In vitro stability and functional properties of nitrate reductase from the ascomycete Sphaerostilbe repens

Mycol. Res. 94 ( 7 ) :985-992 (1990) Printed in Great Britain

985

In uitro stability and functional properties of nitrate reductase from the ascomycete Sphaerostilbe repens

A. ESSGAOURI A N D B. BOTTON* Uniuersiti de Nancy I, Laboratoire de Physiologie Vkgktale et Forestiire, B.P. 239, F-54506 Vandoeuvre-les-Nancy Cedex, France

In vitro stability and functional properties of nitrate reductase from the ascomycete Sphaerostilbe repens. Mycological Research 94 ( 7 ) : 985-992 (1990). The composition of the buffer for extraction of nitrate reductase (EC 1 . 6 . 6 . 3 ) from Sphaerostilbe repens was studied in order to obtain optimal and stable activity. A good extraction buffer consisted of 100 mM K-phosphate buffer, pH 7 5 , 1 % casein, 1 mMEDTA, 10 VM-FAD,1 PM-sodium molybdate and 5 % fresh weight PVPP. In spite of these protectants, nitrate reductase in crude extracts or submitted to gel filtration proved to be very unstable. Thus, fungal extracts were stable at -25 OC for about 3 d but at O0 the activity was reduced by half within 24 h and at 20' enzyme activity was completely lost in less than one hour. Stability was greatly improved when the enzyme was extracted in the presence of 1 m-PMSF and partially purified by a twostep procedure including ammonium sulphate fractionation and DEAE-Trisacryl chromatography. The optimal pH value for the reduction on nitrate was about 75. The enzyme was NADPH-specific and required the addition of flavin adenine dinucleotide for maximum activity. The Michaelis constants for nitrate and NADPH were 1.4 and 0032 mM respectively. Nitrate reductase activity was induced by nitrate and repressed by ammonium. The induction resulted in an atypical curve characterized by minor increase of activity at the 8th min of incubation on nitrate and a major increase 40 min later. words: Ascom~cete.Casein, Induction, Nitrate reductase, Repression, Sphaerosti/be repens, stability, Nitrate reductase, the first enzyme in the pathway of nitrate assimilation, is generally found t o be a molybdoflavoprotein that in fungi utilizes N A D P H as the preferred electron donor (McDonald & Coddington, 1974) or N A D H in some yeasts (Guerrero & Gutierrez, 1977). In spite of nitrate reductase having been extensively studied in a few fungi such as Neurospora crassa (Garrett & Nason, 1969), Aspergillus nidulans (McDonald & Coddington, 1974) and Hebeloma cylindrosporum (Plassard, Mousain & Salsac, 1984), there is very little information o n the properties of the enzyme from other fungi. This deficiency is in part due t o difficulties experienced in purifying the enzyme. Indeed, nitrate reductases in fungi, as in plants, generally appear t o be unstable. In order t o prevent loss of activity during extraction, numerous protectants have been incorporated in media for extraction of nitrate reductases. Addition t o the standard extraction medium of bovine serum albumin or casein (Schrader et al., 1974; Robin, 1979; Plassard et al., To whom reprint requests should be addressed. Abbreviations used in this account are as follows: DEAE: diethylaminoethyl; EDTA: ethylenediaminetetraacetic acid; FAD: flavin adenine dinucleotide; PMSF: phenylmethylsulphonyI fluoride; PVPP: polyvinylpolypyrrolidone.

1984) o r phenylmethylsulphony1 fluoride (Wallace, 1975) can improve the stability of the enzyme indicating that instability of nitrate reductase may be due t o proteolytic degradation. Sulphydryl protectants such as cystein, mercaptoethanol o r dithiothreitol have also been shown t o be efficient (Robin, 1979; Hageman & Hucklesby, 1971). In vitro stability of nitrate reductase has also been improved b y polyvinylpolypyrrolidone (Stulen, Koch-Bosmatt & Koster, 1971). Nitrate reduction is frequently stimulated by addition of FAD (McDonald & Coddington, 1974; Guerrero & Gutierrez, 1977) and phosphate; the effect of this latter compound has been attributed t o the requirement of a phosphomolybdate complex for the reduction process (Nicholas & Stevens, 1955). In most of the organisms, including fungi, nitrate reductase is generally regarded as a substrate-inducible enzyme. W h e n the nitrogen source is switched from nitrate t o ammonium, nitrate reductase usually decreases t o extremely low levels (Beevers & Hageman, 1980). Because of this behaviour, nitrate is often described as an inducer and ammonia a repressor of synthesis of the enzyme even though the molecular mechanisms are largely unknown (Guerrero, Vega & Losada, 1981). In several yeasts such a s Candida nitratophila and Hansenula wingei, the induction of enzyme activity is essentially

Nitrate reductase in Sphaerosfilbe repens due to de novo synthesis and changes in nitrate reductase activity levels in response to nitrogen sources could be correlated with amounts of nitrate reductase protein (Cannons, Ali & Hipkin, 1986; Jones, Wray & Kinghom, 1987). In Neurospora, it has been proposed that the role played by nitrate is not that of a true inducer. In fact, nitrate appears not to be essential for the transcription of nitrate reductase genes, but its presence results in both enhancement of transcription and protection of the enzyme against inactivation (Premakumar, Sorger & Gooden, 1979). It has also been shown that in green algae and fungi, nitrate reductase can exist in viva in two interconvertible forms, active and inactive, depending on the state of the molybdenum centre (Guerrero & Gutierrez, 1977; Funkhouser, 1980; De La Rosa, Gomez-Moreno & Vega, 1981; Minagawa & Yoshimoto, 1985). The antagonistic effect of ammonium does not always seem to be the same and significant differences are found in this regard between fungi. Usually, ammonium overrides any stimulating effect of nitrate and enzyme activity is totally absent (or present only at basal levels) both in ammonium and in ammonium-nitrate containing medium (Guerrero ef al., 1981; Jones ef al., 1987). However, the basidiomycete Hebeloma cylindrosporurn grown in presence of either ammonium or nitrate has the same nitrate reductase activities, suggesting that the enzyme is not induced by nitrate in this organism (Plassard et al., 1986). In this work we examined the effect of nitrogen sources on the appearance of nitrate reductase activity in Sphaerostilbe repens after having developed an extraction buffer for stabilizing activity during extraction and assay of the enzyme. As we d o not know the molecular mechanisms which underlie such changes, we used the terms ' induction ' and ' repression ' as operational terms to define either an increase or decrease in enzyme activity. Sphaerosfilbe repens grown in pure culture differentiates aerial synnemata and immersed rhizomorphs. These two structures are anatomically continuous and are formed of juxtaposed hyphae growing in the same direction (Botton, 1983). Preliminary results have shown that nitrate, unlike reduced nitrogen molecules, was a poor differentiating compound. Moreover, once differentiated, rhizomorphs proved to be more adapted to nitrate uptake than vegetative mycelium (Essgaouri, 1984). It appeared then of importance to study this organism nitrate reductase which is the first enzyme involved in the pathway of nitrate assimilation.

MATERIALS A N D M E T H O D S Fungus culture and enzyme extraction

Sphaerosfilbe repens (strain CBS 275.60) was grown at 28' on Bertrand basal liquid medium modified so that sodium nitrate was the sole nitrogen source (Botton & Msatef, 1983). Nitrogen concentration was, unless specified, maintained constant at 1 g 1-' throughout the experiments. Material was harvested after 8 d incubation and immediately macerated in the presence of acid-washed sand with 6 ml buffer g-' fresh material in a chilled mortar and pestle. The macerate was immediately squeezed through two layers of cheesecloth, and centrifuged for 20 min at 40000 g. The supernatant could

986 serve as crude extract for enzyme assays but was usually desalted with Sephadex G 25 prior to assay. All extraction procedures were carried out at 0 to 3'. The initial extraction buffer was 0.1 M K-phosphate, pH 7.5 containing 1% casein, 1 m~-EDTA,10 VM-FAD,1 VM-N~ molybdate and I % PVPP. For subsequent studies, the extraction buffer was sequentially modified to provide maximal nitrate reductase stability. Enzyme purification Ammonium sulphate fracfionation. The crude extract was brought to 20 % ammonium sulphate saturation. After stirring for 10 min the solution was centrifuged at 40000 g for 20 min to sediment precipitated proteins which were discarded. The supernatant was brought to 80% saturation by stepwise addition of ammonium sulphate. The precipitate was dissolved in the extraction buffer containing 1 m ~ - P M S Fdissolved in a minimum volume of isopropyl alcohol. DEAE-Trisaql chromatography. Fractions with the highest activities were pooled, desalted by diafiltration on AMICON PM 10 (AMICON Corporation, Lexington) and washed with the extracting buffer. The volume was applied to a DEAE-Trisacryl column (Pharmacia) (170 x 60 mm) equilibrated with 0.01 M Tris-HC1 buffer at pH 7.5. After washing the column with the same buffer until all unabsorbed proteins were removed, nitrate reductase was eluted with a linear in ~Tris-HC1 buffer. Fractions gradient from 0 to 0.4 M - N ~ C were collected in tubes containing 2 ml of extraction buffer with 1mM PMSF. The total volume of the fractions was 4 ml and the flow rate was 20 ml h-'. Enzyme assay

Activity of nitrate reductase (NADPH) was measured calorimetrically by estimating nitrite formation with the diazo coupling procedure described by Nicholas & Nason (1957) slightly modified. The assay mixture comprised 1ml of 1% (w/v) sulphanilic acid in 3 N-HCl, 1 ml of 0.02% (w/v) N-(1naphtyl) ethylenediamine dihydrochloride and nitrite extract to give a final volume of 3 ml. The colour intensity was determined at 540 nm after 20 min at 20'. The presence of added reduced pyridine nucleotides at usual enzymic assay levels did not interfere with colour development. The reaction mixture giving rise to nitrite contained 50 mM K-phosphate buffer, pH 7-5, 10 m ~ - N a N O , ,0.4 m ~ - N A D P H and 200 ~1 of fungal extract in a total volume of 1.1 ml. Incubation was for 30 min at 30'. The reaction was terminated by adding 100 pl of 1 M-ZnSO,. After centrifugation for 20 min at 5000g, 1 ml of supematant was used for nitrite determination. Under these standard assay conditions it was determined that nitrite reductase activity was negligible and no inhibitor of this enzyme was added to the reaction mixture. Procedure for transfer experiments

The fungus was grown for 8 d in 250 ml Erlenmeyer flasks containing 130 ml of liquid medium to which was added sodium nitrate or ammonium chloride to give 1 g nitrogen I-'.

A. Essgaouri and B. Botton Mycelia were harvested, washed with distilled water and transferred under sterile conditions to fresh medium containing the appropriate nitrogen source. The fungus was harvested at various times after the transfer and the results were based on a sample of four colonies at each time.

98 7 Table I. Effect of two extraction buffers on stability of nitrate reductase from 5. repens Extraction buffers T i e after extraction

K-phosphate 100 m ~ pH , 75

Tris-HCI 100 m ~ pH , 7.5

Protein estimation

Protein in the eluates from chromatography columns was estimated by absorbance at 280 nm. Protein in the most active fractions was measured according to Bradford (1976) using bovine serum albumin as a standard.

RESULTS AND DISCUSSION

6 g of fresh material were homogenized in 2 0 ml of either 100 mM Kphosphate buffer, pH 7.5, or 100 mM Tris-HC1 buffer, pH 7 5 . Both buffers included optimal concentrations of casein, EDTA, FAD, Na molybdate and PVPP. The crude extract was filtered through a Sephadex G 25 column before being assayed as described in the experimental part. Extracts were maintained at -ZOO between assays. Results are expressed in m o l e s NO,- released g fresh mass-' h-'.

Stability and partial purification Influence of protective agents. K-phosphate buffer 100 mM, Increasing polyvinylpolypyrrolidone (insoluble PVP) conpH 7.5 proved to be a better extraction buffer than Tris-HCI used at the same molarity and pH. Indeed, as shown in Table centrations from 0 to 5% of the fresh weight of material led to a doubling of enzyme activity (Fig. IF). Higher con1, nitrate reductase activity, immediately after extraction was doubled when the enzyme was extracted with phosphate centrations did not adversely affect the activity. Nitrate reductase was rapidly inactivated in extracts buffer.Furthermore, this buffer prevented to a large extent the rapid decline in enzyme activity. Thus, after one day at - 20°, maintained at 20°, no activity being found after 1 h (Table 2). 60% of the initial activity was retained by using phosphate At O0 the rate of inactivation was slower but it was only at buffer, while only 8-8% was recovered in the presence of Tris- - 25O that the enzyme activity was maintained at a high level for several days; in this case 70% of the activity was retained HCl buffer. Nitrate reductase activity was significantly improved by 72 h after extraction. In order to improve the stability of nitrate reductase, PMSF, increasing phosphate buffer concentrations from 10 to 100 mM (Fig. IA). Higher concentrations slightly reduced enzyme an inhibitor of serine proteases was used at several concentrations in the extraction medium. Although PMSF did activity. Evaluation of the extraction buffer pH indicated that pH 7.5 allow some increase in the recovery of nitrate reductase, it resulted in the most active nitrate reductase and optimal only partially prevented its inactivation in vitro (Table 3). The enzyme stability was also found to be at this pH value (not percentage of recovery increased from 39% in the absence of shown). PMSF to 55 % in the presence of I m ~ - P M S F .Higher levels M in the The addition of sodium molybdate at , were not completely of protease inhibitor (up to 10 m ~ )which extraction buffer increased nitrate reductase activity by 48% soluble, only slightly improved the recovery and stability of (Fig. 1B). Higher and lower concentrations were less efficient. nitrate reductase in preparations of Sphaerostilbe repens. These This is in agreement with earlier reports which clearly results are similar to those obtained by Wallace (1975) in demonstrated the requirement for molybdenum for nitrate maize root samples where PMSF gave also a ~ a r t i aprotection l reduction (Beevers & Hageman, 1980). of nitrate reductase. Nitrate reductase activity increased with increasing casein concentrations in the extraction buffer until a maximal value Partial purification and effect on stability. The first step of of 1% (w/v) and decreased rapidly beyond this concentration purification with ammonium sulphate fractionation proved to (Fig. IC). In absence of casein, only 17% of the enzyme be efficient as the nitrate reductase peak appeared with a salt activity was retained. This compound can act as an alternate concentration of 30 to 50% while maximum proteins substrate for proteolytic enzymes (Wallace, 1975). However, precipitated between 50 and 60% of saturation (Fig. 2). the stabilizing effect of casein has, in addition, been diversely The four peak fractions with the highest activities were interpreted. It may also protect the enzyme molecule from pooled and loaded on a DEAE-Trisacryl column. Only one dissociation into subunits or may form hydrogen-bond peak of nitrate reductase appeared which was eluted at complexes with phenolic compounds, thus removing them as 0.15 ~ - N a c(Fig. l 3). In this second step of purification, nitrate inhibitors of nitrate reductase (Robin, 1979; Guerrero et al., reductase was purified 20-fold with 10% of initial total 1981). activity. This overall recovery, which was very low, is likely EDTA concentrations ranging from 1 to 2 mM provided due to the instability of the enzyme during the purification maximum nitrate reductase activity while other concentrations steps. However, the three peak fractions collected from the did not modify the enzyme activity (Fig. ID). FAD column in the presence of protectants and combined, showed M and M provided maximum concentrations at that the partially purified nitrate reductase was much more stabilizing effect during nitrate reductase extraction (Fig. IE). stable than the crude extract (Table 4). After 80 h of The effect of exogenous FAD may be attributed to maintaining incubation at o0 the enzyme retained 20% of its catalytic prosthetic group integrity (Beevers & Hageman, 1980). activity and it was very stable at -25'. It may be that partial

Nitrate reductase in Sphaerostilbe repens

988

Fig. 1. Effects of different compounds used in the extraction buffer on the activity of nitrate reductase of Sphclerostilbe repens. A. Effect of phosphate buffer concentrations. Fungal material was ground in different concentrations of K-phosphate buffer, pH 7 5 , each molybdate and PVPP (5% of the fresh weight of material). B. Effect of sodium containing 1 % casein, I m ~ - E D T A ,10 DM-FAD,I D M - N ~ molybdate in the extraction buffer on the activity of nitrate reductase. The extraction buffer contained 100 mM phosphate buffer pH 7.5 with different concentrations of sodium molybdate and other components as in (A). C. Effect of casein on the activity of the enzyme. The extraction buffer contained 100 mM phosphate buffer pH 7.5 and variable concentrations of casein as indicated. Other conditions as in (A). D. Effect of EDTA concentration on nitrate reductase activity. The extracts were made in 100 mM phosphate buffer pH 7.5 in the presence of varying concentrations of EDTA as indicated. Other conditions as in (A). E. Effect of FAD o n the enzyme activity. The extracts were made in 100 m u phosphate buffer pH 7.5 in the presence of varying concentrations of FAD as indicated. Other conditions as in (A). F. Effect of PVPP on the enzyme activity. The extracts were made from 6 g of fresh material ground in 100 m~ phosphate buffer pH 7 5 containing variable concentrations of PVPP expressed in percent of fresh weight. Other conditions as in (A).

100 200 300 400 Phosphate buffer concn ( m ~ )

1o-I0

Casein % in the extraction buffer

IO-~

lo-'

10-6 FAD concn

10-5 (M)

104

lo4 lo4 Molybdate concn (M)

EDTA concn ( m ~ )

10

20 30 40 PVPP (% of fresh wt)

50

A. Essgaouri and B. Botton Table 2. In vitro decay of nitrate reductase activity in extracts of S. repens maintained at 20'. O0 and -25O

Time after extraction (h)

20°

0°

- 25O

Nitrate reductase activity

Nitrate reductase activity

Nitrate reductase activity

YO

%

%

The enzyme was extracted in the presence of K-phosphate buffer, casein, EDTA, FAD, PVPP and sodium molybdate. The crude extract was passed through a Sephadex G-25 column before being assayed as described in the experimental part. Nitrate reductase activities are expressed in moles NO,- released g fresh mass-' h-1.

Fig. 2. Recovery of nitrate reductase and protein using ammonium

Table 3. Effect of PMSF in extraction buffer on activity and stability of nitrate reductase of S. repens

sulphate fractionation. T h e crude extract containing 300 m g protein was precipitated between 2 0 and 8 0 % saturation by increasing steps of 5 % saturation. Proteins were estimated according t o Bradford (1976).

Nitrate reductase activity (moles NO,- released g fresh mass-' h-l) PMSF concn (m~)

At the time of extraction

24 h after extraction

f-$LI .-

m

A

6

9

-rb" HZ, -2 2

.-

5 g of fresh material were homogenized in 20 ml of 100 mM K-phosphate bdfer including optimal concentrations of casein, EDTA, FAD, PVPP and sodium molybdate. The crude extract was passed through a Sephadex G25 column before being assayed as described in the experimental part. Extracts were maintained at O0 between assays.

23

25

35

45

55

65

75

Ammonium sulphate (% saturation)

Fig. 3. Elution profile of nitrate reductase from DEAE-Trisacryl. The column was loaded with 1 0 ml of the resuspended 30 t o 5 0 % C~ ammonium sulphate fraction containing 46.4 m g protein. Nitrate reductase was eluted with a linear gradient from 0 t o 0.4 M - N ~ in Tris-HC1 buffer. 400

-

:.

.-

1

300

U

m ,

a -i

2 =

5 b" 200 F a 0 E g 3 loo

-

V1

10

20

30

40

Fraction number

50

60

70

990

Nitrate reductase in Sphaerostilbe repens Table 4. In vitro decay of nitrate reductase activity from extracts of S. repens partially purified - 25O

0°

Time after extraction

(h)

Nitrate reductase activity

%

Nitrate reductase activity

YO

The enzyme was extracted under optimal conditions and partially purified by ammonium sulphate fractionation and DEAE-Trisacryl chromatography. Fractions with the highest activities, collected from the column and mixed up with the protectants were assayed as indicated in the experimental part. Nitrate reductase activities are expressed in pmoles NO,- released g fresh mass-' h-l.

Fig. 4. Effect of pH on nitrate reductase activity. The enzyme was extracted from 8-d-old thalli under optimal conditions, partially purified by using ammonium sulphate fractionation and DEAETrisacryl chromatography and assayed as described in the methods section. Maximum activities were about 300 umoles NO,-released. h-'.

purification protected the enzyme by removing inactivating or proteolytic enzymes. A few experiments have shown that thiols (mercaptoethanol, dithiothreitol), NADPH and nitrate improved the stability of the enzyme only slightly (not shown).

Functional properties

The apparent Km value for nitrate was found to be 1.4 mM. Although the same value has been reported by Nason & Evans (1953) in Neurospora crassa, this Michaelis constant is ten times greater than most of those previously reported. However, assuming an even distribution in the cells, nitrate concentration in S~haerostilberepens was estimated to range from 4 to 8 mM, depending on the nitrogen sources (nitrate or ammonium nitrate) and on the tissues (vegetative mycelium or rhizomorphs) (Essgaouri, 1984). This nitrate accumulation is three to six times higher than those estimated in several basidiomycete fungi (Plassard et al., 1984). In view of these estimations, it seems likely that the enzyme can operate in vivo in spite of lower affinity for nitrate. The Km value for NADPH was found to be 0-032 mM and is in agreement with numerous values found in other fungi (Beevers & Hageman, 1980). With respect to cofactor requirements, NADPH was an effective electron donor for nitrate reductase. No activity was found when NADH was used as cofactor.

Induction and repression of nitrate reductase activity. After 8 d of culture on ammonium nitrogen, Sphaerostilbe repens mycelia were transferred onto fresh media to which 0 to 120 mM sodium nitrate was added as sole nitrogen source. Nitrate reductase activity increased with increasing concentrations of nitrate up to 20 mM then slightly declined at higher concentrations (Fig. 5); half-maximal induction was obtained at a concentration of approximately 8 mM. At molar concentrations of 0.06 and 0.12, nitrate reductase activities were, respectively, 88 and 80% of the value obtained at optimum concentrations. Such an optimal concentration has already been reported for Neurospora crassa, but it was much lower (4.7 m ~ )moreover, ; the half-maximal induction found in this organism was at a concentration of 0.1 m ~ - N a N o , (Kinsky, 1961). No nitrate reductase activity was detected when the fungus was grown in presence of ammonium as sole N-source. Figure 6 shows the effect of incubation time on the induction of nitrate reductase. Enzyme activity was detected after a lag period of approximately 8 min; thereafter the

Fig. 5. Effect of NaNO, concentration on nitrate reductase induction. The growth medium was modified to contain the final concentrations of nitrate indicated on the abscissa. After 2 h incubation, thalli were removed and extracts prepared under optimal conditions then assayed for nitrate reductase as described in the experimental part.

Optimum pH. With nitrate reductase extracted under optimal conditions and partially purified, the optimum pH for the enzyme activity was in the range 7.2 to 7.6 (Fig. 4). These values are in agreement with those required by the extracting buffer. The pH value of 7.5 adopted throughout this work was also optimal for nitrate reductase activity in Neurospora crassa (Nason & Evans, 1953) and in Aspergillus niger (Thiery, 1977). ~ i n e f i cconstants for nitrate reduction. The substrate saturation curves were analysed by Lineweaver and Burk plots.

NaN03 concn (mM)

A. Essgaouri and B. Botton

991

Fig. 6. Time course of enzyme induction. Thalli previously grown for 8 d on ammonium were transferred on media containing 78 mM sodium nitrate. After incubation for various times, thalli were harvested and extracts prepared and assayed for nitrate reductase under optimal conditions as indicated in the methods section. I

Induction time (min) Fig. 7. Time course of enzyme repression by ammonium. Thalli were grown for 8 d on media containing sodium nitrate (*-a) or ammonium nitrate (0-0).then transferred on media containing 78 mM ammonium chloride. After incubation for 24 and 48 h, thalli were harvested and extracts prepared and assayed for nitrate reductase under optimal conditions as indicated in the methods section.

previously grown in the presence of sodium nitrate, was transferred into media containing ammonium chloride as a sole N-source, nitrate reductase specific activity decreased approximately exponentially with a half-life of about 1 7 h. This value is considerably higher than those reported for other fungi and higher plants (Cove, 1966; Guerrero e f al., 1981). A s already suggested for the induction of the enzyme, the heterogeneity of the fungal tissues due t o the presence of rhizomorphs, might explain a slow diffusion of ammonium towards the internal cells and consequently the enzyme repression which was spread over several hours. In the presence of ammonium nitrate, the initial rate of nitrate reductase activity was reduced b y 7 % and declined slowly with more than 5 0 % of the activity remaining after 48 h (Fig. 7). It appears therefore, that not only the synthesis of active enzyme, but also the rate of its decline was dependent o n the nitrogen sources in the culture medium. Nitrate reductase from Sphaerosfilbe repens appears t o b e extremely labile but its stability can be greatly improved with a mixture of protectants. These investigations, especially those with PMSF and casein, suggest that nitrate reductase instability is almost certainly the result of protease action. Nitrate reductase appearance with its lag period of 8 min is similar t o that observed in other filamentous fungi, although the induction curve with t w o peaks of activity is different from results so far reported. Nitrate reductase in Sphaerosfilbe repens appears not only t o b e induced b y nitrate but also t o b e repressed b y ammonium. However it is not known whether nitrate is essential t o induce this capacity. The development of the fungus in nitrogenstarved culture would be useful t o obtain a better understanding of the regulation of this enzyme in Sphaerostilbe repens. W e are grateful to Georges Durand and Jacques Banvoy for technical assistance during the investigation and Mrs Lysiane Patard for typing the manuscript.

0

24

48

Incubation time (h) induction was found t o b e discontinuous as a function of time. There was a rapid increase of activity at the 8th min of induction, then after 3 0 min, enzyme activity resumed maximum activity. A similar result was obtained in Aspergillus (= Emericella) nidulans which was found to be unable to maintain continuous enzyme synthesis in the presence of inducer (Cove, 1967). In Sphaerostilbe repens it has been shown b y using mycelial and rhizomorphic mutants that rhizomorphs are much more adapted to nitrate nutrition than vegetative mycelium (Essgaouri, 1984). Consequently, a possible explanation might be that the initial rate of nitrate reductase activity corresponded t o the induction of the vegetative mycelium, while the resumption of activity which occurred from the 50th hour, was the result of the rhizomorph induction. However, additional experiments are needed t o explain this discontinuity of nitrate reductase activity in Sphaerosfilbe repens. The time course of repression of nitrate reductase by ammonium is shown in Fig. 7. When Sphaerostilbe repens,

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CANNONS, A,, ALI, A. H. & HIPKIN, C. R. (1986). Regulation of nitrate reductase synthesis in the yeast Candida nitratophila. lournal of General Microbiology 132,2005-2011. COVE, D.1. (1966).The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochimica et Biophysics Acta 113, 51-56.

COVE, D. J. (1967). Kinetic studies of the induction of nitrate reductase and cytochrome c reductase in the fungus Aspergillw nidulans. Biochemical Journal 104, 1033-1039.

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(Received for publication 17 March 1989)

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