Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.4.1.2 (glutamate dehydrogenase)
4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

UTP, labeled with 15N and 13C (at all carbon atoms of the ribose moiety), was obtained enzymatically from [15N]uracil and [13C6]glucose. Eleven enzymes and suitable substrates reconstituted a metabolic pathway in which glucose was first transformed to 5-phosphoribosyl-1-pyrophosphate. The latter compound plus uracil yielded UMP in a second step by the reaction catalyzed by uracil phosphoribosyltransferase. UMP was subsequently phosphorylated to the corresponding di- and triphosphate. ATP, required for five phosphorylation reactions, was regenerated from creatine phosphate, whereas NADP+ necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and alpha-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N, 13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy.
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PMID:Chemienzymatic synthesis of uridine nucleotides labeled with [15N] and [13C]. 874 75

A new autosomal dominant syndrome in a Swedish pedigree is described. Five patients were affected with cerebellar ataxia and sensorineural deafness. Four of these patients had symptoms of narcolepsy. Optic atrophy, other neurological abnormalities and psychiatric symptoms developed with increasing disease duration. Three patients had non-neurological disease in addition, including diabetes mellitus in two and hypertrophic cardiomyopathy in one. Autopsy with neuropathological examination was performed in one case. Molecular studies focused on the short arm of chromosome 6, including the HLA DR2 locus associated with narcolepsy and the (CAG)n repeat at the spinocerebellar ataxia type 1 (SCA1) locus. Biochemical investigation of muscle biopsy of one case indicated mitochondrial dysfunction with selective decrease in ATP production for substrates that normally give the highest rates. The activity of glutamate dehydrogenase was reduced, indicating a low mitochondrial density. We postulate an autosomal dominant genetic factor responsible for this syndrome. Linkage was excluded to HLA DR2, and a normal sized SCA1 repeat was observed. We conclude that a locus predisposing to ataxia, deafness and narcolepsy exists outside this region of chromosome 6.
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PMID:Autosomal dominant cerebellar ataxia deafness and narcolepsy. 874 54

Fructose-1,6-bisphosphate (FBP), an intermediate of glucose metabolism, is neuroprotective in brain hypoxia or ischemia. Because the mechanisms for this protection are not clear, we examined the effects of FBP on two important events in brain ischemia, i.e., loss of ATP and release of the excitatory neurotransmitter glutamate. Glutamate release from cortical brain slices was measured fluorometrically (glutamate dehydrogenase-catalyzed conversion of glutamate to alpha-ketoglutarate) during hypoxia (PO2 15 mm Hg) or hypoxia plus 100 microM cyanide. FBP (3.5 mM, with glucose 20 mM) reduced glutamate release during hypoxia by 55% and during hypoxia/cyanide by 46% (p < 0.005), and prevented a significant fall in [ATP]. [ATP] was maintained in oxygenated glucose-free conditions with 20 but not 3.5 mM FBP, and fell to < 20% of normal with hypoxia. Despite the drop in [ATP], 3.5 or 20 mM FBP without glucose decreased hypoxia-evoked glutamate release. We conclude (1) FBP present without glucose preserves normal [ATP] only when oxygen is available, suggesting limited uptake and metabolism; and (2) FBP decreases hypoxia-evoked glutamate release by processes independent of [ATP]. These results suggest protective actions of FBP that are separate from augmentation of anaerobic energy production, as previously proposed.
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PMID:Effects of fructose-1,6-bisphosphate on glutamate release and ATP loss from rat brain slices during hypoxia. 885 28

A 50-kDa membrane protein corresponding to a membrane-bound isoform of glutamate dehydrogenase was proposed as a molecular species that could mediate lysosome-microtubule interactions. This protein, isolated from purified lysosome membranes, is a peripheral membrane protein with an ATP-dependent microtubule binding activity. We have produced antibodies against the purified 50-kDa protein to investigate its role in the association of lysosomes to microtubules using a cell-free reconstitution assay and cell microinjection. Pretreatment of purified lysosomes with the antibodies inhibited the association of these vesicles to microtubules. The blocking effect of antibodies was demonstrated by a differential sedimentation method and negative staining electron microscopy, allowing us to quantify the amount of microtubules interacting with lysosomes and the proportion of lysosomes bound to microtubules, respectively. Affinity-purified antibodies microinjected into intact cells altered the distribution of lysosomes that appeared less clustered in the vicinity of nuclei. The antibody-induced lysosome dispersion was assessed by quantitative videomicroscope analyses. These data show that the 50-kDa membrane protein could act, through its microtubule binding activity, as a molecule of attachment of lysosomes to microtubules. This membrane-bound isoform of glutamate dehydrogenase could be involved in the microtubule-dependent perinuclear localization of lysosomes.
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PMID:Involvement of a membrane-bound form of glutamate dehydrogenase in the association of lysosomes to microtubules. 893 30

Glial cells transform glucose to a fuel substrate taken up and used by neurons. In the honeybee retina, photoreceptor neurons consume both alanine supplied by glial cells and exogenous proline. Ammonium (NH4+) and glutamate, produced and released in a stimulus-dependent manner by photoreceptor neurons, contribute to the biosynthesis of alanine in glia. Here we report that NH4+ and glutamate are transported into glia and that a transient rise in the intraglial concentration of NH4+ or of glutamate causes a net increase in the level of reduced nicotinamide adenine dinucleotides [NAD(P)H]. Biochemical measurements indicate that this is attributable to activation of glycolysis in glial cells by the direct action of NH4+ and glutamate on at least two enzymatic reactions: those catalyzed by phosphofructokinase (PFK; ATP:D-fructose-6-phosphotransferase, EC2.7.1.11) and glutamate dehydrogenase (GDH; L-glutamate:NAD oxidoreductase, deaminating; EC1.4.1.3). This activation leads to an increase in the production and release of alanine by glia. This signaling, which depends on the rate of conversion of NH4+ and glutamate to alanine and alpha-ketoglutarate, respectively, in the glial cells, raises the novel possibility of a tight regulation of the nutritive function of glia.
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PMID:Ammonium and glutamate released by neurons are signals regulating the nutritive function of a glial cell. 906 99

We developed a new simple assay for potassium ion in serum using urea amidolyase (UAL) from yeast sp. The method is based on activation of the enzyme by potassium ion. We eliminated endogenous ammonium ion by use of glutamate dehydrogenase (GLDH), and then monitored the production of ammonium ion by UAL, urea, ATP, bicarbonate and magnesium ions. Ammonium ion was produced proportional to the potassium ion concentration and was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH. We monitored the change of absorbance at 340 nm. The inhibitory effect of calcium ion to this assay was eliminated by adding glycoletherdiamine-N, N, N', N'-tetraacetic acid to the reaction. The within-assay coefficients of variation (CV) of this method were 0.9-1.55% (n = 10) at 3.32-6.18 mmol/L. Day-to-day CVs ranged from 1.49% to 2.46%. The analytical recovery was 96-108%. The correlation coefficient between the values obtained by our method (y) and those by the ion-selective electrode (ISE) method (x) was 0.994 (y = 1.032x-0.166 mmol/L, Syx = 0.110, n = 100). The presence of bilirubin, haemoglobin or other ions did not affect this assay, confirming the usefulness of this assay for clinical purposes.
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PMID:New enzymatic assay with urea amidolyase for determining potassium in serum. 924 70

The ADP-receptor on the surface of human platelets and cells of megakaryocytic lineage has been classified as P2T purinergic receptor for which ADP is an agonist and ATP is an antagonist. Although it is one of the earliest identified of the important cellular receptors, it has neither been purified nor cloned. We have developed an immunoaffinity method for rapidly identifying the platelet ADP-receptor and this method can be extended to the purification of the receptor. A polyclonal antibody to glutamate dehydrogenase (GDH) covalently modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) recognized neither FSBA nor glutamate dehydrogenase. Immunoblot of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized FSBA-labeled platelets showed the presence of a protein band at 100 kDa and this band was absent in the immunoblots of platelets that were preincubated with ADP and ATP or covalently modified by the chemically reactive ADP-affinity analogs, 2- and 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (2- and 8BDB-TADP) and 2-(3-bromo-2-oxopropylthio)adenosine-5'-diphosphate (2-BOP-TADP), prior to treatment with FSBA. FSBA as well as 2- and 8-BDB-TADP and 2-BOP-TADP have been previously shown to inhibit ADP-induced platelet responses by selectively and covalently modifying aggregin (100 kDa), an ADP-receptor in intact human blood platelets. The results show that polyclonal antibody to FSBA-labeled GDH is capable of recognizing FSBA-labeled aggregin on platelets and, thus, could be used to purify aggregin by immunoaffinity column chromatography. The immunoaffinity method was found to be far more sensitive than the radiochemical methods to identify aggregin previously developed in our laboratory. Since FSBA is also capable of reacting with enzymes that require ATP for their catalytic function, the polyclonal antibody may be used to identify and purify other P2-type purinergic receptors that require binding of ATP before eliciting cellular responses.
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PMID:Immunoaffinity method to identify aggregin, a putative ADP-receptor in human blood platelets. 936 34

Hyperstimulation with the cholecystokinin analogue cerulein induces mild edematous pancreatitis in rats. It is believed that an impaired energy metabolism diminishes the cellular defense capacity in the inflamed pancreatic tissue and, therefore, contributes to the injuries in acinar cells. In the present study, changes in the capacity of oxidative phosphorylation were quantified within the first 24 h after subcutaneous cerulein injections. Serum amylase level and pancreatic water content were maximally elevated 5 h after the first injection. The capacity of mitochondrial respiration was reduced in isolated acinar cells to 69 and 44% at 5 and 24 h, respectively, compared to that in saline controls. Simultaneously, glutamate dehydrogenase (GLDH) activity dropped to 70 and 46%. The respiration rates of acinar cells and of isolated mitochondria related to GLDH activities were not different from controls. This suggests that the major portion of the mitochondrial population within the acinar cell is inactivated in the course of cerulein treatment. After 24 h, the reduced population of functionally intact mitochondria restricted the rate of phosphorylating respiration in acinar cells (52%), which resulted in a diminution of cellular ATP to 57%. It is concluded that cerulein hyperstimulation induces a drastic and long-lasting reduction of the capacity for mitochondrial ATP production which may adversely affect energy-requiring reactions of the gland during regeneration.
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PMID:Effect of supramaximal cerulein stimulation on mitochondrial energy metabolism in rat pancreas. 943 68

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

The ability of alpha-ketoisocaproate (KIC) to induce ATP production in isolated mitochondria from pancreatic beta-cells was examined with a bioluminometric method. There was no ATP production from KIC when tested alone or in combination with malate (1 mmol/l), nor did DL-beta-hydroxybutyrate induce mitochondrial ATP production, whereas palmitoyl-carnitine and pyruvate were efficient stimulators of mitochondrial ATP production in the presence of an equimolar concentration of malate. However, KIC stimulated the mitochondrial ATP production when tested in combination with glutamate (10 mmol/l). The concentration necessary to obtain half-maximal stimulation was approximately 50 micromol/l KIC, and maximal activity, comparable to that obtained with fatty acids, was reached at 1 mmol/l KIC. Higher KIC concentrations inhibited the mitochondrial ATP production, whereas a plateau was attained at 1 mmol/l KIC in the presence of glutamine. Ca2+ stimulated the maximal mitochondrial ATP production induced by KIC. Maximal stimulation was obtained with 300 nmol/l Ca2+ in the presence of 0.3 mmol/l KIC. Ca2+ reduced the concentration of KIC necessary for half-maximal stimulation to <30 micromol/l. Leucine stimulated the mitochondrial ATP production in the presence of glutamate to the same extent as KIC. Half-maximal stimulation was observed with 2 mmol/l leucine. There were no additive effects on mitochondrial ATP production when KIC and leucine were tested in combination. The results demonstrate that KIC by itself is not a mitochondrial substrate for ATP production. KIC must transaminate with glutamate or glutamine to yield alpha-ketoglutarate and leucine. Since leucine allosterically activates glutamate dehydrogenase, which also produces alpha-ketoglutarate, the insulinogenic effect of KIC may in part be due to the intramitochondrial generation of alpha-ketoglutarate. Since KIC-induced ATP production reaches a plateau already at micromolar concentrations (i.e., far below the concentrations at which KIC induces insulin release), it is proposed here that the catabolism of KIC may induce additional signals related to insulin release.
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PMID:Alpha-ketoisocaproate is not a true substrate for ATP production by pancreatic beta-cell mitochondria. 951 37


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