A highly sensitive glutamic acid biosensor based on the determination of NADH enzymically generated by l-glutamic dehydrogenase

In this article, a sensitive and stereo-selective biosensor for l-glutamic acid (l-Glu) based on the electrochemiluminescence (ECL) of Ru(bpy)32+ has been designed by applying l-glutamic dehydrogenase (GLDH) for enzymatic generation of NADH in situ.

The Behavior of Proteins on Reversed-Phase Supports During High-Performance Liquid Chromatography and Prediction of Protein Retention Time on Ymc Columns

Protein molecules present more complex analytical challenges than conventional low molecular weight pharmaceutical compounds, and thus need powerful analytical approaches for the entire pharmaceutical development and quality control process. We used high-performance liquid chromatography to investigate the reversed-phase chromatographic behavior of eleven proteins (albumin, carbonic anhydrase, cytohrome c, L-glutamic dehydrogenase, enolase, α-lactoglobulin, Lysozyme, myoglobin and ribonuclease. By using a water/organic solvent/trifluoroacetic acid system the influence of experimental parameters was examined, and chromatographic results from two C4- chain-length supports were found to be comparable. The model enables prediction of initial conditions from two experimental data points for different types of reversed-phase columns with water-acetonitrile-TFA, water-methanol-TFA, and water-2-propanol-TFA mobile phases

Vitamin E-Induced Changes in Glutamate and GABA Metabolizing Enzymes of Chick Embryo Cerebrum

Vitamin E exists in eight different forms, four tocopherols and four tocotrienols. It forms an important component of our antioxidant system. The structure of Vitamin E makes it unique and indispensable in protecting cell membranes. α-tocopherol, one of the forms of Vitamin E, is also known to regulate signal transduction pathways by mechanisms that are independent of its antioxidant properties. Vitamin E compounds reduce the production of inflammatory compounds such as prostaglandins. Swollen, dystrophic axons are considered as the hallmark of Vitamin E deficiency in the brains of rats, monkeys, and humans. The present work aimed to study the Vitamin E- (α-tochopherol acetate-) induced alterations of enzymes involved in metabolism of Glutamate and GABA during developmental neurogenesis of cerebrum. Therefore, cytosolic and crude mitochondrial enzyme activities of glutamine synthetase, aspartate transaminase, alanine transaminase, GABA transaminase, succinic Semialdehyde dehydrogenase, glutamic dehydrogenase, and α-Ketoglutarate dehydrogenase were analysed. Vitamin E induced significant changes in these enzymes thus altering the normal levels of glutamate and GABA during developmental neurogenesis. Such changes are surely to disturb the expression and/or intensity of neurotransmitter signaling during critical periods of brain development.

Aspartate aminotransferase isotope exchange reactions: implications for glutamate/glutamine shuttle hypothesis

Aspartate aminotransferase (AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate (Asp) to a keto acid acceptor—oxaloacetate (OA) or α-ketoglutarate (KG), respectively. Data presented here show that AAT catalyzes two partial reactions resulting in isotope exchange between3H-labeled Glu or 3H-labeled Asp and the cognate keto acid in the absence of the keto acid acceptor required for the net reaction. Tritiated keto acid product was detected by release of 3H2O from C-3 during base-induced enolization. Tritium released directly from C-2 (or C-3) by the enzyme was also evaluated and is a small fraction of that released because of exchange to the keto acid pool. Exchange is dependent on AAT concentration, time-dependent, proportional to the amino-to-keto acid ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor. Enzymatic conversion of [3H]KG to Glu by glutamic dehydrogenase (GDH) or of [3H]OA to malate by malic dehydrogenase (MDH) “protects” the label from release by base, showing that base-induced isotope release is from keto acid rather than a result of release during the exchange process. AAT isotope exchange is discussed in the context of the glutamate/glutamine shuttle hypothesis for astrocyte/neuron carbon cycling.

Use of Near Infrared Spectroscopy (MRS) to Monitor Changes in the Composition of Rumen Fluid

To obtain more information about rumen fluid composition, levels of different components are determined by a range of techniques which are often slow and cumbersome. This study was established to develop calibration equations for the rapid determination of the composition of rumen fluid with a view to monitoring changes in composition with time.Forty-three strained rumen fluid samples, taken throughout a 7 h period from three lactating cows offered silage either alone or with fish meal or rapeseed meal/urea twice daily, were used. Immediately upon withdrawal from the rumen each sample was analysed for pH, then acidified using concentrated sulphuric acid before storing at -18°C. Following centrifugation at 11600 G concentrations of volatile fatty acids and ammonia-nitrogen were determined in the supernatant by packed column gas chromatography and enzymatically using glutamic dehydrogenase respectively.

Osmoregulation in Rhizobium meliloti: characterization of enzymes involved in glutamate synthesis

Rhizobium meliloti, like many bacteria, accumulates elevated levels of glutamate when osmotically stressed. The biochemical basis for this increase in glutamate production was investigated. Enzymes involved in glutamate synthesis, including glutamine synthetase, glutamate synthase, and glutamic dehydrogenase, were characterized in dialyzed crude cell-free extracts. A transaminase activity, which uses branched chain amino acids for the amination of 2-ketoglutaric acid, was also characterized. With the exception of glutamic dehydrogenase, the specific activity of the enzymes did not vary more than 4-fold in response to the available source of nitrogen or supplemental glutamate. Glutamic dehydrogenase activity was 13-fold greater when cells grew with 10 mM [Formula: see text] than when cells grew with 0.5 mM [Formula: see text]. Glutamate synthase was repressed 2-fold when cells grew with supplemental glutamate. Conversely, this enzyme was derepressed 2× when cells grew with 0.5 mM [Formula: see text] or nitrate. Growing cells in minimal defined medium with 400 mM NaCl to cause osmotic stress had little effect on the specific activity of any of the enzymes. The addition of K+ to the reactions stimulated heat-stable glutamine synthetase activity, but inhibited the other enzymes. Glutamate synthase was inhibited to a limited extent by several intermediates in the Krebs' cycle and very severely by glyoxylate. The addition of 10 mM glutamate to the reaction inhibited glutamate synthase 20%, but had no effect on the other enzymes. Key words: enzymes, glutamate synthesis, osmotic stress.