Glutamate Dehydrogenase from the Nonheterocystous ...

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aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the ... EC 1.4.1.4) catalyzes the reductive amination of 2-oxogluta-.
JOURNAL

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BACTERIOLOGY, Oct. 1988, p. 4897-4902

Vol. 170, No. 10

0021-9193/88/104897-06$02.00/0 Copyright © 1988, American Society for Microbiology

Induction, Isolation, and Some Properties of the NADPH-Dependent Glutamate Dehydrogenase from the Nonheterocystous Cyanobacterium Phormidium laminosum MERTXE MARTINEZ-BILBAO, AURORA MARTINEZ, INAKI URKIJO, MARIA J. LLAMA, AND JUAN L. SERRA* Departamento de Bioquirmica y Biologia Molecular, Facultad de Ciencias, Universidad del Pat's VascolEuskal Herriko Unibertsitatea, Apartado 644, E48080 Bilbao, Spain Received 14 March 1988/Accepted 27 June 1988

The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-flxing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30°C it showed a specific activity of 32.9 gimol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (K,m = 1.25 mM) and NADPH (Km = 64 ,uM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [SJ0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60°C (Ea = 5.1 kcal/ mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.

The NADPH-dependent glutamate dehydrogenase (GDH; EC 1.4.1.4) catalyzes the reductive amination of 2-oxoglutarate, providing the cell with L-Glu. The reaction is reversible, but the direction in which it proceeds physiologically has been and still is a subject for controversy. The possible anabolic or catabolic function of the enzyme might be significantly different among species, or, in addition, more than one GDH, with distinct and specifical functions, could be present in the same organism (13, 23). In previous years it was thought that GDH was the major enzyme responsible for ammonium assimilation in higher plants and microorganisms. However, its relevance drastically decreased after the finding of the glutamine synthetaseglutamate synthase cycle, because the Km values reported for the characterized GDH were very much higher than those found for glutamine synthetase (31, 40). Today it is well documented that GDH is responsible for ammonium assimilation in yeasts (43) and fungi (2). However, in cyanobacteria (31, 37), as well as in higher plants and algae, ammonium assimilation is widely considered to take place through the glutamine synthetase-glutamate synthase cycle. In the literature, contradictory results on the existence of GDH in cyanobacteria have been reported. Whereas several workers reported that the enzyme was absent or present only at very low level (15, 36), other groups reported that it was responsible for L-Glu synthesis in Anacystis nidulans (30) or that under certain conditions of nitrogen supply it could be an alternative pathway to the glutamine synthetaseglutamate synthase cycle for ammonia assimilation (10, 27). In a recent paper (27) we reported the presence of an NADPH-dependent GDH in cell-free preparations of both nitrate- and ammonium-grown cells of the non-nitrogen*

fixing cyanobacterium Phormidium laminosum. Moreover, when nitrate-grown cells were transferred to an ammoniumcontaining medium, an increase of GDH specific activity occurred. Here we report further details on the inducible nature of the NADPH-GDH activity and some relevant properties of the purified enzyme. MATERIALS AND METHODS Materials. Mono-Q and Sephacryl S-300 were obtained from Pharmacia Biotechnology, Uppsala, Sweden. DEAEcellulose (DE-52) was from Whatman, Maidstone, United Kingdom. Molecular weight markers and nucleotides were from Boehringer GmbH, Mannheim, Federal Republic of Germany. 2',5'-ADP-agarose, 2-oxoacids, L-amino acids, methylamine, and other biochemicals were from Sigma Chemical Co., London, United Kingdom. All other chemicals were from E. Merck AG, Darmstadt, Federal Republic of Germany. Organism and growth conditions. P. laminosum OH-1p.C1 was originally obtained from R. W. Castenholz, University of Oregon, Eugene. Cells were grown photoautotrophically for 6 days in pure culture at 45°C in 25-liter polyethylene carboys containing 20 liters of modified medium D (6) supplemented with 6 mM NaHCO3 as described previously (27). The nitrogen source was 5 mM NH4Cl instead of the nitrate used in the original medium. To increase the cellular level of the enzyme, we supplemented the cultures with more 5 mM ammonium for 24 h before harvesting. Enzyme isolation. All the purification steps took place at 4°C in 50 mM Tris hydrochloride (pH 8.0) (standard buffer) unless otherwise stated. Cells were collected by centrifugation (Kubota RC-1A continuous-flow rotor), washed and suspended in buffer (1 g [wet weight]/10 ml), and disrupted

Corresponding author. 4897

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by sonication (MSE sonicator; 1 min/10 ml) in an ice bath. The sonic extract was centrifuged for 1 h at 105,000 x g, and the blue supernatant was applied to a DE-52 column (2.5 by 14 cm) equilibrated with buffer. The column was washed with equilibration buffer containing 0.1 M KCl until most of the blue pigments were removed (about 10 column volumes), and the activity was eluted with 0.2 M KCl-containing buffer. The active fractions were pooled, diluted with 1 volume of buffer, and applied to a 2',5'-ADP-agarose column (1 by 5 cm) equilibrated with buffer. After the column had been washed with buffer containing 0.4 M KCl until no A280 could be detected in the eluate, the enzyme was eluted with the above buffer supplemented with 0.5 mM NADPH. The active fractions were combined, dialyzed, and applied to a Mono-Q HR 5/5 column equilibrated with 20 mM buffer at a flow rate of 1 ml/min and fitted to Fast Protein Liquid Chromatography (Pharmacia) equipment. After the column had been washed with the equilibration buffer supplemented with 0.2 M KCl, the GDH activity was eluted with a 20-min linear gradient of KCl (from 0.2 to 0.5 M) in buffer. Enzyme assays. All the enzyme assays were measured spectrophotometrically at 30°C by recording the variations of A340 by the method of Hooper et al. (16). Aminating NADPH-dependent GDH activity (EC 1.4.1.4) was routinely assayed, unless otherwise stated. The cuvette contained (in a final volume of 1 ml) 50 mM Tris hydrochloride (pH 8.0), 15 mM 2-oxoglutarate, 0.8 mM CaCl2, 150 mM NH4Cl, 0.08 mM NADPH, and an appropriate amount of enzyme. The reaction was started by adding NADPH, which was omitted in the reference cuvette. One unit of enzyme catalysed the oxidation of 1 ,umol of NADPH per min. In all cases, the data represent the average of at least three separate experiments.

Analytical methods. Protein was determined by the method of Bradford (3), with crystalline bovine serum albumin as the standard. Polyacrylamide gel electrophoresis was carried out by the method of Davis (8) with 7% (wt/vol) acrylamide gel slabs. NADP-GDH activity was located in the gels by the method of Talley et al. (42), and protein bands were visualized by using a silver stain. The molecular mass of the native enzyme was estimated by molecular sieve chromatography on a Sephacryl S-300 column (2.2 by 90 cm) calibrated with ferritin (450 kilodaltons [kDa]), catalase (220 kDa), aldolase (158 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), and cytochrome c (12.5 kDa). RESULTS Cellular levels and induction of NADPH-dependent glutamate dehydrogenase. When nitrate-grown cultures were harvested and resuspended in fresh medium D containing different nitrogenous compounds as the sole nitrogen source, considerable differences in the cellular levels of NADPHGDH activity were observed after 24 h of growth (Table 1).

TABLE 1. Effect of the nitrogen source on the cellular levels of NADPH-GDH in P. laminosum"

Nitrogen source (concn) None

(mU/mgSpofactprotein)

................................

. ........................ NaNO3 (5 mM) NaNO3 (5 mM) + MSX (100 I.LM) ............. . ........................ NaNO2 (5 mM) NH4Cl (5 mM) ..................................... NH4Cl (100 mM) ..................................... NH4Cl (5 mM) (in darkness) ................. NH4Cl (5 mM) + NaNO3 (5 mM) ................... NH4Cl (5 mM) + NaNO2 (5 mM) ...................

L-Glc (5 mM) L-Gln (5 mM)

CH3NH3 (5 mM)

. .......................... . .......................... . ........................

2.7 ± 3.7 ± 7.7 ± 9.4 ± 11.4 ± 13.8 + 12.8 ± 15.3 ± 15.2 ± 30.0 ± 20.9 ± 37.8 ±

1.1 2.0 1.6 2.0 1.9 2.8 2.3 2.9 2.0 3.6 5.2 7.0

a Cells growing in nitrate containing-medium D (showing a specific activity of 2.5 ± 0.5 mU/mg of protein) were transferred to fresh medium containing the indicated nitrogenous compounds as the sole nitrogen source, and after 24 h of growth the NADPH-dependent GDH activity in cell extracts was measured.

Ammonium ions were the best inorganic inducer of the activity, even in darkness; and similar specific activity values (about 11 mU/mg of protein) were routinely measured in cells grown in either 1 or 5 mM ammonium chloride, although slightly higher values occurred at 100 mM ammonium ion. The presence of 5 mM nitrate or nitrite did not decrease the inductory effect caused by ammonium ions, but, instead, a slight stimulatory effect was observed in both cases. The organic nitrogenous compound methylamine and the amino acids L-Glu and L-Gln were better inducers of P. laminosum NADPH-GDH than was ammonium ion by itself. Enzyme purification. The results of a typical purification procedure are given in Table 2. The enzyme was purified >4,400-fold with an 11% recovery and showed a specific activity of 32.9 U/mg of protein. In crude extracts and unpurified preparations, both NADH- and NADPH-dependent GDH activities were detectable. However, the NADPH-GDH/NADH-GDH ratio significantly increased on purification (Table 2), and no NADH-dependent activity could be measured after the Mono-Q chromatographic step. The electrophoretogram in polyacrylamide gels of one sample after the last step of purification showed a band of protein coincidental with a single band of activity and a band of contaminant protein. Catalytical properties. The purified NADPH-GDH showed no aminotransferase activity and was unable to utilize L-Gln as an alternative substrate to ammonium ion. At 150 mM L-Glu, it catalyzed the amination of 2-oxoglutarate 12- to 15-fold more strongly that the reverse reaction. The optimum pH values for the aminating activity of GDH in phosphate buffer and Tris hydrochloride were 7.5 and 8.0, respectively.

TABLE 2. Purification of NADPH-GDH from P. laminosum Step

105,000 x g supernatant DE-52 eluate 2',5'-ADP-agarose eluate Mono-Q (HR 5/5) eluate

Amt of protein

(mg) 1,740 228.6

0.57 0.044

Activity" (U)

12.9 7.8 2.1 1.5

" One unit of enzyme catalyzed the oxidation of 1 FLmol of NADPH per min.

Sp act (U/mg of protein)

0.0074 0.034 3.7 32.9

% Yield

100 60 16 11

Purification

(-fold) 1 4.6 495

4,446

NADPH-GDH NADH-GDH

6 9 28

Xi

P. LAMINOSUM NADPH-DEPENDENT GLUTAMATE DEHYDROGENASE

VOL. 170, 1988

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100 2.0 I-

a-

4J

P0 1.5

-

F _

0)

C]

E

UJ

n

a n

6I 1.0 _

wi

304

I~-

2

0

5 15 10 TIME (min) FIG. 1. Thermal inactivation of NADPH-GDH from P. laminosum. The enzyme was incubated at the indicated temperatures in standard buffer in sealed tubes. At intervals, the samples were cooled in an ice bath and the residual activity was determined by the aminating assay at 300C. Each point represents the average values from at least two separate experiments. The 100% level of activity corresponded to 2.3 U/mg of protein.

Molecular mass determination. The molecular mass of the native enzyme estimated by gel filtration was 280 + 5 kDa. Enzyme stability. The enzyme stability drastically decreased with further purification. Enzyme preparations of the 105,000 x g supernatant were stable at either 4 or -200C, and more than 75% of the initial activity could be routinely measured after 8 weeks of storage at -20°C. By contrast, the purified enzyme was unstable when stored frozen at -20°C for a few hours, even in the presence of 50% (vol/vol) glycerol. Effect of temperature. The effect of temperature on the stability of the purified GDH is shown in Fig. 1. The enzyme retained more than 75% of the initial activity after 15 min of incubation at temperatures up to 65°C, and almost total inactivation occurred at 72°C. The presence of substrates did not significantly protect the enzyme against thermal inactivation. From an Arrhenius plot, an activation energy of 5.1 kcal/mol (21.3 kJ/mol) and a thermal coefficient (Qlo) of 1.3 were calculated for the aminating NADPH-GDH activity. Effect of ionic strength and urea. The NADPH-GDH activity was slightly activated (about 15%) by the presence in the reaction mixture of low concentrations (ca. 100 mM) of either NaCl or KCl. However, ionic strengths higher than 0.1 led to a progressive decrease in the activity, which was negligible in the presence of 2 M chloride. Similar inactivation was caused by urea, and no activity could be detected when the enzyme was assayed in the presence of 6 M urea. After the complete removal of salts or urea by dialysis against standard buffer, more than 50% of the initial activity could be recovered in the dialysate. Substrate affinities. The purified enzyme showed high specificity for the 2-oxoacid and the electron donor used for activity. Very low rates ( 0.99), apparently denoting hyperbolic kinetics. However, when the enzyme was assayed at ammonium concentrations ranging from 1 to 75 mM, nonhyperbolic kinetics, denoting negative cooperativity, could be observed (Fig. 2). When these data were analyzed by using a Hill plot, a [S]O5 of 18.2 mM for ammonium and a Hill coefficient, h, of 0.89 were calculated. Kinetic mechanism. The aminating NADPH-GDH activity of the enzyme was assayed by fixing the concentration of one substrate (100 ,uM NADPH, 15 mM 2-oxoglutarate, and 150 mM ammonium) and varying the concentrations of the other two. The results obtained for this "pseudobisubstrate" reaction were consistent with a random mechanism with independent sites (data not shown). Effectors. The enzyme was not apparently affected by the presence of 1 mM thiol reagents, 5 mM thiol compounds, 5 mM nucleotides, 1 mM divalent cations, 20 mM L-methionine-DL-sulfoximine (MSX), and most L-amino acids at 20 mM. Only glutaric acid and L-Glu caused about 25% inhibition at 20 mM; L-His and L-Trp caused 15% inhibition at the same concentration. DISCUSSION As reported earlier (27), cell extracts of nitrate-grown cells of P. laminosum showed detectable levels (about 2.5 to 3.5 mU/mg of protein) of the NADPH-dependent GDH. These

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levels could be significantly increased if the cells were grown with ammonium ions or other sources of inorganic or organic nitrogen. Similar results were reported for the inducing effect of ammonium ions on GDH from fungi (14), green algae (35, 44), and higher plants (5, 22) and the inability of nitrate or nitrite to counterbalance the effect of ammonium (18). However, in the eucaryotic algae Chlorella sorokiniana (32, 35) and Euglena gracilis (9), the inducing effect of ammonium on GDH was apparent only in the light. The well-known specific inhibitor of glutamine synthetase, MSX, also increased the specific activity of the NADPH-GDH of cell-free preparations of nitrate-grown P. laminosum. This result could be explained if GDH partially substituted for the irreversibly MSX-inhibited glutamine synthetase (27) or if the ammonium produced from nitrate (26), which cannot be assimilated by the cell, were directly responsible for the GDH induction. Although the apparent ammonium-inducible nature of the enzyme would suggest that GDH may play a role in the assimilation of ammonium at high concentrations of this ion (14), the fact that amino acids lead to higher cellular levels of GDH than ammonium does is consistent with a deaminating function of the enzyme. However, in E. gracilis (9), as well as in other sources in which GDH plays a catabolic rather than an assimilatory role, the enzyme was induced by L-Glu but not by ammonium ions. Although under laboratory conditions P. laminosum is able to grow in medium D with L-Glu as the sole nitrogen source, it seems unlikely that amino acids would be available as the main nitrogen source in its natural habitat. Therefore, the physiological significance of this finding remains unclear. However, because of its kinetic properties, Botton and Msatef (2) suggested that GDH plays an important assimilatory role in Sphaerostilbe repens, although the enzyme is L-Glu-inducible. Although methylamine, an ammonium analog, was the best inducer of GDH activity in terms of specific activity measured in cell extracts, P. laminosum cultures on methylamine were not able to develop properly, and the cells underwent a rapid bleaching. The increase of specific activity is probably due to a drastic decrease in the cellular protein content rather than to a true increase in the total amount of the enzyme. Because of the contradictory findings reported in the literature on the existence of GDH activity in cyanobacteria (7, 10, 29, 30, 41), and to characterize further the possible assimilatory role of the enzyme, it seemed necessary to isolate the activity. Typical results of the isolation of the NADPH-GDH from P. laminosum are summarized in Table 2. Although the P. laminosum enzyme was purified more than 4,400-fold, the preparation was not homogeneous when analyzed by polyacrylamide gel electrophoresis, indicating that this protein was present in a very low concentration even in ammonium-grown cells. However, the purified enzymes from other sources showed lower specific activities (between 3.5 and 8.5 U/mg of protein) (11, 17) and were less highly purified (78- and 6-fold, respectively). Our enzyme recovery compares to that of the enzyme from the fungus Cenococcum graniforme (25) but is much lower than that of Chlorella spp. (80%) (48) or Synechocystis sp. strain PCC 6803 (101%) (10). The enzyme appeared quite specific for its substrates, and, in contrast with the GDH characterized from other sources (24), the P. laminosum enzyme was completely unable to utilize L-Gln as the amino-group donor to synthesize L-Glu. The calculated molecular mass of the native enzyme (about 280 kDa) is higher than that reported for the Synechocystis sp. strain PCC 6803 enzyme (208 kDa) (10) but is of

J. BACTERIOL.

the same order of magnitude as that of other GDHs characterized from different sources (1, 20, 34). Owing to the presence of a contaminant protein and because of the small amount of purified protein recovered in the Mono-Q preparation, we were unable to determine the subunit composition of the enzyme. Because of the molecular mass of the native GDH, like the enzyme characterized from other sources, the P. laminosum enzyme would probably also be composed of several subunits. This idea was supported by the fact that the enzyme activity was strongly affected by high concentrations of either salts or urea, which classically dissociate oligomeric proteins into subunits. The reversible nature of this effect would be in agreement with the possible existence of a dissociation-association equilibrium between subunits, which would be directly related to enzyme activity. As reported for the enzyme isolated from other sources (4, 21), the K,JM of the P. laminosum enzyme for ammonium depended on the ammonium concentrations used. Several possible explanations for the kinetic behavior of GDH with respect to ammonium were reported in the literature. Thus, to explain the kinetics of the enzyme isolated from the fungus Neurospora crassa or bovine liver, several workers invoked the possible hysteretic nature of the enzyme (33). The possible existence of two binding sites, with differential affinities for ammonium, was proposed for the enzyme from Azospirillum brasilense (28). However, the biphasic kinetics found for ammonium with the enzyme from Nitrobacter agilis could be explained by the existence of different aggregation states of the enzyme (21). Finally, the possible existence of negative cooperativity (h = 0.66) would explain the behavior of the enzyme from Sphaerostilbe repens (2). The kinetic data obtained for the P. laminosum enzyme could be explained by assuming negative cooperativity, although the possible existence of association and dissociation among subunits would not be disregarded. The enzyme was not apparently hysteretic for ammonium (data not shown), and the possible existence of two binding sites, with high and low affinities for ammonium, seems unlikely. As stated above, both nitrate-grown and ammoniumgrown P. laminosum cells showed some NADPH-GDH activity. To find whether both activities corresponded to the same protein, we determined the affinities for ammonium of the purified enzyme from both preparations. The enzyme from each preparation was purified separately by an identical procedure, and the same K,,, value for ammonium was obtained for both enzyme preparations when they were analyzed for exactly the same range of ammonium concentrations. These results confirmed that the enzyme present in nitrate-grown cells possessed the same physicochemical and kinetic properties as that present in ammonium-grown cells, strongly suggesting that both enzymes corresponded to the same protein. In contrast with the NADPH-GDH activity characterized from other sources (21, 38), the P. laminosum enzyme was affected by neither thiol reagents nor thiol compounds. As with the enzyme from bacterial (28, 45) or higher plant (39) sources, only high concentrations of glutaric acid or L-Glu inhibited the enzyme to some extent. Moreover, the presence of Ca2+ or other divalent cations did not affect the P. laminosurm enzyme, whereas this cation was essential for the activity of other aminating GDHs (20, 47) or the deaminating activity of Synechocystis sp. strain PCC 6803 (10). As in higher plants, algae, and microorganisms, ammonium assimilation in P. laminosum probably proceeds through the so-called glutamine synthetase-glutamate synthase cycle. However, some evidence of the ammonium-

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inducible nature of the NADPH-GDH, together with the ammonium-repressible nature of glutamine synthetase activities in P. laminosum, has been reported (27). Moreover, nitrogen-starved cells of P. laminosum take up ammonium from the external medium faster than they take up nitrate or nitrite (M. Martinez-Bilbao and J. L. Serra, unpublished results). In addition, the in vitro aminating activity was significantly higher than the deaminating activity. On the other hand, the affinity of GDH for ammonium was higher at low than at high ammonium concentrations, at which toxic cellular levels would be achieved, and, hence, the efficiency of the enzyme in playing a possible detoxicant role (12) would be very low. These data again suggest that GDH, would play, at least, a minor role in ammonium assimilation (27), which would be strictly regulated. In this way, Kane et al. (19) and Vierula and Kapoor (46) suggested that the catabolic GDH would be regulated by the carbon source, whereas the anabolic enzyme would be regulated by the nitrogen source. However, the L-Glu-inducible nature of the enzyme (27) and the ammonium release to the external medium by MSX-treated cells (26) also support a possible catabolic role of the GDH in P. laminosum. ACKNOWLEDGMENTS This work was partially supported by grants from the Comisi6n Asesora de Investigaci6n Cientffica y Tecnica (grant 928/84) and the Basque Country Government (grant X-86. 049). M.M.B. was the recipient of a scholarship (PFPI) from the Spanish Ministry of Education and Science. LITERATURE CITED 1. Blumenthal, K. M., and E. L. Smith. 1973. Nicotinamide ade2. 3. 4.

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nine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. J. Biol. Chem. 248:6002-6008. Botton, B., and Y. Msatef. 1983. Purification and properties of the NADP-dependent glutamate dehydrogenase from Sphaerostilbe repens. Physiol. Plant. 59:438 444. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. Camardella, L., G. Di Prisco, F. Garofano, and A. M. Guerrini. 1976. Purification and properties of a NADP-dependent glutamate dehydrogenase from yeast nuclear fractions. Biochim. Biophys. Acta 429:324-330. Cammaerst, D., and M. Jacobs. 1985. A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana. Planta 163:517-526. Castenholz, R. W. 1970. Laboratory culture of thermophilic cyanophytes. Schewiz. Z. Hydrol. 32:538-551. Dharmawardene, M. W. N., A. Haystead, and W. D. P. Stewart. 1973. Glutamine synthetase of the nitrogen-fixing alga Anabaena cylindrica. Arch. Mikrobiol. 90:281-295. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404427. Fayyaz-Chaudhary, M., A. C. Cannons, and M. J. Merret. 1984. Photoregulation of NADPH-glutamate dehydrogenase in regreening cultures of Euglena gracilis. Plant Sci. Lett. 34:89-94. Florencio, F. J., S. Marques, and P. Candau. 1987. Identification and characterization of a glutamate dehydrogenase in the unicellular cyanobacterium Synechocystis PCC 6803. FEBS Lett. 223:37-41. Gayler, K. R., and W. R. Morgan. 1976. An NADP-dependent glutamate dehydrogenase in chloroplasts from the marine green alga Caulerpa simpliciuscula. Plant Physiol. 58:283-287. Guerrier, G., F. Beaujard, and J. D. Vienont. 1985. Influence of in vivo nutrient concentration in Erica darleyensis nitroplant transfer in perlite media. 2. Relations between free nitrogenous form (NO2-, N03-, NH4+) and nitrate reductase or glutamate dehydrogenase activities. Plant Soil 84:337-345.

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13. Hemmings, B. A., and A. P. Sims. 1977. The regulation of glutamate metabolism in Candida utilis. Evidence for two interconvertible forms of NADP-dependent glutamate dehydrogenase. J. Biochem. 80:143-151. 14. Hernandez, G., R. Sanchez-Pescador, R. Palacios, and J. Mora. 1983. Nitrogen source regulates glutamate dehydrogenase NADP synthesis in Neurospora crassa. J. Bacteriol. 154:524528. 15. Hoare, D. S., S. L. Hoare, and R. B. Moore. 1967. The photoassimilation of organic compounds by autotrophic bluegreen algae. J. Gen. Microbiol. 49:351-370. 16. Hooper, A. B., J. Hansen, and R. Bell. 1967. Characterization of glutamate dehydrogenase from the ammonium-oxidizing chemoautotroph Nitrosomonas europea. J. Biol. Chem. 242:288296. 17. Hornby, D. P., and P. C. Engel. 1984. Characterization of Peptostreptococcus asaccharolyticus glutamate dehydrogenase purified by dye-ligand chromatography. J. Gen. Microbiol. 130: 2385-2394. 18. Israel, D. W., R. M. Gronostajski, A. T. Yeung, and R. R. Schmidt. 1977. Regulation of accumulation and turnover of an inducible glutamate dehydrogenase in synchronous cultures of Chlorella. J. Bacteriol. 130:793-804. 19. Kane, J. F., J. Wakim, and R. S. Fischer. 1981. Regulation of glutamate dehydrogenase in Bacillus subtilis. J. Bacteriol. 148: 1002-1005. 20. Kindt, R., E. Pahlich, and I. Rasched. 1980. Glutamate dehydrogenase from pea: isolation, quaternary structure and influence of cations on activity. Eur. J. Biochem. 112:533-540. 21. Kumar, S., and D. J. D. Nicholas. 1984. NAD+- and NADP+dependent glutamate dehydrogenases in Nitrobacter agilis. J. Gen. Microbiol. 130:967-973. 22. Laurirre, C., and J. Daussant. 1983. Identification of the ammonium-dependent isoenzyme of glutamate dehydrogenase as the form induced by senescence or darkness stress in the first leaf of wheat. Physiol. Plant. 58:89-92. 23. Loyola-Vargas, V. M., and E. Sanchez de Jimenez. 1984. Differential role of glutamate dehydrogenase in nitrogen metabolism of maize tissues. Plant Physiol. 76:536-540. 24. Male, K. B., and R. B. Storey. 1983. Kinetic characterization of NADP-specific glutamate dehydrogenase from the sea anemone Anthopleura xanthogrammica: control of amino acid biosynthesis during osmotic stress. Comp. Biochem. Physiol. 76:823-829. 25. Martin, F., Y. Msatef, and B. Botton. 1983. Nitrogen assimilation in mycorrhizas. I. Purification and properties of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of the ectomycorrhizal fungus Cenococcum graniforme. New Phytol. 93:415-422. 26. Martinez, A., A. Alaiia, M. J. Llama, and J. L. Serra. 1987. Sustained photoproduction of ammonia from nitrate or nitrite by free and immobilized cells of Phormidium laminosum, p. 220222. In W. R. Ullrich, P. J. Syrett, F. Castillo, and P. J. Aparicio (ed.), Inorganic nitrogen metabolism. Springer-Verlag KG., Berlin. 27. Martinez-Bilbao, M., A. Alana, J. M. Arizmendi, and J. L. Serra. 1987. Inorganic nitrogen assimilation in the non N2-fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH-dependent glutamate dehydrogenase. Physiol. Plant. 70:697-702. 28. Maulik, P., and S. Ghosh. 1986. NADPH/NADH-dependent cold-labile glutamate dehydrogenase in Azospirillum brasilense. Eur. J. Biochem. 155:595-602. 29. Meeks, J. C., C. P. Wolk, W. Lockau, N. Schilling, W. Shaffer, and W. S. Chien. 1978. Pathways of assimilation of ['3N]N2 and 13NH4' by cyanobacteria with and without heterocysts. J. Bacteriol. 134:125-130. 30. Meeks, J. C., C. P. Wolk, J. Thomas, W. Lockau, P. W. Shaffer, S. M. Austin, W. S. Chien, and A. Galonsky. 1977. The pathways of assimilation of 13NH4' by the cyanobacterium Anabaena cylindrica. J. Biol. Chem. 252:7894-7900. 31. Miffin, B., and P. Lea. 1976. The pathway of nitrogen assimilation in plants. Phytochemistry 15:873-885. 32. Molin, W. T., T. P. Cunningham, N. F. Bascomb, L. H. White,

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