NAD-linked Isocitrate Dehydrogenase Research Articles

Effect of Physical Training on the Activity of the Low Km Phosphodiesterase in Heart Ventricular and Skeletal Muscle Tissues of Normal and Diabetic Rats


 
 
 
 This study assessed the effect of physical training on the low Michaelis constant cyclic AMP phosphodiesterase (low Km PDE) activity in heart ventricular tissue and three different skeletal muscle tissues of normal and diabetic rats. Rats were rendered diabetic with streptozotocin (45 mg/kg i.v.) and either kept sedentary (SD, n=16) or submitted to a progressive 10-week treadmill running program (TD, n=17). Two groups of nondiabetic rats served as trained (TC, n=17) and sedentary controls (SC, n=15). The activity of NAD-linked isocitrate dehydrogenase was significantly increased (p < 0.001) in the gastrocnemius muscle of trained animals, confirming that they were adequately trained. Plasma glucose levels were elevated in SD rats (18.8 ± 1.7 mmol/l) compared to those in SC rats (7.7 ± 0.2 mmol/l ; mean ± standard error of the mean [SEM], p < 0.001). These levels were partially normalized following training (12.7 ± 1.7 pmol/l; p < 0.01 vs. SD rats). Plasma insulin levels were significantly reduced in TC rats (223 ± 16 pmol/l) compared with those in SC rats (306 ± 13 pmol/l; p < 0.01). Similarly, the levels in SC rats were significantly different when compared with SD rats (155 ± 15 pmol/l; p < 0.01). In TD rats, plasma insulin levels (156 ± 14 pmol/l) were similar to those of SD rats. This suggests that mild diabetes mellitus in the rat can be improved by endurance training and that improved glycemic control may be mediated by an increase in insulin sensitivity. The low Km PDE activity in the membranes prepared from the four different muscle tissues was not modified by diabetes. Similarly, physical training in normal and diabetic rats did not induce any significant changes in the low Km PDE activity in any of the tissues tested. This suggests that improvements in myocardial contractile function and in glucose homeostasis, as seen in diabetic rats submitted to endurance training, are not associated with changes in PDE.
 
 
 

Detailed kinetics and regulation of mammalian NAD-linked isocitrate dehydrogenase

A mathematical model is presented to describe the catalytic mechanism of mammalian NAD-linked isocitrate dehydrogenase (NAD-IDH), a highly regulated enzyme in the tricarboxylic acid cycle, a crucial pathway in energy metabolism and biosynthesis. The mechanism accounts for allosteric regulation by magnesium-bound isocitrate and EGTA and calcium-bound ATP and ADP. The developed model is used to analyze kinetic data for the cardiac enzyme and to estimate kinetic parameter values. Since the kinetic mechanism is expressed in terms of chemical species (rather than biochemical reactants), the model explicitly accounts for the effects of biochemical state (ionic strength, pH, temperature, and metal cation concentration) on the kinetics. Because the substrate isocitrate competes with allosteric activators (ATP and ADP) and an inhibitor (EGTA) for metal ion cofactors (Ca 2+ and Mg 2+), the observed kinetic relationships between reactants, activator and inhibitor concentrations, and catalytic flux are complex. Our analysis reveals that under physiological conditions, the ADP/ATP ratio plays a more significant role than Ca 2+ concentration in regulating the enzyme's activity. In addition, the enzyme is highly sensitive to Mg 2+ concentration in the physiological range, pointing to a potential regulatory role of [Mg 2+] in mitochondrial energy metabolism.

Effects of thiabendazole (TBZ) on mitochondrial function in renal cortex of ICR mice

The effects of thiabendazole (TBZ) on mitochondrial function of the renal cortex were investigated in ICR mice. Mice were given 1000 or 2000 mg TBZ/kg body weight by gavage and mitochondria were isolated from the renal cortex for the measurement of respiratory rates. The state 3 and DNP-uncoupled respiratory rates of renal cortical mitochondria were dose-dependently depressed at 6 hours after dosing. The depression of these respiratory rates of renal cortical mitochondria was more marked at 16 hours after dosing. There was no depression in these respiratory rates of renal cortical mitochondria at 3 hours after dosing, although renal cortical concentrations of TBZ were higher than those at 6 or 16 hours after dosing. Histochemical examination revealed that NAD-linked isocitrate dehydrogenase, a marker enzyme of mitochondria, was inhibited in renal cortical tubules at 16 hours after dosing of 1000 or 2000 mg TBZ/kg body weight. Furthermore, renal cortical ATP level was significantly decreased at 16 hours after dosing of 1000 or 2000 mg TBZ/kg body weight. The results indicate that administration of TBZ caused mitochondrial dysfunction in renal cortical tubules of mice.

Novel NADP-linked isocitrate dehydrogenase present in peroxisomes of n-alkane-utilizing yeast, Candida tropicalis: comparison with mitochondrial NAD-linked isocitrate dehydrogenase

Novel NADP-linked isocitrate dehydrogenase present in peroxisomes of n-alkane-utilizing yeast, Candida tropicalis: comparison with mitochondrial NAD-linked isocitrate dehydrogenase

Metabolic profiles of white and red-intermediate spinal motoneurons in the zebrafish.

To study the interactions between the citrate cycle and amino acid metabolism in zebrafish spinal motoneurons, we composed enzyme histochemical profiles from the activities of NAD-linked isocitrate dehydrogenase (NAD-ICDH), glutamate dehydrogenase (GDH), succinate dehydrogenase (SDH) and glucose 6-phosphate dehydrogenase (G6PDH). The enzyme assays were performed on serially-sectioned motoneuron somata. The motoneurons were identified by retrograde tracing from the trunk muscle and classified, on the basis of their location in the motor column, as those innervating the white, fast glycolytic fibers (WMNs) or those innervating the red and intermediate slow oxidative fibers (RIMNs). We found the following relationships between enzyme activities in WMNs: GDH correlates with G6PDH activity (r = 0.31; p = 0.02) and NAD-ICDH correlates with GDH activity (r = 0.37; p < 0.01); correlations between NAD-ICDH and SDH and between SDH and GDH are not significant. In RIMNs we found correlations between NAD-ICDH and SDH (r = 0.34; p = 0.03), between NAD-ICDH and GDH (r = 0.41; p < 0.01) and between GDH and SDH (r = 0.50; p < 0.01); the correlation between GDH and G6PDH is not significant. The differences in metabolic profiles between WMNs and RIMNs can be explained in the following way: in WMNs, alpha-ketoglutarate is drawn off from the citrate cycle and is used in amino acid metabolism whereas in RIMNs the removal of alpha-ketoglutarate from the cycle is balanced by formation of alpha-ketoglutarate. The data suggest that the functional role of the citrate cycle differs in the two motoneuron populations: in RIMNs energy generation predominates but in WMNs a role in biosyntheses seems most important.

In situ measurements of enzyme activities in the brain

The present review focuses on enzymes involved in the metabolism of amino acid neurotransmitters and the microphotometric determinations of their activities in various layers of the rat hippocampus. The enzymes are NAD-linked isocitrate dehydrogenase (NAD-ICDH), glutamate dehydrogenase (GDH), and GABA transaminase (GABAT), all of which are localized in mitochondria. GDH seems to be restricted to astrocytes, whereas NAD-ICDH and GABAT are localized in neurons as well as in astrocytes. NAD-ICDH is an important enzyme of the tricarboxylic acid cycle and may deliver alpha-ketoglutarate for the formation of glutamate and GABA, which serve as neurotransmitters in the hippocampus. GDH catalyses the interconversion of alpha-ketoglutarate and glutamate, whereas GABAT is the important GABA-degrading enzyme and requires alpha-ketoglutarate for its activity. While differing in their cellular distribution and activity levels, NAD-ICDH, GDH and GABAT are significantly correlated in their hippocampal distribution. Furthermore, developmental and pharmacohistochemical studies suggest that the distribution and activity of astrocytic GDH is correlated with amino-acidergic neurotransmission in the hippocampus. The data reported give further evidence for a metabolic relationship between neurons and astrocytes in the turnover and metabolism of glutamate and GABA.

Mitochondrial enzymes related to glutamate and GABA metabolism in the hippocampus of young and aged rats: a quantitative histochemical study.

Quantitative histochemistry (scanning microphotometry) was used to determine the activities of the mitochondrial enzymes NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), L-glutamate dehydrogenase (EC 1.4.1.3) and GABA transaminase (EC 2.6.1.19) in various layers of the hippocampus (middle one third) of young (3-4 months old) and memory-impaired aged rats (28-30 months old). For comparison, determinations of cytochrome c oxidase (EC 1.9.3.1) as a marker for mitochondria and energy metabolism were also performed. The study showed that there was a layered reaction pattern in the hippocampus and that the cellular distribution and the levels of enzyme activity were different. However, the activities of the different enzymes (excepting GABA transaminase and cytochrome c oxidase) were significantly correlated in the hippocampus in both age groups. Age-dependent changes were only observed for NAD-linked isocitrate dehydrogenase and GABA transaminase (significant increases of activities in some layers of the hippocampus, preferentially in the terminal field of the perforant path). From the present study it is concluded that, 1. the enzymatic complement of mitochondria in neurons and glia depends upon layer specific metabolic processes of the hippocampus (also with respect to glutamatergic and GABAergic terminal fields) indicating a layer specific interaction of the enzymes studied to produce or catabolize glutamate and GABA, and 2. the age dependent changes of the studied enzymes are very restricted.

Enhancement of NAD-linked isocitrate dehydrogenase activity in rat liver by clofibrate feeding

After feeding rats for 3 weeks with clofibrate specific activities of the liver mitochondrial enzymes glycerol-3-phosphate dehydrogenase (G3PDH) and NAD-linked isocitrate dehydrogenase (NAD-ICDH) were found to be increased in a large particle fraction 6-fold and 1.6-fold, respectively, whereas the activity of NADP-linked isocitrate dehydrogenase (NADP-ICDH) remained unchanged. Possibly these effects contribute to the hypolipidemic action of clofibrate.

Eubacterial isocitrate dehydrogenase with dual specificity for NAD and NADP fromRhodomicrobium vannielii

Cell-free extracts of the photosynthetic eubacterium Rhodomicrobium vannielii contained both NADP and NAD-linked isocitrate dehydrogenase activities. Apparent Km values of 12 μM for NADP, 0.75 mM for NAD, 9.3 μM for isocitrate (NADP utilising) and 8.2 μM for isocitrate (NAD utilising) were determined in such extracts. Four lines of evidence indicated that one enzyme was responsible for the two activities; (i) non-additivity of reaction rates in the presence of both NADP and NAD (ii) the presence of one band which stained for activity with both cofactors on non-denaturing polyacrylamide gels (iii) identical heat inactivation kinetics for both activities (iv) co-elution of both activities after ion-exchange and hydrophobic interaction chromatography. This is the first report of a eubacterial isocitrate dehydrogenase with dual cofactor specificity.

Purification and characterization of NAD-linked isocitrate dehydrogenase from Methylophilus methylotrophus

Purification and characterization of NAD-linked isocitrate dehydrogenase from <i>Methylophilus methylotrophus</i>

The effect of pH on production of citric and gluconic acid from beet molasses using continuous culture

The effect of pH on the production of citric and gluconic acid, from beet molasses byAspergillus niger, was studied using continuous culture. At pH values above 2.5 gluconic acid was the major product, citric acid being the predominant product at low pH values. The optimum specific activities of citrate synthase, aconitase, NAD-linked isocitrate dehydrogenase, and NADP-linked isocitrate dehydrogenase occurred at pH 4 and of glucose oxidase at pH 5.

Regulation of 2-oxoglutarate dehydrogenase and NAD-linked isocitrate dehydrogenase within toluene-permeabilized mitochondria

Regulation of 2-oxoglutarate dehydrogenase and NAD-linked isocitrate dehydrogenase within toluene-permeabilized mitochondria

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.

Differences in some of the metabolic properties of mitochondrial isolated from cerebral cortex and olfactory bulb of the rat

The metabolic properties of mitochondria from rat cerebral cortex and olfactory bulb were investigated. The pyruvate-supported oxygen uptake rates by olfactory bulb mitochondria were significantly lower than those by cerebrocortical mitochondria. This is consistent with the differences in pyruvate dehydrogenase complex activities between these mitochondrial preparations. Pyruvate dehydrogenase kinase, NAD-linked isocitrate dehydrogenase, and hexokinase activities in olfactory bulb mitochondria were significantly lower than those in cerebrocortical mitochondria. However, NADP-linked isocitrate dehydrogenase, and NAD-linked and NADP-linked glutamate dehydrogenase activities in olfactory bulb mitochondria were significantly higher than those in cerebrocortical mitochondria. The differences between these two mitochondrial preparations in terms of the activities of these energy-metabolizing enzymes reflect the differences detected in the homogenates of these regions.

Energy metabolism in the developing larval stages of Ancylostoma tubaeforme and Haemonchus contortus: glycolytic and tncarboxylic acid cycle enzymes

The activities of glycolytic and related enzymes and the tricarboxylic acid cycle enzymes were measured in freshly isolated 1st- (Li), 2nd- (L2) and 3rd-stage (L3) larvae of bothAncylostoma tubaeformeandHaemonchus contortus. All enzymes of the glycolytic pathway were present in all developmental stages of both strongylid nematodes although higher levels of activities were obtained in the pre-infective 1st- and 2nd-stage larvae than in the infective 3rd stage. However, the pre-infective larvae contained lower levels of pyruvate kinase (PK) than the infective larvae. Consequently, the pyruvate kinase to phosphoenolpyruvate carboxykinase (PEPCK) ratios were 0·23 and 0·26 for the L1s and L2s forA. tubaeformeand 0·36 and 0·21 for those ofH. contortusrespectively. High levels of activity of glucose-6-phosphate dehydrogenase obtained in the bacteriophagous pre-infective larvae were consistent with high rates of morphogenesis and substrate synthesis characteristic of the pre-infective stages. All the tricarboxylic acid cycle enzymes were present in the infective larvae of both nematodes while in the pre-infective Li and L2 stages, the enzymes at the beginning of the cycle, namely aconitate hydratase and NAD-linked isocitrate dehydrogenase, were not detected. A scheme was proposed for the energy metabolism of these developing larvae. In this scheme, the pre-infective larvae were shown to operate an anaerobic metabolic pathway involving the carboxylation of phosphoenolpyruvate (PEP) by phosphoenolpyru vate carboxykinase (PEPCK) to form oxaloacetate (OAA), whereas in the infective larvae the metabolic pathway favouring the direct dephosphorylation of PEP, as in vertebrate tissues, was followed.

Inhibition by alloxan of mitochondrial aconitase and other enzymes associated with the citric acid cycle

Considerable variations were found in the in vitro effect of alloxan on mouse liver enzymes associated with the citric acid cycle. The following approximative alloxan concentrations induced 50% inhibition of enzyme activity: 10 −6 M for aconitase, 10 −4 M for NAD-linked isocitrate dehydrogenase, glutamate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase and fumarase, and 10 −3 M for citrate synthase and NADP-linked isocitrate dehydrogenase. Pyruvate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were not inhibited by 10 −3 M alloxan. The inhibition of aconitase was competitive both when using mouse liver and purified porcine heart enzyme. The K i values for the purified enzyme in the presence of 5 μM alloxan were 0.22 μM with citrate, 4.0 μM with cis-aconitate and 0.62 μM with isocitrate as substrate. The high sensitivity of aconitase for inhibition by alloxan probably plays a prominent role for the toxic effects of alloxan.

The Activities of Some Energy‐Metabolising Enzymes in Nonsynaptic (Free) and Synaptic Mitochondria Derived from Selected Brain Regions

The enzyme complement of two different mitochondrial preparations from adult rat brain has been studied. One population of mitochondria (synaptic) is prepared by the lysis of synaptosomes, the other (non-synaptic or free) by separation from homogenates. These populations have been prepared from distinct regions of the brain: cortex, striatum, and pons and medulla oblongata. The following enzymes have been measured: pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), fumarase (EC 4.2.1.2), NAD-linked malate dehydrogenase (EC 1.1.1.37), D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and mitochondrially bound hexokinase (EC 2.7.1.1) and creatine kinase (EC 2.7.3.2). The nonsynaptic (free) mitochondria show higher enzyme specific activities in the regions studied than the corresponding values recorded for the synaptic mitochondria. The significance of these observations is discussed in the light of the different metabolic activities of the two populations of mitochondria and the compartmentation of the metabolic activities of the brain.

Regional enzyme development in rat brain. Enzymes of energy metabolism.

The development of several key enzymes of pyruvate and 3-hydroxybutyrate metabolism and of the tricarboxylic acid cycle was studied in six regions (cerebellum, medulla oblongata and pons, hypothalamus, striatum, mid-brain and cortex) of the neonatal, suckling and adult rat brain (2 days before birth to 60 days after birth). The enzymes whose developmental patterns were studied were: pyruvate dehydrogenase (EC 1.2.4.1), 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and fumarase (EC 4.2.1.2). Citrate synthase, isocitrate dehydrogenase and pyruvate dehydrogenase develop as a cluster in each region, although the pyruvate dehydrogenase appears to lag slightly behind the others. As with the glycolytic-enzyme cluster [Leong & Clark (1984) Biochem. J. 218, 131-138] the timing of the development of the activity of this group of enzymes varies from region to region; 50% of the adult activity developed first in the medulla oblongata, followed by the hypothalamus, striatum and mid-brain, and then in the cortex and cerebellum respectively. The 3-hydroxybutyrate dehydrogenase activity also develops earlier in the medulla oblongata than in the other regions. The results are discussed with respect to the neurophylogenetic development of the brain regions studied and the importance of the development of the enzymes of aerobic glycolysis in relationship to the development of neurological maturation.

Purification and characterization of NAD-dependent isocitrate dehydrogenase from Dictyostelium discoideum

Isocitrate dehydrogenase ( threo- d s-isocitrate: NAD oxidoreductase (decar☐ylating) EC 1.1.1.41) from Dictyostelium dicoideum was purified 161-fold. The purified enzyme was NAD specific and required Mn 2+ for activity. Isocitrate consumption and 2-oxoglutarate and NADH production were stoichiometric; no NADH oxidase or glutamate dehydrogenase activities were detected. The pH optimum range for activity was pH 7.5–8.5. Reductive car☐ylation of 2-oxoglutarate with NADH could not be demonstrated. Lineweaver - Burk plots of data from initial velocity studies were linear. There was no evidence of allosteric control by reported effectors (AMP, ADP, citrate) of isocitrate dehydrogenase activity. The reaction was inhibited by NADH. The inhibition by NADH was competitive when either isocitrate or NAD was the variable substrate. 2-Oxoglutarate was not inhibitory at concentrations below 4 m m. The Michaelis constant ( K m ) and dissociation constant ( K ib ) for isocitrate were 0.16 m m; and K m and dissociation constant ( K ia ) for NAD were 0.34 m m. The inhibition constant for NADH was 0.02 m m. The data are consistent with a rapid equilibrium random bi-bi reaction mechanism (Cleland nomenclature). The NAD-linked isocitrate dehydrogenase activity was also demonstrated in crude extracts of isolated mitochondria.

Interrelationships among nucleotide binding sites of pig heart NAD-dependent isocitrate dehydrogenase.

Binding of NADH and NADPH to NAD-linked isocitrate dehydrogenase from pig heart (which contains three types of subunits of similar molecular weight in the ratio 2:1:1) was studied by use of the enhancement of nucleotide fluorescence. NADH and NADPH bind independently and, for both reduced nucleotides, 0.5 binding sites/average subunit or two binding sites for every four subunits were detected. Binding of NADH is unaffected by metal ion, isocitrate, or NAD+. ADP is also not competitive with NADH binding but reduces the strength of binding 3-fold. In contrast, ATP (KI = 9 microM ATP4-) appears to be competitive with NADH. Evidence for an NADH site with higher dissociation constant (KD greater than 200 microM) than that obtained from the fluorescence measurements (KD = 2.8 microM at pH 6.1) is indicated by competition of NADH with [14C]NAD+ binding. NADPH binding is enhanced by the presence of manganese. The metal dependence is consistent with NADPH (KD = 8.1 microM at pH 6) binding to the same site as Mn-NADPH2- (KD = 0.9 microM). Dissociation constants for both species increase with increasing pH in the range 6-8. NADPH binding is competitively inhibited by ADP, ATP, ADP-ribose, and isocitrate. This inhibition of NADPH binding is probably indirect since measurements of [14C]ADP in the presence and absence of NADPH showed little inhibition of binding by NADPH concentrations sufficient to saturate the fluorescent site. These studies extent the number of ligands for isocitrate dehydrogenase for which there are fewer binding sites than there are subunits.