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

---

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

A flow injection analysis sensor for the measurement of glucose/lactate/glutamate is reported. The glucose oxidase/glutamate oxidase/lactate oxidase was immobilized on silanized controlled pore glass particles and packed into a Teflon column (i.d., 1.2 mm; length, 40 mm) to give a bed for glucose/lactate/glutamate. The hydrogen peroxide formed by the enzymatic reaction in the packed bed was monitored by a horseradish peroxidase- and tetracyanoquinodimethane (TCNQ)- modified graphite paste electrode at 50 mV vs Ag/AgCl. The glucose oxidase/lactate oxidase/glutamate oxidase were regenerated in the packed bed, whereas peroxidase was regenerated in the TCNQ-mediated graphite paste electrode by the oxidation of TCNQ. The oxidized TCNQ was electrochemically reduced at 50 mV vs Ag/AgCl. The cathodic current obtained by the reduction of TCNQ determined the concentration of the injected analytes in the packed bed. The system showed very rapid response. Response curves for the analysis of peroxide, glucose, lactate, and glutamate are reported.
...
PMID:Peroxidase- and tetracyanoquinodimethane-modified graphite paste electrode for the measurement of glucose/lactate/glutamate using enzyme-packed bed reactor. 771 Jan 4

Glutamate dehydrogenase is one of the main enzymes involved in the formation and metabolism of the neurotransmitter glutamate. In the present study we investigated the enzyme ultrastructurally in the cerebellar cortex, a region rich in well defined glutamatergic neurons, by pre-embedding immunocytochemical staining (peroxidase-antiperoxidase), as well as by post-embedding immunogold labelling employing a new system for quantitation and for specificity testing under the conditions of the immunocytochemical procedure. A new antiserum against immunologically purified bovine liver glutamate dehydrogenase or antibodies isolated from this by affinity chromatography were used in rats fixed by perfusion with aldehydes. The pre-embedding method displayed peroxidase reaction preferentially in mitochondria of astroglial cells (including the Bergmann glia). Mitochondria of neuronal tissue elements were usually free of peroxidase-reaction product. Extra-mitochondrial staining was not observed. The post-embedding immunogold method was employed to overcome penetration problems and allow semiquantitative analysis of localization and specificity. The highest densities of gold particles were found over the mitochondria in astroglial cell elements (including the Bergmann glia). Mitochondria in cell bodies of Bergmann glia had a lower particle density than those in astrocytic processes. In the latter, analysis of frequency distribution revealed no evidence of a population of mitochondria lacking glutamate dehydrogenase, but suggested the presence of populations with different levels of immunoreactivity. Comparison with the labelling of embedded bovine liver glutamate dehydrogenase indicated that the enzyme constitutes a high proportion (10%) of the total matrix protein of these mitochondria. A weaker but significant labelling was found in oligodendrocytes of the white matter. The labelling of mitochondria in neuronal elements including glutamatergic mossy fibre terminals was of the order of 15% of that in astroglial mitochondria. No difference was detected between glutamatergic neurons (mossy and parallel fibres, granular cells) and non-glutamatergic neurons (Purkinje cells). The particle density over non-mitochondrial areas was very close to background over empty resin. The results, obtained with different methods of tissue and antibody preparation, agree to show that the present form of glutamate dehydrogenase is restricted to mitochondria and preferentially localized in astrocytes.
...
PMID:Quantitative ultrastructural localization of glutamate dehydrogenase in the rat cerebellar cortex. 775 71

The neurotoxic amino acid, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-ODAP,ODAP) was oxidized by immobilized glutamate oxidase (GlOD) to produce hydrogen peroxide. The peroxide reacts with Trinder reagent in a reactor with immobilized horseradish peroxidase to form a red-colored quinone imine dye, which was detected spectrophotometrically at 512 nm. Determinations were made in a flow injection (FI) setup consisting of four packed-bed enzyme reactors containing GlOD (20 microL), catalase (20 microL), GlOD (250 microL), and peroxidase (50 microL) in series. Glutamate is oxidized quantitatively in the first reactor, but the hydrogen peroxide is destroyed in the second so that interferences from this substrate are removed. This step destroys only a few percent of the ODAP in the sample. Most of the remaining ODAP is oxidized in the third reactor. Injections of 20-microL ODAP standards gave a response curve which was linear within the range 10-650 microM. Phosphate buffer extracts of grass peas (lathyrus sativus) were purified by centrifugation and membrane filtration. Samples were injected into the FI setup to assay the toxin at a rate of 20 samples per hour. The beta-ODAP content of a batch of dry seed corresponded to 0.74% (w/w) with a relative standard deviation of 2.8%. Thermal treatment of ODAP standards at 80-90 degrees C reduced the response to 62% of that before heating. The decrease is due to beta<-->alpha isomerization, and the experiment thus confirms that the method is selective for the toxic beta-isomer.
...
PMID:Flow injection assay for the neurotoxin beta-ODAP using an immobilized glutamate oxidase reactor with prereactors to eliminate glutamate interferences. 780 62

An online sensor with a low detection limit for L-glutamate was developed in order to monitor the change in the extracellular L-glutamate concentration as a result of stimulated release from cultured nerve cells. The sensor consisted of a microdialysis (MD) probe fixed at the manipulator, a small-volume L-glutamate oxidase enzymatic reactor (0.75 mm i.d. and 2.5 cm long), and an electrochemical detector in a thin-layer radial flow cell with an active volume of 70-340 nL. Glassy carbon bulk or carbon film ring-disk electrodes were used as detectors by modifying them with Os poly(vinylpyridine) mediator containing horseradish peroxidase. The overall efficiency of L-glutamate detection with the sensor is 94% under optimum conditions, due to an efficient enzymatic reaction in the reactor and a high conversion efficiency in the radial flow cell. As a result, we achieved a sensitivity of 24.3 nA/muM and a detection limit of 7.2 nM (S/N = 3). The effect of interferents such as L-ascorbic acid can be minimized effectively by applying a low potential to the electrode for hydrogen peroxide detection (O mV) and via the ring-disk electrode geometry by using the disk for preoxidation. In the in vitro experiment, an MD probe for sampling was connected to a manipulator that controls distance between the probe and the stimulated cells. The cells were stimulated by KCl in a glass capillary or electrically with microarray film electrodes fabricated on a substrate. By using the sensor, we can monitor L-glutamate concentration changes at the submicromolar level caused by KCl stimulation of a single nerve cell and micromolar L-glutamate concentration increases caused by electrical stimulation of a brain slice. An increase in L-glutamate concentration can also be measured by positioning the probe near the cell that is connected synaptically to the stimulated cell.
...
PMID:Concentration of extracellular L-glutamate released from cultured nerve cells measured with a small-volume online sensor. 868 11

A small volume L-glutamate online sensor was developed in order to monitor changes in the local concentration of L-glutamate released from cultured nerve cells. Syringe pump in the suction mode is used to sample extracellular fluid continuously from a glass micro-capillary and the concentration of L-glutamate can be determined by using a glassy carbon (GC) electrode modified with an Os-polyvinylpyridine mediator bottom film containing horseradish peroxidase and a bovine serum albumin top layer containing L-glutamate oxidase. The overall efficiency of L-glutamate detection with a sensor is 71% under optimum conditions due to an efficient enzymatic reaction at the modified electrode in the thin layer radial flow cell. As a result, we achieved a detection limit of 7-15 nM and a linear range of 50 nM to 10 microM. In an in vitro experiment, the extracellular fluid near a particular nerve cell can be sampled with this micro-pipet and continuously introduced into the modified GC electrode in the radial flow cell via suction provided by a syringe pump. The nerve cells are stimulated by the KCl in a glass capillary and the L-glutamate concentration change can be monitored by changing the distance between the sampling pipet and the nerve cells.
...
PMID:Continuous monitoring of L-glutamate released from cultured nerve cells by an online sensor coupled with micro-capillary sampling. 917 16

We report the first on-line electrochemical sensor for the continuous measurement of gamma-aminobutyric acid (GABA), which is a well-known inhibitory neurotransmitter in the nervous system. The sensor is composed of a glutamate oxidase (GluOx) and catalase immobilized small-volume enzymatic reactor and a glassy carbon (GC) electrode modified with a top layer film consisting of gabase and GluOx coimmobilized bovine serum albumin and an Ospoly(vinylpyrridine) bottom layer film containing horseradish peroxidase. The response of the sensor depends on the alpha-ketoglutarate concentration and is almost saturated when its concentration is 100 times higher than GABA. The sensor exhibits a sensitivity of 1.56 nA/microM for GABA under optimized conditions and shows almost no response when 10 microM glutamate is continuously injected. A detection limit of 0.1 microM is obtained with a linear range of 0.1-10 microM. GABA can be measured in the absence of alpha-ketoglutarate when there is L-glutamate in the sample solution, which is a typical condition for the extracellular measurement of cultured nerve cells.
...
PMID:Small-volume on-line sensor for continuous measurement of gamma-aminobutyric acid. 943 68

Several proteins (avidin, carboxypeptidase B, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, maltase, and peroxidase) composed of one to six subunits were irradiated in the frozen state. Each irradiated protein was examined by size-exclusion chromatography (SEC) and by denaturing gel electrophoresis (SDS-PAGE). All these proteins eluted from SEC as a single peak even though SDS-PAGE showed cleavage of the polypeptide backbone of the monomers. Thus, fragmentation of the subunits did not result in dissociation of the oligomeric structure.
...
PMID:Radiation effects on the native structure of proteins: fragmentation without dissociation. 958 17

A continuous spectrophotometric assay for determining low levels of L-glutamate is described. The assay, which involves the enzymes L-glutamate oxidase and glutamic-pyruvic transaminase, is based on the recycling of L-glutamate into alpha-ketoglutarate, with the concomitant appearance of one molecule of hydrogen peroxide in each turn of the cycle. This is subsequently reduced by means of a peroxidase-coupled reaction, using 2, 2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) as substrate. In this way the interference observed in the cyclic assay using glutamic-oxalacetic transaminase, which is due to the fact that L-aspartate is also a substrate of L-glutamate oxidase, is eliminated. A kinetic study of the system is presented, with the accumulation of chromophore being seen to undergo a transient phase, which is dependent both on the cycling rate and on the auxiliary enzyme concentration. The kinetic parameters characterizing the system have been determined, making it possible to optimize costs with respect to the enzymes involved in the cycle, since the minimum amount needed for a given rate constant of the cycle can be calculated.
...
PMID:A continuous spectrophotometric method based on enzymatic cycling for determining L-glutamate. 961 6

A new enzyme-immobilization reaction by means of L-ascorbic acid (ASA) is described using NH(2) polymers based on cellulose or poly(vinyl alcohol) with the example of oxidoreductase enzymes. In this way, enzyme proteins such as glucose oxidase (GOD), glutamate oxidase, lactate oxidase, urate oxidase and peroxidase can be covalently fixed with a high surface loading to ultrathin and transparent NH(2)-polymer films if their surfaces are previously treated with an ASA solution, in, for example, N,N-dimethyl acetamide, DMSO or methanol. ASA then obviously reacts like a diketo compound with amino groups of the NH(2)-polymer film and enzyme protein, forming dehydroascorbic acid derivatives with neighbouring Schiff's-base structures. In a subsequent fragmentation reaction, the latter presumably form stable oxalic acid diamide derivatives as coupling structures between enzyme protein and NH(2)-polymer film, as suggested by results from investigations of the ASA reaction with n-butylamine. The immobilized enzymes can be stored at 4 degrees C in bidistilled water for at least 1 month without becoming detached from the NH(2)-polymer film and without diminished enzyme activity. The apparent K(m) values of the immobilized enzymes are in part clearly smaller than those of the dissolved enzymes or those found in other immobilization processes such as the diazo coupling or the bifunctional glutardialdehyde reaction. For example, the K(m) value of the immobilized GOD with different NH(2) polymers as the matrix structure is smaller by a factor of approx. 20 than that of the dissolved enzyme.
...
PMID:A novel efficient enzyme-immobilization reaction on NH2 polymers by means of L-ascorbic acid. 1051 95

Enzymatic formation of glutamate is critical to numerous biological pathways. However, current methods for assaying the activities of glutamate-forming enzymes are not particularly suitable for high-throughput screening in drug discovery. We present a continuous-read, fluorometric assay for high-throughput analysis of glutaminases. This assay is adapted to a microplate format and employs glutamate oxidase and horseradish peroxidase to couple glutamate formation to production of the fluorescent reporter molecule, resorufin, for enhancement of sensitivity (M. Zhou, Z. Diwu, N. Panchuk-Voloshina, and R. P. Haughland, 1997, Anal. Biochem. 253, 162-168). Described herein is the selection of suitable levels of coupling enzymes for optimal kinetic response and lag time of the reporter system, based on the kinetic characteristics of the individual coupling enzymes. Finally, implementation of the assay in a format for high-throughput kinetic analysis of glutaminases is demonstrated for Escherichia coli carbamoyl phosphate synthase. Derived kinetic constants are comparable to literature values determined using a variety of assay techniques.
...
PMID:Implementation of a continuous, enzyme-coupled fluorescence assay for high-throughput analysis of glutamate-producing enzymes. 1096 23


<< Previous 1 2 3 4 5 Next >>

---