Investigations Into Enzymes of Nitrogen Metabolism of The

Home , Suillus bovinus

Investigations into Enzymes of Nitrogen Metabolism of the Ectomycorrhizal Basidiomycete, Suillus bovinus Norbert Grotjohann3, Wolfgang Kowallik3*, Yi Huangb, Andrea Schulte in den Bäumen3 a Lehrstuhl für Stoffwechselphysiologie, Fakultät für Biologie, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany. Fax: 0521-106-6039. E-mail: [email protected] b Department of Urban and Environmental Sciences, Peking University, Beijing, 100 871, P. R. China * Author for correspondence and reprint requests Z. Naturforsch. 55c, 203-212 (2000); received November 24/December 15, 1999 Dedicated to Professor Andre Pirson on the occasion of his 90th birthday Suillus bovinus, Glutamate Dehydrogenase, Glutamine Synthetase/Glutamate Synthase, Aminotransferases, Urease Axenic mycelia of the ectomycorrhizal basidiomycete, Suillus bovinus, were grown in liquid media under continuous aeration with compressed air at 25 °C in darkness. Provided with glucose as the only carbohydrate source, they produced similar amounts of dry weight with ammonia, with nitrate or with alanine, 60-80% more with glutamate or glutamine, but about 35% less with urea as the respectively only exogenous nitrogen source. In crude extracts of cells from NH 4+-cultures, NADH-dependent glutamate dehydrogenase exhibited high aminating (688 nmol x mg protein -1 x min-1) and low deaminating (21 nmol x mg protein -1 x min-1) activities. Its /Cm-values for 2-oxoglutarate and for glutamate were 1.43 mM and 23.99 mM, respectively. pH-optimum for amination was about 7.2, that for deami nation about 9.3. Glutamine synthetase activity was comparatively low (59 nmol x mg pro tein-1 x min-1). Its affinity for glutamate was poor ( K m = 23.7 mM), while that for the NH4+ replacing NH2OH was high ( K m = 0.19 mM). pH-optimum was found at 7.0. Glutamate syn thase (= GOG AT) revealed similar low activity (62 nmol x mg protein -1 x min-1), K m-v alues for glutamine and for 2-oxoglutarate of 2.82 mM and 0.28 mM, respectively, and pH-optimum around 8.0. Aspartate transaminase (= GOT) exhibited similar affinities for aspartate ( K m = 2.55 m M ) and for glutamate ( Km = 3.13 m M ), but clearly different /Cm-values for 2-oxoglutar ate (1.46 m M ) and for oxaloacetate (0.13 m M ). Activity at optimum pH of about 8.0 was 506 nm ol x mg protein-1 x min-1 for aspartate conversion, but only 39 nmol x mg protein-1 x min-1 at optimum pH of about 7.0 for glutamate conversion. Activity (599 nmol x mg pro tein -1 x min-1), substrate affinities (Km for alanine = 6.30 m M , for 2-oxoglutarate = 0.45 m M ) and pH-optimum (6.5-7.5) proved alanine transaminase (= GPT) also important in distribu tion of intracellular nitrogen. There was comparatively low activity of the obviously constitutive enzyme, urease, (42 nm ol x mg protein -1 x min-1) whose substrate affinity was rather high (K m = 0.56 mM). Nitrate reductase proved substrate induced; activity could only be measured after exposure of the mycelia to exogenous nitrate. Routes of entry of exogenous nitrogen and tentative significance of the various enzymes in cell metabolism are discussed.

Introduction ous advantages from each other. A very important In mycorrhiza, the symbiotic association of plant interdependence is the exchange of nutrients (see: roots and fungal mycelia, both partners take vari- e.g. Harley and Smith, 1983; Cairney and Cham bers, 1997). The fungus profits by provision with photosynthetically produced carbohydrates, while Abbreviations: DEAE, diethylaminoethyl; EDTA, ethy- the tree root benefits from a special supply with lenediaminetetraacetic acid; HEPES, 4-(2-hydroxy- inorganic, but also organically bound nitrogen (e.g. ethyl)-l-piperazineethanesulfonic acid; TEAE, tri- Bowen and Theodorou, 1973; Lewis, 1976; Pate- ethanolamine hydrochloride; TRICINE, (N-Tris- [hydroxymethyljmethylglycine); TRIS, 2-amino-2-(hy- man and Kinghorn, 1976; Martin et al., 1987; Tin droxymethyl)-!, 3-propanediol. ker et al., 1994).

For growth of isolated mycorrhizal fungi, vari Material and Methods ous exogenous nitrogenous sources have been Axenic cultures of Suillus bovinus (L. ex Fr.) O. found suitable. Among them there are inorganic Kuntze, Boletaceae, were used. Mycelia, isolated molecules, such as ammonia and nitrate, and or from fruiting bodies collected at Sennefriedhof/ ganic molecules, such as urea, proteins and several Bielefeld by Dr. U. Röder, Department of Ecol amino acids, as well (e.g.: Peeler and Mullins, 1981; ogy, University Bielefeld, Germany, were kept on France and Reid, 1984; Littke et al., 1984; Abuzi- 1 .2 % agar plates prepared with nutrition solution nadah et al., 1986; Read et al., 1989; Finley et al., (see below) at room temperature in the dark (= 1992; Sarjala, 1999). For de novo synthesis of cellu stock cultures). lar proteins, nitrogen of all these sources has to be incorporated into amino acids, of course, and Growth conditions amino acids have also been found to be transfer red to the tree root. Among them, glutamine has Mycelia were grown in liquid media. Samples of been reported on first place repeatedly (Harley stock cultures were transferred to nutrition solu and Smith, 1983; Martin et al., 1987). Also transfer tion after Kottke et al., (1987) containing 10 g/1 of alanine, glutamate and aspartate has been ob glucose as carbon source: (NH 4 )2 H P 0 4, 500 mg/1; served (Melin and Nilsson, 1953; Martin et al., KH 2P 0 4, 500 mg/1; M gS0 4 x 7 H 2 0 , 150 mg/1; 1986). From this, enzymes, such as glutamate de CaCl2, 50 mg/1; KC1, 37.28 mg/1; NaCl, 25 mg/1; hydrogenase, glutamine synthetase, and also gluta H 3 B O 3 , 15.46 mg/1; M nS0 4 x H 2 0 , 8.45 mg/1; Z nS 0 4 x 7 H 2 0 , 5.75 mg/1; C uS0 4 x 5 H zO, mate synthase (= glutamine- 2 -oxoglutarate amino transferase), have been examined intensively by 1.25 mg/1; FeCl 3 x 6 H 2 0 , 1 mg/1; (NH 4 )6Mo 7 0 24 x various authors in the past (e. g. Pateman and Kin- 4 H 2 0, 0.18 mg/1; thiamine hydrochloride, 0.1 mg/1. ghorn, 1976; Stewart et al., 1980; Ahmad and Hel- Nitrogen was supplied either as an inorganic lebust, 1991; Botton and Chalot, 1995). Beside (NH4+ or N 03'), or as an organic (alanine, gluta these ammonia metabolizing enzymes, also those mate, glutamine or urea) molecule. pH of the me catalyzing processes which produce NH4+ for in dia was set to 5.0 in the beginning. It dropped with corporation, such as nitrate and nitrite reductases time in all cases except of glutamate. The auto- and urease for hydrolytic cleavage of urea, have claved (5 min at 1.2 bar) media were inoculated been under investigation (Ho and Trappe, 1980; with suspended hyphae and the resulting suspen Plassard et al., 1984a,b; Sarjala, 1990). sions filled in sterilized (4 h at 150 °C) culture The various literature data available result from tubes with an inlet for aeration at their bottom. examinations of several different organisms, i. e., Length of tubes was 45 cm, 0 4 cm. For growth, as far as we know, there is no comprehensive in tubes were placed in a water bath of 25 °C in the formation yet on the respective enzymatic equip dark and the suspensions were aerated continu ment of one species. Therefore, we decided to take ously with compressed air. up over-all investigations into the enzymatic equ ipment for basic carbohydrate and protein metab Preparation for enzymatic analysis olism of the ectomycorrhizal partners, Suillus bovi- 3-4 days after inoculation, mycelia were har nus and Pinus sylvestris. Starting with the isolated vested on filter paper in a Buchner funnel. The fungus, we proved existence and elucidated some resulting pellet was resuspended in 0 . 1 m phos- kinetic properties of all glycolytic enzymes, re phate-buffer pH 7.5 and cells were broken by cently (Kowallik et al., 1998). Studies on the fate grinding with sea sand in a mortar under cooling of pyruvate, the endproduct of glycolysis, are un with liquid nitrogen. After separation from sand der way. In this paper, we present data on ammo and large fragments by filtration through 4 layers nia liberating and ammonia incorporating en of cheese cloth, the resulting homogenates were zymes of Suillus bovinus, sustaining the fungus’ centrifuged for 20 min at 20 000 x g and 4 °C (Sor- growth, but also producing nitrogenous com vall RC-5 Superspeed Refrigerated Centrifuge). pounds for potential transfer to its mycorrhizal The resulting supernatants were used as crude ex partner. tracts. N. Grotjohann et al. ■ Enzymes of Nitrogen Metabolism of Suillus bovinus 205

Enzyme assays stop reagent (0.2 m trichloroacetic acid, 0.67 n HC1, 0.37 m FeCl3) after 30 min at 30 °C. Within All enzyme assays, based on literature as indi 30 min, 5 min centrifugation at 4 300 x g and de cated below, were optimized for the Suillus crude termination of absorbance at 546 nm in superna extract; i.e., maximum in vitro activities (= capaci tant had to be performed. ties) were determined. Optimum concentrations Glutamate synthase (NADH) ( = glutamine-2- for substrates and cofactors, optimum pH, and ap oxoglutarate aminotransferase) propriate concentrations of auxiliary enzymes and (L-glutamate:NAD+ oxidoreductase [transami- of artificial electron donors and acceptors had to nating], EC 1.4.1.14) (after Miller and Stadtman, be determined. These data will not be presented 1972; Kowallik and Neuert, 1984) here in detail. It shall only be mentioned that Absorbance changes at 334 nm resulting from activities of all enzymes could be improved over NADH oxidation due to reductive transfer of the those determined with literature tests developed amido group of glutamine to 2 -oxoglutarate by the for different living materials. enzyme were determined. Glutamate dehydrogenase (L-glutamate: Assay: 220 mM phosphate-buffer pH 8.0, 100 mM NAD(P)+ oxidoreductase, EC 1.4.1.3) (after glutamine, 2 . 0 mM 2 -oxoglutarate (start), 1 1 0 [ig Schmidt, 1974; Smith et al., 1975) crude cell extract protein/ml test volume, 0.2 mM Enzyme activity has been determined in both NADH. directions. Aminating action was analyzed by Aspartase (- fumaric aminase) (L-aspartate-am- following decrease in absorbance at 334 nm result monia lyase, EC 4.3.1.1) (after Tokushige, 1985) ing from NADH oxidation due to reductive ami- Absorbance changes at 240 nm resulting from nation of 2-oxoglutarate by the enzyme. Deami- fumarate produced by deamination of aspartate by nating action was analyzed by following increase the enzyme were determined. in absorbance at 334 nm resulting from NAD+ re Assay: 100 mM TRIS-HCl-buffer pH 7.9, 6 6 mM duction due to 2 -oxoglutarate oxidation resulting Na-aspartate, 27 [ig crude cell extract protein/ml from oxidative deamination of glutamate by the test volume (start). For control, accumulation of enzyme. the second reaction product, NH4+, was also deter Assay 1: 50 mM HEPES-buffer pH 7.25, 200 mM mined at up to 300 [ig crude cell extract protein/ NH 4C1, 10 mM 2-oxoglutarate (start), 110 [ig crude ml test volume. cell extract protein/ml test volume, 0.4 mM Procedure: Enzyme reaction was stopped by ad NADH. dition of 40 [il 3 m trichloroacetic acid/ml test vol Assay 2: 100 mM TRIS-glycine-buffer pH 9.35, ume. After centrifugation (10 min, 15 000 x g), su 100 mM glutamate (start), 145 [ig crude cell extract pernatant was neutralized with NaOH and 0.5 ml protein/ml test volume, 0.3 mM NAD+ . samples were tested for NH4+ by absorbance Glutamine synthetase (glutamate-ammonia li- changes at 334 nm dependent on reductive anima gase [ADP-forming], EC 6.3.1.2) (after Rowe et tion of 2 -oxoglutarate with glutamate dehydroge al., 1970; Meister, 1974) nase (Boehringer test, cat. no. 1112732). The so-called „synthetic reaction“ (see Miflin Aspartate transaminase (- glutamate-oxaloace- and Lea, 1982; Brun et al., 1992) using hydroxyl- tate transaminase [GOT]) (L-aspartate:2-oxoglu- amine instead of NH4+ and resulting in y-glutamyl- tarate aminotransferase, EC 2.6.1.1) (after Berg- hydroxamic acid instead of glutamine formation meyer and Bernt, 1974a) by the enzyme has been used, y-glutamylhydroxa- Action of the enzyme has been examined in mic acid was determined colorimetrically at both directions. Transfer of the amino group of 546 nm after formation of a yellow-brown com aspartate to 2 -oxoglutarate - leading to oxaloace- plex with FeCl 3 (see Mecke, 1985). tate and glutamate - was followed by decrease in Assay: 100 mM HEPES-TRICINE-buffer pH 7.0, absorbance at 334 nm resulting from NADH oxi 12.5 mM M gS04, 137.5 mM L-glutamate, 10 mM dation by oxaloacetate reduction catalyzed by ATP, 150 [ig crude cell extract protein/ml test vol added malate dehydrogenase. The opposite reac ume, 12 mM hydroxylamine (start). Reaction was tion, transfer of the amino group of glutamate to stopped with equal volume as assay volume of oxaloacetate, was analyzed by increase in absor- 206 N. Grotjohann et al. ■ Enzymes of Nitrogen Metabolism of Suillus bovinus bance at 334 nm resulting from NAD+ reduction Assay: 100 mM K-phosphate-buffer p H 7.4, 1.8 by oxidation of 2 -oxoglutarate by added 2 -oxoglu- m M K N 0 3 (start), 0.2 m M NADH, 300 jig crude tarate dehydrogenase. Formation of NADH by ox cell extract protein/ml test volume. For nitrite de idative deamination of glutamate by endogenous termination, above assay mixture was incubated glutamate dehydrogenase and concomittant con for 15 min at 30 °C. Thereafter, reaction was sumption of NADH from reduction of oxaloace- stopped by rapid addition of 1.25 ml sulfanil- tate by endogenous malate dehydrogenase had to amide-N-(l-naphthyl)ethylenediamine reagent (1 be considered. vol. 0.5 g sulfanilic acid in 100 ml 2 n acetic acid Assay 1: 220 m M phosphate-buffer p H 8.0, 100 plus 2 vol. 1 0 0 mg naphthylethylenediaminedi- mM aspartic acid, 2 0 mM 2 -oxoglutarate (start), chloride in 200 ml 2 n acetic acid) to 0.5 ml of assay 25 jig crude cell extract protein/ml test volume, 5 mixture. After 10 min, absorbance of colour devel units/ml malate dehydrogenase (GOT-free!), 0.2 oped was determined. mM NADH. Assay 2: 100 mM phosphate-buffer pH 7.0, 100 Soluble protein m M glutamate, 2.0 m M oxaloacetate (start), 117 [ig crude cell extract protein/ml test volume, 0.4 units/ Soluble protein in crude extracts was deter ml 2-oxoglutarate dehydrogenase, 0.3 mM NAD+. mined according to Lowry et al. (1951) with bovine Alanine transaminase (= glutamate-pyruvate serum albumin as reference. transaminase [GPT]) (L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) (after Bergmeyer Fast performance liquid chromatography (FPLC) and Bernt, 1974b) Absorbance changes at 334 nm resulting from 0.5 ml of crude cell extract, filtered through a NADH oxidation due to pyruvate reduction by 0.2 (im sterile filter (Sartorius GmbH, Göttingen added lactate dehydrogenase were determined. Germany), were applied to a Superose 6 (gluta Pyruvate resulted from transfer of the amino mine synthetase) or Superose 12 (glutamate syn group of alanine to 2 -oxoglutarate by the enzyme. thase) column (HR 10/30, Pharmacia, Uppsala Assay: 100 m M phosphate-buffer p H 7.5, 207 m M Sweden). The column was equilibrated and eluted alanine, 20 mM 2-oxoglutarate (start), 25 fig crude with 100 mM HEPES-buffer p H 7.5 in case of glu cell extract protein/ml test volume, 0.4 units/ml tamine synthetase and with 220 m M phosphate- lactate dehydrogenase, 0.2 m M NADH. buffer p H 8.0 in case of glutamate synthase, respec Urease (urea amidohydrolase, EC 3.5.1.5) (after tively. Fractions of 250 \i\ were collected at a flow Schlegel and Kaltwasser, 1974) rate of 1 ml x min - 1 at room temperature. For mo Absorbance changes at 334 nm resulting from lecular weight determination, the column was cal NADH oxidation due to reductive amination of 2- ibrated with following standards: thyroglobulin di oxoglutarate by ammonia liberated from urea by mer 1338 kDa, thyroglobulin 669 kDa, ferritine the enzyme were determined. 440 kDa, catalase 232 kDa, aldolase 158 kDa, al Assay: 100 m M TEAE-buffer pH 8.0, 500 m M bumin 67 kDa, hemoglobin 64.5 kDa, Chymotryp urea, 26 m M 2-oxoglutarate, 9 units/ml glutamate sin 25 kDa, myoglobin 17.2 kDa, and cytochrome dehydrogenase + 0.4 m M ADP (activator), 0.2 m M c 12.2 kDa. NADH, 110 jig crude cell extract protein/ml test volume (start). Ion exchange chromatography Nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) (after Hagemann and Reed, Crude cell extract was separated on DEAE 1980) Sephacel (column 2.2 x 8 cm) using 50 m M TRIS- Enzyme activity was determined by 2 methods - HCl-buffer p H 7.8 + 6 m M dithiothreitol + 1 m M 1. by absorbance changes at 334 nm resulting from EDTA for elution. The column was washed with NADH oxidation due to nitrate reduction to nit 2 bed volumes of buffer and developed with a lin rite by the enzyme; 2. by absorbance at 540 nm of ear KC1 gradient (0-0.6 m ). 1.6 ml fractions were a coloured product resulting from a reaction of collected at a flow rate of 45 ml x h_1. KC1 densitiy nitrite produced with Griess-Ilosvay’s reagent. in fractions was calculated from index of refraction N. Grotjohann et al. ■ Enzymes of Nitrogen Metabolism of Suillus bovinus 207

(Abbe universal refractometer, Schmid and operatively. Deaminating activity of the enzyme is Haensch, Berlin Germany). found extremely low and its affinity for glutamate is more than 15 times smaller than that for 2-oxo Results glutarate. In both directions, the enzyme is strictly dependent on NADH/NAD+. Growth of mycelia Glutamine synthetase , determined by the so- In axenic liquid cultures of mycelia of Suillus called „synthetic reaction“, shows high affinities bovinus, various exogenous nitrogen sources led to for ATP and for hydroxylamine, but low affinity significant, but clearly different increases in dry for glutamate. Similar results for glutamate and for weight. Table I shows the data obtained 4 days af NH4+ have already been reported by Stewart et al. ter inoculation. They are slightly (see Kowallik et (1980) for another fungus. There is indication for al., 1998) affected by different changes in pH dur positive cooperativity of hydroxylamine (n = 1.67), ing growth, but probably also by different access but this does not necessarily include positive coo to nutrients and oxygen because of variable aggre perativity of the true substrate, NH4+. There are gation. The earlier described (Kowallik et al., no indications for isoforms, known to exist in 1998) yellowish-white, soft „balls“ of hyphae ex higher plants and in algae, (e.g. Hirel and Gadal, isted at growth with NH4+, with nitrate and with 1980; Meya and Kowallik, 1995; Mäck, 1998). alanine, but were replaced by fine white downy Fig. 1 (upper) shows only one peak of glutamine flakes at growth with glutamate and with glut synthetase activity after separation on DEAE- amine. Sephacel column, Fig. 1 (lower) the same after FPLC on Superose 6 of crude cell extracts. The Table I. Increase in dry weight of axenic cultures of my celia of Suillus bovinus supplied with different nitrogen sources and pH of media at harvest. Growth conditions: liquid inorganic medium with glucose as sole carbon source and with indicated nitrogen sources, initial pH 5.0, 10 0 continuous aeration with compressed air, 25 °C, dark ness, (n = 6). 80

Nitrogen source Relative increase in pH at harvest 60 dry weight [%] 40 ( n h 4)2h p o 4 100 3.11 ± 0.06 Nitrate 116 ± 8 3.96 ± 0.08 Alanine 107 ± 7 4.67 ± 0.05 p\^ 20 Glutamate 182 ± 10 5.55 ± 0.09 > 0 Glutamine 160 ± 18 3.65 ± 0.04 0 Urea 65 ± 9 4.57 ± 0.17 ro

Table II. Activities, apparent K m- or S0.s-values and Hill coefficients of enzymes in nitrogen metabolism of Suillus bovinus. Growth conditions: liquid inorganic medium with glucose as sole carbon source and ammonia as sole nitrogen source, initial pH 5.0, continuous aeration with compressed air, 25 °C, darkness. For preparation of crude cell extracts and enzyme assays see Material and Methods.

Enzyme Activity Km(S0.5) Hill [nmol x mg protein -1 x min-1] [mM ] coefficient

Glutamate dehydrogenase (NADH): 2-oxoglutarate: 1.43 ± 0.09 1.08 aminating 688.4 ± 33.3 NH4+: 5.98 ± 0.40 0.94 deaminating 21.3 ± 3.2 glutamate: 23.99 ± 1.28 1.04 Glutamate dehydrogenase no activity detected (NADPH)

Glutamine synthetase glutamate: 23.71 ± 1.96 1.19 synthetic reaction 59.4 ± 5.1 hydroxylamine: 0.19 ± 0.01 1.67 ATP: 1.53 ± 0.18 1.03

Glutamate synthase (=GOGAT) 62.3 ± 3.7 glutamine: 2.82 ± 0.18 1 .0 0 (NADH) 2-oxoglutarate: 0.28 ± 0.011.02 Glutamate synthase no activity detected (NADPH) Glutamate synthase no activity detected (Ferredoxin)

Aspartase no activity detected

Aspartate transaminase (=GOT) aspartate: 2.55 ± 0.17 0.99 NH2-donor = aspartate 506.2 ± 28.7 2-oxoglutarate: 1.46 ± 0.08 1.08 NH2-donor = glutamate 39.1 ± 4.8 glutamate: 3.13 ± 0.08 1.04 oxaloacetate: 0.13 ± 0.02 0.99

Alanine transaminase 599.4 ± 36.0 alanine: 6.30 ± 0.64 0.97 (=GPT) 2-oxoglutarate: 0.45 ± 0.03 1.10

Urease 41.5 ± 2.7 urea: 0.56 ± 0.03 0.97

Nitrate reductase no activity detected

enzyme of Suillus bovinus exhibits an apparent inhibitor, azaserine. Whether they represent native molecular mass of 595 ± 25 kDa. species or whether the heavier form is an aggrega Glutamate synthase could only be detected de tion product of preparation, cannot be decided. pendent on NADH. The, in general, additionally Aspartate transaminase (= glutamate-oxaloace- known electron donors NADPH or reduced ferre- tate transaminase) exhibits more than 1 0 times doxin never revealed any measurable activity in higher activity with aspartate than with glutamate crude extracts of Suillus. Total maximum activity as NH 2 -donor. At no big differences in the affini of the NADH-dependent enzyme corresponds ties and no indications for any homotropic cooper- with that of glutamine synthetase, indicating the ativity, this corresponds with the generally as generally assumed close connection of both these sumed preferred aspartate converting function of enzymes also in this organism. Separation on Su- the enzyme (but see Discussion). Comparable re perose 1 2 revealed two peaks of active protein sults have been obtained by Khalid et al. (1988) showing molecular masses of about 300 kDa and for Cenococcum geophilum. of about 1000 kDa. Both of them exhibited com Alanine transaminase (= glutamate-pyruvate parable sensitivity towards the glutamate synthase transaminase) shows also rather high activity N. Grotjohann et al. ■ Enzymes of Nitrogen Metabolism of Suillus bovinus 209 and - as in all other cases - lower affinity for its Suillus bovinus, revealed activity o f nitrate reduc nitrogenous substrate than for the NH 2 accepting tase which was exclusively NADPH-dependent. 2 -oxocarbonic acid. In the artificial buffer systems used, all enzymes Activity and affinity of urease are comparatively exhibited pH-optima between pH 7 and pH 8. Only low. Both were not improved by prolonged culti deamination by glutamate dehydrogenase took vation with urea. The enzyme, therefore, appears place best at pH 9.35. In most cases, pH-depen- to be constitutive. dences exhibited rather sharp optima, i. e. enzyme Absence of nitrate reductase activity - inspite of activity dropped by 50% at pH changes of less than growth with this nitrogen source (see Table I) - is one order. Less pronounced optima existed for al puzzling on first view. However, as in higher plants anine transaminase and - towards alkaline val and also in some fungi, the enzyme turned out to ues - for glutamine synthetase (Fig. 2). be induced by its substrate. When grown with ni trate as the only nitrogen source, crude extracts of

100 - aminating ✓Q deaminating A - glutamate • aspartate - converting J

J t 0 \ * T i i i I i I i i i > i i I i 1 t O' 100 > % 80 ’■M O 60 glutamine CO synthetase <1) 40 (GS)

CÜ 20 CD cr 0 -i___ L

100

pH pH Fig. 2. pH-dependences of the activities of enzymes in nitrogen metabolism of axenic mycelia of the ectomycorrhizal basidiomycete, Suillus bovinus. Growth conditions: liquid inorganic medium with glucose as sole carbon source and (NH4)2H P 0 4 as sole nitrogen source, initial pH 5.0, continuous aeration with compressed air, 25 °C, darkness. For enzyme assays see Material and Methods. Buffers used: HEPES = glutamate dehydrogenase (aminating), HEPES- TRIS = glutamine synthetase, phosphate = glutamate synthase, aspartate transaminase, alanine transaminase, nitrate reductase, phosphate and TRIS-glycine = glutamate dehydrogenase (deaminating), TEAE = urease. 210 N. Grotjohann et al. ■ Enzymes of Nitrogen Metabolism of Suillus bovinus

Discussion The high aminating ( 6 8 8 nmol x mg protein“ 1 x min-1) and low deaminating ( 2 1 nmol x mg pro The strict dependence on NADH/NAD+ of glu tein - 1 x min-1) activities of glutamate dehydroge tamate dehydrogenase of our Suillus bovinus iso nase - together with the enzyme’s good affinity lates is most remarkable different from respective for 2-oxoglutarate ( K m = 1.43 m M ) and poor affini- reports in literature which describe either NADH- tiy for glutamate (K m = 23.99 m M ), clearly point - plus NADPH-dependent or only NADPH-depen- although its affinity for NH4+ is rather poor dent glutamate dehydrogenases for various mycor- (5.98 mM) - to NH4+ incorporating rather than rhizal fungi ( Sphaerostilbe repens, Botton and glutamate degrading action of the enzyme in Suil Msatef, 1983; Cenococcum graniforme, Martin et lus. pH optima of about 7.2 for amination and of al., 1983, 1988; Laccaria bicolor , Ahmad et al., about 9.3 for deamination support this assump 1990; Chalot et al., 1991; Stropharia semiglobata , tion. Glutamine synthetase exhibits much better Schwartz et al., 1991). Rudawska et al. (1994) re affinity for the NH4+ replacing NH2OH ( K m = 0.19 port the NADPH-dependent enzyme species even m M ), indeed. Therefore, calculating arbitrary num for Suillus bovinus. Using isolates from Poland, bers for intracellular binding of ammonia of both this discrepancy can only be taken as another ex enzymes by deviding activities into /Cm-values, ample of the repeatedly discussed metabolic dif leads with relative numbers of about 1 0 0 for gluta ferences in various isolates of presumably identical mate dehydrogenase and about 300 for glutamine fungal species. synthetase to preferred involvement of the latter. Proof of constitutive existence of urease appears However, respective NH4+ acceptors might not to be novel, too. At least, we are not aware of any equally be available in both cases. While 2-oxoglu- respective detailed report in literature. tarate ought to be produced sufficiently by tricar In contrast, observation of the inductive charac boxylic acid cycle (unpublished data), glutamate ter of nitrate reductase is not new; it is just another might be scarce because of the rather low activity example for this property also in mycorrhizal fungi of glutamate synthase at the presumably intracel (Plassard et al., 1984a,b; Sarjala 1990). In our ex lular pH of about 7. Since the affinity for gluta periments, 2-3 days exposure to nitrate yielded mate of glutamine synthetase is rather poor ( K m = marked increase in cell mass and also nitrate re 23.71 m M ), efficiency of the latter might be ad ductase activity. Absence of isoforms of glutamine versely affected. Summing up, there is good reason synthetase has also been reported before ( Lacca to assume that in Suillus bovinus, NADH-depen- ria bicolor , McNally and Hirel, 1983). The molecu dent glutamate dehydrogenase is mainly responsi lar mass of the Suillus enzyme corresponds to that ble for glutamate formation and not for glutamate of some bacteria (Miller and Stadtman, 1972) and degradation, as discussed in literature (Ahmed cyanobacteria (Srivastava and Amla, 1997); it is and Helleburst, 1991). Glutamate ought to be ad however almost two times larger than that re ditionally produced by the glutamine synthetase/ ported for higher plants (Hirel and Gadal, 1980; glutamate synthase system. Distribution of incor Mäck, 1998), unicellular algae (Meya and Kowal poration of exogenous nitrogen among both en lik, 1995) and the fungus Neurospora crassa (Ka zymes cannot reliably be decided. poor et al, 1969). The comparatively high aspartate-dependent ac Concerning respective significances of the vari tivity of aspartate transaminase (506 nmol x mg ous enzymes in vivo, the data obtained do not lead protein - 1 x min-1) certainly points to the already to unequivocal conclusions. Being determined in mentioned preferred action of aspartate, not gluta the same crude cell extract, i.e. based on identical mate conversion by the enzyme. But search for en total protein contents however, they should allow zymes producing aspartate as product of NH4+ in relative comparison of enzyme actions. A widely corporation was unsuccessful. Neither amination and controversially discussed question concerns of fumarate, nor reductive amination of oxaloace- incorporation of inorganic nitrogen, NH4+, into or tate could be detected. Therefore, inspite of the ganic molecules of an organism. Reductive anima much smaller glutamate-dependent activity tion of 2 -oxoglutarate and amidation of glutamate (39 nmol x mg protein - 1 x m in-1), we have to as are the respective reactions. sume mainly glutamate as NH2-donor for the en- N. Grotjohann et al. • Enzymes of Nitrogen Metabolism of Suillus bovinus 211 zyme, in vivo. The much better affinity for oxalo- mate in basic nitrogen metabolism of Suillus bovi acetate (K m = 0.13 mM) than for 2-oxoglutarate nus - as it is known for various other organisms - (K m = 1.46 m M ), and the pH-optimum for gluta is certainly indicated by all data obtained. mate conversion at about 7.0, and for aspartate conversion at about 8 .0 , certainly support this as Acknowledgements sumption which is also not contradicted by similar /Cm-values for aspartate and for glutamate. Y. H. is indebted to Prof. G. H. Schmid for finan Unfortunately, technical reasons prevented us cial support within the frame of a cooperation be from examination of glutamate dependence of ala tween Deutsche Forschungsgemeinschaft/Bundes- nine transaminase. We, therefore, can only assume, minister für Wissenschaftliche Zusammenarbeit that also this enzyme may function preferrably and National Natural Science Foundation of glutamate converting. A central position of gluta- China.

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