Plant Physiol. (1983) 71, 582-5870032-0889/83/7 1/0582/06/$00.50/0

Reversible Inactivation of Nitrate Reductase by NADH and theOccurrence of Partially Inactive Enzyme in the Wheat Leaf'

Received for publication October 6, 1982 and in revised form November 8, 1982

ARUN P. ARYAN, RICHARD G. BATT, AND WILLIAM WALLACEAgricultural Biochemistry Department, Waite Agricultural Research Institute, University ofAdelaide,Glen Osmond, South Australia 5064, Australia

ABSTRACT

Nitrate reductase from wheat (Tititum aestivum L. cv Bindawarra)leaves is inactivated by pretreatment with NADH, in the absence of nitrate,a 50% loss of activity occurring in 30 minutes at 25C with 10 micromolarNADH. Nitrate (50 micromolar) prevented inactivation by 10 micromolarNADH while cyanide (1 micromolar) markedly enhanced the degree ofinactivation.A rapid reactivation of NADH-inactivated nitrate reductase occurred

after treatment with 0.3 mlllimolar ferricyanide or exposure to light (230milliwatts per square centimeter) plus 20 micromolar flavin adenine dinu-cleotide. When excess NADH was removed, the enzyme was also reacti-vated by autoxidation. Nitrate did not influence the rate of reactivation.

Leaf nitrate reductase, from plants grown for 12 days on 1 milolarnitrate, Isolated in the late photoperiod or dark period, was activated byferricyanide or light treatment. This suggests that, at these times of theday, the nitrate reductase in the leaves of the low nitrate plants is in apartially inactive state (NADH-inactivated). The nitrate reductase frommoisture-stressed plants showed a greater degree of activation after lighttreatment, and inactive enzyme in them was detected earlier in the photo-period.

It has been established that Chlorella NR2 is converted into areduced inactive state when incubated in vitro with NADH, in theabsence of nitrate (for review, see Ref. 28). The inactivation ismediated by the binding of a low level of cyanide (.l0-10M) tothe molybdenum of the reduced enzyme, blocking electron trans-fer to nitrate. Rapid reactivation ofNR occurs when it is oxidizedby ferricyanide (17) or exposed to blue light in the presence ofFAD (2). A slow reactivation is obtained by incubation withnitrate (17). This inactivation process and the binding of cyanidehas been shown to occur in vivo in Chlorella (13).A similar inactivation of NR from higher plants has been

demonstrated (12, 19, 23, 29) and the involvement of cyanideimplicated (12, 29). Reactivation by ferricyanide (12, 19, 29), light+ FAD (2), or trivalent manganese ions (15) has been demon-strated but reactivation by nitrate has not been reported. Further,the isolation of an inactive form of NR which can be reactivatedby oxidants and has been recorded in Chlorella (13, 21) has notbeen demonstrated in higher plants. It is likely that, under certainconditions (20, 30), the flux of nitrate to the leaf cell might beinterrupted and the NR converted to a reduced inactive state,

'Supported by a scholarship (A. P. A.) from University of AdelaideResearch Grant.

2Abbreviations: NR, nitrate reductase; FAD, flavin adenine dinucleo-tide.

especially in plants grown on limited nitrate.In this paper, the physiological significance of the inactivation

of wheat leaf NR by NADH is evaluated. We have determinedthe level of NADH required to inactivate NR, the requirementsfor its reactivation, and the occurrence of inactive NR in the wheatplant.

MATERIALS AND METHODS

Plant Material. Wheat (Triticum aestivum L. cv Bindawarra)was grown on washed sterilized sand, supplied daily with 0.25strength Hoagland nutrient solution (8) containing 10 mm nitrate(2.5 mm Ca[NO3J2, and 5 mM KNO3). In some experiments, asindicated, the nitrate level was 1 mm. The plants were grown in aphytotron with light (4000E m 2 s-' PAR) from 7.00 to 19.00 hat 20°C and in dark at 15°C. Plants were harvested at 12 d (secondleafjust appearing). Unless stated, the harvest was at 5 h after thestart of the photoperiod.

Isolation and Partial Purification ofNR. The extraction medium(5 ml/g sample fresh weight) as 0.1 M K-phosphate (final pH 7.5),5 mM EDTA, 1 mM DTT, 10 tiM FAD, 3% (w/v) casein (Ham-marsten grade), and 2.5% (w/v) Polyclar AT (insoluble PVP).After maceration with a mortar and pestle, the sample was centri-fuged at 20,000g (5 min). Unless stated, the supernatant waspassed through a Sephadex G-25 column equilibrated with 50 mmK-phosphate (pH 7.5) containing 0.1 mm DTT and 10 ,UM FAD.Sephadex G-25 (coarse grade) was obtained from Pharmacia andprepared according to the manufacturers instructions. For anenzyme sample of 15 ml, a 4.3-cm2 x 21-cm column was used.The precipitate with 40%o saturation of (NH4)2SO4 was obtainedby adding 0.226 g/ml of salt (Mann 'ultra pure' grade) to theenzyme sample, mixing for 20 min, and centrifuging at 20,000g(10 min). The pellet fraction was resuspended in one fifth oforiginal volume with 50 mm K-phosphate (pH 7.5) containing 0.1mM DTT and 10 iLm FAD and finally passed through a SephadexG-25 column as described above. All operations were performedin the range of 0°C to 4°C.

Preincubation and Assay of NR. Desalted enzyme sample (usu-ally diluted x 5) was incubated at 25°C (0.1 ml) with 0.1 ml 50mm K-phosphate (pH 7.5) containing NADH and other reagentsas indicated. For the NR assay, 0.8 ml of the following reactionmixture was added: 0.5 ml 0.1 M K-phosphate (pH 7.5), 0.1 ml0.05 M KNO3, 0.1 ml H2O, and 0.1 ml 2 mm NADH. After 15 minat 25 °C, the reaction was terminated, excess NADH oxidized, andnitrite determined as described by Shannon and Wallace (22). Inone study where initial reaction rate was determined, the sameNR assay mixture was used and the rate of NADH oxidationmeasured at 340 nm. Levels ofNADH and nitrate included in thepreincubation mixture were shown not to interfere with the sub-sequent assay of the enzyme. All NADH solutions were freshlyprepared in 25 mm K-phosphate (pH 7.5).

Procedure for Reactivation of NR. In the experiments on the582

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INACTIVATION OF WHEAT LEAF NITRATE REDUCTASE BY NADH

reactivation of in vitro inactivated NR or occurrence of inactiveNR in the plant, all operations were performed in diffuse redlight. To test for light reactivation ofNR, the FAD content of theenzyme sample was increased to 20 LM and the sample exposed tolight at 230mw cm 2 (PAR) for 10 min in a glass cuvette, jacketedwith circulating iced water. The light source was a standard KodakCarousel projector (24 v, 150 w lamp) and the sample was placedjust in front of the main lens (focus = 150 mm). The temperatureofthe enzyme sample was maintained below 6°C. For ferricyanidereactivation, 0.1 ml NR sample was incubated with 0.05 mL 0.9mM K3Fe(CN)e for 5 min at 25°C before assay of NR activity.The ferricyanide solution was prepared in 0.2 M K-phosphate (pH7.5) and this buffer was also used in the subsequent assay of NR.

RESULTS

Inactivation of Wheat Leaf NR In Vitro with NAD(P)H. Theinclusion of casein, DTT, and FAD in the extraction mediumstabilized NR in crude extracts of wheat leaves (Fig. 1). AdditionofNADH resulted in an exponential loss ofNR activity. With apartially purified sample of NR (40% [NH4J2SO4 ppt, l10-foldpurification) the addition of 10 jtM NADH gave a 50% loss ofNRactivity in 30 min at 250C (Fig. 2). A much higher level ofNADPH (Q70 ,UM) was required to produce the same effect. Theactivity of the wheat leafNR with NADPH was only 3% of thatwith NADH. It is also shown in Figure 2 that a small fraction ofthe enzyme (-0%) was not inactivated, even when relatively high

120 1

4-1

~o04

a4

I-

0

z

0 20 40 60

Time ( min )FIG. 1. Inactivation of wheat leafNR by NADH. A crude extract was

prepared and passed through a Sephadex G-25 column as described in

"Materials and Methods." It was preincubated at 25°C with NADH (0.3mm) added as indicated. The initial NR activity was 0.34 Amol N02produced min-' g-' fresh weight.

~g200

NA D PH

E 00

70 NADH

'0 50 o0~~~~z o0s30

0 100 200 500

NAD(P)H (j,M)FIG. 2. Inactivation by NADH and NADPH. The NR sample was the

precipitate fraction of the leaf extract obtained with 40% saturation with(NH4)2S04 and finally passed through a Sephadex G-25 column. Theinitial extraction medium, final enzyme buffer, and preincubation proce-dure for 30 min were as described in "Materials and Methods.

2.5

2.0

N

x0

Xa4cz

o3 .02

0.8

0.6. * A

0 20 40 60 80

Time (min )FIG. 3. Nitrate modulation of inactivation of NR by NADH. The

enzyme sample prepared as described in Figure 2 was preincubated at25°C with 0 (0) or 10 pm NADH and either nil N03- (A), 5 pM N03(0), 10 pM NO3 (3), or 25 pA NO3 (0). At times indicated, 0.2 ml wastested for NR activity by the rate ofNADH oxidation.

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Plant Physiol. Vol. 71, 1983

00

801

4

w

C')

w

wI-

4

60

401

20 [

0

FIG. 4. Cyanide enhancement of inactivation of NR by NADH. The enzyme sample was a desalted crude extract prepared as described in Figure1. It was preincubated at 25°C for 30 min with the NADH and KCN levels shown, before assay ofNR activity remaining. A sample incubated in thesame way without NADH or cyanide had an activity of 0.42 ,umol N02- produced min-' g-' fresh weight.

Table I. Reactivation of In Vitro Inactivated NR by Ferricyanide andLight

A desalted crude extract of a leaf sample from I mM nitrate-grownplants was prepared as described in "Materials and Methods." Afterpretreatment with NADH for 30 min at 25°C, excess NADH was removedon a Sephadex G-25 column equilibrated with 50 mm K-phosphate (pH7.5) containing 10IOM FAD. The ferricyanide treatment was 5 min at 25°Cand light exposure was 10 min at 230 mw cm-2 (PAR) and 6°C. Fulldetails are given in "Materials and Methods."

NR Activity

Pretreatment After treatment withwith NADH (0.3 mM) Initial

Ferricyanide Light + 20(0.3 mM) pM FAD

nmol NO2- produced min-' g-'fresh wt215 214 212

+ 54 199 172

levels of NAD(P)H were used.To study the effect of nitrate on NADH pretreatment of NR,

the activity of the enzyme was measured by the initial rate ofNADH oxidation (Fig. 3). It is again shown that the enzyme was

quite stable during incubation at 25°C for I h. When 10 ,MNADH was added, there was a rapid loss of activity which wasdecreased with the inclusion of nitrate. Even in the presence of 25«tm nitrate, there was still a 10%o loss of activity. The inactivationof NR by 10 UM NADH was prevented by 50 ,LM nitrate. When

the NADH-inactivated NR (pretreated with 10 ,UM NADH) wasincubated for up to 3 h with 100 ,LM nitrate, no reactivation of theenzyme was observed (data not shown).The effect of exogenous cyanide on the inactivation of NR in

a crude extract, which had been passed through a Sephadex G-25column, is shown in Figure 4. Low levels of cyanide (1-3 uM)resulted in a small inhibition ofNR but markedly stimulated thedegree of inactivation by 5 ,UM NADH. Similarly, the portion ofNR not inactivated by relatively high levels of NADH (-20%o inFig. 4) was reduced to a low level by the addition of cyanide.

Reactivation of NADH-Inactivated NR. In this study, the NRsample was preincubated with NADH for 30 min at 25°C andexcess NADH removed by passing the sample through a SephadexG-25 column (a control was run in which the NR sample waspreincubated in the absence of NADH). It is shown in Table Ithat treatment of the NADH-inactivated NR with 0.3 mm ferri-cyanide for 5 min at 25°C resulted in almost complete recovery ofenzyme activity. A high level of reactivation was also found onexposing the inactive enzyme to an intense source of light for 10min in the presence of 20 ,UM FAD. The conditions specified fortreatment with ferricyanide and light were found to be optimalfor reactivation. Ferricyanide reactivation also occurred in thepresence of NADH.

After excess NADH was removed by gel filtration, a slowreactivation occurred in the dark at 25°C (Fig. 5). This reactiva-tion, presumably resulting from an autoxidation of the NR en-

zyme, was slower in the presence of 0.1 mM DTT. Treatment withferricyanide after incubation for 1 h at 25°C showed that the sameamount of NR was present in the DTT and DTT-free samples(Fig. 5), and the NR activity was now higher than in the initialenzyme sample. DTT at 0.1 mm did not interfere with the acti-vation ofNR by 0.3 mm ferricyanide. Complete reactivation hadalso occurred in both samples, after 24 h at 0°C in the dark. Therate of reactivation at 25°C and 0°C was not enhanced by I mmnitrate.

Partialy Inactive NR in Wbeat Leaves. Wheat plants suppliedwith a low level of nitrate (1 mM) were investigated, and all

NADH (,uM) cyanide (pM)

5 pM lOOm 1 2 3 510NADH NADH

5 + +

10 cyanide cyanide50

qu~(m) (PM)

500 1 2 3I.I-1I____TT2 F3]

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INACTIVATION OF WHEAT LEAF NITRATE REDUCTASE BY NADH

I. I

C6

o T. 0I. M

0z

0

0 1 5 30 45 60

TIME mn )FIG. 5. Autoxidation of NADH-inactivated NR. The enzyme sample

was prepared and treated with NADH as described in Table I. In theSephadex G-25 step to remove excess NADH, part of the sample was

passed through a column with 0.1 mM DTT included in the buffer. Bothsamples were then incubated at 25°C for 60 min followed by treatmentwith 0.3 mm ferricyanide as described in "Materials and Methods." NRactivity of initial enzyme sample was 172 nmol NOI- produced min-' g-'fresh weight.

Table II. Activation of NRfrom Wheat Leaf and Root Extracts

Plants grown on 1 mm nitrate were harvested at 22.00 h. The initialextraction medium and Sephadex column buffer are described in"Materials and Methods" except that no thiol was used in the columnbuffer. Light and ferricyanide treatments were as in Table I.

NR Activity

SampleAfter treatment with

Initial Ferricyanide Light + 20

(0.3 mM) pm FAD

nmol N02- produced min-' g-'fresh wt

LeafCrude extract 112 112 1IISephadex-treated 132 234 189

RootCrude extract 12 11Sephadex-treated 20 20

enzyme extraction and assay procedures were performed underred light. Evidence for the occurrence of inactive NR in theseplants is shown in Table II. With the initial crude extract, it wasnecessary to add I mm DTT to stabilize NR. Under these condi-tions, ferricyanide and light treatment did not increase the enzymeactivity (Table II). When the thiol was omitted from the Sephadexcolumn buffer, the gel-filtered enzyme was reactivated by ferri-cyanide and light. The increase in the initial activity after Sepha-

Table III. Occurrence of Partially Inactive NR in Wheat Leaves atDifferent Times of the Day

Plants grown on I mm nitrate were harvested at the times indicated-the photoperiod being 07.00 to 19.00 h. Nutrient solution was supplied atthe start of the dark period, on the day prior to the enzyme extraction, butwas omitted from one batch of plants investigated subsequently as'stressed.' Initial NR activity was measured on the crude extract, whileferricyanide and light activation were tested immediately after this extractwas passed through a Sephadex G-25 column as described in Table I.

Activation by Treatment withTime Initial NR Activity

Ferricyanide Light

nmol N02- produced %min-' g-lfresh wt

06.00 56 29 1310.00 85 23 214.00 103 31 116.00 85 31 417.30 60 55 2320.30 52 81 3524.00 39 54 22

Stressed14.00 40 62 4417.30 46 89 70

dex treatment may have resulted from the removal of someinhibitory compound(s) or autoxidation of the enzyme. Althougha similar increase in activity of root extracts was obtained afterSephadex treatment, there was no additional activation by ferri-cyanide. In a further study (Table III), the enzyme was isolated atdifferent times of the day and tested for activation with ferricya-nide and light. Plants subjected to moisture stress were alsoexamined. The NR activity of crude extracts reached a maximumat 14.00 h followed by a decrease in the late photoperiod whichcontinued into the dark period (Table III). Ferricyanide treatmentresulted in an increase in enzyme activity of all leaf extracts, butits effect was more pronounced during the later stages of thephotoperiod and early dark period. The greater ferricyanide acti-vation at these times was correlated with an increase in activityresulting from light treatment (Table III). The plants that weremoisture-stressed had a reduced NR activity but showed a highlevel of activation, by ferricyanide and light, which was alsoapparent at 14.00 h.The activity of the NR from plants grown on 10 mm nitrate and

harvested at 21.30 h was not increased by ferricyanide or lighttreatment (data not shown). It was also checked, with the lownitrate plants, that when 10 mm nitrate was included in theextraction medium the amount of inactive NR detected was notaltered.

DISCUSSION

There were conflicting reports as to whether preincubation ofhigher plant NR with NADH, in the absence of nitrate, stabilizes(10, 11, 24, 26) or inactivates the enzyme (12, 19, 23, 29). We haveconfirmed that with NR of wheat leaf both phenomena can bedemonstrated. NADH protection of NR activity could only bedemonstrated in crude leaf extracts, prepared either with no thioladded or with cysteine, in the presence of 0.1 mm NADH. WithDTT, casein, and FAD in the extraction medium, the wheat leafenzyme was relatively stable, but it was very susceptible to inac-tivation by NADH. DTT did not influence the rate of inactivationby NADH (date not shown). After preincubation of either thecrude extract or partially purified enzyme for 30 min at 25°C with1O iLM NADH, about half of the enzyme activity was lost.The level of NADH shown to inactivate NR (10 lsM) is in the

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concentration range reported for this reduced nucleotide in theleaf cell (16). Sherrard and Dalling (23) with a purified NR fromwheat leaves, reported a Km for NADH of 33 AM, while a lowervalue (-10 Am) has been found for a range of other plants (3).Thus, if nitrate supply to the NR enzyme was interrupted, theNADH level in the cytoplasm would appear to be sufficient toinactivate it. Even with low levels of nitrate (<50 .M cf Km 200!LM [3], some inactivation would still occur. Maldonado et al. (14)with spinach leaf NR reported inactivation of the enzyme whenNADH at 250 AM was in excess of nitrate at 150 AM.A low level of cyanide that gave a small inhibition of the wheat

leaf NR, markedly enhanced its inactivation in the presence ofNADH. This indicates that the NADH inactivation of wheat leafNR could be mediated by the binding of cyanide to the molyb-denum of the reduced enzyme as has been demonstrated forChlorella (28). A very low level of cyanide was required (1 nM),and pathways for its generation have also been described inChlorella (28).

Reactivation of NADH-treated wheat leaf NR was demon-strated by treatment with ferricyanide or, after removal of excessNADH, by exposure to light in the presence of FAD. In a studyon the reactivation of spinach leafNR, it was established that bluelight was most effective (2). It has been proposed (1) that the FADin the NR complex absorbs the blue light and after light excitation,it oxidizes the enzyme complex. We also found that autoxidationand activation of NADH-inactivated wheat enzyme can occur andthat the rate is reduced in the presence of DTT. Leong and Shen(12) found that NADH-CN-treated rice NR was reactivated bypassing it through a Sephadex column; however, this did not occurwith the enzyme from Chlorella (25) and Ankistrodesmus (4). WithNR from Chlorella (25) and Nitrobacter (6), additional incubationwith nitrate resulted in a reactivation of the NADH-treated en-zyme. We found no evidence for nitrate reactivation of the inactivewheat enzyme, and even after the removal of excess NADH therate of autoxidation was not enhanced by nitrate.

It is well established that NR in Chlorella is inactivated in vivoby NADH and cyanide either as a result of treatment withammonium (13) or due to incubation with 02 and light (21).Trinity and Filner (27) reported activation of NR extracted fromtobacco cells during incubation at 20°C or storage at lowertemperature but did not check whether the enzyme could beactivated by oxidants. Leong and Shen (12) reported that theywere unable to isolate inactive NR from rice seedlings treated withammonium.We have demonstrated in this paper that with wheat plants

grown on a low nitrate supply, the leaf NR isolated in the latephotoperiod and dark period was activated by treatment witheither ferricyanide or light. This indicates that at these time periodspart of the NR in the leaf is present in an inactive form (NADH-inactivated).'Studies on the diurnal fluctuations in the uptake andtransport of nitrate to the shoot (20, 30) indicate that at this timeof the day nitrate supply to the leaf will be minimal, thus coincid-ing with maximum inactivation ofNR by NADH.With the wheat plants grown on 1 mM nitrate (but not 10 mm

nitrate), some activation ofthe leafNR by ferricyanide was alwaysobtained (20-30%). This suggests that a portion of the enzyme inthe low-nitrate plants is in the inactive state. An increase in thedegree of activation ofNR by ferricyanide was usually correlatedwith the amount of light activation that could be demonstrated.Some inconsistencies in this relationship do, however, requirefurther investigation, eg. the leaf sample harvested at 06.00 h orfrom stressed plants at 17.30 h (Table III).When the plants were subjected to moisture stress and presum-

ably reduced flux of nitrate to the leaf, an increase in the amountof inactive NR was detected even at an earlier time in thephotoperiod. In a study on the effect of moisture stress on NR inwheat leaves, Heuer et al. (7) postulated that in addition to loss of

NR activity due to a reduced rate of enzyme synthesis there wasa direct inhibition of the enzyme. This inhibition could be ex-plained by the NADH-mediated inactivation of NR followinglimitation of the nitrate supply.

It has been reported for Chlorella (9) that NADH-inactivatedNR represents a more stable form of the enzyme, less susceptibleto degradation by trypsin. In the wheat leaf, when nitrate flux tothe metabolic pool is interrupted, synthesis of NR would cease(18). Should degradation of the existing enzyme be reduced, thenit could be reactivated in response to a renewed supply of nitrate.Reactivation would involve oxidation of the enzyme either bylight or by an oxidizing agent such as trivalent Mn ions, whichcan be generated in the chloroplast (15). Should the NADH levelbe maintained, then autoxidation is unlikely to occur.

Acknowledgments-We wish to thank Professor D. J. D. Nicholas for helpfuldiscussion during the course of the work and for critical reading of the manuscript.The comments of Dr. A. Oaks and Dr. B. T. Steer were also appreciated.

LITERATURE CITED

1. APARICIO PJ, JM MALDONADO 1979 Regulation of nitrate assimilation in pho-tosynthetic organisms. In EJ Hewitt, CV Cutting, eds, Nitrogen Assimilationof Plants. Academic Press, London, pp 207-215

2. APARICIO PJ, JM ROLDAN, F CALERO 1976 Blue light photoreactivation ofnitratereductase from green algae and higher plants. Biochem Biophys Res Commun70: 1071-1077

3. BEEVERS L, RH HAGEMAN 1980 Nitrate and Nitrite reduction. In PK Stumpf, EEConn, eds, The Biochemistry of Plants, Vol 5. Academic Press, New York, pp115-167

4. DE LA ROSA MA, C GOMEZ-MORENO, JM VEGA 1981 Interconversion of nitratereductase from ankistrodesmus braunii related to redox changes. BiochimBiophys Acta 662: 77-85

5. Deleted in proof6. HERRERA J, DJD NICHOLAS 1974 Inactivation of nitrate reductase by NADH in

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12. LEONG CC, TC SHEN 1979 Reversible inactivation of the nitrate reductase of riceplants. Experentia 35: 584-585

13. LORIMER GiH, HS GEWITZ, W VOLKER, LP SOLOMONSON, B VENNESLAND 1974The presence of bound cyanide in the naturally inactivated form of nitratereductase of Chlorella vulgaris. J Biol Chem 249: 6074-6079

14. MALDONADO JM, BA NOTTON, EJ HEWITT 1978 Inhibitory effects of ferrocyto-chrome c on NADH-nitrate reductase activity of spinach. Plant Sci Lett 13:143- 150

15. MALDONADO JM, BA NOTTON, EJ HEWITr 1980 The reactivation of nitratereductase from spinach, inactivated by NADH and cyanide, using trivalentmanganese either generated by illuminated chloroplasts or as mangani-pyro-phosphate. Planta 150: 242-248

16. MANN AF, DP HUCKLESBY, EJ HEWITT 1979 Effect of aerobic and anaerobicconditions on the in vivo nitrate reductase assay in spinach leaves. Planta 146:83-89

17. MORENO CG, PJ APARICIO, E PALACIAN, M LOSADA 1972 Interconversion of theactive and inactive forms of Chlorella nitrate reductase. FEBS Lett 26: 11-14

18. OAKS A, W WALLACE, D STEVENS 1972 Synthesis and turnover of nitratereductase in corn roots. Plant Physiol 50: 649-654

19. PALACIAN E, F DE LA ROSA, F CASTILLO, CG MORENO 1974 Nitrate reductasefrom Spinacia oleracea. Reversible inactivation by NAD(P)H and by thiols.Arch Biochem Biophys 161: 441447

20. PEARSON CJ, BT STEER 1977 Daily changes in nitrate uptake and metabolism inCapsicum annuum Planta 137: 107-112

21. PISTORIUS EK, H-S GEwITz, H Voss, B VENNESLAND 1976 Reversible inactiva-tion of nitrate reductase in Chlorella vulgans in vivo. Planta 128: 73-80

22. SHANNON JD, W WALLACE 1979 Isolation and characterisation of peptidehydrolases from the maize root. Eur J Biochem 102: 399-408

23. SHERRARD JH, MJ DALLING 1979 In vitro stability of nitrate reductase fromwheat leaves. I. Stability ofhighly purified enzyme and itscomponent activities.Plant Physiol 63: 346-353

24. SLurrERs-SCHOLTEN CMTH 1975 Photosynthesis and the induction of nitratereductase and nitrite reductase in bean leaves. Planta 123: 175-184

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26. TiscHLER CR, AC PURVIS, WR JORDAN 1978 Factors involved in in vitrostabilization of nitrate reductase from cotton (Gossypium hirsutum L.) cotyle-dons. Plant Physiol 61: 714-77

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NITRATE REDUCTASE BY NADH 587

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