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PubMed1998

NifA-dependent expression of glutamate dehydrogenase in Rhizobium etli modifies nitrogen partitioning during symbiosis.

Constitutive expression of foreign glutamate dehydrogenase in Rhizobium etli inhibits bean plant nodulation (A. Mendoza, A. Leija, E. Martínez-Romero, G. Hernández, and J. Mora. Mol. Plant-Microbe Interact. 8:584-592, 1995). Here we report that this inhibition is overcome when controlling gdhA expression by NifA, thus delaying the GDH activity onset after nodule establishment. Expression of gdhA modifies the nitrogen partitioning inside the bacteroid, where newly synthesized ammonia is preferentially incorporated into the amino acid pool instead of being exported to the infected cells. As a consequence, the fixed nitrogen transport to the leaves, measured as the ureides content in xylem sap, is significantly reduced. Nitrogenase activity, although not nifHDK expression, is significantly reduced in bacteroids expressing gdhA, probably due to the utilization of energy and reducing power for nitrogen assimilation. Here we show that ammonia assimilation inside R. etli bacteroids is active, albeit at low levels, and when enhanced is deleterious to the symbiotic performance. This leads us to believe that further reduction of the basal nitrogen metabolism in the bacteroid might stimulate the nitrogenase activity and increase the nitrogen supply to the plant.

Bacterial Proteins↗
PubMed2004

RNA synthetic activity of glutamate dehydrogenase: determination of enzyme purity, RNA characteristics, and deamination/amination ratio.

The activity of glutamate dehydrogenase (GDH), an important enzyme in carbon and nitrogen metabolism, is routinely assayed by photometry. The RNA synthetic activity of the enzyme provides new technologies for assaying its activity. The enzyme was made to synthesize RNAs in the absence of DNA and total RNA but with different mixes of the four nucleoside triphosphates (NTPs) in order to investigate the RNA characteristics. RNase VI (hydrolyzes base-paired residues) digested the poly (U,A) RNA completely because the U and A residues were evenly distributed to produce many base-paired regions. Therefore, the synthesis of RNA by GDH was by random addition of NTPs. The RNA synthetic activity of the enzyme was at least 50-fold more active in the deamination than in the amination direction, thus providing a robust technology for assay of the enzyme's activity. cDNAs prepared from the RNAs were subjected to restriction fragment differential display polymerase chain reaction analyses. Sequencing of the cDNA fragments showed that some of the RNA synthesized by GDH shared sequence homology with total RNA. Database searches showed that the RNA fragments shared sequence homologies with the G proteins, adenosine triphosphatase, calmodulin, phosphoenol pyruvate (PEP) carboxylase, and PEP carboxykinase, thus explaining the molecular mode of their functions in signal transduction.

Amination↗
PubMed1985

Denaturation of bovine liver glutamate dehydrogenase by guanidine hydrochloride. Correlation between enzymatic activity and molecular state.

Bovine liver glutamate dehydrogenase (GDH), a hexameric enzyme, undergoes subunit dissociation, denaturation, and inactivation in the presence of guanidine hydrochloride (GdnHCl), depending on the denaturant concentration. The correlation between the enzymatic activity and the molecular state of GDH, and the reconstitution of native hexamer from subunits after the removal of GdnHCl were examined by measuring the enzymatic activity and CD spectrum in the far ultraviolet region. It was found that only the hexameric form of GDH has enzymatic activity, and the reconstitution of the hexamer with full enzymatic activity from the trimeric form which has native polypeptide chain structure can be achieved by the removal of GdnHCl. On the other hand, the recovery of enzymatic activity from the dissociated form in more concentrated GdnHCl solution where unfolding of the polypeptide chain takes place showed an exponential decrease with increasing incubation time in the GdnHCl solution. The time constant for the decay of enzymatic activity with respect to the incubation time was almost the same as that for unfolding of the polypeptide chain (followed by CD spectroscopy). It is suggested on the basis of these experimental results that the failure of reconstitution of GDH hexamer from subunits produced at high denaturant concentration is due to failure in the refolding of the unfolded subunit to the correct three-dimensional structure of the polypeptide chain rather than in the reassociation process from subunits.

Animals↗
PubMed1980

The binding of oxidised coenzyme to bovine-liver glutamate dehydrogenase studied by circular-difference spectroscopy.

1. The binding of NAD+ to glutamate dehydrogenase may be followed quantitatively by titration, using high-sensitivity circular dichroism (CD) difference spectroscopy. 2. The CD of the bound coenzyme in the binary complex E . NAD closely resembles that of bound ADP, although the affinity is much lower, being 350-fold less for NAD+ at 20 degrees C in 0.1 M phosphate, pH 7. 3. A family of CD spectra may be analysed by unconstrained linear regression assuming only three components: free enzyme, free coenzyme, and a single binary complex, E . NAD. 4. Taking the molar CD of bound ADP as representing the molar CD of the adenine chromophore of bound NAD+, the linear regression shows the formation of a simple 1 : 1 complex E . NAD with Kd = 0.72 mM in a simple binding process without positive or negative cooperativity. 5. NADP+ binding is more than 10-fold weaker than NAD+ binding. 6. From the similarity of the CD of bound ADP and bound NAD+ it is probable that NAD+, in forming a simple binary complex, binds preferentially at the regulatory (adenine nucleotide) binding site (site II). 7. Direct evidence has been obtained for the binding of a second molecule of NAD+ to the ternary complex E . NAD . glutarate. This process occurs with low affinity and is probably also located at the adenine regulatory site. 8. This second-site binding of NAD+ may contribute to the phenomena of non-Michaelis-Menten kinetics and apparent negative homotropic interactions in the binding of NAD+, previously attributed to subunit-subunit cooperative interactions.

Adenosine Diphosphate↗
PubMed1992

Time course of renal glutamate dehydrogenase induction during NH4Cl loading in rats.

To study mechanisms involved in renal glutamate dehydrogenase (GDH) regulation in response to systemic acid loading, we have measured blood pH, ammonium excretion, renal GDH mRNA levels, and GDH activity in rats. Acid intake (0.28 M NH4Cl in drinking water for 3 days) increased GDH mRNA levels in the renal cortex, but had no effect in the outer stripe of the outer medulla, inner stripe of the outer medulla, or the inner medulla. Rats were subjected to a step change in acid intake by alkali loading for 3 days (7.2 meq NaHCO3 per day in food slurry) and shifting to acid loading for up to 7 days (7.2 meq NH4Cl in food slurry). Ammonium excretion rose rapidly, increasing by 14-fold in the first 24-h period and 38-fold in the second 24-h period. Cortical GDH mRNA levels were increased relative to alkali-loaded values by 3.7-fold in 24 h, 4.3-fold in 4 days, but only 2.2-fold in 7 days. GDH activity was unchanged after 24 h of acid intake, but was significantly increased after 48 h. We concluded the following: 1) GDH mRNA is present in all regions of the kidney, but levels increase in response to acid loading only in the renal cortex; 2) GDH mRNA levels increase within 1 day after the initiation of acid loading, but the associated increase in functional enzyme activity takes 2 or more days; and 3) the large increases in ammonium excretion that occur in the first day after initiation of acid loading are not dependent on increased GDH activity.

Ammonium Chloride↗
PubMed1982

The binding of NADH and NADPH to bovine-liver glutamate dehydrogenase. Spectroscopic characterisation.

1. The binding of NADH and NADPH to bovine liver glutamate dehydrogenase has been studied using circular dichroism (CD) and fluorescence spectroscopy, under comparable conditions of enzyme concentration. Spectroscopic titrations have been analysed using non-linear least-squares techniques to determine dissociation constants and parameters for the different chromophoric portions of the bound coenzymes, according to different models for the binding equilibria. 2. For NADPH, enhancement of dihydronicotinamide fluorescence and positive CD at 340 nm reflect binding of the coenzyme at the active site (site I) via the dihydronicotinamide moiety with a dissociation constant K1 of 50 microM; negative CD at 260 nm reflects the binding of a second molecule of coenzyme at the non-active site (site II) via the adenine moiety with a KII of 650 microM. 3. For NADH, the same spectroscopic assignment is made; however, site II affinity is much greater (KII 20 microM) and exceeds the affinity at the active site (KI 50 microM). 4. In no case was spectroscopic evidence obtained with either coenzyme to support the concept of non-identical active site binding as three sites IA and three sites IB per hexamer. 5. Coenzyme affinities are approximately twofold higher in triethanolamine buffer (0.05 + 0.1 M KCl, pH 7.6) compared with phosphate buffer (0.1 M at pH 7.0). 6. ADP enhances the CD of bound NADPH at 340 nm by up to twofold; phosphate ion also enhances the CD and binds with a Kd of 60-80 mM. GTP causes strong negative CD at 340 nm but only for the NADPH bound at site II. These results are consistent with independent binding sites for ADP (site II) and GTP (site III); phosphate ion is apparently able to affect certain properties of all three sites.

Animals↗
PubMed1987

Control of mitochondrial Ca2+ retention by ADP-stimulated glutamic dehydrogenase.

The protective effect of ADP on unspecific Ca2+ release and collapse of the transmembrane potential was analyzed in mitochondria from kidneys of rats. The presence of ADP in the incubation mixture prevents Ca2+ leakage and collapse of delta psi in sucrose-containing medium, but fails to do so in KCl medium. The effect of the adenine nucleotide in sucrose media correlates with an increase in the level of reduced pyridine nucleotides; the increase was due to a stimulatory effect on the activity of glutamic dehydrogenase. It also was observed that in KCl media, in the presence and in the absence of ADP the rate of NADH oxidation through the respiratory chain was higher than in sucrose; in this latter medium a high level of reduced pyridine nucleotides was found, in comparison to KCl media. It is proposed that the role of ADP is to increase glutamic dehydrogenase activity and in consequence to provoke a higher rate of formation of NADH which in turn controls Ca2+ release.

Adenosine Diphosphate↗
PubMedDate not supplied

Reactive amino acid residues involved in glutamate-binding of human glutamate dehydrogenase isozymes.

In the present study, the cassette mutagenesis at several putative positions (K94, G96, K118, K130, or D172) was performed to examine the residues involved in the glutamate-binding of the human glutamate dehydrogenase isozymes (hGDH1 and hGDH2). None of the mutations tested affected the expression or stability of the proteins. There was dramatic reduction in the catalytic efficiency in mutant proteins at K94, G96, K118, or K130 site, but not at D172 site. The K(M) values for glutamate were 4-10-fold greater for the mutants at K94, G96, or K118 site than for the wild-type hGDH1 and hGDH2, whereas no differences in the K(M) values for NAD(+) were detected between the mutant and wild-type enzymes. For K130Y mutant, the K(M) value for glutamate increased 1.6-fold, whereas the catalytic efficiency (k(cat)/K(M)) showed only 2-3% of the wild-type. Therefore, the decreased catalytic efficiency of the K130 mutant mainly results from the reduced k(cat) value, suggesting a possibility that the K130Y residue may be involved in the catalysis rather than in the glutamate-binding. The D172Y mutant did not show any changes in k(cat) value and K(M) values for glutamate and NAD(+), indicating that D172Y is not directly involved in catalysis and substrates binding of the hGDH isozymes. For sensitivity to ADP activation, only the D172Y mutant showed a reduced sensitivity to ADP activation. The reduction of ADP activation in D172Y mutant was more profoundly observed in hGDH2 than in hGDH1. There were no differences in their sensitivities to GTP inhibition between the wild-type and mutant GDHs at all positions tested. Our results suggest that K94, G96, and K118 residues play an important role, although at different degrees, in the binding of glutamate to hGDH isozymes.

Adenosine Diphosphate↗
PubMed2000

Analysis of negative cooperativity for glutamate dehydrogenase.

The empirical equation, which describes negative cooperativity in the enzyme kinetics, has been proposed. The equation is obtained from the Michaelis-Menten equation where the Michaelis constant is replaced by the effective Michaelis constant, which is a linear function of the v/Vmax ratio (v is the rate of the enzymatic reaction and Vmax is the limiting value of v at saturating concentrations of substrate). The equation allows the limiting values of the Michaelis constant at v/Vmax --> 0 and V/Vmax --> 1 to be estimated, K0 and Klim, respectively. The Klim/K0 ratio is considered as a quantitative characteristic of negative cooperativity. The applicability of the equation has been demonstrated for the kinetic data obtained for glutamate dehydrogenases from various sources (negative kinetic cooperativity for coenzyme). The negative cooperativity for the functions of saturation of protein by ligand is also analyzed. The data on binding of spin-labeled NAD, NADH, and NADPH by beef liver glutamate dehydrogenase are used as examples.

Animals↗
PubMed1993

Glutamate dehydrogenase improves binding of [3H]CGP39653 to NMDA receptors in the autoradiographic assay.

The novel high-affinity competitive NMDA receptor antagonist, CGP39653, is employed as radioligand to autoradiographically label the NMDA receptor in rat and human brain. Glutamate dehydrogenase (GlDH; E.C.1.4.1.3) was added to the incubation buffer to degrade residual endogenous L-glutamate, which was not entirely removed from the section after the prewashing step and interfered with [3H]CGP39653 binding. At 20 nM [3H]CGP39653, GLDH increased specific binding as much as 5 times, depending on the dose of GlDH and the presence of NAD+, and hydrazine. Scatchard plots of binding data revealed that this increase was due to a decrease of the KD from 148 nM to 33 nM in the absence and the presence of GlDH, respectively. Addition of GlDH in the NMDA receptor autoradiographic assay may be of importance in quantitative studies with human brain tissue which may contain variable levels of endogenous L-glutamate.

2-Amino-5-phosphonovalerate↗
PubMed1995

Effects of glutamate dehydrogenase, choline oxidase, and glucose-6-phosphatase on 67Ga accumulation in lysosome.

AIM: To clarify the effects of the activities of hepatic enzymes in liver, hepatoma, and malignant tumor on 67Ga accumulation in lysosome. METHODS: 67Ga-citrate solution was prepared from carrier-free 67Ga-citrate solution 0.08 mol.L-1 and sodium citrate solution 0.08 mol.L-1, and was injected iv 0.4 ml to the rats. Subcellular fractions of the liver were measured for radioactivity of 67Ga by a well-type scintillation counter (Aloka JDC-701). Glutamate dehydrogenase, choline oxidase, and G-6-P activities were calculated as described by Shimizu H, Ikuta S, and Baginski E, respectivily. RESULTS: 67Ga radioactivity in normal liver lysosome (55%) was significantly higher than those in hepatoma AH 109A (32%) and Yoshida sarcoma (18%). Glutamate dehydrogenase activities were 1830 +/- s 320 U.L-1 in normal liver while 23 +/- s 6 U.L-1 in hepatoma AH 109A, and 7 +/- s 2 U.L-1 in Yoshida sarcoma. Choline oxidase activities were 46 +/- s 10 U.L-1 for normal, 25.0 +/- s 0.4 U.L-1 for hepatoma AH 109A, and 2.0 +/- s 0.4 U.L-1 for Yoshida sarcoma. G-6-P activities were 2550 +/- s 180 U.L-1 in normal, 84 +/- s 14 U.L-1 in hepatoma AH 109A, and 78 +/- s 13 U.L-1 in Yoshida sarcoma. CONCLUSION: Lysosome of normal rat liver in which hepatic enzymes work actively played a major role in the tissue concentration of 67Ga, but the role diminishes with the neoplastic transformation into hepatoma. The lysosome of Yoshida sarcoma does not play any role in 67Ga accumulation because it does not possess any features of liver.

Alcohol Oxidoreductases↗
PubMed1982

The role of chloroplast and cytoplasm in the NADP-glutamate dehydrogenase and glutamine synthetase synthesis in Ankistrodesmus cells.

The effects of transcription and translation inhibitors on NADP-glutamate dehydrogenase and glutamine synthetase synthesis in nitrogen-starving Ankistrodesmus braunii cells have been studied. Considering the results obtained one can suggest that both enzymes are coded in a chloroplast genome and that during nitrogen starvation specific mRNA's are partly transferred from chloroplast into cytoplasm and can be translated there on 80S ribosomes.

Chloramphenicol↗
PubMed1982

The role of chloroplast and cytoplasm in the NADP-glutamate dehydrogenase and glutamine synthetase synthesis in Ankistrodesmus cells.

The effects of transcription and translation inhibitors on NADP-glutamate dehydrogenase and glutamine synthetase synthesis in nitrogen-starving Ankistrodesmus braunii cells have been studied. Considering the results obtained one can suggest that both enzymes are coded in the chloroplast genome and that during nitrogen starvation specific mRNA's are partly transferred from the chloroplast into the cytoplasm and can be translated there on 80S ribosomes.

Chloramphenicol↗
PubMedDate not supplied

[Effect of reproduction of the LPP-3 cyanophage on glutamate dehydrogenase and glutamine synthetase activity in the cyanobacterium Plectonema boryanum].

The effect of cyanophage LPP-3 reproduction on glutamate dehydrogenase and glutamine synthetase (GS) in P boryanum cells have been studied. It was determined that the both reactions are intensified by 135% and 220%, accordingly. Isoenzymes of GS were purified from native and infected cell of cyanobacteria. Their physical-and-chemical properties are different. The cyanophage development probably causes specific modification of the cell enzymes.

Bacteriophages↗
PubMed2001

Construction, separation and properties of hybrid hexamers of glutamate dehydrogenase in which five of the six subunits are contributed by the catalytically inert D165S.

In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase. C320S and D165S mutant enzymes were chosen to construct the hybrid proteins. The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine. D165S is capable of binding both glutamate and NAD(+) but is catalytically inactive. The mutant proteins were denatured separately in 4 M urea, mixed in a 5 : 1 (D165S/C320S) ratio and diluted into a refolding mixture composed of 2 mM NAD(+), 1 M fluoride and artificial chaperones (4 mM polyoxyethylene 10 lauryl ether and 1.6 mM beta-cyclodextrin). Under these conditions approximately 50% refolding was achieved for both mutant proteins separately. The renatured mixture was concentrated and separated from denatured proteins and the components of the refolding mixture by ultrafiltration and ion-exchange chromatography. Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), which binds close to the NAD(+) binding site, thus abolishing coenzyme binding in the wild-type enzyme, also reacts with D165S but has no effect on C320S. Modification by DTNB was coupled with dye-ligand affinity chromatography on a Procion Red HE-3B column in order to separate the hybrid mixture into fractions of defined composition. An optimized procedure based on salt gradient elution was developed. DTNB-modified 5 : 1 hybrids, with only one subunit capable of binding coenzyme, showed classical Michaelis-Menten kinetics when the NAD(+) concentration was varied, whereas removal of the thionitrobenzoate moieties that blocked the other five coenzyme binding sites in the hexamer reinstated nonlinear behaviour, suggesting that 'nonlinear' behaviour of the native enzyme and the hybrid with six coenzyme binding sites depends on binding to multiple sites. When assayed at high pH with increasing glutamate concentration, the sample with only one active subunit showed reduced sigmoidicity in the dependence of reaction rate on glutamate concentration (h = 3.0) compared with native C320S with six active subunits (h = 5.2) suggesting that the interaction between the subunits was reduced but not abolished completely. Catalytically silent subunits can thus still contribute to cooperativity.

Allosteric Regulation↗
PubMed1984

[Identification of a glutamate dehydrogenase amino acid residue modified by 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl. Study of the type of inactivation of a catalytically active enzyme oligomer in modification].

It has been shown that 2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl selectively blocks epsilon-amino group of Lys126 residue in bovine liver glutamate dehydrogenase (L-glutamate NAD(P) oxydoreductase, EC 1.4.1.3). Modification of this residue in one of the six promoters of catalytically active hexamer is accompanied by the loss of about half of the enzymatic activity. The enzyme inactivation caused by modification has a cooperative character.

Amino Acid Sequence↗
PubMed1984

Differential role of glutamate dehydrogenase in nitrogen metabolism of maize tissues.

Both calli and plantlets of maize (Zea mays L. var Tuxpeño 1) were exposed to specific nitrogen sources, and the aminative (NADH) and deaminative (NAD(+)) glutamate dehydrogenase activities were measured at various periods of time in homogenates of calli, roots, and leaves. A differential effect of the nitrogen sources on the tissues tested was observed. In callus tissue, glutamate, ammonium, and urea inhibited glutamate dehydrogenase (GDH) activity. The amination and deamination reactions also showed different ratios of activity under different nitrogen sources. In roots, ammonium and glutamine produced an increase in GDH-NADH activity whereas the same metabolites were inhibitory of this activity in leaves. These data suggest the presence of isoenzymes or conformers of GDH, specific for each tissue, whose activities vary depending on the nutritional requirements of the tissue and the state of differentiation.

Journal Article↗