PURIFICATION AND SOME PHYSICOCHEMICAL PROPERTIES OF NITRATE REDUCTASE ISOLATED FROM WINTER TRITICALE SEEDLINGS

Cezary Sempruch, Antoni P. Ciepiela, Iwona Sprawka, Grzegorz Chrzanowski
Department of Biochemistry and Molecular Biology, University of Podlasie, Siedlce, Poland

ABSTRACT

Nitrate reductase (NR, EC 1.6.6.1-3) is a key enzyme of assimilative reduction of nitrate in plant tissues. NRs present in higher plants are differed in structure and physicochemical properties. In literature there are information concerning properties of the enzymes isolated from tissues such cereals as wheat, maize, rice and barley, but not triticale. Because of that, the aim of the research was to purify nitrate reductase isolated from the leaves of seven-day-old seedlings of winter triticale cultivar Moreno and to analyze its physicochemical properties.

The research results showed that NR obtained from the winter triticale seedlings is a homogeneous protein fraction of the molecular weight of approximately 190000 Da. The absorbency of the purified enzyme reached its maximum values at 244, 463 and 491 nm. NR was specific in relation to NADH₂ as well as to NADPH₂ although it showed lower specific activity in the presence of the latter coenzyme. NR isolated from the seeds of the winter triticale also showed other activities such as MVH (methylviologen) – NR, FMNH₂ – NR, NADH-ferricyanide reductase and NADH-cyt c reductase, which is the evidence of the enzyme partial ability to catalyze reactions other than the reduction of nitrate to nitrite in the presence of NADH₂ or NADPH₂ as the electron donors.

Key words: nitrate reductase, winter triticale, assimilative reduction of nitrate.

INTRODUCTION

Nitrate reductase of higher plants is a olygomeric protein catalyzing the electron flow from NADH₂ or NADPH₂ to nitrate through the prosthetic groups to which FAD, the heme (the cytochrome b-557) and the molybdenum cofactor (MoCo) belong [14]. The effect of this process is an assimilative reduction of nitrate, which is a basic source of nitrogen for majority of plants as it dominates among nitrogen soil forms [14]. It is generally thought that 2-electron reduction of nitrate conditions the rate of the metabolism of all nitrogen compounds which occur in plant organisms.

The available literature sources contain a lot of information concerning different purification methods and physicochemical properties of NRs isolated from tissues of cereals [1,3,9,11,12,17,18]. The enzymes obtained from various plant species, cultivars and even organs, are claimed to vary in molecular weight, number of subunits and specificity with relation to NADH₂ or NADPH₂ as electron donor [7,12,18]. NR has not been analyzed yet in winter triticale which is an important component of cereal crops in Poland. Moreover, the triticale is a very interesting new cereal species, obtained artificially by crossing of wheat and rye and its biochemical and physiological properties are still not clear.

Therefore, this research aimed at purifying NR isolated from shoots of seven-day-old seedlings of winter triticale cultivar Moreno and then analyzing some of its physicochemical properties.

MATERIAL AND METHODS

Plants:
The studied plants were cultivated in horticultural soil in laboratory conditions at the temp. of 20±5°C, relative humidity of 70% and photoperiod 14 h:10 h.

Enzyme extraction:
The procedure of the analyses was described by Sherrard and Dalling [17]. The enzyme was extracted from the fresh plant material by homogenization with 25 mM phosphate buffer of pH 7,5, with addition of 5 mM of EDTA and 5 mM of cysteine (extraction buffer). The homogenate obtained was impressed through two layers of blotting-cloth, and then centrifuged at 25000 x g for 15 minutes at 4°C. For the purpose of the enzymatic analyses, the supernatant was collected as an extract.

Enzyme purification:
Solid ammonium sulfate was slowly added to the enzymatic extract which was stirred continuously. The fractions precipitated at 30 to 45 % saturation were re-dissolved in an ice-cold extraction buffer.

The ammonium sulfate fraction was loaded onto a column (2.3 x 8.0 cm) of blue dextran – Sepharose 4B (prepared according to Sherrard and Dalling [17]) equilibrated with the extraction buffer. NR was eluted from the gel by 5 µM NADH solution in the extraction buffer.

All steps of the extraction and purification of NR were conducted at 4°C.

Enzyme assay:
NADH (or NADPH)-NR. A reaction mixture of 2 cm³ in total volume consisted of 100 µM K-phosphate buffer (pH 7.5), 10 µM potassium nitrate, 0.4 µM NADH₂ (or NADPH₂), and enzymatic extract. After 15 min. of incubation at 25°C, the reaction was stopped by the mixture of 0.5% (w/v) sulfanilamide and 0.1% (w/v) of N-1-naphthylethylene diamine dichloride in 1.5 M HCl. Nitrate content was determined after 15 min. by measuring the absorbance (A) at 540 nm with the HP 8453 UV-Vis spectrophotometer (made by Hewlett-Packard).

MVH-NR. The reaction mixture of 0.9 cm³ in total volume contained 100 µM K-phosphate buffer (pH 7.7), 10 µM potassium nitrate, 1 µM reduced methylviologen (MVH), and enzymatic extract. The reaction was started by adding freshly prepared solution of 10 mg · cm^-3 sodium dithionite dissolved in 95 mM sodium bicarbonate. After 15 min. of incubation in 25°C, the reaction was terminated by vortexing with oxidized MVH and remaining dithionite. Nitrate was estimated according to NADH-NR.

FMNH₂-NR. The reaction mixture of 0.9 cm³ in total volume contained 50 µM Tris-HCl buffer (pH 7.4), 0.7 µM potassium ferricyanide, 1.5 µM reduced FMN, and enzymatic extract. The reaction was started and terminated as described for the MVH-NR. Nitrate was estimated according to NADH-NR.

NADH-ferricyanide reductase. The reaction mixture of 1.0 cm³ in total volume contained 40 µM Tris-HCl buffer (pH 8.5), 0.7 µM potassium ferricyanide, and enzymatic extract. The reaction was started by adding of NADH solution of 4 mg · cm^-3 NADH. The mixture was incubated in 25°C and rate of ferricyanide reduction was estimated by A changes at 420 nm.

NADH-cyt C reductase. The reaction mixture of 1.0 cm³ in total volume contained 80 µM potassium phosphate buffer (pH 7.5), 1.0 mg cyt c, 0.3 µM NADH, and enzymatic extract. The rate of cyt c reduction was estimated at 25°C by A changes at 550 nm. The assay was carried out only with enzyme prepared in the absence of cysteine.

Protein assay:
The content of protein in enzymatic extract was analyzed in accordance with the Lowry et al. [10] method.

Gel electrophoresis:
Electrophoretic analyze of highly purified NR extract was conducted with the plate technique in 15% polyacrylamide gel containing SDS according to Laemmli [8]. Obtained fraction was stained with coumassi brillant blue. Molecular weight of NR was stated on the basis of standard curve prepare accordance with Weber and Osborn [21] method.

RESULTS AND DISCUSSION

Precipitation with ammonium sulphate and chromatography on the column of blue dextran – Sepharose 4B made possible 2481.8 fold purification of NR from shoots of seedlings of winter triticale Moreno cultivar (Table 1). The recovery of the method amounted 20%, and the specific activity of highly purified enzyme reached 27.3 µM of NO₂^- mg^-1 of protein · hour^-1 (unit · mg^-1 of protein) at 1.20 · 10^-2 mg of the content of protein. Obtained results confirm the opinion of Nakagawa et al. [12] which give, that blue dextran – Sepharose 4B chromatography is a most efficient method of NADH-NR purification. Performed analyses are also nearing to results obtained by Sherrard and Dalling [17] which purified and analyzed of the physicochemical properties of NR isolated from leaves of wheat (Triticum aestivum L.). These authors with 21% recovery obtained 2573 fold purified enzyme with the specific activity of 23.1 unit × mg^-1 of proteins. However Oji et al. [13] which for purification of NR from leaves of barley (Hordeum vulgare L.) used precipitating with ammonium sulphate, Phenyl Sepharose CL-4B chromatography, Hydroxyapatite chromatography, Blue sepharose CL-6B chromatography CL-6B and HPLC purified enzyme 4260 fold with the recovery of 2.1%.

Results of electrophoresis in 15% polyacrylamide gel containing SDS showed, that NR from winter triticale seedlings is a homogeneous protein fraction with the molecular weight about 190000 Da (Fig. 1). Hoff et al. [6] showed, that this enzyme in higher plants is a dimer construct from two single subunits contain from 881 (in the pea) to 926 (in the spinach) the amino acid residues. According to Barbier et al. [2] the mass of one monomer amounts about 100000 Da, and the all enzyme 200000 Da [18]. Nakagawa et al. [12] ascertained however that NR from leaves of the maize can occur in two molecular forms in the dependence on the eluent of used to washing of its from the chromatographic column. In the case when elution was managed by NADH₂ solution, the molecular weight of the enzyme amounted 160000 Da while after using of nitrate – 433000 Da. In the opinion of authors [12] this difference was caused by different degree of the polymerization of the enzymatic protein.

Spectroscopic analyses showed that highly purified nitrate reductase from shoots of winter triticale seedlings had absorption maxima at 244, 463 and 491 nm (Fig. 2). This results proved that obtained NR occurred in oxidized form [12]. Ho and Tamura [5] state however, that the enzyme extracted from spinach leaves had maxima at 276, 388 and 573 nm. Moreover, the results obtained and those from of earlier studies suggest that the absorption spectra of NR can themselves differ significantly depending on the plant species, its organs, and also the conditions of the analyses.

NR from shoots of winter triticale seedlings was specific with relation to NADH₂ as well as NADPH₂, though it showed lower specific activity in presence of second coenzyme (Table 2). Similar results obtained Kleinhofs and Warner [7], in who opinion cereals such as maize, barley and rice contain NADH-NR as well as NADPH-NR. Moreover roots of barley, maize and oat contain monospecific NADH-NR and bispecific NAD(P)H-NR [15].

NR isolated from the seeds of winter triticale shows additional activities such as MVH (methylviologen) – NR, FMNH₂ – NR, NADH-ferricyianide reductase and NADH-Cyt c reductase (Table 3). Hoff et al. [6] stated that in vitro condition NR is clever to the catalysis of some reaction discordant with a general schema of her functions. These reactions demand presences of additional prosthetic groups and use artificial donors and acceptors of electrons.

Presented results evidence of partial ability of studied enzyme to the catalysis of other reactions than the reduction of nitrate to nitrite with the utilization NADH₂ or NADPH₂ as electrons donors. Different activities of the enzyme in the presence of various coenzymes may have significant metabolic importance, because the ability of nitrate reduction is a fundamental process for the metabolism of the plant nitrogen. This reaction makes it possible to obtain a basic mineral nitrogen form (nitrate) and include this component in the organic compounds. The rate of the assimilative reduction of nitrate limits the rate of transformation of all plant nitrogenous compounds, i.e. proteins, nucleic acids, nucleotides, and in consequence it conditions the quantity and quality of the crop plant yield.

CONCLUSIONS

Nitrate reductase from winter triticale seedlings is homogenous protein fraction with molecular weight about 190000 Da. The enzyme is partially able to catalyze reactions other than the reduction of nitrate to nitrite in presence NADH2 or NADPH2 as electron donors.

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Accepted for print: 4.01.2008

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