Asterias rubens. 2. 2. Translocation of acetyl CoA is performed only by the citrate route and not by the acetate or acetylcarnitine route. 3. 3. In the cytoplasm, NADPH is produced by glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of the pentose phosphate shunt and by an active NADP-dependent isocitrate dehydrogenase. 4. 4. NADP-dependent malic enzyme seems to be absent in the sea star. 5. 5. The implications of the possible replacement of malic enzyme by isocitrate dehydrogenase will be discussed.

Effects of buffer salts on NADP-dependent isocitrate dehydrogenase inhibition by long chain free fatty acids.

The cytoplasmic NADP-dependent isocitrate dehydrogenase inhibition by long chain free fatty acids and palmityl-CoA was revealed: 1. In a Tris-citrate buffer, palmityl-CoA inhibited strongly but the free fatty acids hardly at all; whereas, the converse occurred in a glycylglycine buffer. 2. In both instances, the inhibitory effects were competitive with the substrate.

Effect of Proline, Betaine and Some Other Solutes on the Heat Stability of Mitochondrial Enzymes

When isolated plant mitochondria are heated, isocitrate dehydrogenase, malate dehydrogenase and fumarase lose activity at different rates. The rate of loss of activity of each enzyme is reduced if the mitochondria are heated in the presence of proline, betaine or some other solutes; protection by proline or betaine against heat inactivation is also evident with these enzymes when they are solubilized. NAD-isocitrate dehydrogenase in pea mitochondria and NADP-dependent isocitrate dehydrogenase of pea chloroplasts are also protected by proline and betaine against inactivation when the isolated organelles are heated.