Some Properties of Glutamate Dehydrogenase from the Marine Red Alga Gracilaria sordida ( Harv . )

Glutamate dehydrogenases (GDH, EC 1.4.1.2-4) catalyse the entry of ammonium into the organic cycle via amination of α-ketoglutarate and its release via deamination of Lglutamate. NAD-GDH facilitates NAD-/NADP-dependent deamination while NADPH-GDH facilitates NADH-/NADPH-dependent amination reactions. In the study reported here, GDHs were extracted from the red alga Gracilaria sordida and purified 10to 180-fold before examining their amination and deamination reaction properties. NAD-/NADPand NADH-/ NADPH-dependent activities were the order of 11:1 and 1:1.8, respectively. The pH optima for amination and deamination were 8.2 and 8.4 under NADHand NADPHand 8.4 and 9 under NADand NADPdependent activities, respectively. Whereas both NADand NADP-dependent deamination activities were activated by calcium ions (Ca ), only NADPH-dependent amination was activated. The K m values (in mM) were 3.0–3.6 for ammonia, 2.0–3.3 for α-ketoglutarate, 0.00286 for NADH, 0.0033 for NADPH),1.7–2.1 for L-glutamate, 0.344 for NAD and 0.476 for NADP. It appears that the GDHs in G. sordida are dominated by NAD-dependent deamination enzyme and that in NADH-NADPHdependent amination reactions, NADPH is more preferred. It is suggested that NAD-GDH’s role in G. sordida could be as a catabolic shunt facilitating respiration. Anabolic functions could be assimilation of ammonia released during photorespiration and synthesis of N-rich transport compounds.


INTRODUCTION
The inorganic nitrogen acquired by plants is converted to ammonia before being assimilated into organic molecules.Amination of αketoglutarate to glutamic acid is the major route of ammonia assimilation in plants.This reversible reaction may be catalysed by (1) glutamate dehydrogenases (GDH) and (2) glutamine synthetase (GS)/ glutamate synthase (GOGAT).In the GS/ GOGAT route, ammonia is first incorporated into glutamine by the action of GS and subsequently into glutamic acid by GOGAT.The GS/ GOGAT route is a dominant pathway in higher plants (Lea & Miflin, 2003).The major hindrance to the GDH involvement in the mentioned organisms is low affinity to ammonia.The reported K m (ammonia) for GDH are generally higher than 5 mM while tissue concentration of ammonia is in the range close to 1.0 mM.At about 2 mM ammonia concentration, chloroplasts are uncoupled (Lea & Miflin, 2003).
In this paper, some properties of GDH from the marine red macroalga Gracilaria sordida are reported.This information contributes to the knowledge base of amination and deamination pathways in algae.

Materials
Gracilaria sordida (Harv.)W. Nelson was collected in Hanukau Harbour, New Zealand, in December 1983 and maintained in monoculture in cylinder systems as detailed in Lignell et al. (1987).Its growth rate per day during the pre-cultivation period was around 25% in natural seawater with salinity of 33‰, NH 4 Cl content of 820 µM, NaH 2 PO 4 of 30 µM, inorganic carbon 2.5 mM, pH 7.8 ± 0.1, temperature 26 ± 0.5 ºC, and light intensity of 800 µmole photons/m 2 /s.Seawater was changed every 2 weeks.For enzyme extraction, epiphyte-free samples were used after rinsing with distilled water and mild centrifugation to get rid of excess salts and water, respectively.All the chemicals used were purchased from Sigma Chemicals Co.

Extraction and partial purification
Two hundred and two grammes (202 g) of the algal sample were pulverised in liquid nitrogen using a mortar and pestle.All the procedures thereafter were carried out at 0-5 ºC unless otherwise stated.Extraction was effected in 200 mM Tris-HCl (pH 8.0) containing 14 mM mercaptoethanol and 5% (w/v) polyvinylpyrrolidone (PVP) for 30 minutes.The turbid red extract was filtered through four layers of cheesecloth and centrifuged at 10,000 g for 30 minutes.

Purification by ammonium sulphate
Saturated ammonia sulphate solution was added to the supernatant to give a final concentration of 40%, the suspension was stirred for 30 minutes and centrifuged at 20,000 g for 30 minutes.The supernatant was adjusted to 60% saturation with ammonia sulphate solution.After stirring for 30 minutes the precipitate was removed by centrifuging at 25,000 g for 30 minutes.As the precipitate had some enzyme activity, it was homogenised in the extraction buffer (without PVP) and re-extracted for 30 minutes while adjusted at 60% saturation with ammonia sulphate solution and treated as above to obtain the supernatant.The combined supernatant was finally adjusted to 75% saturation with ammonia sulphate solution, stirred for 30 minutes and the precipitate was obtained by centrifuging at 25,000 g for an hour.The precipitate was dissolved in the extraction buffer (without PVP) and dialysed in 10 mM Tris-HCl (pH 7.4) containing 14 mM mercaptoethanol for 12 hours and used for enzyme analysis.

Purification by affinity precipitation and filtration by Centricon 30
Affinity precipitation was achieved by using N 2 N 2adipodihydrazido-bis-(N 6 -carbonylmethyl-NAD) (Bis-NAD) as detailed in Flygare et al. (1983).The resultant precipitate was dissolved by addition of 0.5 ml of 5 mM NADH and 1 ml of 20 mM αketoglutarate.Centriprep 10 (with molecular weight cutoff point (MWCO) of 10,000) was used to remove Bis NAD and excess NADH and glutarate.Finally filtration using Centricon 30 (MWCO 100,000) was used following a native gel, which showed a band near 100,000.

Enzyme assay
The activity of the extracted GDH was determined spectrophotometrically by measuring at 340 nm (using HITACHI model U-2000 double beam spectrophotometer) the initial rate of oxidation of NADH or NADPH or reduction of NAD or NADP at 30 ºC.Before the assay, reaction mixtures were maintained at 30 ºC for 10 minutes.The standard amination reaction medium contained 200 mM Tris-HCl (pH 8.2 for NADH-and 8.4 for NADPHmediated reactions, respectively), 50 mM ammonium chloride, 4 mM α-ketoglutarate and 18 mM NADH or 35 µM NADPH.The deamination reaction medium used the same buffer concentration (pH 8.4 for NAD and 9.0 for NADP mediated reactions, respectively), 50 mM L-glutamate, 40 µM calcium ions (Ca 2+ ) and 6 mM NAD or NADP.The coenzymes were adjusted to pH 7 before use.All other substrates were prepared in assay buffer.Substrate saturation activities were assayed by varying the individual substrate and the initial velocities were recorded.Blank controls had individual substrates omitted.In assessing the effect of pH on enzyme activity, assay mixtures were prepared using 200 mM Tris-HCl regulated to the pH of interest.
The effect of 140 µM dimethylsulfoxide (DMSO) was used to establish the absence of phenols in the crude extract.Specific enzyme activity was expressed in units per mg protein.One unit is defined as the amount of enzyme required to form 1 µmole of co-enzyme (NADH/NADPH, NAD/NADP, respectively) per minute at 30 ºC.Protein content in the extracts was determined according to Peterson (1983).Assays were repeated at least twice and their mean results were recorded.

GDH co-enzyme specificity
The enzyme partially purified by Bis NAD precipitation and filtration (MWCO 100,000) exhibited NADH-, NADPH-, NAD-and NADPdependent activities with specific activities of 55.511 and 102.559 for the amination and 5625.499 and 490.389 for the deamination, respectively (Table 1).The activities with NAD as co-enzyme were in the order of 11.4-fold higher in the deamination and that of NADH was 1.8-fold lower in the amination.The crude extract also exhibited NADH-, NADPH-, NAD-and NADP-dependent activities.However, NAD-dependent specific activity was 33% lower than NADP-dependent activity, while NADPH-dependent activity was 4.4 higher than NADH-dependent activity.
The GDH was partially purified as the NADH, NADPH-, NAD-and NADP-dependent specific activity for the crude extract and the dialysed Bis-NAD product were about 120, 50, 180 and 10-fold purified, respectively.Enzyme recovery in the purification process was about 120, 50, 190 and 10% for the NADH-, NADPH-, NAD-and NADPdependent enzymes, indicating that the crude extract contained some inhibitory factors for NADH-and NAD-dependent activities.

Effects of calcium ions on the GDH activity
The relationship between calcium ion concentration and NADH-, NADPH-, NAD-and NADP-influenced activity of GDH were examined (Fig. 1).In the presence of 50 µM Ca 2+ the NADPHdependent activity maximally increased by 2.4-fold and any further increase of the concentration decreased the activity.The NADH-dependent activity gradually decreased with an increase in Ca 2+ concentration (Fig 1a).
In the deamination reaction, at 40 µM Ca 2+ the NAD-dependent activity was increased 3-fold while further increase in the concentration decreased the activity.The NADP-dependent activity was increased 1.3-fold at 25 µM Ca 2+ , but unlike NAD-GDH, further increase in Ca 2+ was accompanied by an insignificant decrease until the Ca 2+ was above 75 mM (Fig. 1b).

Optimal pH
Amination for NADH-mediated activity was maximal at pH 8.1 and for NADPH-mediated activity it was maximal at pH 8.4 (Fig. 2a).Towards the acidic values the profiles of both curves did not decline as steeply as towards the basic pH.Maximum deamination NADPdependent activity was at pH 9.0.Towards both acidic and basic pH values, the decline of the activities were steep.NAD-dependent deamination  activity was maximal at pH 8.4, but there was another small peak of activity at pH 9.4.Between the pH 8.4 and 9.4 peaks, the decline of activity was as steep as away from them.

Substrate saturation kinetics
The substrate saturation curves were analysed by Lineweaver and Burk plots and K m values are presented in Table 2 and Fig. 3.All the K m s were  below 3.6 mM.The affinity for glutamate (as shown by K m 1.670-2.130)were higher than ammonia (Km 3.077-3.610)and it was NADdependent activity that had the highest affinity for glutamate (1.670 mM).In amination reactions, NADPH-dependent activity was (K m 3.077) higher than NADH-dependent activity (K m 3.610).

Effect of the products of the reaction on the catalysis velocity
L-Glutamate, NAD and NADP were added to the assay mixture during amination, and ammonia, αketoglutarate, NADH and NADPH during deamination.The deamination products were more inhibitory to the catabolic reaction than the amination products were to the anobolic reaction (Fig. 4).Whereas the catabolic activities could not be detected in the presence of less than 1mM of its products, anabolic activities were even at >1 mM of its products.

DISCUSSION
Terrestrial plant glutamate dehydrogenases (GDH) may be mitochondrial (for deamination) or chloroplastic (for amination or amino acid synthesis) with NAD-GDH and NADPH-GDH dominating in the first and second organelle, respectively.Both enzymes have dual co-enzyme specificity to some extent.In the present study, both GDHs appear to be present in the crude extract and the semi-purified product (Table 1).In another red alga Porphyra yezoensis, NADPH-GDH was detected but NADH-GDH was not, and the extracted NADPH-GDH had an insignificant NADH-dependent activity (Sato et al., 1984).The NAD-/NADP-and NADH-/NADPH-dependent activities reported in this study for the crude extract and the Bis-NAD product (in parentheses) were of the order of 1:1.5 (11:1) and 1:4.4 (1:1.8),respectively.
The shown NADH-/NADPH-dependent activities differ significantly from those reported for the green alga Caulerpa simpliciuscula where NADH-/NADPH-dependent activities in the crude extract and the purified enzyme were 1:9 and 1:75 respectively (Gayler & Morgan, 1976).The pH optima for amination and deamination in this study were 8.2 and 8.4 under NADH-and NADPH-and 8.4 and 9 under NAD-and NADP-dependent activities, respectively.Although the shown optimum pH for NADP-dependent activity is closer to that of Porphyra yezoensis GDH (8.8), that for NADPH-dependent activity is much higher than that for Porphyra (pH 7.7) (Sato et al., 1984).It is possible that NAD-, NADP-, NADH-and NADPH-dependent activities vary with species and/or growth conditions.Growth in high ammonium or low nitrate media caused a large de novo synthesis of NADPH-dependent GDH in Chlorella (Ahmad & Hallebust, 1984;Bascomb et al., 1987;Bascomb & Schmidt, 1987).In the present study, NAD-GDH dominated despite growing G. sordida in ammonia.
The studies reported here have shown that both NAD-and NADP-dependent deamination activities were activated by calcium ions (Ca 2+ ).In the amination reactions, only the NADPHdependent reaction was activated.This is contrary to what has been reported from some terrestrial plant GDHs (Chou & Splittstoesser, 1972;Joy, 1973;Furuhashi & Takahashi, 1982) where amination but not deamination reaction appears to be activated by Ca 2+ .
A comparison of the affinity for substrates of previously characterised plant GDHs with the enzyme purified from G. sordida shows that the enzyme in the present study does not have an apparent affinity for ammonium ions as does that from green alga Caulerpa simpliciuscula (K m 0.4-0.7,Gayler & Morgan, 1976) and red alga Porphyra yezoensis (Sato et al,. 1984).The K m for ammonia reported in this study shows that GDH in G. sordida could be NAD-specific with a catabolic role as reported in higher plants (Lea & Miflin, 2003 and references therein).Its anabolic role, e.g.assimilation of ammonia released during photorespiration and synthesis of N-rich transport compounds (Lea & Miflin,203) is not ruled out if ammonia concentration in mitochondria is in the range of 5-10 µM (Yamaya et al., 1984).Its molecular weight is so far not known, however its retention in a Cetricon 100 shows that its molecular weight is equal to or above 100,000.In Chlorella sorokiniana, mitochondrial and chloroplastic GDHs have been shown to be tetrameric and hexameric, respectively.Whereas the mitochondrial GDH had identical subunits with MW 45,000 each, the chloroplastic one had αand βisoenzymes with subunit MW of 55,500 and 53,000, respectively (Bascomb et al., 1987;Bascomb & Schmidt, 1987;Meredith et al., 1978).

CONCLUSION
The glutamate dehydrogenases (GDHs) in Gracilaria sordida have an affinity for ammonia (Km 3.0-3.6).Establishing ammonia concentrations in algal mitochondria and chloroplasts is crucial to elucidating the role of GDH in the alga.However, assuming that ammonia concentration in G. sordida chloroplasts is in the same ranges as in higher plants (Lea & Miflin, 2003), it is possible that GDHs in G. sordida play an insignificant role in glutamate formation.Their role could be that of facilitating a catabolic shunt in mitochondria if ammonia concentrations are similar to what is reported in corn shoot (Yamaya et al., 1984).Anabolic functions, e.g. the assimilation of ammonia released during photorespiration and synthesis of N-rich transport compounds are also possibilities (Lea & Miflin, 2003).

Fig. 1 .
Fig. 1.Mean calcium ions (Ca 2+ ) activity profiles for NADPH-GDH (A) and NAD-GDH (B) from G. sordida as influenced by NADPH, NADH, NADP and NADP, respectively.Filled square, circle and triangle or their respective open symbols at each point along the profiles represent results for the three assays; mean values were used to generate the profiles.Standard amination and deamination reaction mixtures were used except for the [Ca 2+ ], which were varied as shown in the figures

Fig. 2 .Fig. 3 .
Fig. 2. Mean pH activity profiles for NADPH-GDH (a) and NAD-GDH (b) from Gracilaria sordida as influenced by NADPH, NADH, NADP and NADP, respectively.Filled square, circle and triangle or their respective open symbols at each point along the profiles represent results for the three assays; mean values were used to generate the profile.Standard amination and deamination reaction mixtures were used except for the pH, which was varied as shown in the figures.

Fig
Fig. 4. Amination and deamination product activity profile of NADPH-GDH (A1-A2) and NAD-GDH (D1-D3).During deamination the influence of different concentrations of ammonia (NH4), α α α α α-ketoglutarate (KG), NADH (NADH) and NADPH (NADPH) were measured when GDH was influenced by NAD (NAD-INF) or NADP (NADP-INF).In each case, respective standard amination or deamination reaction mixtures were used in the presence of the shown product concentration.Filled square, circle and triangle or their respective open symbols at each point along the profiles represent results for the three assays; mean values were used to generate the profiles

Table 1 . Purification of glutamate dehydrogenase: Shown values of protein concentration and enzyme activity are mean values of at least two determinations. The degree of purification was calculated as a ratio of the specific activities (units of enzyme activity/mg protein) before and after the purification procedures shown. Percentage recovery of the enzyme activities was corrected for the actual proportion of the initial tissue extract used in each purification step
TA, total activity; SA, specific activity; OP, overall purification (fold); OR, overall recovery.+ Bis-NAD = N 2 N 2 -adipodihydrazido-bis-(N 6 -carbonylmenthl-NAD).

Table 2 . Michaelis Menten constants for substrates of the enzyme GDH from Gracilaria sordida in its amination and deamination reactions under NADH/NADPH and NAD/NADP respectively. The constants were determined as shown for L-glutamate in Fig. 3.
+ at 50 mM Ca 2+ .