Glutamate Dehydrogenase

A screen-printed three-electrode amperometric biosensor based on urease and the nicotinamide adenine dinucleotide hydrogen (NADH)–glutamic dehydrogenase system was developed and applied to the screening of heavy metals in environmental samples. The development of an amperometric sensor for the monitoring of urease activity was feasible by coupling the urea breakdown reaction catalysed by urease to the reductive ammination of ketoglutarate catalysed by glutamic dehydrogenase (GLDH). The ammonia provided by the urea conversion is required for the conversion of ketoglutarate to glutamate with the concomitant oxidation of the NADH cofactor. NADH oxidation is monitored amperometrically at 0.3 V (vs. Ag/AgCl) after urease immobilization onto the screen-printed three-electrode configuration. Immobilization of urease on the surface of screen-printed electrodes was performed by entrapment in alginate gel and adsorption on the electrode in a nafion film. Low sensitivity to inactivation by metals was recorded after urease entrapment in alginate gel with detection limits of 2.9 and 29.8 mg L−1 for Hg(II) and Cu(II), respectively. The use of the negatively charged nafion film created a more concentrated environment of cations in proximity to the enzyme, thus enhancing the urease inhibition when compared to gel entrapment. The calculated detection limits were 63.6 and 55.3 μg L−1 for Hg(II) and Cu(II), respectively, and 4.3 mg L−1 for Cd(II). A significant urease inactivation was recorded in the presence of trace amounts of metals (μg L−1) when the enzyme was used free in solution. Analysis of water and soil samples with the developed nafion-based sensor produced inhibition on urease activity according to their metal contents. The obtained results were in agreement with the standard methods employed for sample analysis. Nevertheless, the use of the amperometric assay (with free urease) proved more feasible for the screening of trace amounts of metals in polluted samples.

Phosphate depletion (PD) in vivo causes a sundry of abnormalities in pancreatic islets including a rise in cytosolic calcium, low ATP content, reduced Ca2+ ATPase and Na(+)-K+ ATPase activity, and impaired insulin secretion in response to glucose or potassium. L-Leucine is a strong secretagogue that triggers insulin secretion by deamination to alpha-ketoisocaproic acid (KIC) and the subsequent metabolism of the latter to ATP and by the activation of glutamate dehydrogenase (GLDH), which acts on glutamate to generate alpha-ketoglutarate, the metabolism of which results in ATP production. The generation of ATP triggers events that lead to insulin secretion. It is not known whether PD impairs leucine-induced insulin secretion, and the cellular derangements that are involved in such an abnormality are not defined. These issues were studied in PD rats and in pair-weighed normal animals as controls. D-Leucine uptake by islets from PD rats is normal, but both leucine- and KIC-induced insul...

... Barbara Riond & Marina L. Meli & Ueli Braun & Peter Deplazes & Kaspar Joerger & Rudolf Thoma & Hans Lutz & Regina ... may occur, which, in conjunction with hypoxia, may lead to hepatic cell degen-eration and glomerular dysfunction (Allbritton and Seger 1962; Allen and ...

Blood samples from silver foxes experimentally infected with Opisthorchis felineus and Metorchis bilis, respectively, were examined for the activity of liver enzymes. The average activities of aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), ...

1H/15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15N]glutamine, [5-15N]glutamine, [2-15N]glutamate, 15NH4Cl, [2-15N]alanine, and [1-13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amido-transfer reaction. In glutamine-free media 15NH4+ was consumed and incorporated into alanine. 15NH4+ was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1-13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.

To assess the effects of subordinate social status on digestive function, metabolism, and enzyme activity in salmonid fish, juvenile rainbow trout Oncorhynchus mykiss were paired with size-matched conspecifics (<1.5% difference in fork length) for 5 d. Fish that were fasted for 5 d and fish sampled directly from the holding tank were used as control groups. Both subordinate and fasted fish experienced significant decreases in intestine mass (P = 0.043), and the gall bladder showed marked and significant changes in both size (P = 0.004) and appearance. These findings suggest that the negative effect of social subordination on digestive function reflects in large part a lack of feeding. Hepatic phosphoenolpyruvate carboxykinase activity was significantly higher in subordinate fish relative to dominants, whereas subordinate hepatic pyruvate kinase activity was significantly lower; activities of both enzymes were significantly correlated with plasma cortisol concentrations and behavi...

This study was undertaken to determine the effects of long-distance running on serum bilirubin fractions. Thirteen male ultramarathon runners participating in a 100-km race volunteered for the study. Venous blood samples were obtained shortly before and immediately after the race. Decreased serum haptoglobin levels (-66%) indicated the presence of hemolysis. After accounting for plasma-volume loss, significant post-race increases were found for creatine kinase (+20-fold), creatine kinase-MB (+252%), alanine aminotransferase (+42%), aspartate aminotransferase (+193%), gammaglutamyl transpeptidase (+56%), and glutamate dehydrogenase (+58%) serum activities, suggesting that running causes alterations of both muscle and liver tissues. Serum concentration of total bilirubin was significantly elevated (+106%) following the race, with changes corresponding to both unconjugated (+96%) and conjugated esters (+283%) of the pigment and significant increases in the ratio of esterified to total bilirubin. Our data show that long-distance running causes increases in the different serum bilirubin fractions which can be accounted for both hemolysis and hepatic disturbances.

A pBR322-derived plasmid (pCGL107) that carries the Corynebacterium melassecola ATCC 17965 analogue of Escherichia coli gdhA gene (encoding glutamate dehydrogenase), was introduced into the related strain, Brevibacterium lactofermentum CGL2002, by electroporation and integrated into its chromosome by homologous recombination. However, pCGL107 cannot integrate into C. melassecola, since the host restriction prevents successful electroporation by E. coli-modified DNA. Nevertheless, B. lactofermentum-modified replicative plasmid DNA can be transformed by electroporation into C. melassecola; thus pCGL519-2, a shuttle plasmid that carries the C. melassecola analogue of E. coli ghA (encoding citrate synthase), was extracted from the former host and electroporated into the latter. Rare restriction sites conveniently placed in pCGL519-2 were used to recover a replicon-less cartridge called ‘integron’, that contains a selectable marker and gtlA within a single fragment. Integron prepared from pCGL519-2 DNA which had been extracted from C. melassecola, and thus, was capable of eluding the C. melassecola restriction barrier(s), was successfully electroporated into this host. The molecular analysis of the resulting transformants suggests that they result from the integration of a single circular integron molecule by homologous recombination between the gltA regions of the host genome and the integron. These transformants were stable for 30 generations in the absence of selection.

Genetic variation in growth performance was estimated in 26 families from two commercial strains of Arctic charr Salvelinus alpinus. Physiological determinants of growth and metabolic capacities were also assessed through enzymatic assays. A relatedness coefficient was attributed to each family using parental genotypes at seven microsatellite loci. After 15 months of growth, faster growing families had significantly lower relatedness coefficients than slower growing families, suggesting their value as indicators of growth potential. Individual fish that exhibited higher trypsin activity also displayed higher growth rate, suggesting that superior protein digestion capacities can be highly advantageous at early stages. Capacities to use amino acids as expressed by glutamate dehydrogenase (GDH) activities were lower in the liver of fast-growing fish (13–20%), whereas white muscle of fast-growing fish showed higher activities than that of slow-growing fish for amino acid metabolism and aerobic capacity [22–32% increase for citrate synthase (CS), aspartate aminotransferase (AAT) and GDH]. The generally higher glycolytic capacities (PK and LDH) in white muscle of fast-growing fish indicated higher burst swimming capacities and hence better access to food.