NADH-GDH Activity Research Articles

NADH-GOGAT Overexpression Does Not Improve Maize (Zea mays L.) Performance Even When Pyramiding with NAD-IDH, GDH and GS.

Maize plants overexpressing NADH-GOGAT were produced in order to determine if boosting 2-Oxoglurate production used as a carbon skeleton for the biosynthesis of amino acids will improve plant biomass and kernel production. The NADH-GOGAT enzyme recycles glutamate and incorporates carbon skeletons into the ammonium assimilation pathway using the organic acid 2-Oxoglutarate as a substrate. Gene pyramiding was then conducted with NAD-IDH and NADH-GDH, two enzymes also involved in the synthesis of 2-Oxoglurate. NADH-GOGAT overexpression was detrimental for shoot biomass production but did not markedly affect kernel yield. Additional NAD-IDH and NADH-GDH activity did not improve plant performance. A decrease in kernel production was observed when NADH-GDH was pyramided to NADH-GOGAT and NAD-IDH. This decrease could not be restored even when additional cytosolic GS activity was present in the plants overexpressing the three enzymes producing 2-Oxoglutarate. Detailed leaf metabolic profiling of the different transgenic plants revealed that the NADH-GOGAT over-expressors were characterized by an accumulation of amino acids derived from glutamate and a decrease in the amount of carbohydrates further used to provide carbon skeletons for its synthesis. The study suggests that 2-Oxoglutarate synthesis is a key element acting at the interface of carbohydrate and amino acid metabolism and that its accumulation induces an imbalance of primary carbon and nitrogen metabolism that is detrimental for maize productivity.

The relationship between carbon and nitrogen metabolism in cucumber leaves acclimated to salt stress.

The study examines the effect of acclimation on carbon and nitrogen metabolism in cucumber leaves subjected to moderate and severe NaCl stress. The levels of glucose, sucrose, NADH/NAD+-GDH, AspAT, AlaAT, NADP+-ICDH, G6PDH and 6GPDH activity were determined after 24 and 72 hour periods of salt stress in acclimated and non-acclimated plants. Although both groups of plants showed high Glc and Suc accumulation, they differed with regard to the range and time of accumulation. Acclimation to salinity decreased the activities of NADP+-ICDH and deaminating NAD+-GDH compared to controls; however, these enzymes, together with the other examined parameters, showed elevated values in the stressed plants. The acclimated plants showed higher G6PDH activity than the non-acclimated plants, whereas both groups demonstrated similar 6PGDH activity. The high activities of NADH-GDH, AlaAT and AspAT observed in the examined plants could be attributed to a high demand for glutamate. The observed changes may be required for the maintenance of correct TCA cycle activity, and acclimation appeared to positively influence these adaptive processes.

Nitrogen metabolism-related enzymes in Mesembryanthemum crystallinum after Botrytis cinerea infection

We compared C3 and CAM (crassulacean acid metabolism) states in Mesembryanthemum crystallinum, a facultative CAM species, with respect to the involvement of phosphoenolpyruvate carboxylase (PEPC) and nitrogen metabolismrelated enzymes in plant response to Botrytis cinerea infection. The enzyme activities were monitored both in pathogeninoculated 2nd leaf pair and non-inoculated 3rd leaf pair. The control activities of most studied enzymes were dependent on the mode of photosynthesis. Compared to C3 plants, those performing CAM exhibited higher PEPC, nitrate reductase (NR), and deaminating glutamate dehydrogenase (NAD-GDH) activities but lower glutamine synthetase (GS) and alanine aminotransferase (ALT) activities. Regardless of the mode of photosynthetic carbon assimilation, the plants responded to infection with enhancement of PEPC and inhibition of NR activities in the inoculated leaves. Whereas the activity of GS remained unaffected, those of all glutamate-yielding enzymes, namely ferredoxin-dependent glutamate synthase (Fd-GOGAT), aspartate aminotransferase (AST), ALT, and aminating glutamate dehydrogenase (NADHGDH) were altered after infection. However, the time-course and extent of the observed changes differed in C3 and CAM plants. In general, CAM plants responded to infection with an earlier increase in PEPC and Fd-GOGAT activities as well as later inhibition of NR activity. Contrary to C3 plants, in those performing CAM the activities of PEPC, Fd-GOGAT, NADH-GDH, and AST in the non-inoculated 3rd leaf pair were similarly influenced by infection as in leaves directly inoculated with the pathogen. This implies that the local infection induced an alteration of carbon/nitrogen status in healthy upper leaves. This reprogramming resulting from changes in PEPC and nitrogen metabolism-related enzymes was C3- and CAM-specific.

Adaptation to rhizosphere acidification is a necessary prerequisite for wheat (Triticum aestivum L.) seedling resistance to ammonium stress

Because soil acidification accompanies ammonium (NH4+) stress, the tolerance of higher plants to ammonium is associated with their adaptation to root medium acidification. However, the underlying mechanisms of this adaptation have not been fully elucidated. The objective of this study was thus to elucidate the effect of rhizosphere pH on NH4+ tolerance in different winter wheat cultivars (Triticum aestivum L.). Hydroponic experiments were carried out on two wheat cultivars: AK58 (an NH4+-sensitive cultivar) and XM25 (an NH4+-tolerant cultivar). Four pH levels resembling acidified (4.0, 5.0, 6.0 and 7.0) were tested and 5 mM NH4+ nitrogen (AN) was used as a stress treatment, with 5 mM nitrate nitrogen used as a control. The addition of AN led to a severe reduction in biomass and an increase in free NH4+, amino acids, and the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in the shoots and roots of the two wheat cultivars. Further decreases in growth medium pH led to further increases in free NH4+, but decreases in total amino acids and the activities of GS and NADH-dependent glutamate synthase (NADH-GDH). However, there was less of an increase in free NH4+ and less of a reduction in the activities of GS and NADH-GDH in the cultivar XM25 compared with AK58. In addition, total soluble sugar content and the root-to-shoot soluble sugar ratio were also decreased by AN treatment, except in the shoots of XM25. Decreasing pH resulted in lower root-to-shoot soluble sugar ratios with greater reductions in the AK58 cultivar. These results indicate that wheat growth was inhibited significantly by the addition of NH4+ combined with low pH. Low medium pH reduced the capacity for nitrogen assimilation and interrupted carbohydrate transport between the shoot and root. The NH4+-tolerant cultivar XM25 was better adapted to low rhizosphere pH due to its increased capacity for assimilating NH4+ efficiently and thereby avoiding toxic levels of intracellular NH4+ at low medium pH.

Nitrogen Source Differently Regulates Barley (Hordeum vulgare) Response to NaCl Stress At Seed Germination and Early Seedling Development Stages

Nitrogen (N) acts as nutrient and signaling molecule in plants all over their development stages. The involvement of various N forms in the regulation of seed germination response to salt stress was assessed in the present work. Nitrogen sources (NO, NO2−, NO3−, NH4+, glutamine and glutamate) were added at 1mMto the germination medium of barley (Hordeum vulgare, cv Ardhaoui) in combination or not with NaCl stress (14 g.L−1). The application of nitrogen monoxide (NO) alleviated by about 20% the NaCl-induced germination capacity decrease. However, the addition of ammonium ions (NH4+) and glutamic acid (Glu) accentuated the inhibitory effects of NaCl, decreasing germination capacity by about 50% compared to the control. The levels of malondialdehyde (MDA), which is an indicator of membrane lipid peroxidation by stresses, were increased by salinity in seeds treated with nitrite (NO2−), NO3−, Glu and Gln. In N-free medium, NaCl stress induced a severe nitrate reductase activity (NR, EC 1.6.1.6) inhibition. Such an effect was alleviated by the application of N treatments. Glutamate dehydrogenase (GDH, EC 1.4.1.2) aminating activity (NADH-GDH) of seedlings was inhibited by NaCl stress in the presence of NO, Glu and Gln. Conversely, there was stimulation by salt stress of NADH-GDH activity in seedlings treated with NaCl and NH4+. Deaminating GDH activity (NAD-GDH) was found to be enhanced by salt stress in NO2− and NO3− treatments. The differential effects of applied N forms on germination and early seedling development processes in this grass probably underlines different regulatory actions within N mobilization and assimilation.

Differential capacity of nitrogen assimilation between apical and basal leaf portions of a tank epiphytic bromeliad

The leaf is the main organ of the vegetative body of tank epiphytic bromeliads, which may be subdivided into apical and basal parts. Little is known about the existence of morphological, anatomical or physiological differences between these two leaf portions. The objective of this study was to verify the existence of a differential capacity to assimilate nitrogen from urea between basal and apical leaf portions of Vriesea gigantea, a tank epiphytic bromeliad. Plants were cultivated in vitro in the presence of 5 mM urea for 15 days. During this period, the activities of the enzymes glutamine synthetase (GS, EC 6.3.1.2) and NADH-dependent glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) were quantified, as well as the densities of trichomes and stomata found at the leaf surface of both regions. The highest activities of GS and NADH-GDH were always detected at the top portion, and on the 3rd day of in vitro cultivation an increase in these activities was detected. An inverse correlation between number of trichomes and stomata in these two leaf portions was verified: the top region presented 30% less trichomes and twice as many stomata as the basal region. This suggests that the upper region of the leaf may be preferentially involved with the assimilation of nitrogen from urea via GS and NADH-GDH, whereas the basal region may be involved with its absorption. This is the first study to report that ammonium assimilation may take place preferentially in a specific portion of tank-forming bromeliad leaves.

Glutamine synthetase and glutamate dehydrogenase contribute differentially to proline accumulation in leaves of wheat ( Triticum aestivum) seedlings exposed to different salinity

To investigate the roles of ammonium-assimilating enzymes in proline synthesis under salinity stress, the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) were determined in leaves of wheat (Triticum aestivum) seedlings exposed to salt stress at 150 and 300 mM NaCl for 5d. At the lower salinity, only GS activity increased markedly. At 300 mM NaCl, however, NADH-GDH activity increased while GS activity decreased. A significant accumulation of proline was found only at high-salinity exposure while glutamate, a proline precursor, increased dramatically under both low and high salinity. These data suggests that GS-catalysis might be the main glutamate synthesis pathway under low salinity. At 300 mM NaCl, glutamate seems to be preferentially produced through the process catalyzed by NADH-GDH. The increase of ammonium in salinity-stressed wheat seedlings might have resulted from increased photorespiration, which is responsible for the higher NADH-GDH activity. The activity of Delta(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) was significantly enhanced at 300 mM NaCl but remained unchanged at 150 mM. Delta(1)-Pyrroline-5-carboxylate synthetase (P5CS) activity did not show a specific response, indicating that P5CR might be the limiting step in proline synthesis from glutamate at high salinity.

Effect of selected compounds on the activity of glutamate dehydrogenase from triticale roots

The influence of selected factors on the activity of highly purified GDH in triticale roots was investigated in vitro. In the presence of 2-ME, NADH-GDH activity increased by 400 %, while NADPH-GDH activity rose by 500 %. No effect of reducing factors on NAD(P)+-GDH reaction was detected. The sulphydryl groups inhibitors, such as p-chloromercuribenzoate (p-CMB) and iodoacetamide, proved the strongest inhibitors of the aminative reaction. Metal-binding compounds: ethylenediaminetetraacetic acid disodium salt (EDTA) and Zinkov also considerably inhibited NAD(P)H-GDH activity. Diisopropylfluorophosphate (DFP) and pepstatin A, the inhibitors specific for -OH serine and COO− aspartic acid groups respectively, caused a non-significant NAD(P)H-GDH activity decrease. Cd2+, Co2+, Hg2+, Mg2+, Pb2+ and Zn2+ ions strongly inhibited the amination reaction, whereas their inhibiting effect upon NAD+-GDH activity was negligible. Among the applied ions, only Ca2+ activated NADH-GDH.

Response of Zea mays to the Inoculation with Azospirillum on Nitrogen Metabolism under Greenhouse Conditions

The maize (Zea mays L.) plants inoculated by N2-fixing bacterium Azospirillum showed increased activity of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) in root cells free extracts over uninoculated control plants. Maximum differences in NADH-GDH activity were observed during the second and third weeks after sowing. The specific activity of GS showed a greater increase at the end of the assay. The percentage of nitrogen in leaves, root and foliage length, total fresh mass and nitrogenase activity were higher in inoculated plants than in the control ones.

The NAD(P)H-dependent glutamate dehydrogenase activities of Prevotella ruminicola B(1)4 can be attributed to one enzyme (GdhA), and gdhA expression is regulated in response to the nitrogen source available for growth.

Prevotella ruminicola B(1)4 possesses both NADPH- and NADH-linked glutamate dehydrogenase (GDH) activities, with the greatest specific activity being measured from ammonia-limited cultures. Relative to cells grown in the presence of 1 mM ammonium chloride, the NADPH-dependent activity was decreased approximately 10-fold when peptides were provided as a nitrogen source. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was used to visualize the GDH protein(s) in cell extracts of P. ruminicola. For all growth conditions tested, only one GDH protein was detectable, and its relative abundance, as well as its reactivity with either NAD(P)+ or NAD(P)H, correlated well with the specific activities measured from whole-cell assays. Consistent with the findings from enzyme assays and PAGE activity gels, Northern (RNA) blot analysis revealed that expression of a gene encoding NAD(P)H-GDH activity was greatest in ammonia-grown cultures and that GDH activity is regulated in response to nitrogen source (ammonia versus peptides), probably at the level of transcription. A gene encoding the NAD(P)H-utilizing GDH activity (gdhA) was cloned, and its nucleotide sequence was determined and shown to contain an open reading frame of 1,332 bp which would encode a polypeptide of 48.8 kDa. The deduced amino acid sequence possesses three highly conserved motifs typical of family I GDHs, but several unique amino acid substitutions within these motifs were evident. These results are discussed within the context of ruminal nitrogen metabolism and the growth efficiency of succinate- and propionate-producing anaerobic bacteria.

Effect of NaCI on nitrate reductase, glutamate dehydrogenase and glutamate synthase inVigna radiata calli

The effect of NaCI stress on the activities of nitrate reductase (NR), glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT) in callus lines ofVigna radiata which differ in salt resistance, was studied at weekly intervals upto 28 d of growth. After 28 d, the NaCI resistant callus (selected at 300 mM NaCI) at NaCI concentrations higher than 200 mM maintained higher NR activity than non-selected line. NaCI stress also affects aminating and deaminating activities of GDH. The NADH-GDH activity in the presence of NaCI was higher in the resistant than non-selected line. On the other hand, NAD-GDH activity in both the lines was completely inhibited after 7 d of growth. The increased activity of NADH-GDH in resistant calli may play a vital role in protecting the cells from toxic effect of increased endogenous level of ammonia which probably accumulates due to efficient NO3 - reduction. NADH-GOGAT activity was found to decrease under salt stress in both the callus lines. Nitrogen assimilation in salt-resistant calli under salt stress was found to be characterized by high NR and NADH-GDH activities, concomitantly with low GOGAT activity.

Effect of DTNB on glutamate dehydrogenase activity in root and shoot extracts of maize seedlings

NADH specific glutamate dehydrogenase (GDH) activity was examined in roots and shoots of maize seedlings grown in half-strength Hoagland’s solution containing NH4NO3 as sole nitrogen source under irradiance of 60 W m−2 and temperature of 25±2°C. When 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB) was supplied to the assay mixture, it inhibited NADH-GDH activity in both roots and shoots, irrespective of whether the enzymes were extracted from light- or dark-treated roots and shoots. In each case the inhibition increased with the increase in DTNB concentration. At the maximum concentration of DTNB used (20 μM) the inhibition of shoot NADH-GDH was more pronounced than inhibition of root enzyme. This indicated differences in shoot and root NADH-GDH.

Mechanical Slicing-Induced Alteration of GDH Isoenzyme Patterns in Brassica napus Leaf Protoplasts

Summary Mechanical slicing applied to the leaves to facilitate the penetration of a cellulase-pectinase enzyme mixture induces the synthesis of glutamate dehydrogenase (GDH, EC 1.4.1.2) isoenzyme 7 that predominantly inheres in Brassica napus leaf protoplasts. The induction of GDH 7 and anodal isoenzymes were inhibited by Cycloheximide, but not by Actinomycin D and α-Amanitin. Slicing of leaves increased NADH-GDH activity and reduced the amount of soluble protein, amino acids and ammonia in the leaf strips. These results may indicate that ammonia released from the sliced leaf strips is responsible for the increased NADH-GDH activity resulting from the synthesis of the GDH 7 and other anodal isoenzymes, and suggest that the protoplasts isolating from the sliced leaf strips seem to be senescent before culture.

Immunocharacterization of NADH-Glutamate Dehydrogenase from Vitis vinifera L

Rabbit antiserum was raised against NADH-glutamate dehydrogenase (GDH) isoenzyme 1, purified from leaves of Vitis vinifera L. cv Soultanina and its specificity was tested. This antiserum was used for immunocharacterization of the GDH from leaf, shoot, and root tissues. The antiserum recognized the seven isoenzymes of NADH-GDH and precipitated all the enzyme activity from the three tissues tested. Western blot following SDS-PAGE revealed the same protein band for the three tissues, with a molecular mass of 42.5 kilodaltons corresponding to NADH-GDH subunit. Results, based on the immunological studies, revealed that NADH-GDH from leaf, shoot, and root tissues are closely related proteins. Furthermore, addition of ammonium ions to the culture medium of in vitro grown explants resulted in a significant increase in NADH-GDH activity in root, shoot, and leaf tissues.

Aktivitäten der Glutaminsäuredehydrogenase (GDH), Glutaminsynthetase (GS) and Glutamatsynthase (GOGAT) in Suspensionskulturen von Beta vulgaris (Zuckerrübe) und Chenopodium album (Gänsefuß): Activities of Glutamate Dehydrogenase (GDH), Glutamine Synthetase (GS) and Glutamate Synthase (GOGAT) in Suspension Cultures of Beta vulgaris (Sugar Beet) and Chenopodium album (Goosefoot)

Summary In extracts of suspension cultures of Beta vulgaris and Chenopodium album the activities of NADH-GDH, GS and NADH-GOGAT were determined. Nitrogen assimilation occurs both via reductive amination of 2-oxoglutarate (GDH-pathway) and the route via glutamine synthetase (GS/GOGAT-pathway). In the course of exponential growth of the cell cultures, which were cultured for about 14 days, the level of the enzymes did not change. GDH and GS activities reached a high level, the value of GOGAT was found to be ca. 1/12 the amount of GS. In cases of the absence of ammonia and the addition of twice the amount of nitrate to the suspension medium, the growth was depressed, the GS was stimulated ca. 20-fold, and the GDH activity dropped and vice versa, i.e. while nitrate was eliminated and twice the amount of ammonia added, the growth of cells was stopped, the GDH rose and GS dropped to zero. The cultures of Beta vulgaris and Chenopodium album did not differ from each other in their enzyme dynamics.

Effect of Nitrate and Ammonium on NADH-dependent Glutamate Dehydrogenase Activity of Detached Roots and Cotyledons of Citrullus vulgaris L.

Roots and cotyledons of watermelon seedlings, grown in darkness for six days in a media with nitrate or ammonium as nitrogen source, were detached and incubated for 24 h in media of these nitrogen sources with or without actinomycin D or D-chloramphenicol. A continous protein synthesis at the organellular and the cytoplasmic level was required for maintaining NADH-GDH activity levels, regardless of the nitrogen source. When ammonium was present in the nutrient solution, the increase of activity observed seemed to be due to an elevated organellular and cytoplasmic protein synthesis in the cotyledons, while in the roots only the organellular synthesis was noted. The addition of nitrate decreased the GDH activity. In the cotyledons this effect seemed to be due to a decreased organellular protein synthesis, while in the roots, the effect was probably produced by an inactivation at the cytoplasmic level. Electrophoretic patterns of GDH varied depending on the plant organ and on the nitrogen source.

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.

Characteristics of Glutamate Dehydrogenase in Mitochondria Prepared from Corn Shoots

The amination of alpha-ketoglutarate (alpha-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca(2+). The NADH-GDH purified 161-fold with ammonium sulfate, DEAE-Toyopearl, and Sephadex G-200 was also activated by Ca(2+) in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca(2+) had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca(2+).About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca(2+) had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca(2+). In a simulated in vitro system using concentrations of 6.4 millimolar NAD, 0.21 millimolar NADH, 5 millimolar alpha-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca(2+), 10 and 50 millimolar NH(4) (+), and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation.

Occurrence of glutamate dehydrogenase isoenzymes during growth of Oocystis alga

Oocystis sp., a unicellular green alga, contained two glutamate dehydrogenase isoenzymes: one was specific for NADH and the other for NADPH. Activity staining after gel electrophoresis indicated that one component in NADH-GDH was not specific for the cofactor and three components in NADPH-GDH. The optimal concentration of substrate, purification procedure and kinetic properties of both glutamate dehydrogenase (GDH) enzymes in vitro are presented. The kinetics of growth, nutrient removal and enzyme activities for Oocystis growing in wastewater showed that ammonia was preferentially utilized over nitrate and the medium was depleted before the maximum population was obtained in indoor culture. There was a sharp increase in NADPH-GDH activity following the exhaustion of ammonia from the medium but NADH-GDH activity remained unchanged. The NADPH-GDH activity at the outset increased exponentially with time in greenhouse culture but then decreased sharply accompanied by a rapid increase in biomass and nitrite concentration. The K(m) values for ammonia in this algal GDH was high, while glutamate synthase activity was not detected; this suggests that Oocystis may adapt to conditions of ammonia limitation by producing large quantities of NADPH-GDH instead of using glutamate synthase pathway.

Ammonia utilization by a proposed bacterial pathogen in human periodontal disease, Capnocytophaga ochracea

Capnocytophaga ochracea strain 25 was originally isolated from a patient with severe juvenile periodontitis. An NAD-specific glutamate dehydrogenase (GDH) (EC 1.4.1.2.) was found in cell-free extracts from this organism. The NADH-dependent reductive, or ammonia-assimilating activity (NADH-GDH), of the enzyme was 8–10-fold higher than its NAD-dependent oxidative, or ammonia-releasing activity (NAD-GDH), suggesting that the primary physiological role of the GDH is ammonia-fixing. Capnocytophaga ochracea GDH was purified approximatsly 39-fold by a rapid, single-step purification procedure using DEAE-cellulose (DE52) ion-exchange column chromatography which gave 90 per cent recovery of total enzyme units. Paper chromatography of an NADH-GDH assay mixture containing the partially purified enzyme showed that glutamate was, indeed, a product of the ammonia-assimilating reaction. The pH optimum for the NAD-GDH reaction was 9.0; that for the NADH-GDH reaction was 7.5. Although a number of mono- and divalent cations were tested, none had a large effect on either NAD-GDH or NADH-GDH activity. The NAD-GDH reaction showed a hyperbolic kinetic response to glutamate and NAD and the K m values for glutamate and NAD were 2.44 and 0.083 mM respectively. The kinetic response of the NADH-GDH reaction to NADH, α-ketoglutarate and ammonium chloride also obeyed Michaelis-Menten kinetics and their respective K m values were 0.069, 1.44 and 3.33 mM. Of a number of biologically-active compounds tested for their ability to modulate GDH activity, only ADP and NAD exerted much effect. The NADH-GDH activity showed a negative hyperbolic kinetic response to both ADP and NAD and Dixon plot-analysis of the NAD and ADP saturation data gave K i values for ADP and NAD of 4.0 and 0.46 mM respectively. Both NAD and ADP appeared to exert their negative effects on NADH-GDH activity by completely inhibiting the binding of the reduced coenzyme, NADH, to the enzyme.