Subcellular localization of gamma-aminobutyrate transaminase and glutamate dehydrogenase in adult rat brain. Evidence for at least two small glutamate compartments in brain.

The subcellular localizations of gamma-aminobutyrate transaminase (EC 2.6.1.19) and glutamate dehydrogenase (EC 1.4.1.2) in brain tissue of adult rats were compared with each other and with those of NAD+-isocitrate dehydrogenase (EC 1.1.41) and monoamine oxidase (EC 1.4.3.4; kynuramine as substrate). Crude mitochondrial fractions from brain tissue were centrifuged in continuous sucrose density gradients. gamma-Aminobutyrate transaminase and glutamate dehydrogenase were always found at a higher density than NAD+-isocitrate dehydrogenase and monoamine oxidase. When centrifuged for 1 h at 53 000gav., there was a slight difference between the distribution profiles of glutamate dehydrogenase and gamma-aminobutyrate transaminase. This difference was larger when the centrifugation time was only 15 min. It is concluded that there are subpopulations of brain mitochondria with differing proportions of gamma-aminobutyrate transaminase and glutamate dehydrogenase. The results are discussed in relation to evidence obtained with labelled precursors in vivo that there are at least two small glutamate compartments in adult brain.     

Enzyme Pattern and Aerobic Growth of Saccharomyces cerevisiae Under Various Degrees of Glucose Limitation

The enzyme pattern of Saccharomyces cerevisiae was followed during batch growth and in continuous culture in a synthetic medium limited for glucose under aerobic conditions. Seven enzymes were measured: succinate-cytochrome c oxidoreductase, malate dehydrogenase, nicotinamide adenine dinucleotide-linked glutamate dehydrogenase, malate synthase, isocitrate lyase, aldolase, and nicotinamide adenine dinucleotide phosphate (NADP(+))-linked glutamate dehydrogenase. During fermentation of glucose and high growth rate (mu) during the first log phase in batch experiments, the first five enzymes (group I) were repressed, and aldolase and NADP(+)-linked glutamate dehydrogenase (group II) were derepressed. During growth on the accumulated ethyl alcohol and lower mu, the group I enzymes were preferentially formed and the other two were repressed. A sequence of derepression of the group I enzymes was found during the shift from glucose to ethyl alcohol metabolism, which can be correlated with a strong increase in the percentage of single (nonbudding) cells in the population. A correlation between the state of cells in the budding cycle and enzyme repression and derepression is suggested. In continuous culture, the enzyme pattern was shown to be related to the growth rate. The group I enzymes were repressed at high growth rates, while the group II enzymes were derepressed. Each enzyme exhibits a different dependence. The enzyme pattern is shown to depend on the rate of substrate consumption as well as on the type of metabolism and to be correlated with the budding cycle. The enzyme pattern is considered to be controlled by changes of intracellular catabolic or metabolic conditions inherent in the division cycle.     

Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5''-phosphate.

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5'-phosphate and this inactivation increased 3--4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5'-phosphate.     

Crystallographic studies of glutamate dehydrogenase. Preliminary crystal data.

Native and pyridoxal phosphate modified rat liver glutamate dehydrogenase crystals have been obtained and used for a preliminary x-ray crystallographic analysis. The space group is P6222 (P6422) having unit cell dimensions a = b = 101 A, c = 724 A and gamma = 120 A. The unit cell contains 36 subunits (six hexameric molecules) of molecular weight 56,000 and there is one half-molecule, i.e. three subunits, in the asymmetric unit. Packing considerations suggest that the glutamate dehydrogenase molecule has the point group symmetry 32 and that each subunit can be represented as a particle with approximate dimensions of 45 x 45 x 60 A.     

Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep

The potential of GST as a vaccine candidate against liver fluke infection in ruminants was studied by vaccinating sheep (n = 9) with GST purified from adult worms of Fasciola hepatica and challenging with 500 F. hepatica metacercariae. The immunization induced a high antibody response to GST in contrast to the poor or undetectable response to this Ag observed in naturally infected sheep. Throughout the trial, the progress of the fluke infection was monitored by measuring RBC hemoglobin levels, the extent of liver damage and the fecal egg output in the sheep. This analysis indicated that a subpopulation (n = 4) of the GST vaccinated animals exhibited no anemia, reduced liver damage and a lower mean fecal egg count relative to the infected control group suggesting a lower fluke burden in these animals. Worm burdens in the livers of the GST vaccine group (107 +/- 22) were 57% lower than in the infected control group (250 +/- 25). The subpopulation of the GST vaccine group demonstrated a 78% reduction in mean worm burdens relative to the control group. These results show that GST of adult F. hepatica is a novel Ag that can significantly protect sheep against liver fluke infection. The results suggest that the immune response to GST is directed to the juvenile worm reducing the number of worms that can establish in the liver of the vaccinated animals.     

GABA metabolism in venezuelan equine encephalomyelitis virus infection

Mice infected with the Venezuelan equine encephalomyelitis virus showed a significant decrease in the GABA content of cerebral hemispheres. Activity of the enzyme which synthetizes GABA, glutamate decarboxylase, is also reduced in whole cerebral hemispheres, neostriatum, and frontal cortex of infected animals, as compared to values obtained from the same regions of control mice. No significant difference was demonstrated in the activities of GABA transaminase, glutamate dehydrogenase, lactate dehydrogenase, succinate dehydrogenase and NAD-malate dehydrogenase in any of the regions studied. The results suggest that the viral infection produced an alteration in the mechanism of GABA synthesis.     

固定化谷氨酸棒杆菌T6—13细胞的酶学性质研究

比较研究了固定化谷氨酸棒杆菌细胞和自然细胞的谷氨酸脱氢酶、异拧檬酸脱氢酶,葡萄糖-6-磷酸脱氢酶的一些性质。最适pH、温度对二者酶促反应速度的影响基本相似;pH、热稳定性固定化细胞高于自然细胞;底物表观米氏常数谷氨酸脱氢酶,异柠檬酸脱氢酶有所增大,而葡萄糖-6-磷酸脱氢酶则有所下降;辅酶表观米氏常数均有所增大。这些是影响固定化细胞应用的主要因素。     

Immune response of goats immunised with glutathione S-transferase and experimentally challenged with Fasciola hepatica

Glutathione S-transferase (FhGST) purified from Fasciola hepatica adult worms was used to immunise goats against F. hepatica in an experimental infection; the level of protection, in terms of fluke burden, faecal egg counts and hepatic damage was determined, as well as the humoral and cellular immune response elicited. Animals were allocated into three groups of six animals each: group 1 (immunised with FhGST and infected), group 2 (unimmunised and infected), and group 3 (unimmunised and uninfected). There was no significant reduction of fluke burden (9.3%) or faecal egg counts; hepatic damage was also similar in both infected groups. However, immunisation with FhGST induced the development of a well-defined immune response, characterized by the production of specific-FhGST antibodies as well as the appearance of circulating IL-4.     

Coregulation of oxidized nicotinamide adenine dinucleotide (phosphate) transhydrogenase and glutamate dehydrogenase activities in enteric bacteria during nitrogen limitation

The relationship between oxidized nicotinamide adenine dinucleotide (phosphate) [NAD(P)+] transhydrogenase (EC 1.6.1.1) and NAD(P)+ glutamate dehydrogenase in several enteric bacteria which differ slightly in their regulation of nitrogen metabolism was studied. Escherichia coli strain K-12 was grown on glucose and various concentrations of NH4Cl as the sole nitrogen source. In the range of 0.5 to 20 mM NH4Cl, the energy-independent transhydrogenase increased two to threefold. Comparable changes occurred in NAD(P)+-linked glutamate dehydrogenase. NH4Cl concentrations of 20 to 60 mM resulted in relatively constant specific activities for both enzymes. Higher exogenous NH4Cl, however, led to a decline in both activities. Isocitrate dehydrogenase, another potential source of cellular NADPH, was insensitive to NH4Cl limitation. Similar studies in the presence of glutamate and different exogenous NH4Cl concentrations again showed concerted effects on both enzymes. Growth on glutamate as the sole nitrogen source led to severe repression of both transhydrogenase and glutamate dehydrogenase. In Salmonella typhimurium, both enzymes were unaffected by limiting NH4Cl or growth on glutamate as the sole nitrogen source. Both were, however, repressed by growth on aspartate, a potential source of cellular glutamate. Coordinate changes in glutamate dehydrogenase and transhydrogenase were also evident in Klebsiella aerogenes, particularly under conditions in which glutamate dehydrogenase was regulated inversely to glutamate synthetase. Coordinate changes in glutamate dehydrogenase and transhydrogenase in enteric bacteria are discussed in terms of the possible involvement of the latter enzyme as a direct source of NADPH in the ammonia assimilation system.     

Thermodynamic resolution of cytochrome P450 and characterization of an iron-sulfur center in rat liver microsomal preparations.

Binding of 8-anilinonaphthalene sulfonate (ANS) to glutamate dehydrogenase results in enzyme inhibition and a marked increase in the fluorescence of ANS. Perphenazine and GTP increase the fluorescence of ANS-glutamate dehydrogenase secondary to their known ability to alter the conformation of this enzyme. Aspartate aminotransferases, which form enzyme-enzyme complexes with glutamate dehydrogenase, produce a slight decrease in the fluorescence of ANS-glutamate dehydrogenase.While ANS and perphenazine are allosteric inhibitors of reactions catalyzed by free glutamate dehydrogenase, they do not inhibit reactions catalyzed by aminotransferaseglutamate dehydrogenase complexes. This is in spite of the fact that the aminotransferase does not prevent either ANS or perphenazine from being bound to glutamate dehydrogenase. Therefore, reactions catalyzed by the enzyme-enzyme complex are apparently not inhibited by ANS or perphenazine because binding of the aminotransferase to glutamate dehydrogenase prevents these ligands from altering the conformation of glutamate dehydrogenase. This is consistent with the fact that the aminotransferase also prevents perphenazine from enhancing the fluorescence of ANS-glutamate dehydrogenase.Reactions catalyzed by the enzyme-enzyme complex are inhibited by GTP and the aminotransferase does not prevent GTP from enhancing the fluorescence of ANS-glutamate dehydrogenase. Therefore, binding of the aminotransferase to glutamate dehydrogenase does not prevent GTP from altering the conformation of glutamate dehydrogenase.The fact that the aminotransferase completely prevents perphenazine from increasing the fluorescence of ANS-glutamate dehydrogenase suggests that in the enzymeenzyme complex each glutamate dehydrogenase polypeptide chain can be bound to an aminotransferase polypeptide chain. This would mean that three aminotransferase molecules can be bound to each monomeric unit (M_r 3 × 10⁵) of glutamate dehydrogenase.     

Dual-enzyme assay of glutamate in single cells based on capillary electrophoresis

A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Glutamate dehydrogenase and glutamic pyruvic transaminase were used to catalyze the glutamate reaction. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. Glutamate dehydrogenase catalyzed the formation of NADH, and glutamic pyruvic transaminase drives the glutamate dehydrogenase reaction by removing a reaction product and regenerating glutamate. The detection limit of glutamate is down to the 10⁻⁸ M level, which is 1 order of magnitude lower than previously reported detection limits based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most amino acids. The glutamate content in single human erythrocytes and baby rat brain neurons were determined with this method and the results agreed well with literature values.     

The regulation of ammonia assimilating enzymes in Lemma minor

Summary Lemna minor has the potential to assimilate ammonia via either the glutamine or glutamate pathways. A 3-4 fold variation in the level of ferredoxindependent glutamate synthase may occur, when plants are grown on different nitrogen sources, but these changes show no simple relationship to changes in the endogenous pool of glutamate. High activities of glutamate synthase and glutamine synthetase at low ammonia availability suggests that these two enzymes function in the assimilation of low ammonia concentrations. Increasing ammonia availability leads to a reduction in level of glutamate synthase and glutamine synthetase and an increase in the level of glutamate dehydrogenase. Glutamine synthetase and glutamate dehydrogenase are subject to concurrent regulation, with glutamine rather than ammonia, exerting negative control on glutamine synthetase and positive control on glutamate dehydrogenase. The changes in the ratio of these two enzymes in response to the internal pool of glutamine could regulate the direction of the flow of ammonia into amino acids via the two alternative routes of assimilation.Abbreviations GS Glutamine synthetase - GDH Glutamate dehydrogenase - GOGAT Glutamate synthase     

Regulation of NAD-dependent glutamate dehydrogenase by protein kinases in Saccharomyces cerevisiae

The active NAD-dependent glutamate dehydrogenase of wild type yeast cells fractionated by DEAE-Sephacel chromatography was inactivated in vitro by the addition of either the cAMP-dependent or cAMP-independent protein kinases obtained from wild type cells. cAMP-dependent inhibition of glutamate dehydrogenase activity was not observed in the crude extract of bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase. The cAMP-dependent protein kinase of CYR3 mutant cells, which has a high K alpha value for cAMP in the phosphorylation reaction, required a high cAMP concentration for the inactivation of NAD-dependent glutamate dehydrogenase. An increased inactivation of partially purified active NAD-dependent glutamate dehydrogenase (Mr = 450,000) was observed to correlate with increased phosphorylation of a protein subunit (Mr = 100,000) of glutamate dehydrogenase. The phosphorylated protein was labeled by an NADH analog, 5'-p-fluorosulfonyl[14C]benzoyladenosine. Activation and dephosphorylation of inactive NAD-dependent glutamate dehydrogenase fractions were observed in vitro by treatment with bovine alkaline phosphatase or crude yeast cell extracts. These results suggested that the conversion of the active form of NAD-dependent glutamate dehydrogenase to an inactive form is regulated by phosphorylation through cAMP-dependent and cAMP-independent protein kinases.     

Effects of the changes in enzyme activities on metabolic flux redistribution around the 2-oxoglutarate branch in glutamate production by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

An experimental method for metabolic control analysis (MCA) was applied to the investigation of a metabolic network of glutamate production by Corynebacterium glutamicum. A metabolic reaction (MR) model was constructed and used for flux distribution analysis (MFA). The flux distribution at a key branch point, 2-oxoglutarate, was investigated in detail. Activities of isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), and 2-oxoglutarate dehydrogenase complex (ODHC) around this the branch point were changed, using two genetically engineered strains (one with enhanced ICDH activity and the other with enhanced GDH activity) and by controlling environmental conditions (i.e. biotin-deficient conditions). The mole flux distribution was determined by an MR model, and the effects of the changes in the enzyme activities on the mole flux distribution were compared. Even though both GDH and ICDH activities were enhanced, the mole flux distribution was not significantly changed. When the ODHC activity was attenuated, the flux through ODHC decreased, and glutamate production was markedly increased. The flux control coefficients of the above three enzymes for glutamate production were determined based on changes in enzyme activities and the mole flux distributions. It was found that the factor with greatest impact on glutamate production in the metabolic network was obtained by attenuation of ODHC activity.     

Immunocytochemical Characterization of Glutamate Dehydrogenase in the Cerebellum of the Rat

The immunocytochemical distribution of glutamate dehydrogenase was studied in the cerebellum of the rat using antibodies made in rabbit and guinea pig against antigen purified from bovine liver. Antiserum was found to block partially enzymatic activity both of the purified enzyme and of extracts of the rat cerebellum. Using immunoblots of proteins of rat cerebellum, a major immunoreactive protein and several minor immunoreactive proteins were detected with antiserum. Only a single immunoreactive protein was detected using affinity-purified antibody preparations. This protein migrates with a molecular weight identical to that of the subunit of glutamate dehydrogenase. Further evidence that the antibodies were selective for glutamate dehydrogenase in rat cerebellum was obtained through peptide mapping. Purified glutamate dehydrogenase and the immunoreactive protein from rat cerebellum generated similar patterns of immunoreactive peptides. No significant cross-reaction was observed with glutamine synthetase. Immunocytochemistry was done on cryostat- and Vibratome-cut sections of the cerebellum of rats that had been perfused with cold 4% paraformaldehyde. Glial cells were found to be the most immunoreactive structures throughout the cerebellum. Most apparent was the intense labeling of Bergmann glial cell bodies and fibers. In the granule cell layer, heavy labeling of astrocytes was seen. Purkinje and granule cell bodies were only lightly immunoreactive, whereas stellate, basket, and Golgi cells were unlabeled. Labeling of presynaptic terminals was not apparent. These findings suggest that glutamate dehydrogenase, like glutamine synthetase, is enriched in glia relative to neurons.     

Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes

Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes--lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase--were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.     

Isolation and enzymological characterization of infected and uninfected cell protoplasts from root nodules of Glycine max

Infected and uninfected cell protoplasts were isolated from soybean ( Glycine max L. Merr. cv. Akisengoku) root nodules and purified by the use of nylon mesh filters and discontinuous Percoll gradients. Activities of the enzymes involved in carbon and nitrogen metabolism were measured in cytoplasmic fractions of purified protoplasts, as well as in the bacteroids isolated from infected cell protoplasts and in the cortical tissues after enzymatic digestion of the central zone of the nodules.
A high degree of purity of both infected and uninfected cells was demonstrated by microscopic observations and assays of β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and uricase (EC 1.7.3.3) activities and leghemoglobin contents.
As a whole, higher specific activities of enzymes of glycolysis were found in the cortical and uninfected cells than in the infected cells. The activities of glycolytic enzymes were extremely low in the bacteroids. Invertase (EC 3.2.1.26) was highly localized in the cortex. However, activity of sucrose synthase (EC 2.4.1.13) was highest in the cytosol of infected cells. Alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27) activities were much higher in uninfected than in infected cells. Specific activities of enzymes for nitrogen assimilation, that is, glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.14), aspartate (EC 2.6.1.1) and alanine (EC 2.6.1.2) aminotransferase were several-fold higher in uninfected cells than in the infected cells.
The results are discussed in relation to the possible cellular organization of carbon and nitrogen metabolism in soybean root nodules.     

Vaccination with cathepsin L phage-exposed mimotopes,single or in combination,reduce size,fluke burden,egg production and viability in sheep experimentally infected with Fasciola hepatica

Fascioliasis is a worldwide emergent zoonotic disease that significantly constrains the productivity of livestock. In this study, fluke burdens, liver fluke size and biomass, faecal eggs counts, serum levels of hepatic enzymes and immune response were assessed in sheep vaccinated with peptide mimotopes of cathepsin L and infected with metacercariae. A total of 25 sheep were allocated randomly into five groups of five animals each, and experimental groups were immunised with 1 × 10¹³ filamentous phage particles of cathepsin L1 (CL1) (TPWKDKQ), CL2 (YGSCFLR) and mixtures of CL1 + CL2 mimotopes, in combination with Quil A adjuvant, and wild-type M13KE phage in a two-vaccination scheme on weeks 0 and 4. The control group received phosphate-buffered saline. All groups were challenged with 300 metacercariae two weeks after the last immunisation and euthanised 16 weeks later. The CL1 vaccine was estimated to provide 57.58% protection compared with the control group; no effect was observed in animals immunised with CL2 and CL1 + CL2 (33.14% and 11.63%, respectively). However, animals receiving CL2 had a significant reduction in parasite egg output. Vaccinated animals showed a significant reduction in fluke length and width and wet weights. In the CL1 group, there was a significant reduction in the total biomass of parasites recovered. Egg development was divided into seven stages: dead, empty, unembryonated, cell division, eyespot, hatched and hatching. The highest percentage of developmental stages was detected for vaccinated sheep administered CL1 + CL2 with cell division, and the lowest percentage was observed in the hatching stage. Furthermore, a significant difference in all developmental stages was observed between vaccinated animals and the control group (P < 0.01). The levels of anti-phage total IgG in immune sera increased significantly at four weeks after immunisation and were always significantly higher for cathepsin L vaccine group than in the challenged control group. Total IgG was inversely and significantly correlated with worm burden in the CL1 group.