characterization nitrate reductasefrom corn leaves (zea … · plantphysiol. (1984) 75, 285-289...

Plant Physiol. (1984) 75, 285-2890032-0889/84/75/0285/05/$01.00/0

Characterization of Nitrate Reductase from Corn Leaves (Zeamays cv W64A x W182E)ITWO MOLECULAR FORMS OF THE ENZYME

Received for publication August 8, 1983 and in revised form January 26, 1984

HIROKI NAKAGAWA2, MICHEL POULLE, AND ANN OAKS*Biology Department, McMaster University, Hamilton, Ontario L8S 4K1 Canada

ABSTRACr

The primary kaves from corn seedlings grown for 6 days were har-vested, frozen ith liquid N2 and exbacted in a Tris buffer (pH 85, 250millimolar) contining I millmolar dithithreitol, 10 millioar cysteine,1 millimolar EDTA, 20 o r favi adenine dinwleotide and 10%(v/v) glycerol. Nitrate reductse (NR) in the crude extct was stable forseveral days at 0°C and for several mouths at -WC. The ewyme waspurified using (NH S04 fractatin, brushite-hydroxyl-patite chro-matography and blesepharose affity chrotoaphy. The enzymewas elated from the blue-ephare column with a iHnear graient ofNADH (0-100 mkromolar) or with 0.3 molar KNO3. About 10% of theoriginl activity was recovered with NADH (NADH-NR). Ithad aspecif activity of about 60 to 70 umits (miromoles NO2- per minuteper mi protein). A sequ l elutio with NADH followed byKNO3 (0.3 molar) orKa (0.3 molar) yielded 2 peaks. Rech togaphyof each peak gave two peas agip. Tbese malts indicate that we aredeaflig with two forms of the same enzyme rather than two diMerent NRprotei. The two NRs had different molecuar weights as judged bychromatoaphy on Toyopearl. The NADH-NR was more senitive thanthe NOj-NR to antibody prepard agat bariey lef NR. In Oachter-loay assays a single precipitin line, with completely fsWed ,was observed.

Nitrate reductase (EC 1.6.6.1) in Zea mays catalyzes anNADH dependent reduction of nitrate to nitrite. Solomonson(26) successfully purified the enzyme from Chlorella vulgarisusing blue dextran sepharose affinity chromatography (the chro-mophore of blue dextran being Cibacron Blue F3G-A) and thistechnique has also proven to be a simple and rapid techniquefor the isolation of NR3 from higher plants (3, 8, 9, 10, 12, 16,21, 24). However, attempts to purify NR to homogeneity havebeen relatively unsuccessful because of the instability of theenzyme and the relatively low levels ofNR in higher plant tissues.Recently, Kuo et al. (13) have developed a buffer system whichstabilizes the NR obtained from barley leaves. Using a similarbuffer in conjunction with affinity chromatography they have

' Supported by the Natural Sciences and Engineering Research Coun-cil of Canada (Grnt No. A2818). H. N. supported by the NaturalSciences and Engineering Research Council (Canada) visiting scientistprogram, and by a travel grant from the Ministry of Education ofJapan.

2 Prent address: Faculty of Horticulture, CHIBA University, 648Matsudo, Matsudo City, CHIBA 271, Japan.

3Abbreviations: NR, nitrate reductase; BS, blue-sepharose; 2-ME, 2-mercaptoethanol; FAD, flavin adenine dinucleotide.

been able to purify the barley leafNR and to obtain an enzymewith a single subunit with a mol wt of about 10,000 D (12).The main objective of the present research was to develop a

procedure for the purification of corn leafNR using BS affinitychromatography and to characterize the enzyme. We have beenable to show that two molecular forms ofNR (NADH-NR andN03 -NR) can be eluted from a BS column and that these twoforms are interconvertible.

MATERIAIS AND METHODS

Plant Material. Corn (Zea mays cv W64A x W182E), pur-chased from the Wisconsin Seed Foundation, Madison, WI, wasplanted in vermiculite and sand, and grown for 6 d. The plantswere watered daily with a one-tenth Hoagland solution whichcontained 10 mM KNO3. The growth chamber was maintainedon 16 h days with a light intensity of about 150 ;LE m-2 s-'. Theday and night temperatures were 28 and 26°C, respectively.

Preparation of Crude Extract. Primary leaves from 6-d seed-lings were frozen with liquid N2, ground to a powder with amortar and pestle, and then stored at -80°C. The powder wasthawed for 10 min at 4C and homogenized in a blender in 250mM Tris-HC1 buffer, pH 8.5, containing 10 mM cysteine, 1 mMEDTA, 20 pM FAD, 1 mM DTT, and 10% (v/v) glycerol (13).The homogenate was centrifuged at 10,OOOg for 30 min.

Activity Measuements. During enzyme purification, activitywas measured as the rate ofNO2- production at 28C. The assaymixture contained, KNO3 (10 mM), Hepes (0.065 M; pH 7.0),NADH (0.5 mM) in phosphate buffer (0.04 M; pH 7.2) andenzyme in a final volume of 1.5 ml. The reaction was started byadding the NADH. After 15 min it was terminated by adding 1ml of 1 N HCI solution containing 1% sulfanilamide followed by1 ml of 0.02% aqueous N-l-naphthylethylene-diamine-dihydro-chloride (NED). The A was read at 540 nm after 10 min. Nitriteconcentrations were calculated from a standard curve whichcontained NO3-, NADH, and phosphate in addition to N02-.One unit ofNR activity was defined as the amount of enzymecatalyzing the formation ofone pmol ofNO2- per min.

Electophoresis. Analytical disc gel electrophoresis ofNR wasperformed at 4°C using the Tris-glycine buffer system of Davis(6). Samples were applied to 7.5% acrylamide gels, and electro-phoresis was carried out first at 1 mamp/gel for 20 min and thenat 2 mamp/gel until the tracking dye front was close to thegel bottom (about 2 h). Proteins were located by staining with0.25% Coomassie Brilliant Blue R-250 in 7% acetic acid for 1 hfollowed by diffusion-destaining with several changes of 7%acetic acid. NR was detcted after electrophoresis either in thegel by the method of Solomonson et al. (27) or in sliced gelsections (2.5 mm) with the standard NADH-NR assay.

Determination of Stokes Radius. The Stokes radius was deter-285

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NAKAGAWA ET AL. Plant Physiol. Vol. 75, 1984

mined as described by Siegel and Monty (25). We used a Toyo-pearl WH-55 column (2.5 x 50 cm) equilibrated with K-phos-phate (250 mm; pH 7.5), containing EDTA (1 mm), 2-ME (1mm) either in the presence or absence of KCI (0.1 m). Two mlsamples were layered on the top of the column and were elutedat a flow rate of 22 mi/h. cm'. Elution volume (Ve), void volume(Vo), and total volume (Vt), were determined in the usualmanner (25). Standards ofknown Stokes radius were: ovalbumin,2.7 nm; BSA, 3.5 nm; yeast alcohol dehydrogenase, 4.6 nm;

catalase, 5.2 nm; ferritin, 7.8 nm; fibrinogen, 10.7 nm.

Sensitivity of Maize NRs to Antibodies Prepared againstBarley Leaf NR. The behavior of NADH- and N03-NRs was

studied using an immunoprecipitation procedure. Constantamounts ofenzyme were incubated for 3 h on ice with increasingconcentrations of antibody in a total volume of 0.5 ml. Thepreparation was then centrifuged in an Eppendorf Microfuge for5min and the activity remaining in the supernatant solution wasassayed. The antibody was a gift of D. A. Somers, Tsung-iinKuo, and R. L. Warner, Washington State University, PullmanWA.

Gel Immunodiffusion. NADH- and N03--NRs (eluted fromthe BS column) were assayed in the presence of antibodies byimmunodiffusion (5) inagar plates (1% agarose, in 50 mm Tris-citrate buffer, pH 8.5). After 48 h, the plates were stained for 15mmn with 0.5% amido black in methanol:acetic acid:water(50:l0:4OXv/v/v). The plates were subsequently destained withthe methanol:acetic acid:water (50:l0:4OXv/v/v).

Protein Determination. Protein concentrations were deter-mined by the Bio-Rad dye-binding assay (standard assay proce-

dure) for crude preparations,(NH4)4SO4, and hydroxylapatitefractions. Protein obtained at later stages of purification, was

determined by the Bio-Rad dye-binding assay (microassay pro-

cedure).Chemicals and Supplies. FAD, NADH, catalase and alcohol

dehydrogenase (yeast) were purchased from Sigma. Blue-sepha-rose CL-6B (BS), purchased from Pharmacia Fine Chemicals,was regenerated according to the method of Noland et al. (1 5).Toyopearl WH-55 was supplied by Toyo Soda Mfg. Co. Ltd.,Tokyo, Japan. Hydroxylapatite and brushite were prepared bythe methods ofLevin (1 4) and Rice et al. (22). All other chemicalsused were the purest available from commercial sources.

A

. 2.0

Ec

U3

X 1.0co0

-C

Ir-

IIli

1%"

0~~70 06D 9-0 -1 60FRACTION NUMBER

FIG. 1. BS affinity chromatography of corn NR. NR was absorbedon a BS column (2.5 x 6 cm) and washed overnight with 50 mM Tris-

HCI buffer (pH 8.3) containing EDTA, 2-ME, FAD, and glycerol. Arrowsindicate where the different elution buffers were applied: A, 50mM Tris-

HCI, 1 mm EDTA, I mM 2-ME, and 20 iLM FAD (pH 8.3); B, a 0 to 100

AiM NADH linear gradient in the original buffer (pH 8.3); C, 300 mMKNO3 in the original buffer (pH 8.3). Flow rate was adjusted to about50 ml/h cm2. NADH-NR activity was expressed as A at 540 mm (0)

and the protein concentrations were measured at 595 nm by the standard(@) or microbiord (A) assays.

RESULTS

The Effect of Age on the Stabiity of NR Activity. In the earlyexperiments, leaves were harvested at daily intervals fromS to10 d after planting. The NR activity in the(NH4SS04 fraction(27.5-50% saturation) was measured either immediately or afterthe preparation had been stored on ice for 24 h. Preparations,from leaves harvested 6 d after planting, had the highest initialactivities and were the most stable. Hence, in subsequent extrac-tions, primary leaves from6-d-old plants were used. Crudeextracts were also prepared with Hepes (100 mm, pH 7.4), K-phosphate (50 mm; pH 7.5 or 8.5), or Tris-HCI (250 mm; pH8.5) buffers and the activity was measured either immediately or

at various times after storage at 4C. Tris-buffer yielded both themost active and the most stable preparations. It was, therefore,used in subsequent extractions.

Purification of Nitrate Reductase. Solid(NH4S SO was addedto the crude extract to 27.5% saturation. After removing theprecipitate by centrifugation, solid(NH4)SS0 was added again

to give 50% saturation. The precipitate thus formed was collectedby centrifugation and dissolved in a minimal amount of 10 mmK-phosphate (pH 7.5), containing mm EDTA, mm 2-ME,and 10% (v/v) glycerol. The solution was dialyzed for a total of4 h against 2 L of the 10 mm K-phosphate which was changed 2times. After centrifugation, the dialyzedsupernatant solutionwas applied to a hydroxylapatite brushite (1:3 v/v) column (5 x

5 cm) equilibrated with mm K-phosphate (pH 7.5) containingmm EDTA, mm 2-ME, and 10% glycerol. After washing the

column with 750 ml of the same buffer, elution was performedwith 100 mm K-phosphate (pH 7.5) containing mm EDTA,mm 2-ME, and 10% glycerol. The fractions having high NRactivities were combined and applied to a BS column (2.5 x 6cm) equilibrated with 10 mm K-phosphate (pH 7.5) containingmm EDTA,mm 2-ME, 20 Am FAD, and 10% (v/v) glycerol.

The column was then washed overnight with 50 mm Tris-HCIbuffer (pH 8.3) containing mm EDTA, mm 2-ME, 20 mM

FAD, and 10% glycerol. The flow rate was 50 ml/hmcm2 and 7

ml fractions were collected. Adding 0.3 m Tris-HCI (pH 8.3) afterthis treatment failed to elute NR activity. When a linear concen-

tration gradient ofNADH(0-1000M) in 50 mm Tris-HCI buffer(pH 8.3) containing the usual additives, was used, a major peakofNR activity was eluted with Mm NADH (Fig. 1). To preventinactivation of this NR, fractions were conlected into tubes whichcontained 0.1 ml of m KN03. Subsequently, the BS columnwas washed with 50 mM Tris-HCl buffer (pH 8.3) plus the usualadditives, and then with the same buffer containing 0.3M KNO3.A second NR peak (NO3-NR) was eluted with 0.3M KNO3(Fig. 1). Adding 0.3M KCI instead of KNO3 alsoresulted in a

peak of NR activity. However, adding1 M KCI after KNO3resulted in no further elution of NR. The two or three fractionspossessing high NR activities were combined and concentratedby means of an immersible-membrane obtained from Millipore.Once the purification procedure was established, the specific

activities recovered routinely had about 70 units of NADH-NRactivity per mg protein. The two NR peaks were also obtainedwhen the dialyzed crude enzyme or the (NH)2SO0 precipitatedNR was transferred directly to the BS column. Redinbaugh andCampbell (20) and Oji et al. (18) have also reported two formsof NR obtained from root tissue after affinity chromatography.Usually, however, only one form of the enzyme has been reported(12, 16, 24).

Charcterization of Nitrate Redutase Proteins. (a) Polyacryl-amide Gel Electrophoresis. NADH-NR on 7.5% acrylamide gelsat pH 8.3 yielded one major and two minor protein bands. Themajor protein band was also associated with methyl viologen-NR activity (Fig. 2). The N03 -NR had several protein bandsand two bands associated with methyl viologen-NR activity. TheRm, value for NADH-NR was 0.21 and for N03--NR, 0.20, and

286



CHARACTERIZATION OF NITRATE REDUCTASE

b b a

4-

4-

FIG. 2. Separation ofNADH- (A) andN03- (B) NRs on polyacrylamide gels.Activity bands determined with methylviologen (a) and protein bands (b) ofthe affinity-column purified NRs arecompared. The arrows indicate the po-sition of the protein or NR activitybands Difference in RF values are re-lated to amounts of extract required togive protein or activity bands.

B

0.24.(b) Toyopearl Chromatography. The NADH-NR and N03--

NR were eluted from a calibrated Toyopearl column at a positioncorresponding to a Stokes radius of 5.8 and 7.1 nm, respectively(Fig. 3) when calculated according to the method of Siegel andMonty (25); of4.9 and 6.0 nm according to the protocol currentlyrecommended by Pharmacia (Gel filtration calibration kit in-struction manual for protein mol wt determinations by gelfiltration). Thus, the apparent mol wt for the two forms ofNRare different.

(c) InterconvertabilityofNADH-NR (NR-I) andN03-NR (NR-II). A sequential elution from the BS column with NADH

followed by KNO3 yielded 2 peaks (Figs. 1 and 4), designatedNR-I and NR-II, respectively (Fig. 4). When fractions from eachpeak were pooled, dialyzed, and rechromatographed on BS, weagain recovered two peaks (Fig. 4, B and C). Thus, NR recoveredfrom the primary leaf of corn exists in two forms which areinterconvertible.

(d) Reaction with Antibody Prepared against Barley LeafNR.Preliminary tests showed that corn NR in crude preparationswas inactivated by antibody prepared against barley leafNR andthat the antibody-NR complex could be removed by centrifuga-tion (A. Oaks, T.-M. Kuo and R. L. Warner, unpublished data).When equal amounts of NR protein as determined by NR

a

A

287



Plant Physiol. Vol. 75, 1984

1.1 F

1.0pV0.9 F

o0

0.8

0.7 I

NADH-NR;

BAt

II -i~~~~~~~~~~~~~~~~~~~~~102 4 6 8

STOKES RADIUS (nm)

FIG. 3. Gel filtration of corn NR on a Toyopearl WH-55 column.The column (2.5 x 50 cm) was equilibrated and developed with 250 mmK-phosphate, pH 7.5, containing 1 mM EDTA, 1 mM 2-ME, either inthe presence (0) or absence (0) of 0.1 M KC. The arrows show thepositions of the NRs. Standards of known Stokes radius were: A, ova-bumin, 2.75 nm; B, BSA, 3.5 nm; C, yeast alcohol dehydrogenase, 4.6nm; D, catalase, 5.2 nm; E, ferritin, 7.8 nm; F, fibrinogen, 107 nm.

500

c2 400t

t 300

> 200

100z

FRACTION NUMBER

-I

c

o.

z5 10 15 5 10 15

FRACTION NUMBER

FIG. 4. Interconvertibility of NADH- and N037-NR. NR obtainedfrom the hydroxylapatite brushite column was applied to a BS column(0.7 x 3 cm) equilibrated with the 100 mm K-phosphate, pH 7.5containing 1 mM EDTA, 1 mM 2-ME, and 10 gm FAD (buffer A). TheBS was washed successively with 25 ml buffer A, with a linear NADHconcentration gradient from 0 to 100 Ad (20 ml) in buffer A and finallywith 0.3 M KNO3 in buffer A. Tubes containing each NR were pooledand concentrated to about 2 ml, were dialyzed for 3 h against buffer A,and the dialyzed solutions were reapplied to a BS column (0.7 x 2 cm)equilibrated with buffer A. Arrows indicated where the different elutionbuffers were applied: A, buffer A; B, 0 to 100 pM NADH in buffer A; C,0.3 M KNO3 in buffer A. NR activity was express as nmol NO2-produced/min*fraction.

FIG. 5. Interaction of NR with antibody pear from barley leafNR: A, the incubation mixture included 50 id enzyme, 50 PI Tris-buffer(pH 8.5,250 mm), 100 id BSA (3% in Tns-buffer), and 100 d ofantibodyor water. Original undiluted serum was given a value of 100. A relativescale from 0 to 100 includes the various dilutions. 0, N03--NR: 100 =92.1;-, NADH-NR: 100 = 44.3 pmol NOi- per reaction. B, Ouchterlonydouble-immunodiffusion ofNADH- and NO--NRs. Wells 1 and 2 were

filed with 8 of N037-NR (65 pg ofprotein); wells 3 and 4 were filledwith 8 A ofNADH-NR (16 4g protein). Center well was filled with 8 1ofcrude serum.

activity are titrated against this antibody, the NADH-NR is muchmore sensitive to the antibody (Fig. 5A). The equivalence pointis reached at about 16 units of antibody for NADH-NR and atabout 43 units for NO3--NR. This result could be explainedeither by a higher relative level ofNR protein in the NO3--NRfraction, by different confiurations of the two proteins, or bydifferent antigenic properties. The complete fusion of singleprecipitin bands indicates similar antgenic properties in the twoNRs (Fig. SB).

DISCUSSIONThe NR examined in several plant species has a relatively high

turnover rate in vivo (1, 17, 23, 28). The enzyme from cornleaves, for example, has a half-life of 6 h (1, 23). Ths instability

is also seen after extraction and has made purification of theenzyme difficult. Recently, buffers introduced by Kuo et al. (12,13) have ameliorated this problem. The use of affinity chroma-tography with BS has also enhanced the ease with which NR canbe purified (4, 20). Other affinity gels have also been usedsuccessfully in purifying NR from various sources (3, 7, 10, 12,16, 18, 21, 24, 26).There have been previous reports ofNR being eluted from BS

b

4

288 NAKAGAWA ET AL.



CHARACTERIZATION OF NITRATE REDUCTASE

by high salt (1.0 M KCG) or high N03- (0.3 M KNO3). Campbelland Smarrelli (4) found, for example, that NR from squash andcorn could be eluted from BS specifically with NADH and either0.3 M KNO3 or some other salt. In our experiments, both thedifference in the Stokes radius between the NADH- and NO3--NR and the difference in distance moved by the NR proteins inthe acrylamide gels suggest that there are two different configu-rations of NR in corn leaf extracts. In Chlorella vulgaris thereare oxidized and reduced forms ofNR (26, 27) which appear tobe related to the metabolic status of the cells (19). However, it isdifficult to demonstrate reversible oxidized and reduced formsofNR prepared from corn leaves and, hence, we suspect that thetwo forms ofNR that we see are not related to this phenomenon.

Recently Howard and Solomonson (11) have been able tostabilize Chlorella NR in various states of polymerization froma monomer to a tetramer. The degree of polymerization de-pended on the dilution of the protein. Since the two forms ofNR obtained from corn leaves have different apparent mol wt,we could also be dealing with different levels of polymerization.For example, the approximate mol wt of the NADH-NR (usingthe Pharmacia method of quantitation) is about 160,000 D, ofthe N03--NR is about 433,000 D..Results from barley (12) orChlorella (11) suggest a monomer of about 100,000 D as thebasic subunit of the NR protein. Our preliminary results alsoindicate a similar subunit size at least in the N03 -NR (M. Poulleand A. Oaks, unpublished data). Thus, the NADH-NR could bea polymer oftwo basic subunits and the N03 -NR a polymer offour basic subunits. This interpretation is supported both by thedifferent mol wt and by the different sensitivities of the twoforms of NR to antibody prepared against barley leaf NR (Fig.5A). It remains to be seen whether the two forms ofNR obtainedfrom corn leaves reflect a particular physiological status of thecells.

LITERATURE CITED

1. ASLAM M, A OAKS 1976 Comparative studies on the induction and inactivationof nitrate reductase in corn roots and leaves. Plant Physiol 57: 572-576

2. BEEVERS L, RH HAGEMAN 1969 Nitrate reductase in higher plants. Annu RevPlant Physiol 20: 495-522

3. CAMPBELL WH 1976 Separation of soybean nitrate reductases by affinitychromatography. Plant Sci Lett 7: 239-247

4. CAMPBELL WH, J SMARRELLI 1978 Purification and kinetics of higher plantNADH:nitrate reductase. Plant Physiol 61: 611-616

5. CLAUSEN J 1974 Immunochemical techniques for the identification and esti-mation of macromolecules. In TS Wak, E Wak, eds, Laboratory Techniquesin Biochemistry and Molecular Biology, Vol I, part III. Elsevier-North

Holland Publishing Co, New York, pp 599-5726. DAvis BJ 1964 Disc electrophoresis Method and application to human serum

protein. Ann NY Acad Sci 121: 404-4077. DE LA ROSA M, J DIEz, JM VEGA,M LOADA 1980 Purification and properties

ofassimilatory nitratereductase from Ankistrodesmus brauch. EurJ Biochem106: 249-256

8. GUERRERO MG, K JETSCHMANN, W VOLKER 1977 The stereo-specificity ofnitrate reductase for hydrogen removal from reduced pyridine nucleotides.Biochim Biophys Acta 482: 19-26

9. GUERRERO MG, JM VEGA, M LOADA 1981 The assimilatory nitrate reducingsystem and its regulation. Annu Rev Plant Physiol 32: 169-204

10. HEIMER YM, S KRASHIN, E RIKLIS 1976 The use of affinity chromatographyfor the purification of nitrate reductase. FEBS Lett 62: 30-32

11. Howard WD, LP Solomonson 1982 Quaternary structure of assimilatoryNADH:nitrate reductase from Chlorella. J Biol Chem 257: 10243-10250

12. Kuo T-M, A KLEINHOFS, RL WARNER 1980 Purification and partial charac-terization of nitrate reductase in barley leaves. Plant Sci Left 17: 371-38 1

13. Kuo T-M, RL WARNER, A KuINHOFs 1982 In vitro stability of nitratereductase from barley leaves. Phytochemistry 21: 531-533

14. LEVIN 0 1962 Column chromatography of proteins on calcium phosphate.Methods Enzymol 5: 27-32

15. NOLAND BJ, RE AREBALA, E HANSBURY, TJ SCALLEN 1980 Purification andproperties of sterol carrier protein. J Biol Chem 255: 4282-4289

16. NorroN BA, RJ FiDo, EJ HEwIrr 1977 The presence of functional haem in ahigher plant nitrate reductase. Plant Sc Lent 8: 165-170

17. OAKs A, W WALLACE, D STEVENs 1972 Synthesis and turnover of nitratereductase in corn roots. Plant Physiol 50: 649-654

18. OJI Y, Y MIKI, S OKAMOTO 1982 Extraction and affinity purification ofNADH:nitrate reductase from barley (Hordeum distichum L) roots. PlantCell Physiol 23: 1025-1031

19. Pwroiuus EK, HS GEwrrz, H Voss, B VENNESLAND 1976 Reversible inaci-vation of nitrate reductase in Chlorella vulgaris in vivo Planta 128: 73-80

20. REDINBAUGH MG, WH CAMPBELL 1981 Purification and characterization ofNAD(P)H:nitrate reductase and NADH:nitrate reductase from corn roots.Plant Physiol 68: 115-120

21. REDINBAUGH MG, WH CAMPBELL 1983 Purification of squash nitrate reduc-tase by zinc chelate affinity chromatogaphy. Plant Physiol 71: 205-207

22. RICE HV, WR BRIGGS, CJ JACKsON-WHITE 1973 Purification of oat and ryephytochromes. Plant Physiol 51: 917-926

23. SCHRADER LE, GL RITENOUR, GL EuucH, RH HAGEMAN 1968 Some char-acterstics of nitrate reductase from higher plants. Plant Physiol 43: 930-940

24. SHERRARD JH, MJ DALLING 1979 In vitro stability of nitrate reductase fromwheat leaves. I: Stability of highly purified enzyme and its componentactivities. Plant Physiol 63: 346-353

25. SIEGEL LM, KJ MONTY 1966 Determination of molecular weights and fric-tional ratios ofproteins in impure systems by use ofgel filtration and densitygradient centrifugation. Application to crude preparations of sulfite andhydroxylamine reducts. Biochem Biophys Acta 112: 346-362

26. SOLOMONSON LP 1975 Purification of NADH-nitrate reductase by affinitychromatography. Plant Physiol 56: 853-855

27. SOLOMoNSON LP, GH LORIMER, RL HALL, R BORCHERS, JL BAILEY 1975Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorellavulgaris: purification, prosthetic groups, and molecular properties. J BiolChem 250: 4120-4127

28. ZIELKE HR, P FILNER 1971 Synthesis and turnover ofnitrate reductase inducedby nitrate in cultured tobacco cells. J Biol Chem 246: 1772-1779

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characterization nitrate reductasefrom corn leaves (zea … · plantphysiol. (1984) 75, 285-289...

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