Specific Activity Of Glutamate Dehydrogenase Research Articles

Differential Effect of Aluminium on Enzymes of Nitrogen Assimilation in Excised Bean Leaf Segments

Aluminium is a potent toxicant in acidic soils. The present study was taken up to analyze the effects of Al on enzymes of nitrogen assimilation in excised bean (Phaseolus vulgaris) leaf segments so as to gain an insight of the mechanism involved. Supply of 0.001 to 0.1 mM AlCl3 to excised bean leaf segments affected the in vivo nitrate reductase activity differently in the presence of various inorganic nitrogenous compounds, being inhibited with 5 mM ammonium nitrate and 10 mM ammonium chloride but enhanced with 10 mM potassium nitrate. Al effect with 50 mM KNO3 varied with time, showing an increased activity at shorter duration, but decreased at longer duration. Al effect on in vivo NRA was dependent upon the nitrate concentration, thus, inhibiting it at 0, 1 and 50 mM KNO3, while increasing at 2 and 10 mM. Further, saturating and non-saturating effects were observed in the absence and presence of Al. Al supply influenced the in vitro NRA also, being increased at 10 mM, but decreased at 50 mM KNO3. Supply of Al to excised leaf segments substantially inhibited the glutamate dehydrogenase activity in the absence as well as presence of 5 mM NH4NO3 but increased the glutamate synthase activity. Inhibition of specific glutamate dehydrogenase activity by Al supply was also observed. However, specific glutamate synthase activity was increased in the presence of NH4NO3 only. The experiments demonstrated that effect of supply of aluminium on in vivo nitrate reductase activity depended upon nitrogenous source as well as nitrate concentration and it exerted reciprocal regulation of glutamate dehydrogenase and glutamate synthase activities, which depended upon N supply too.

Stress-induced changes in glutamate dehydrogenase activity imply its role in adaptation to C and N metabolism in lupine embryos

The modifying effect of sucrose on glutamate dehydrogenase (GDH) activity and isoenzyme pattern was investigated in isolated embryos of lupine (Lupinus luteus L.), cultured in vitro in a medium with sucrose (+S) or without sucrose (-S) and exposed to cadmium (Cd) and lead (Pb) stress. Sucrose starvation of lupine embryos led to a rapid increase in the specific activity of GDH, immunoreactive beta-polypeptide and it was accompanied by appearance of new cathodal isoforms of enzyme. This suggests that isoenzymes induced in lupine embryos by sucrose starvation combine into GDH hexamers with the predominance of beta-GDH subunits synthetized under GDH1 gene control. The addition of sucrose to the medium caused an opposite effect. Along with upregulation of catabolic activity of GDH by sucrose starvation, activity of proteolytic enzymes was also induced. These data can point to regulatory mechanism implying a sucrose dependent repression of the GDH1 gene according to the mechanism of catabolic repression. Treatment of embryos with Cd(2+) or Pb(2+) resulted in ammonium accumulation in the tissues, accompanied by an increase in anabolic activity of GDH and activity of anodal isoenzymes, in both (+S) and (-S) embryos without new de novo synthesis of alpha subunit proteins. Thus, GDH isoenzyme profiles may reflect the physiological function of GDH, which appears to be an important link of metabolic adaptation in cells, aimed at using carbon sources other than sugar during carbohydrate starvation (catabolic activity of GDH) and protecting plant tissues against ammonium accumulated because of heavy metal stress (anabolic activity of GDH).

Lysine requirement and efficiency of lysine utilization in turbot (Scophthalmus maximus) juveniles

A study was undertaken to estimate dietary lysine requirement and efficiency of utilization in turbot juveniles. Seven isonitrogenous (8.0% N DM) and isolipidic (15% DM) diets were formulated to contain half of dietary nitrogen provided by intact protein and the other half provided by crystalline amino acids. Experimental diets included graded levels of lysine ranging from 2.5 to 6.4 g 16 g−1 N. Step-wise lysine levels were achieved at the expense of sodium glutamate. Except for lysine, overall amino acid profile of the experimental diets was made similar to that of the whole-body composition of turbot juveniles. Each experimental diet was fed by hand, twice daily, to apparent visual satiation, to triplicate groups of 17 fish (initial body weight of 18.1 g) for 8 weeks.Final body weight and weight gain (g kg ABW−1 day−1) of fish increased with the increase of dietary lysine levels. Voluntary feed intake was not affected by dietary lysine level except for group fed the 3.5% lysine diet which was higher than in groups fed the 2.5 and 3.2% lysine diets. At the end of the trial, there were no significant differences among groups in whole-body protein content, AA composition or visceral index. Hepatosomatic index linearly decreased with the increase of dietary lysine level. N retention (g N kg ABW−1 day−1 or % N intake) linearly increased with dietary lysine level but specific activity of glutamate dehydrogenase was not significantly affected by dietary treatments. However, lysine utilization efficiency (expressed as lysine retention in percentage of lysine intake) was inversely related to dietary lysine level, indicating diminishing returns to additional lysine intake as the maximum response was approached.A four-parameter saturation kinetic model was used to fit weight gain (g kg ABW−1 day−1) against dietary lysine level. Based on that model, dietary lysine level meeting 95% of the lysine requirement for maximum weight gain of turbot juveniles was estimated to be 5.00 g 16 g−1 N. Based on this value and on the A/E ratio determined from turbot whole-body amino acid profile an estimation of the EAA requirements of turbot juveniles was calculated.

Effect of the dietary essential amino acid pattern on growth, feed utilization and nitrogen metabolism of European sea bass ( Dicentrarchus labrax)

The aim of this study was to evaluate the influence of the dietary essential amino acid (EAA) pattern on growth performance, feed utilization and nitrogen metabolism of European sea bass. A diet containing only intact protein (fish meal and wheat meal) was used as a control (diet C). Three semi-purified diets were formulated to be isonitrogenous (8% DM) and isolipidic (15% DM) and to contain crystalline-AAs replacing approximately 60% of fish meal protein-bound nitrogen of the control diet. The AA pattern of these diets was adjusted to resemble the overall AA pattern of the fish meal protein (diet FM); the overall AA pattern of the whole-body protein of sea bass (diet WBC) or to meet the ideal protein AA pattern estimated for sea bass (diet REQ). Each experimental diet was fed to juvenile sea bass (7.3 g) in triplicate for 42 days. Weight gain and specific growth rate were highest in fish fed the control diet, followed by FM, WBC and REQ diets. Voluntary feed intake was highest in fish fed the REQ diet, and did not differ within the other groups. Feed efficiency and protein efficiency ratio were similar between fish fed the control and FM diets, while it was lower in the other groups. At the end of the growth trial, whole-body dry matter and protein content were not significantly affected by dietary treatment, while lipid and energy contents were significantly higher in fish fed the control diet than the other diets. Visceral and hepatosomatic indices were significantly lower in fish fed the REQ diet compared to the other groups. Nitrogen retention (g kg BW − 1 day − 1 and % N intake) of fish fed the control and FM diets was significantly higher than that of fish fed the REQ diet. Total non-fecal N excretion (% N intake) was significantly higher in fish fed the REQ diet than in the other groups, among which no significant differences were observed. Specific activity of glutamate dehydrogenase was significantly lower in fish fed the control diet than the REQ diet. Specific activity of alanine and aspartate aminotransferases and glucose-6-phosphate dehydrogenase were not significantly affected by the dietary treatments. Fatty acid synthetase activity was significantly lower in fish fed the REQ diet than in the other groups, among which no significant differences were observed. Results of this study indicate that the best overall growth and feed utilization of sea bass juveniles were obtained with a dietary AA pattern that resembled the overall AA pattern of fish meal. A diet with an AA profile adjusted to meet the ideal protein AA pattern estimated for sea bass significantly depressed growth.

Effect of the dietary essential to non-essential amino acid ratio on growth, feed utilization and nitrogen metabolism of European sea bass ( Dicentrarchus labrax)

A study was undertaken to evaluate if dietary essential to non-essential amino acid ratio (EAA/NEAA) affects growth, feed utilization and nitrogen metabolism of European sea bass. Three semi-purified diets including fish meal and mixtures of crystalline amino acids as the main nitrogen (N) sources were formulated to have a dietary EAA/NEAA ratio of 40/60, 50/50 and 60/40; another diet, fish meal based was used as a control. All diets were formulated to be isolipidic (15% DM) and isonitrogenous (8% DM). Triplicate groups of juvenile sea bass (initial body weight of 7.3 g) were fed each diet, twice daily to apparent satiation, for 42 days. Weight gain and specific growth rate of fish fed the control, 60/40 and 50/50 diets were similar and significantly higher than those of fish fed the 40/60 diet. Voluntary feed intake of fish fed the 60/40 diet was significantly lower than that of fish fed the 50/50 and 40/60 diets. Feed efficiency and protein efficiency ratio significantly improved with the increase of dietary EAA/NEAA ratio. Whole-body composition, visceral and hepatosomatic indices were not significantly affected by diet composition, except whole-body lipid content that was significantly lower in fish fed the 40/60 diet than in the other groups. Daily N retention (g kg ABW − 1 ) was significantly lower with the 40/60 diet than with the other diets. As percentage of N intake, N retention was significantly higher with the 60/40 diet than with the other diets. Energy retention (kJ kg ABW − 1 day − 1 and % of E intake) significantly increased as dietary EAA/NEAA increased. Ammonia-N and urea-N excretion (mg N kg ABW − 1 day − 1 ) did not differ significantly among treatments. Ammonia-N excretion (% N intake) was significantly higher with diet 40/60 than with the other diets. Specific activity of glutamate dehydrogenase was significantly lower in fish fed the control and 60/40 diets than in the other groups. Specific activity of alanine and aspartate aminotransferases and glucose-6-phosphate dehydrogenase did not significantly differ among groups. Fatty acid synthetase activity was significantly higher with the 40/60 diet than with the other diets. Results of this study suggest that a dietary EAA/NEAA ratio of 50/50 is necessary to promote maximum growth performance of juvenile European sea bass. However, this ratio should be increased to 60/40 if maximization of feed, protein and energy utilization is to be considered.

The effect of dietary protein replacement by crystalline amino acid on growth and nitrogen utilization of turbot Scophthalmus maximus juveniles

A study was conducted to evaluate the effect of partial replacement of dietary fish meal by crystalline amino acids on growth performance, feed utilization, body composition and nitrogen utilization of turbot juveniles. Four diets were formulated to be isolipidic (12% DM) and isonitrogenous (8% DM). A fish meal based diet was used as control. In the experimental diets, a crystalline amino acid (AA) mixture was used to partially replace fish meal, corresponding to a non-protein nitrogen content of 19, 37 and 56%, respectively (diets 19AA, 37AA and 56AA, respectively). The overall amino acid profile of the experimental diets resembled that of the whole-body protein of turbot. Each experimental diet was fed to triplicate groups of 20 fish (initial body weight of 31.8 g) twice daily to apparent satiation for 42 days. During the trial water temperature averaged 18 °C. Final body weight, weight gain (g kg ABW − 1 day − 1 ) and specific growth rate were not different between the control and 19AA diet but significantly decreased with the increase of crystalline-AA inclusion from 19 to 56%. Feed intake and feed efficiency of fish fed the control and diet 19AA were similar and significantly higher than those of fish fed the 56AA diet. At the end of the growth trial, there were no significant differences in whole-body composition among groups. Hepatosomatic index was also unaffected by dietary treatments. Nitrogen retention (g kg ABW − 1 day − 1 ) of fish fed the control and the 19AA diets were similar and significantly higher than that of fish fed the other diets. Expressed as a percentage of the nitrogen intake, N retention was significantly higher with the control than with the 37AA and 56AA diets. Daily ammonia excretion (mg kg ABW − 1 day − 1 ) of fish fed the control diet was significantly higher than that of fish fed the 37AA and 56AA diets, while daily urea excretion (mg kg ABW − 1 day − 1 ) did not significantly differ among treatments. Non-fecal nitrogen (ammonia + urea) excretion (mg kg ABW − 1 day − 1 ) was significantly higher for fish fed the control diet than in those fed the 37AA and 56AA diets. However, as percent of N intake, ammonia excretion and non-fecal N excretion were significantly higher in fish fed the 56AA diet than in those fed the control and 19AA diets. Specific activity of glutamate dehydrogenase, alanine and aspartate aminotransferases did not significantly differ among experimental groups. In conclusion, in diets with an overall amino acid profile resembling that of the whole-body protein of turbot, crystalline-AA may replace 19% of dietary protein without negatively affecting growth performances or feed utilization efficiency. However, higher protein replacement levels of protein-bound-AA by crystalline-AA severely depressed growth performance.

Skeletal muscle bioenergetics: a comparative study of mitochondria isolated from pigeon pectoralis, rat soleus, rat biceps brachii, pig biceps femoris and human quadriceps

The metabolism of mitochondria isolated from five functionally different skeletal muscles is compared. Data for a single ectothermic preparation are also reported. The mitochondria were prepared in yields of 44±7% from 50 to 100 mg muscle. The muscle content of mitochondrial protein ranged between 2 and 40 g kg −1. Twelve specific activities of key enzymes and metabolic systems were determined, 10 of these in functional assays with respiratory measurements. The specific activities of glutamate dehydrogenase, α-glycerophosphate dehydrogenase, and exo-NADH oxidase differed considerably among muscle sources. Seven specific activities, including very central reactions, showed low among-muscle variation. The activity of ATP synthesis, for instance, was 1.0–1.3 mmol min −1 g −1 mitochondrial protein, 25 °C. In vitro data were extrapolated to in vivo conditions of the muscles. The calculated rates of respiration and ATP synthesis were in accordance with reported tissue activities. Pigeon pectoralis mitochondria showed a unique cytochrome spectrum and a respiratory chain activity that might effect simultaneous carbohydrate and fatty acid respiration. In mitochondria from the other muscles, the respiratory chain activity balanced the carbohydrate oxidation capacity. In all muscles, the respiratory capacity exceeds that needed for oxidative phosphorylation. This may secure maximal mitochondrial ATP synthesis during maximal work rates and high cellular [Ca 2+].

The involvement of glutamate dehydrogenase in the adaptation of mitochondria to oxidize glutamate in sucrose starved pea embryos

Embryos of pea (Pisum sativum L. cv Sol) deprived of cotyledons were cultured for 3 days in medium with or without sucrose. Respiratory activity of embryos (intact) as well as the ability to oxidize glutamate by mitochondria isolated from embryos were studied. Respiration of intact embryos grown in sucrose supplemented medium was more intensive than in the starved ones. Transfer of the starved embryos to the sucrose-containing medium induced the increase in the intensity of O2 consumption. Mitochondria isolated from both starved and control embryos exhibited respiratory control. Mitochondria isolated from embryos cultured in the absence of sucrose showed higher (about 60 %) ability to oxidize glutamate and α-ketoglutarate than mitochondria from embryos grown in sucrose containing medium. The absence of sucrose in the medium led to a rapid increase in the specific activity of glutamate dehydrogenase (NADH-GDH and NAD-GDH) and it was accompanied by changes in izoenzymatic pattern of enzyme. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase may be responsible for the increase of glutamate oxidation by mitochondria of pea embryos. Electrophoretic separation of glutamate dehydrogenase isolated from embryos cultured in medium without sucrose showed the presence of ca. 17 isoenzymes while in non-starved embryos only 7 isoenzymes were identified. However, the addition of sucrose to starved embryos after 24 hours of cultivation led to a decrease in glutamate dehydrogenase activity (up to 40 %) but it did not cause the changes in isoenzymatic pattern. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase maybe responsible for the increase of glutamate oxidation by mitochondria of pea embryos. The posibility of glutamate dehydrogenase regulation by sucrose is discussed.

Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development

We investigated the effects of genetic modification of nitrogen metabolism via the bacterial glutamate dehydrogenase (GDH) on plant growth and metabolism. The gdhA gene from Escherichia coli encoding a NADPH-GDH was expressed in tobacco plants under the control of the 35 S promoter. The specific activity of GDH in gdhA plants was 8-fold of that in E. coli. Damage caused by spray application of 1.35 mM of phosphinothricin (PPT) herbicide, a glutamine synthetase (GS) inhibitor, was less pronounced in gdhA plants as compared with the control plants which suggests that the introduced GDH can assimilate some of the excess ammonium, at least during GS inhibition. However, gdhA plants were susceptible to 2.7 mM PPT. Biomass production was consistently increased in gdhA transgenic plants grown under controlled conditions and in the field. Total free amino acids and total carbohydrates were increased in gdhA plants grown in the greenhouse suggesting that both nitrogen and carbon metabolism were altered. We conclude that the modifications in transgenic plants may result from both increased nitrogen efficiency and altered gene expression and metabolism.

Ammonia assimilation in Corynebacterium glutamicum and a glutamate dehydrogenase-deficient mutant

In the wild-type of Corynebacterium glutamicum, the specific activity of glutamate dehydrogenase (GDH) remained constant at 1.3 U (mg protein)−1 when raising the ammonia (NH4) concentration in the growth medium from 1 to 90 mM. In contrast, the glutamine synthetase (GS) and glutamate synthase (GOGAT) activities decreased from 1.1 U (mg protein)−1 and 42 mU (mg protein)−1, respectively, to less than 10 % of these values at NH4 concentrations > 10 mM suggesting that under these conditions the GDH reaction is the primary NH4 assimilation pathway. Consistent with this suggestion, a GDH-deficient C. glutamicum mutant showed slower growth at NH4 concentrations ≥ 10 mM and, in contrast to the wild-type, did not grow in the presence of the GS inhibitor methionine sulfoximine. © Rapid Science Ltd. 1998

Potential glutamine synthetase and glutamate dehydrogenase activities in the oligo-mesotrophic Lake Pavin: annual comparison with an indirect estimation of inorganic nitrogen utilization

The annual distribution of potential glutamine synthetase (GS) and glutamate dehydrogenase (GDH) activities was studied in the oligo-mesotrophic Lake Pavin (France, Massif Central). The validity of these enzymatic activies as an index of N-assimilation in field studies was tested by a comparison with the coupling of distributions of inorganic nitrogen uptake rate based on 14 C incorporation into proteins together with several abiotic and biotic parameters. The potential GS and GDH activities recorded during the annual phytoplankton succession were in the same order of magnitude as those reported from the very scanty literature on this subject. Moreover, specific potential GS and GDH activities were significantly correlated with the external NO 3 - -N concentration suggesting that these enzymatic activities reflected nitrate utilization by algae. Specific activities of GDH were about 2.5 fold higher than GS specific activities suggesting that the GDH pathway may play a leading part in inorganic nitrogen assimilation in the oligo-mesotrophic Lake Pavin. However, N-assimilation rates based on the enzymatic approach were lower than those obtained through indirect isotopic determination of N-uptake rates. The reasons for such a discrepancy are discussed.

Aspartate Aminotransferase and Glutamate Dehydrogenase Activities in the Squid Giant Nerve

The present study sought to investigate the presence and distribution of some enzymatic activities involved in the metabolism of glutamate in the giant nerve fiber of the tropical squid Sepioteuthis sepioidea. Specific activities of aspartate aminotransferase and glutamate dehydrogenase were evaluated in homogenates of the isolated giant fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. The activities of both enzymes were present in the tissue. The specific activity of aspartate aminotransferase was similar in axoplasm and sheaths. However, the specific activity of glutamate dehydrogenase was an order of magnitude higher in the sheaths. This finding is discussed in the framework of the hypothesis that proposes that a differential distribution of the enzymes of the glutamatergic system between the axonal and neuroglial compartments forms part of a system of communication between these cells whose neuronal signal may be glutamate.

Glutamate Dehydrogenase Is Not Essential for Glutamate Formation by Corynebacterium glutamicum

Two Corynebacterium glutamicum strains, one being glutamate dehydrogenase (GDH) negative and the other possessing 11-fold-higher specific GDH activity than the parental wild type, were constructed and used to analyze the role of GDH in C. glutamicum. The results indicate (i) that GDH is dispensable for glutamate synthesis required for growth and (ii) that although a high level of GDH increases the intracellular glutamate pool, the level of GDH has no influence on glutamate secretion.

Effect of high concentration of ammonia on cultured hepatocytes of eel ( Anguilla japonica)

1. High concentration of ammonia (13.5 and 23.3 mM) was effective for maintenance of normal morphology of cultured eel hepatocytes. 2. Synthesis of urea did not increase by addition of ammonia to eel hepatocytes. 3. Intracellular protein content in hepatocytes cultured with ammonia was higher than that in control cells. 4. Consumption of essential amino acids, especially Thr, Val, Ile, and Lys, in the medium was reduced by addition of ammonia. 5. A Glu-derivative was synthesized by cultured hepatocytes. The amount of the derivative in the medium was the highest next to that of Gly and increased as the concentration of ammonia in the medium increased. 6. Specific activity of glutamate dehydrogenase (GDH) in cultured hepatocytes increased as the concentration of ammonia increased and the activity of GDH in hepatocytes cultured in the presence of 23.3 mM ammonia was 2.6 times higher than that in the control cells.

Regulation of Glutamate Dehydrogenase Activity in Relation to Carbon Limitation and Protein Catabolism in Carrot Cell Suspension Cultures

Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity.

The effects of carbon starvation on cellular metabolism and protein and RNA synthesis in Gerbera callus cultures

Gerbera jamesonii H. Bolus var. hybrida callus cultures subjected to carbon starvation show alteration in some physiological parameters and protein synthesis. Five days of continuous starvation induce a decrease of protein and carbohydrate contents to respectively, 27% and 12% of the values of the control, while specific activities of glutamate dehydrogenase and proteases reached levels about 5-fold and 2-fold higher than the control ones. Electrophoresis of proteins labelled by in vivo and in vitro translation showed the appearance of several starvation-related proteins (STP). At day 5 from the onset of starvation the induction of STPs was related with levels of in vitro translatable mRNAs, whereas at day 1 the synthesis of STPs depended upon translational regulation. The induction of STPs was also analyzed under conditions of specific protein synthesis inhibition by heat-shock. Protein synthesis in heat-shocked starved cells showed (i) the induction of heat-shock proteins (HSPs), and (ii) the disappearance of all but three STPs of molecular mass 70, 69 and 36 kDa. A similarity between one HSP and one STP is hypothesized on the basis of comigration on two-dimensional gels (70 kDa). During recovery from heat-shock starved cells showed the reappearance of all the STPs and the simultaneous maintenance of most of the HSPs. The data presented support a role of STPs in starvation and the existence of interactions between heat-shock and nutrient deprivation.

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.

Genetical variability of glutamate dehydrogenase activity in monokaryotic and dikaryotic mycelia of the ectomycorrhizal fungus Hebeloma cylindrosporum

Glutamate dehydrogenase (GDH) is the key enzyme of ammonium assimilation by ectomycorrhizal fungi. Its activity might be use as a criterion to select mycelia capable of enhancing the nitrogen nutrition of the host plants. Genetical variability of the GDH activity of the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnesi was studied in an attempt to determine if this enzyme activity could be improved by way of chromosomal genetics. The activity of 11 wild strains was compared with that of 70 mycelia obtained as the progeny of a laboratory fruiting strain HC1. These 70 mycelia were 20 monokaryons (5 for each mating type) and the 50 synthesized dikaryons obtained from all the compatible fusions between these monokaryons. The specific GDH activity of the 11 wild strains ranged from 1.5 to 11.6 nkat mg-1 fungal protein. The activity of the monokaryotic progeny of the HC1 strain was, on average, three times lower (2.85 n kat mg-1 fungal protein) than that of the parental dikaryon. In contrast, synthesized dikaryons originating from these monokaryons were very variable and had an average values similar to that of the parental dikaryon (9.1 nkat mg-1 fungal protein). Eighteen of these synthesized dikaryons contained an activity higher than that of the original HC1 strain. The variation of the GDH activity of these dikaryons involves additive and non additive (interactive) components, each of them accounting for ca. 50% of the genetical variation. The non additive variation could not be explained by a model involving only dominance. These results are discussed with reference to the genetical improvement of mycorrhizal fungi in order to enhance nitrogen nutrition of the host plants.

Glutamate biosynthesis in Bacillus azotofixans. 15N NMR and enzymatic studies.

Pathways of ammonia assimilation into glutamic acid in Bacillus azotofixans, a recently characterized nitrogen-fixing species of Bacillus, were investigated through observation by NMR spectroscopy of in vivo incorporation of 15N into glutamine and glutamic acid in the absence and presence of inhibitors of ammonia-assimilating enzymes, in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and alanine dehydrogenase. In ammonia-grown cells, both the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways contribute to the assimilation of ammonia into glutamic acid. In nitrate-grown and nitrogen-fixing cells, the glutamine synthetase/glutamate synthase pathway was found to be predominant. NADPH-dependent glutamate dehydrogenase activity was detectable at low levels only in ammonia-grown and glutamate-grown cells. Thus, B. azotofixans differs from Bacillus polymyxa and Bacillus macerans, but resembles other N2-fixing prokaryotes studied previously, as to the pathway of ammonia assimilation during ammonia limitation. Implications of the results for an emerging pattern of ammonia assimilation by alternative pathways among nitrogen-fixing prokaryotes are discussed, as well as the utility of 15N NMR for measuring in vivo glutamate synthase activity in the cell.

Role of glutamate dehydrogenase in ammonia assimilation in nitrogen-fixing Bacillus macerans.

Pathways of ammonia assimilation into glutamic acid in Bacillus macerans were investigated by measurements of the specific activities of glutamate dehydrogenase (GDH), glutamine synthetase, and glutamate synthase. In ammonia-rich medium, GDH was the predominant pathway of ammonia assimilation. In nitrogen-fixing cells in which the intracellular NH4+ concentration was 1.4 +/- 0.5 mM, the activity of GDH with a Km of 2.2 mM for NH4+ was found to be severalfold higher than that of glutamate synthase. The result suggests that GDH plays a significant role in the assimilation of NH4+ in N2-fixing B. macerans.