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Query: EC:1.4.3.11 (glutamate dehydrogenase)
 4,437 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ratio of free ATP to free ADP in the mitochondrial matrix [( ATPf]/[ADPf]) has been measured in suspensions of isolated mitochondria under conditions of active phosphorylation of extramitochondrial ADP. These measurements utilized phosphoenolpyruvate carboxykinase which is present in the matrix of mitochondria from the livers of guinea pigs, chickens, and pigeons. Mitochondria isolated from these sources also contain nucleoside diphosphate kinase, malate dehydrogenase, and glutamate dehydrogenase or 3-OH-butyrate dehydrogenase. Together these enzymes catalyze the synthesis of phosphoenolpyruvate and CO2 from oxaloacetate with oxidative phosphorylation as an energy source. These reactions have been shown to be fully reversible in suspensions of mitochondria isolated from the above sources. With oxidative phosphorylation as the source of ATP, phosphoenolpyruvate was synthesized from malate and conversely addition of phosphoenolpyruvate, ADP, and CO2 led to synthesis of malate and ATP. The forward and reverse reactions were allowed to continue until the rate of change of metabolite concentrations was minimal and then the latter were measured. The intramitochondrial [Mg-ATPf]/[MgADPf] was calculated from the equilibrium constants for the reactions and the measured steady state concentrations of the metabolites in both the intra- and extramitochondrial spaces. The value of the intramitochondrial [MgATPf]/[MgADPf] was found to exceed the extramitochondrial value (adjusted to the same free Mg2+ concentration) by a factor (+/- S.E.) of 0.83 +/- 0.22 (n = 17) for the forward reaction and 1.81 +/- 0.54 (n = 11) for the reverse reaction. It is concluded that the adenine nucleotide translocase catalyzes electroneutral exchange of ATP for ADP and that this reaction does not contribute significantly to the energetics of mitochondrial oxidative phosphorylation.
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PMID:Evaluation of the relationship between the intra- and extramitochondrial [ATP]/[ADP] ratios using phosphoenolpyruvate carboxykinase. 688 88

Changes in oxidative metabolism were studied in hepatopancreas, muscle, and hemolymph of the edible crab Scylla serrata, exposed to a sublethal concentration (2.5 ppm) of cadmium chloride. A significant decrease in glycogen, total carbohydrates, and pyruvate and an increase in lactate levels in hepatopancreas and muscle were observed. Hemolymph sugar levels were increased in experimental crabs. An increase in phosphorylase suggested increased glycogenolysis during cadmium toxicity. The decrease in lactate dehydrogenase activity and the increase in lactate content indicated reduced mobilization of pyruvate into the citric acid cycle. Krebs cycle enzymes such as succinate dehydrogenase and malate dehydrogenase were found to be decreased, suggesting impairment of mitochondrial oxidative metabolism as a consequence of cadmium toxicity. Glucose-6-phosphate dehydrogenase activity was increased, suggesting enhanced oxidation of glucose by the HMP pathway. Cytochrome-c oxidase and Mg2+ ATPase activity levels decreased, indicating impaired energy synthesis during cadmium stress. Acid and alkaline phosphatase activities increased, suggesting enhanced breakdown of phosphates to release energy in view of impaired ATPase system during cadmium exposure. A significant decrease in protein and free amino acid and an increase in ammonia, urea, and glutamine levels were observed in the tissues during exposure. An increase in protease, alanine aminotransaminase, and aspartate aminotransaminase suggested increased proteolysis and transamination of amino acids. The increase in glutamate dehydrogenase, AMP deaminase, and adenosine deaminase indicated increased ammonia production. The increased arginase and glutamine synthetase suggested the detoxification or mobilization of ammonia toward the production of urea and glutamine. These results suggest that cadmium affects oxidative metabolism and induces hyperammonemia, and crabs switch over their metabolic profiles toward compensatory mechanisms for the survivability in cadmium-polluted habitats.
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PMID:Changes in oxidative metabolism in selected tissues of the crab (Scylla serrata) in response to cadmium toxicity. 753 86

Rat hippocampal slices preloaded with D-[3H]aspartate, a non metabolizable analogue of L-glutamate, were superfused with artificial CSF. Depolarization was induced by 53.5 mM K+, in the presence of Ca2+ (1.3 mM) or Mg2+ (5 mM) to determine the Ca2+ dependent release. Haloperidol added in the superfusion medium at 100 microM reduced by about 60% the Ca2+ dependent release of D-[3H]aspartate. This drug at 20 microM or 100 microM inhibited the non-activated glutamate dehydrogenase (GDH) but had no effect on GDH activated by ADP (2 mM) or leucine (5 mM). In addition no effect was observed on phosphate activated glutaminase (PAG) in the presence either of 20 mM or 5 mM phosphate. These results indicate that the effect of haloperidol is exerted on presynaptic mechanisms regulating neurotransmitter release.
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PMID:Haloperidol reduces K(+)-evoked Ca(2+)-dependent D-[3H]aspartate release from rat hippocampal slices. 773 54

Kinetic properties of glutamate dehydrogenase (GDH) and the effects on its activity of several putative modulators were examined in mitochondrial extracts of rat pancreatic islets. In the presence of 40 mmol/L NH4Cl and 0.1 mmol/L NADH, stepwise elevation of the 2-oxoglutarate concentration from 0.005 to 0.05 mmol/L increased glutamate formation, whereas further increases led to a progressive decrease of the reaction velocity. Adenosine diphosphate (ADP) at 0.1 mmol/L partially and at 1 mmol/L completely reversed the inhibitory effect of 2-oxoglutarate. The sensitivity to activation by either ADP or leucine was dependent on 2-oxoglutarate concentrations. At higher concentrations of the latter, greater amounts of the activators were needed to attain maximal effect. In the absence of allosteric activators, sulfate or phosphate at 20 mmol/L partially released the inhibitory effect of 2-oxoglutarate levels and increased the maximal velocity (Vmax) for the reaction. In the presence of 0.1 mmol/L ADP, both anions prevented the inhibition by higher concentrations of 2-oxoglutarate, whereas with 1 mmol/L ADP their only effect was a slight increase in the Vmax. Mg2+ and naturally occurring polyamines decreased glutamate formation in a dose-dependent manner; with 0.1 mmol/L ADP, inhibition was seen at all 2-oxoglutarate concentrations studied, whereas with 1 mmol/L ADP, it was noticeable at substrate concentrations higher than 0.5 mmol/L. This inhibitory effect on GDH activity was partially attenuated by sulfate. Addition of either 2 mmol/L spermidine or extra magnesium (final 2.5 or 5 mmol/L) to the perifusion buffer markedly attenuated the insulin release elicited by alpha-ketoisocaproate. It is suggested that naturally occurring polyamines, magnesium, and phosphate act as physiological modulators of GDH activity in pancreatic beta cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of the glutamate dehydrogenase activity in rat islets of Langerhans and its consequence on insulin release. 791 21

Magnesium and the polyamines putrescine, spermidine, and spermine inhibited the activity of glutamate dehydrogenase in permeabilized rat brain mitochondria in a concentration-dependent manner. The inhibitory effect was observed on both the reductive amination of 2-oxoglutarate and oxidative deamination of glutamate, as well as in the presence and absence of ADP and leucine, the allosteric activators of the enzyme. Kinetic studies at various concentrations of substrates showed that inhibition by magnesium and spermine was very pronounced at 2-oxoglutarate concentrations less than 0.5 mM and NADH levels less than 0.08 mM. The presence of the former compounds also accentuated the inhibitory effect of high concentrations of 2-oxoglutarate (> 2.0 mM) and NADH (> 0.32 mM). Addition of magnesium and spermine to suspensions of synaptosomes decreased the amount of ammonia produced from glutamate. It is suggested that polyamines and magnesium, normal constituents of mammalian brain, are responsible, at least in part, for the low glutamate dehydrogenase activity in vivo.
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PMID:Inhibition of glutamate dehydrogenase in brain mitochondria and synaptosomes by Mg2+ and polyamines: a possible cause for its low in vivo activity. 803 99

Photoaffinity labeling with [alpha-32P]8N3GTP and [gamma-32P]8N3GTP was used to identify the guanine binding domain of the GTP regulatory site within glutamate dehydrogenase (GDH). Without photolysis, 8N3GTP mimicked the regulatory properties of GTP on GDH activity with 8N3GTP exhibiting a Ki of 5 microM while the Ki for GTP was about 0.6 microM. Under optimal photolabeling conditions saturation of photoinsertion with 1 microgram of GDH revealed an apparent Kd of 9 +/- 4 microM for [gamma-32P]8N3GTP. Photolabeling with this analog could be competitively inhibited with GTP with an apparent Kd of 12 +/- 2 microM. Other nucleotides such as ATP and NAD(P)H could not reduce the amount of photoinsertion as effectively as GTP. ADP could decrease photoinsertion, but only at much higher concentrations. NAD(P)+, GDP, AMP, and GMP had little effect on photoinsertion. Divalent cations Mg2+ and Ca2+ also reduced photoinsertion significantly while the monovalent K+ and Na+ ions had no effect. Aluminum(III)-chelate or iron(III)-chelate affinity chromatography and reversed-phase HPLC were used to purify photolabel-containing peptides generated with either trypsin or chymotrypsin. This identified a portion of the guanine binding domain within the GTP regulatory site as the region containing the sequence Ile439 to Tyr454. Photolabeling of this peptide was prevented 91% by the presence of 300 microM GTP during photolysis. Lys445 was not identified in sequence analyses of the photolabeled peptides. Also, trypsin was unable to cleave the photolabeled peptide at this site. These results suggest that Lys445 may be the residue modified by [alpha-32P]8N3GTP.
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PMID:Identification of a guanine binding domain peptide of the GTP binding site of glutamate dehydrogenase: isolation with metal-chelate affinity chromatography. 843 45

The role of endogenous mitochondrial Mg2+ as a potential regulator of mitochondrial dehydrogenase activity, and therefore of cellular respiration, was measured in isolated mitochondria containing matrix Ca2+ and Mg2+ levels resembling those occurring in vivo. Ca2+ and Mg2+ depletion was carried out using the cation ionophore A23187 in the presence or absence of the Ca2+ uniporter inhibitor ruthenium red (RR). Divalent cation depletion inhibits the oxidation of alpha-ketoglutarate or pyruvate in states 4 and 3, slows uncoupled respiration and results in decreased membrane potential. Since the addition of Mg2+ could not restore respiration, these dehydrogenases appear not to be regulated by Mg2+. In contrast, similar cation depletion stimulates succinate dehydrogenase (or glutamate dehydrogenase) in state 4 without decreasing membrane potential. The addition of RR caused authentic uncoupling, accompanied by a decrease in membrane potential and an increase in membrane permeability. These effects could be completely reversed by Mg2+. These and other data, showing that Mg2+ depletion results in a change of respiration depending on the substrate oxidized and the metabolic state, indicate that Mg2+ removal may have direct and indirect effects on mitochondrial respiration. A clear direct effect is the stimulation of succinate or glutamate dehydrogenase by decreasing matrix Mg2+. Hence, changes in matrix Mg2+ (in addition to those of Ca2+) could be of great consequence, not only for the control of respiration but also for metabolic pathways affected by changes in concentrations of matrix substrates.
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PMID:Mg2+ control of respiration in isolated rat liver mitochondria. 884 Nov 28

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press
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PMID:Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton 945 44

Many halogenated foreign compounds are detoxified by conversion to the corresponding cysteine S-conjugate, which is N-acetylated and excreted. However, several halogenated cysteine S-conjugates [e.g. S-(1,1,2,2-tetrafluoroethy)-L-cysteine (TFEC)] are converted to mitochondrial toxicants by cysteine S-conjugate beta-lyases. In the present work, we showed that TFEC appreciably inactivated highly purified alpha-ketoglutarate dehydrogenase complex (KGDHC) in the presence of a cysteine S-conjugate beta-lyase. Incubation of PC12 cells (which contain endogenous cysteine S-conjugate beta-lyase activity) with TFEC led to a concentration- and time-dependent loss of endogenous KGDHC activity. A 24-hr exposure to 1 mM TFEC decreased KGDHC activity in the cells by 90%. Although treatment with TFEC did not inhibit intrinsic pyruvate dehydrogenase complex (PDHC) activity, it inhibited dichloroacetate/Mg2+-mediated activation/dephosphorylation of PDHC in the PC12 cells by 90%. To determine the selectivity of enzymes targeted by TFEC, several cytosolic and mitochondrial enzymes involved in energy metabolism [malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase, cytosolic and mitochondrial aspartate aminotransferases (AspAT)] were also assayed in the PC12 cells exposed to 1 mM TFEC for 24 hr. Of these enzymes, only mitochondrial AspAT, a key enzyme of the malate-aspartate shuttle, was inhibited. The present results demonstrate a selective vulnerability of mitochondrial enzymes to toxic cysteine S-conjugates. The data indicate that TFEC may be a useful cellular/mitochondrial toxicant for elucidating the consequences of the diminished mitochondrial function that accompanies numerous neurodegenerative diseases.
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PMID:Inhibition of select mitochondrial enzymes in PC12 cells exposed to S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. 1053 46

Mitochondrial ADP-ribosylation leads to modification of two proteins of approximately 26 and 53 kDA: The nature of these proteins and, hence, the physiological consequences of their modification have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a specific acceptor for enzymatic, cysteine-specific ADP-ribosylation in mitochondria. The modified protein was isolated from the mitochondrial preparation and identified as GDH by N-terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [14C]adenine demonstrated the occurrence of the modification in vivo. Purified GDH was ADP-ribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP-ribose from NAD+ onto the acceptor site. ADP- ribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP-ribose and GDH subunits suggests that modification of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH was reactivated by an Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. GDH, a highly regulated enzyme, is the first mitochondrial protein identified whose activity may be modulated by ADP-ribosylation.
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PMID:Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria. 1135 Sep 29


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