Purification and Characterization of Glutamine Synthetase NADP

Plant Physiol. (1992) 99, 938-9440032-0889/92/99/0938/07/$01 .00/0

Received for publication October 29, 1991Accepted January 23, 1992

Purification and Characterization of Glutamine Synthetaseand NADP-Glutamate Dehydrogenase from the

Ectomycorrhizal Fungus Laccaria laccata'

Annick Brun, Michel Chalot*, Bernard Botton, and Francis MartinLaboratoire de Physiologie Vegetale et Forestiere, Universite de Nancy B.P. 239, 54506 Vandoeuvre-les-Nancy

Cedex, France (A.B., M.C., B.B.); and Laboratoire de Microbiologie Forestiere, Centre de Recherches Forestieres deNancy, Institut National de la Recherche Agronomique, Champenoux, 54280 Seichamps, France (F.M.)

ABSTRACT

Glutamine synthetase (GS) and NADP-dependent glutamate de-hydrogenase (NADP-GDH) play a key role in nitrogen assimilationin the ectomycorrhizal fungus Laccaria laccata (Scop. ex Fr. Cke)strain S 238. The two enzymes were purified to apparent electro-phoretic homogeneity by a three-step procedure involving dieth-ylaminoethyl (DEAE)-Trisacryl and affinity chromatography, andDEAE-5PW fast protein liquid chromatography. This purificationscheme resulted in a 23 and 62% recovery of the initial activity forGS and NADP-GDH, respectively. Purified GS had a specific activ-ity of 713 nanomoles per second per milligram protein and a pHoptimum of 7.2. Michaelis constants (millimolar) for the substrateswere NH4' (0.024), glutamate (3.2), glutamine (30), ATP (0.18),and ADP (0.002). The molecular weight (Mr) of native GS wasapproximately 380,000; it was composed of eight identical subunitsof Mr 42,000. Purified NADP-GDH had a specific activity of 4130nanomoles per second per milligram protein and a pH optimum of7.2 (amination reaction). Michaelis constants (millimolar) for thesubstrates were NH4' (5), 2-oxoglutarate (1), glutamate (26),NADPH (0.01), and NADP (0.03). Native NADP-GDH was a hex-amer with a Mr of about 298,000 composed of identical subunitswith M, 47,000. Polyclonal antibodies were produced against pu-rified GS and NADP-GDH. Immunoprecipitation tests and immu-noblot analysis showed the high reactivity and specificity of theimmune sera against the purified enzymes.

on regulation, physical properties, and kinetic characteristicsof GS and NADP-GDH is derived from studies on higherplants (20, 21, 26), yeasts (7, 25), Neurospora crassa (15, 22,26), and Aspergillus nidulans (13). Conversely, GS and, to alesser extent, NADP-GDH have not been extensively char-acterized in ectomycorrhizal fungi. Only their activity infungal mycelia and ectomycorrhizas has been studied (1, 8,10, 28), and their role in NH4' assimilation by ectomycorrhi-zal fungi and ectomycorrhizas investigated (9, 11, 19). In-deed, the activity and amount of fungal NADP-GDH poly-peptide were strongly suppressed in beech associations (10,19). In contrast, evidence from enzyme activities, electropho-retic patterns, immunocytochemical labeling, and '5N exper-iments consistently showed that fungal NADP-GDH wasinvolved in N-assimilation by spruce ectomycorrhizas (9, 10).Thus, the NH4' assimilation pathways (GDH/GS versus GS/glutamate synthase) appear to differ between mycorrhizalroots of spruce and beech. However, there is still much tolearn about N assimilation in ectomycorrhizas and its regu-lation, particularly in relation to enzyme compartmentation.As a prerequisite to detailed studies on the regulation offungal enzymes and their localization in symbiotic tissues,we report here the purification of GS and NADP-GDH fromthe ectomycorrhizal fungus Laccaria laccata (Scop. ex Fr. Cke)S 238 and present some physical, kinetic, and immunologicalproperties of the purified enzymes.

GS2 (L-glutamate:ammonia ligase, ADP forming, EC6.3.1.2) is the major enzyme for ammonia assimilation inmost higher plant tissues (2, 21, 26). In fungi, primary Nassimilation is brought about by successive activity of NADP-GDH (EC 1.4.1.4) and GS (15, 17, 22). Abundant information

'This work was financially supported by a research grant fromthe Institut National de la Recherche Agronomique (INRA, Actionincitative programm&e Recherches des mecanismes de regulation dumetabolisme des associations mycorhiziennes) to B.B., a scholarshipfrom the INRA and the Centre National Interprofessionnel de l'Hor-ticulture to M.C., and a scholarship from the Ministere de la Re-cherche et de la Technologie to A.B.

2Abbreviations: GS, glutamine synthetase; NADP-GDH, NADP-dependent glutamate dehydrogenase; FPLC, fast protein liquidchromatography; PVDF, polyvinylidene difluoride; IgG, immuno-globulin G.

MATERIALS AND METHODS

Chemicals

All chemicals were purchased from Sigma Chemical Co.and Prolabo. DEAE-Trisacryl, Blue-Sepharose CL-6B, 2',5'-ADP-Sepharose 4B, Sephacryl S-300 HR, PAA (4-30%) pre-cast polyacrylamide gels, Multiphor II and Novablot units,and calibration kits (gel filtration, SDS-PAGE, and gradient-PAGE) were obtained from Pharmacia LKB Biotechnology,Inc. Coomassie Protein Assay Reagent was supplied by PierceChemical Co. The Protein Pak DEAE-5PW column, FPLCsystem and Immobilon PVDF Transfer Membrane were pur-chased from Waters Chromatography Division, MilliporeCorp. The Mini-Protean II apparatus and prestained proteinstandards were obtained from Bio-Rad.

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PURIFICATION OF GLUTAMINE SYNTHETASE AND NADP-GLUTAMATE DEHYDROGENASE

Fungal Strain and Growth Conditions

Laccaria laccata (Scop. ex Fr. Cke) isolate S 238 was main-tained in the collection of ectomycorrhizal fungi at the Lab-oratory of Forest Microbiology (Institut National de la Re-cherche Agronomique, Centre de Recherches Forestieres).The culture was subcultured monthly on 20 mL of Pach-lewski's medium (pH 5.7) containing 25 mm (NH4)2SO4, 110mm glucose, 28 mm maltose, 7 mm KH2PO4, 2.7 mm MgSO4,micronutrients, and 2% (w/v) agar as previously described(18). For large-scale purification, mycelia were cultivated in250-mL Erlenmeyer flasks containing 125 mL of liquid Pach-lewski's medium with 25 mt KNO3 as the sole nitrogensource. Mycelia were grown for 15 d at 240C on a rotaryshaker (125 rpm), harvested in the middle of the rapid phaseof growth, washed with distilled water, and stored at -750Cuntil used for enzyme extraction. These conditions of storagedid not affect the enzyme activities.

Enzyme and Protein Assays

GS activity was determined by a modification of the trans-ferase assay of Shapiro and Stadtman (24). Reactions wereperformed in a final volume of 1 mL containing 50 mm Tris-HCl (pH 7.2), 125 mm Gln, 30 mm NH20H, 20 mM arsenate,4 mm EDTA, 20 mM MgSO4, 0.5 mm ADP, and an appropriateamount of enzyme. The reaction was initiated by the additionof ADP and incubated at 300C for 30 min. The reaction wasstopped by the addition of 1 mL of ferric chloride reagent(0.37 M FeCl3, 0.20 M TCA, and 0.67 N HCl). ADP or Glnwere omitted from controls. The absorbance at 540 nm wasmeasured after 20 min. The spectrophotometric (physiologi-cal) assay and the biosynthetic (nonphysiological) assay de-scribed by Winter et al. (29) were used to determine the Kmvalues of GS for NH4', Glu, and ATP. NADP-GDH wasassayed by reductive amination of 2-oxoglutarate. Extractswere incubated at 300C in 1.15-mL reaction mixtures con-taining 100 mm K-phosphate buffer (pH 7.2), 8.7 mm 2-oxoglutarate, 122 mm (NH4)2SO4, and 0.18 mm NADPH.NADP-GDH activity was determined by following the oxi-dation of NADPH at 340 nm. Controls were conducted withassays lacking 2-oxoglutarate or NH4'. The enzyme prepa-rations were routinely diluted during purification to ensurethat the activity determinations were linear with respect totime. Protein content was determined by a modification ofthe Bradford method (6), using BSA as a standard.

Purification of Enzymes

GS

All purification steps were carried out at 40C except FPLC,which was performed at room temperature. Approximately25 g of mycelium was ground in a chilled mortar and pestlewith 30 volumes of 0.05 M Tris-HCl (pH 7.6) containing 5mM MgSO4, 10% (v/v) glycerol, 2% (w/v) PVP-40, 10% (w/w) insoluble PVP, 2 mm EDTA, 10 mm Glu, and 14 mm 2-mercaptoethanol. The homogenate was filtered through twolayers of cheesecloth and centrifuged at 40,000g for 30 min.The resulting pellet was resuspended and homogenized inan additional 200 mL of extraction buffer and centrifuged as

before. The resulting supematant fractions were pooled andfiltered on a 0.45-um filter (Millipore) and used for enzymepurification. The crude filtrate was applied to a DEAE-Tri-sacryl column (2 x 20 cm) that was preequilibrated withbuffer A (0.05 M Tris-HCl, pH 7.6, 5 mM MgSO4, 10% [v/v]glycerol, 2 mi EDTA, 14 mm 2-mercaptoethanol, and 10 IMGlu). The column was washed with buffer A until A280 of theeluate was close to zero. The enzyme was eluted by a 300-mL linear NaCl gradient (0-0.3 M) and fractions of approxi-mately 2.5 mL were collected at a flow rate of 1.5 mL min-'.Desalting proved unnecessary for subsequent affinity chro-matography. Fractions containing the highest GS activitywere pooled and applied to a Blue-Sepharose CL-6B column(1 x 8 cm) that had been preequilibrated with buffer A. Theenzyme, which is retained under these conditions, was elutedwith 10 mL of 0.5 mm ADP in buffer A at a flow rate of 0.2mL min-'. Fractions containing GS activity were concentratedby ultrafiltration (Amicon PM 10 membrane) to approxi-mately 1 mL and applied to a FPLC-based Protein Pak DEAE-5PW column (0.8 x 7.5 cm) that had been preequilibratedwith buffer A. After injection, the column was washed withbuffer A and the enzyme activity was eluted by a 60-mLlinear NaCl gradient (0-0.1 M) at a flow rate of 1.5 mL min-'.Pooled GS activity fractions were divided into 0.2-mL por-tions, and stored at -750C until used for enzyme character-ization. No loss in activity was seen over a period of 3 monthswhen the enzyme was stored in this manner.

NADP-GDH

Approximately 25 g of mycelium was ground in a mortarand pestle with 20 volumes of 0.05 M Tris-HCl (pH 7.6)containing 10% (v/v) glycerol, 2% (w/v) PVP-40, and 14 mm2-mercaptoethanol. Purification and storage of NADP-GDHwas then performed as described for GS except that buffer B(0.05 M Tris-HCl, pH 7.6, 10% [v/v] glycerol, and 14 mm 2-mercaptoethanol) was used instead of buffer A throughoutthe purification steps. Blue-Sepharose CL-6B affinity chro-matography was replaced by chromotography with a 2',5'-ADP-Sepharose 4B column (1 x 8 cm) preequilibrated withbuffer B, and the elution of NADP-GDH was performed witha linear gradient of NADPH (0-0.25 mM) at 0.2 mL min-'.

Electrophoresis

Nondenaturing PAGE

Uniform-PAGE (6% resolving gels) was carried out in aMini-Protean II unit for 1 h at 200 V as described elsewhere(4). Gradient-PAGE was performed by using polyacrylamide(4-30%) precast gels according to Botton and Msatef (5).Electrophoresis was run for approximately 16 h at 120 V.Gels were stained either for protein using Coomassie blue R-250 or for enzyme activity. Location of GS activity wasdetermined immediately after electrophoresis by bathing thegels for 30 min at 300C in the transferase assay mixture andsubsequently placing them in the ferric chloride reagentaccording to Winter et al. (29). The red-brown color of theferric hydroxamate developed where GS activity was present.GS activity was also located by incubating the gel in the

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biosynthetic assay mixture containing 50 mi CaCl2 at 30°Cuntil a stable band of insoluble CaHPO4 appeared. Stainingfor NADP-GDH activity involved the tetrazolium assay sys-tem (3) to follow the deamination of L-Glu. Gels were incu-bated in a medium containing 30 mM L-Glu, 0.35 mtm NADP,25 mn-m nitroblue tetrazolium, and 12.5 mm phenazine meth-osulfate in 100 mm Tris-HCl (pH 7.8). NADP-GDH bandswere visualized as dark blue zones produced by formazanformation.

SDS-PAGE

SDS-PAGE (15% resolving gels) was performed in a Mini-Protean II electrophoresis unit at 200 V for about 90 minaccording to Laemmli (14), and gels were silver stained asdescribed elsewhere (4).

Mol Wt Determinations

The native Mr was determined on uniform-polyacrylamidegels at various concentrations of acrylamide (6, 7.5, 9, and10.5% T) or with gradient-PAGE as described above. Thenative Mr estimation on uniform-polyacrylamide gels wasderived from a series of Ferguson plots (logio RF versus gelconcentration) and a second plot of retardation coefficientversus the molecular mass of native proteins. Size estimationon gradient-PAGE was performed according to a methoddescribed elsewhere (4, 5). Uniform- and gradient-gels werecalibrated with protein standards of known Mr (thyroglobu-lin, 669,000; ferritin, 440,000; catalase, 232,000; lactate de-hydrogenase, 140,000; BSA, 67,000). The native Mr was alsodetermined by gel filtration on a HiLoad Sephacryl S-300High Resolution column (1.6 x 60 cm) using the WatersFPLC system. The column was equilibrated in 100 mi Tris(pH 7.6) containing 10% (v/v) glycerol and 100 mm NaCl.Blue Dextran 2000 was used to mark the front of elution.Samples (0.5-1 mL) were loaded onto the column first cali-brated with high Mr markers (thyroglobulin, 669,000; ferritin,440,000; catalase, 232,000; aldolase, 158,000; BSA, 67,000).Fractions (1 mL) were collected at a flow rate of 0.3 mL min-'and assayed for A280 or enzyme activity. The native Mr of theenzymes was derived from a plot of partition coefficientversus log Mr. Molecular mass estimations of the subunitswere carried out by SDS-PAGE as described above usingstandards of known Mr (phosphorylase b, 94,000; BSA,67,000; ovalbumin 43,000; carbonic anhydrase, 30,000; soy-bean trypsin inhibitor, 20,100; a-lactalbumin, 14,400). Sizeestimation of the monomer was performed according to amethod described elsewhere (4).

Generation of Polyclonal Antisera

To elicit polyclonal antibodies, enzymes of the pooledDEAE-5PW samples were further purified by preparative-PAGE as previously described (5). Two Fauve de Bourgognerabbits were immunized by two subcutaneous primary injec-tions. One injection (500 ,uL emulsion) was composed of 2501uL of polyacrylamide gel suspension containing the activeenzymes (400 and 200 ,ug for GS and NADP-GDH, respec-tively) and 250 MAL of Freund's complete adjuvant. One

intravenous booster injection, consisting of 200 and 100 Mugof protein for GS and NADP-GDH, respectively, was given1 month later and a second 2 weeks later. Bleedings weretaken from the marginal ear vein 15 d after the last injection.The blood was kept for 12 h at 40C, then the immune serumwas separated by centrifugation. The IgG fraction was pre-cipitated by ammonium sulfate (35% saturation) and col-lected by centrifugation, dissolved in 50 mm borate buffer(pH 8.1) containing 0.9% (w/v) NaCl, then dialyzed againstthe same buffer. This preparation was sterilized by filtrationon a Millipore membrane and kept frozen at -750C untiluse.

Immunoblotting

After SDS-PAGE, the gel was incubated for about 10 minin 48 mm Tris-HCl (pH 8.3), 39 mm glycine, 0.0375% (w/v)SDS, and 20% (v/v) methanol. Polypeptides were then trans-ferred to an Immobilon PVDF Transfer Membrane at a cur-rent of 80 mA per gel for about 1 h under semi-dry horizontalconditions on a Multiphor II Novablot unit. After transfer,the membranes were blocked overnight with 10% (w/v)powdered milk in 10 mm Tris-HCl (pH 7.5) with 150 mmNaCl and reacted with rabbit immune serum diluted 1000-fold in blocking solution for 4 h at room temperature. Mem-branes were washed with the blocking solution and probedfor 1 h with purified goat anti-rabbit IgG antibody coupledto alkaline phosphatase diluted 3000-fold. After washing,membranes were incubated for 10 to 20 min in 0.015% (w/v) 5-bromo-4-chloro-3-indolylphosphate and 0.03% nitro-blue tetrazolium (w/v) in 0.1 M sodium bicarbonate buffer(pH 9.8) with 1 mM MgCl2 at room temperature. Immuno-precipitation tests were performed as described elsewhere(10).

RESULTS AND DISCUSSION

Protein Purification

GS and NADP-GDH were purified using a similar purifi-cation scheme involving separation on DEAE-Trisacryl, affin-ity chromatography, and FPLC-based DEAE-5PW matrices(see 'Materials and Methods'). Mycelia used to purify theenzymes were grown on nitrate as the sole nitrogen source.Growth of the fungus on NH4' resulted in a lower NADP-GDH activity level and a poor, if not undetectable, GS level,which were not suitable for large-scale purification. GS ap-peared to be more labile than NADP-GDH during the ex-traction procedure, but its stability could be greatly improvedby the presence of EDTA, Glu, and Mg2+ throughout purifi-cation. In the presence of these three stabilizing agents, thecrude filtrates could be stored at 40C for at least 3 d withoutany appreciable loss of GS activity. After 10 d at 40C inextraction buffer, GS retains 78% of its initial activity. In theabsence of stabilizing agents, the enzyme loses 100% of itsactivity during this time. GS was purified 31-fold from L.laccata vegetative mycelium with a 23% recovery and aspecific activity of 713 nkat mg-' protein (Table I). The ratioof transferase over biosynthetic activities (3:5) was found tobe similar with crude or purified preparations. The enzymeeluted from the DEAE-Trisacryl column as one sharp activity

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Table I. Purification of GS from L. laccata

Purification Step Total Protein Total Activity Specific Activ- Yield Purificationitymg nkar' nkat/mg protein % -fold

Crude filtrate 116.8 2712 23 100 1DEAE-Trisacryl 7.96 1520 191 56 8Blue-Sepharose CL-6B 1.61 964 599 36 26FPLC-DEAE-5 PW 0.87 620 713 23 31

a nmol/s.

peak at 120 mm NaCl. The anion-exchange column elimi-nated approximately 93% of the total protein with a 56%recovery of GS activity (Table I). The fact that ATP-depend-ent enzymes, including GS, from various sources bind toCibacron-Blue affinity resins (5, 29) prompted us to exploitBlue-Sepharose for L. laccata GS purification (Table I). Ap-proximately 65% of the GS activity was eluted, with a bulkelution using 0.5 mm ADP. The remaining 35% was recoveredin the unbound fraction. Attempts to elute GS from the Blue-Sepharose column with an ADP-gradient proved ineffective,and resulted in a slow release of enzyme in a broad peak(data not shown). Subsequent FPLC-based anion-exchangechromatography on a Protein Pak DEAE-5PW column re-moved minor contaminating proteins as monitored by SDS-PAGE (Fig. 1). The resulting native enzyme preparationyielded a single protein band that co-migrated with GSactivity on nondenaturing PAGE (Fig. 2). The identity of GSon PAGE was further ascertained by its biosynthetic activity,locating the native enzyme by an insoluble CaHPO4 precip-itate (not shown). It was concluded that GS was purified toapparent electrophoretic homogeneity.

kD

94-

67-

1 2 3 4 5

GS>43~ -b__~ - 5<GDHGS._-3000i aatA m4

30) -

20 -

14-

NADP-GDH was purified 688-fold with a recovery of 62%and a specific activity of 4130 nkat mg-' protein (Table II).The enzyme eluted from the DEAE-Trisacryl column as asingle activity peak at 70 mm NaCl. Approximately 95% ofthe total NADP-GDH activity was recovered from the anion-exchange column, whereas 99% of the total protein waseliminated (Table II). The 2',5'-ADP-Sepharose 4B chroma-tography was also an effective purification step (Table II).This affinity matrix was previously used for the purificationof NADP-GDH from the ectomycorrhizal ascomycete Ceno-coccum geophilum (10). After a further purification step onFPLC-based DEAE-5PW, the enzyme preparation was elec-trophoretically homogeneous, as monitored by SDS-PAGE(Fig. 1) or nondenaturing gradient-PAGE (Fig. 2). The finalyield was 62%, whereas a 44% recovery was obtained for Cgeophilum NADP-GDH (F. Martin, unpublished data). Theseyield values are much higher than those obtained for theenzyme from Sphaerostilbe repens, 23% (4), C geophilum, 4%(18), or Saccharomyces cerevisiae, 33% (7), purified by con-ventional methods. Attempts to purify both enzymes fromthe same extract failed because the extraction and purifica-tion buffers used for GS were not suitable for work onNADP-GDH.

U1)

-94-67

GDH>

-2()

-14

Figure 1. SDS-PAGE (15% resolving gel) and immunoblots of GSand NADP-GDH from L. Iaccata. Purified GS (lane 2) and NADP-GDH (lane 4) were subjected to SDS-PAGE and silver stained. Lowmol wt markers are in lane 3. Crude mycelial extracts were sub-jected to SDS-PAGE, transferred to an Immobilon PVDF membrane,and probed with antibodies produced against purified GS (lane 1)and purified NADP-GDH (lane 5). Goat anti-rabbit IgG conjugatedto alkaline phosphatase was used to develop the blots. Each lanecontained between 0.5 and 2 Ag of protein.

1 2 3

ml,

kD

- 669 -

-4( -

- 158-

_i -. 67

<GS

Figure 2. Nondenaturing gradient-PAGE (4-30%) of NADP-GDH(lanes 1 and 2) and GS (lanes 5 and 6) purified from L. laccata. Highmol wt markers are in lanes 3 and 4. Lane 1, NADP-GDH activitystaining; lanes 2 to 5, protein staining with Coomassie blue R-250;lane 6, GS activity staining. Each lane contained between 1 and 3jig of protein.

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Table II. Purification of NADP-GDH from L. laccata

Purification Step Total Protein Total Activity Specific Activ- Yield Purificationitymg nkata nkat/mg protein % -fold

Crude filtrate 316.8 1998 6 l00 1DEAE-Trisacryl 2.03 1913 942 96 1572',5'-ADP-Sepharose4B 0.564 1709 3030 86 505FPLC-DEAE-5 PW 0.301 1243 4130 62 688

a nmol/s.

Characterization of Purified Enzymes

Mol Wt Estimations

The native Mr values of GS and GDH determined by gelfiltration were 365,000 ± 13,000 and 345,000 ± 18,000,respectively, whereas they were 190,000 ± 5,000 and 351,000± 4,000, respectively, when estimated by nondenaturinggradient-PAGE. The subunit Mr values, as determined bySDS-PAGE, were 42,000 ± 1,600 for GS and 47,000 ± 1,000for NADP-GDH (Fig. 1).Based upon the subunit Mr of 42,000 and its relative

mobility in the nondenaturing gradient gel system, L. laccataGS appeared to be a tetramer of identical subunits. Con-versely, GS retention time on gel filtration suggested that theenzyme was octameric. The Mr values of 365,000 and 42,000for the native enzyme and the subunit, respectively, are

within the range of Mr values reported for GS from variouseukaryotes (23, 25-27). However, Stewart et al. (26) reported

1.00 _

to 0.10 _0

0.01

0.4

0

0 0.2

0.1

6 7 8 9 10Acrylamide concentration (%)

0 200 400 600 800

Mol wt

Figure 3. Ferguson plots for the determination of native Mr bynondenaturing PAGE (two experiments). GS and NADP-GDH fromL. laccata and marker proteins were electrophoresed on 6, 7.5, 9,and 10.5% polyacrylamide gels. The relative positions of GS andNADP-GDH are marked on the lower plot with open symbols.

that yeast GS resembles the mammalian enzyme in that itdissociates into two tetramers that are catalytically active.Similarly, Palacios (23) reported native Mr values for N. crassa

GS of 190,000 and 385,000 as estimated by gradient-PAGEand sucrose gradient centrifugation, respectively. Therefore,the L. laccata GS Mr estimated by gel filtration was probablycloser to the actual Mr, suggesting that gradient electropho-resis leads to the dissociation of GS into tetramers. This viewwas further examined by Mr estimation using uniform-PAGEwith various concentrations of acrylamide (6, 7.5, 9, and10.5%) (Fig. 3). Interpolation of the Ferguson plots yielded a

GS Mr of 380,000, which was of the same order as the gelfiltration estimate and exactly twice that from gradient-PAGE. Studies are currently in progress to clarify the in vivostructure of GS.Based on the subunit Mr of 47,000 and the native Mr of

345,000 obtained by gel filtration and 351,000 obtained bygradient-PAGE, NADP-GDH from L. laccata appears to bean octa- or hexameric protein consisting of identical subunits.However, the NADP-GDH Mr derived from Ferguson plotswas 298,000 (Fig. 3). NADP-GDH of microorganisms is a

Table Mll. Properties of GS and NADP-GDH from L. laccataThe Km values were determined by varying one substrate con-

centration while the others were kept near saturation. Results wereanalyzed by Lineweaver-Burk plots, and apparent Km values weredetermined by linear regression. The pH optima were establishedby varying the pH of the reaction mixture and measuring it at thebeginning of the reaction. The temperature optima were obtainedby incubating the reaction mixture and measuring the activity atvarious temperatures. Data shown represent the mean of two tothree experiments.

Property GS NADP-GDH

Km (NH4 ) 24 AM 5 mMbKm (2-oxoglutarate) 1 mMbKm (Glu) 3.2 mMa/5 mMc 26 mMdKm (Gln) 30 mMeKm (ATP) 0.18 mMa/0.54 mMcKm (ADP) 2 AmeKm (NADPH) 10 AMbKm (NADP) 30 AMdpH optimum 7.6c/7.2' 7.2b/8.6dT optimum 330Cc/300Ce 38oCb

a Determined by the spectrophotometric assay. b Deter-mined by the amination reaction. c Determined by the biosyn-thetic assay. d Determined by the deamination reaction.e Determined by the transferase assay.

BSA

LDH

catalase

GDHGSferritin

thyroglobulin

R =0.99thyroglobuln

catalav

°~~~~GS (380,O00)

u.u,

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hexamer composed of identical subunits with Mr rangingbetween 270,000 and 320,000 (3, 5, 10, 18, 26), suggestingthat the NADP-GDH from L. laccata is most likely a hexamer.

Kinetic Parameters

The substrate saturation curves were analyzed by Line-weaver-Burk plots and substrate affinities of GS and NADP-GDH are summarized in Table III. The apparent Km valuesof GS for Glu and ATP were similar to the values previouslyreported (26, 27, 29). The most striking feature was the highaffinity of GS for NH4' (24 Mm). However, use of the coupledspectrophotometric assay gave lower values than assays usingNH20H as substrate (Km NH20H, 0.5 mM). This correlateswith other findings showing that the coupled assay, with itsgreater sensitivity, is advisable if reliable apparent Km valuesare to be obtained (26). The Km values of L. laccata NADP-GDH for 2-oxoglutarate, Glu, and NH4' were similar to thosereported for the enzyme from other fungal sources (2, 5, 18,26). The purified NADP-GDH did not show any activity inthe presence of NADH. L. laccata NADP-GDH did not exhibitbiphasic kinetics for NH4' as reported for the enzyme fromother fungi (2, 5, 18). The Hill number for NH4' was 0.99,arguing against interactions between subunits. In L. laccata,preliminary experiments allowed estimation of a cellular con-centration ranging from 2 to 4 mm for NH4' and from 0.5 to1 mm for glutamate, assuming an even distribution of thesecompounds in the cells. Considering the Km values for NH4'and glutamate, it seems that the dehydrogenase might oper-ate mainly in the direction of glutamate synthesis. Moreover,it has been found, in standard assay conditions, that NH4Iinhibited the catabolic reaction more than glutamate did forthe anabolic one. Indeed, at 4 mm, NH4' ions suppressed68% of the deamination activity, whereas glutamate at thesame concentration did not affect the amination reaction (notshown).The turnover number for GS was 16,300 mol NH4' trans-

formed min-' mol-1 holoenzyme compared with 74,300 forNADP-GDH. However, the apparent affinity of GS for NH4'(24 Mm) was higher than that of NADP-GDH (5 mM), andthe amount of GS g-1 fresh weight (151 jig) was eightfoldhigher than that of NADP-GDH. From these results, it wascalculated that the amount of NH4' reduced min-' g-1 freshtissue was 6.5 Mumol for GS and 4.8 zmol for NADP-GDHunder optimal catalytic conditions. Based on the purificationdata, GS is a highly abundant protein, representing approx-imately 3% (w/w) of the total soluble protein in nitrate-grown mycelia (Table I). Conversely, NADP-GDH representsonly 0.15% (w/w) of the total soluble protein in such mycelia(Table II). This fact, together with the fourfold greater activityof GS over NADP-GDH in extracts of nitrate-grown mycelia(Tables I and II), is consistent with GS being the main routeof NH4' assimilation at low NH4' concentrations. This viewis supported by isotopic studies that demonstrated that[15N]NH4' was mainly incorporated into the amido group ofglutamine in L laccata mycelia (16).

pH and Temperature Optima

Optimum pH values for transferase and biosynthetic ac-tivities of L. laccata GS were 7.2 and 7.6, respectively (Table

III), which agree well with values reported for the enzymefrom eukaryotes (26, 27). The optimum pH values were 7.2and 8.6 for the amination and deamination reactions ofNADP-GDH, respectively (Table III), which are within therange of optimum pH reported for other fungal sources (3,5, 18). At the optimum pH of each reaction, the aminationrate was 10-fold higher than that of the deamination reaction.Considering the cytoplasmic pH value in L. laccata, rangingfrom pH 6.5 to 7 as measured by NMR spectroscopy (F.Martin, unpublished results), such a result emphasizes thepossibility that NADP-GDH may primarily be involved inthe biosynthesis of glutamate. The assay temperature optima,related to the catalytic efficiency, were 30 and 380C for GSand NADP-GDH, respectively.

Immunological Characterization

The effects of anti-GS and anti-NADP-GDH immune seraon the activity of GS and NADP-GDH are shown in Figure4. The amount of anti-GS immune serum required for 50%immunoprecipitation of purified GS activity was about 8 ,Lnkat-1 (Fig. 4A), whereas the amount of anti-NADP-GDHimmune serum required for 50% immunoprecipitation ofpurified NADP-GDH activity was fourfold lower (Fig. 4B).Antibodies raised against GS did not immunoprecipitateNADP-GDH and, similarly, antibodies raised against NADP-GDH did not immunoprecipitate GS. An immunoblot of the

1004 _ 0

A80 - Anti-GS immune serum

0 against NADP-GDH

60 -0 against GS

*f

20

Anti-NADP-GDH immune serum

60 - < 0 againstGS\ againstNADP-GDH

40_

o 50 100 150 200 250Immune serum (gL)

Figure 4. Effect of rabbit anti-GS immune serum (A) and anti-NADP-GDH immune serum (1:10 dilution) (B) on the activities of purifiedGS and NADP-GDH. Constant amounts of antigen (partially purifiedextracts from the DEAE-Trisacryl column) were incubated withincreasing volumes of antisera for 1 h at room temperature andthen stored overnight at 4°C. The antigen-antibody complex wascentrifuged at 10,000g for 10 min and the residual enzyme activitywas determined with an aliquot of the supernatant fraction. Onehundred percent of activity corresponds to 6.4 and 2.7 nkat for GSand NADP-GDH, respectively.

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crude protein extract from L. laccata mycelium probed withanti-GS immune serum displayed one major band migratingwith a Mr of 42,000 (Fig. 1). Protein blot analysis of crudeextracts with the antibodies against NADP-GDH revealedone strong band at 47,000 (Fig. 1). An additional faint im-munoreactive polypeptide was detected in both samples atapproximately 110,000. The immunoprecipitation curves andimmunoblots documented the high reactivity and the mon-ospecificity of the immune sera produced against the purifiedenzymes.

In conclusion, GS, purified for the first time from a basi-diomycete fungus, and L. laccata NADP-GDH appear to bephysically and kinetically similar to the enzymes from otherfungal sources. The separation procedures developed in thepresent study are highly effective and resulted in the purifi-cation to apparent electrophoretic homogeneity of L. laccataGS and NADP-GDH. Because of its low Km for NH4' and itshigh relative abundance, the reaction catalyzed by GS prob-ably represents a major route for the assimilation of NH4+ infungal tissues grown on nitrate. However, the role of NADP-GDH should not be underestimated because "5N experimentswith L. laccata indicated that this enzyme was also operativein N assimilation (16). Future studies must evaluate therespective contribution of these two N-assimilating enzymesin L. laccata mycelia. The contribution of the fungal GS andNADP-GDH in NH4' assimilation by ectomycorrhizas andtheir distribution in the symbiotic tissues await futureexperiments.

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Purification and Characterization of Glutamine Synthetase NADP