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Page 1: Separation of soybean leaf nitrate reductases by affinity chromatography

Plant Science Letters, 7 (1976) 239--247 239 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands

SEPARATION OF SOYBEAN LEAF NITRATE REDUCTASES BY AFFINITY CHROMATOGRAPHY

WILBUR H. CAMPBELL

Environmental Biochem. and Physiol. Program, SUNY, College of Environmental Science and Forestry, Syracuse, N.Y. 13210 (U.S.A.)

(Received March 23rd, 1976) (Accepted May 4th, 1976)

SUMMARY

The NADH: and NADPH: nitrate reductase activities found in soybean leaf extracts were separated into two distinct enzyme components by affinity chromatography on blue dextran-Sepharose. The two soybean nitrate reductases have been designated N AD(P)H: and NADH: nitrate reductase. The NAD(P)H: nitrate reductase, isolated with a specific activity of 650 nmoles/min/mg protein, was characterized by a high Km for KNO3 (4.5 raM) and an ability to utilize both NADPH and NADH as electron donor. The pH optima for NADPH:, NADH:, FADH2 :, and reduced methyl viologen:nitrate reductase activities of the NAD(P)H:nitrate reductase were found to be 6.5; whereas, the pH optima for the NADPH: and NADH:cytochrome c reductase activities of NAD(P)H: nitrate reductase were 7.5. It was suggested that catalysis of the conversion of nitrate to nitrite is the rate limiting step during nitrate reduction by soybean NAD(P)H: nitrate reductase. NADH: nitrate reductase with a specific activity of 360 nmoles/min/mg protein was found to have a pH opt imum of 6.5 for nitrate reduction, a Km (KNO3) of 0.11 mM, and the ability to utilize mainly NADH. The physiological significance of the two soybean nitrate reductases was discussed in terms of their kinetic properties.

INTRODUCTION

Some uncertainty has been associated with the nitrate reduction process in the leaves of soybean. Originally, only NAD(P)H: nitrate reductase (EC 1.6.6.2) was found in soybean leaf extracts [1]. Hageman and coworkers [2,3] have demonstrated that NADH:nitrate reductase (EC 1.6.6.1) is predom- inant in the cysteine stabilized soybean leaf extracts. Jolly et al. [4] have separated the NAD(P)H: and NADH:nitrate reductases from extracts of soy- bean leaves. The soybean NAD(P)H: nitrate reductase has a Sephadex molecular weight of 220 000, pH opt imum of 6.5, Km (KNO3) of 4.5 mM,

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K m (NADPH) of 1.5 #M, Km (NADH) of 3.9 pM, and a ratio of NADPH to NADH activity of 1.5 [4]. The soybean NADH: nitrate reductase has a Sephadex molecular weight of 330 000, pH optimum of 6.5, Km (KNO3) of 0.11 mM, K m (NADH) of 8 uM, Km (NADPH) of 200 uM, and a ratio of NADH to NADPH activity of 2 [4]. NAD(P)H: nitrate reductase was stimulated by FAD and FMN; whereas, NADH: nitrate reductase was not [4].

The DEAE-cellulose separation and purification of soybean nitrate reductases provided only about a four-fold purification and 14 to 20% yield [4]. The application of the affinity medium blue dextran-Sepharose [ 5] to the purification of soybean nitrate reductases, described here, provides preparations of these enzymes with high specific activities and with yields of 40% or greater.

MATERIALS AND METHODS

Soybeans (Glycine max L. Merr-Kanrich) were grown and extracted as previously described [4], except the leaves were extracted with 10 mM potassium phosphate, pH 7.0, 1 mM EDTA, and 10 mM cysteine. The centrifuged crude extract was mixed with blue dextran-Sepharose which had been made according to Ryan and Vestling [6] and had been equilibrated with the extraction buffer. 80 ml of wet settled blue dextran-Sepharose were added to the extract made from 25 to 50 g of primary leaves. After 10 to 15 min of gentle stirring, the blue dextran-Sepharose was recovered by filtration and washed with extraction buffer. The blue dextran-Sepharose was poured into a 2.5 cm diameter column to give a bed height of 15 cm. The column was eluted with 200 ml of 0.05 mM NADPH in extraction buffer. 10-ml fractions were collected and assayed for nitrate reductase using both an assay for NAD(P)H: and NADH:nitrate reductase as previously described [4]. The blue dextran-Sepharose was further eluted with 25{) ml 0.3 M KNO3 in extraction buffer. 10-ml fractions were collected and assayed for both nitrate reductases. The pooled NADPH elution fractions were used without concen- tration. The 0.3 M KNO3 elution fractions were pooled and concentrated by ammonium sulfate precipitation and were desalted by Sephadex G-25 gel filtration [4]. Nitrate reductase assays were done by methods previously described [4]. pH optimum determinations were done as previously described [4]. Cytochrome c reductase assays were done by following the increase in absorbance at 550 nm in 1 ml cuvettes using either a Gilford or Cary 15 spectrophotometer [7]. When cytochrome c reductase assays were done on nitrate reductase preparations, it was necessary to omit cysteine from the extraction buffer [7]. Protein determinations were done by the method of Lowry et al. [8]. All biochemicals were purchased from Sigma Chemical Company, St. Louis, Mo.

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RESULTS

Essentially all the nitrate reductase activity of the crude extract of soybean leaves binds to blue dextran-Sepharose. As shown in Fig. 1, elution of the blue dextran-Sepharose with 0.05 mM NADPH yields a nitrate reductase activity which was most active with NADPH. Subsequent elution of the column with 0.3 M KNO3 yields a second nitrate reductase activity which was much more active with NADH than NADPH (Fig. 1). Therefore, the nitrate reductase activities eluted from blue dextran-Sepharose with NADPH and I~'qOs have been designated NAD(P)H: and NADH:nitrate reductase, respectively. The results of a typical purification are summarized in Table I.

Both NAD(P)H: and NADH:nitrate reductase will accept electrons for reduction of nitrate to nitrite from NADPH, NADH, FADH2, and reduced methyl viologen. The pH optima for all these substrates for nitrate reduction by both enzymes is 6.5. The pH optima for NAD(P)H:nitrate reductase are shown in Fig. 2. However, when NADPH donates electrons to NADH: nitrate

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Page 4: Separation of soybean leaf nitrate reductases by affinity chromatography

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Page 5: Separation of soybean leaf nitrate reductases by affinity chromatography

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reductase, the pH optimum is closer to pH 6.0 and this may not be a direct process [3,4].

The ratio of NADPH supported nitrate reduction to either NADH, FADH2, or reduced methyl viologen supported nitrate reduction for both the NAD(P)H: and NADH:nitrate reductase was essentially the same as was reported for the less purified soybean nitrate reductases [4]. The determination of I(m'S for KNO3, NADPH, and NADH for the NAD(P)H: and NADH: nitrate reductases were also essentially the same as previously reported [4].

Cytochrome c reductase activity can be demonstrated for both soybean nitrate reductases. However, because the presence of sulfhydryl reducing agents interfere with the cytochrome c reductase assay [7] and NADH: nitrate reductase is unstable without reducing agent [1,2,4], I was unable to obtain quantitative data for that enzyme. Although NAD(P)H: nitrate reductase appears to be stabilized by cysteine [2,4], this enzyme is sufficiently stable without cysteine to allow preparations of the blue dextran-Sepharose enzyme and I could study the properties of the cytochrome c reductase of this enzyme.

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244

When soybean primary leaf extracts, prepared wi thout cysteine, were purified using blue dextran-Sepharose, the elution of the column with NADPH resulted in an identical pat tern to that shown in Fig. 1. The elution pattern of NADPH cytochrome c reductase from the column was identical to the NADPH nitrate reductase. Further elution of the column with 0.3 M KNO3 yielded no nitrate reductase activity. The yields of NADPH nitrate reductase and cy tochrome c reductase were 16% and 28% of the crude extract activity, respectively. The specific activities were 90 nmoles of nitr i te/min/mg protein and 1500 nmoles of cy tochrome c reduced/min/mg protein.

FAD stimulated the NADPH nitrate reductase activity of this preparation of NAD(P)H: nitrate reductase by 30%, but FAD did not stimulate the NADPH cytochrome c reductase activity. The ratio of cy tochrome c reductase to nitrate reductase was 8.4 when NADPH was the electron donor at pH 6.5. The ratio of cy tochrome c reductase to nitrate reductase in the crude extract of soybean was 4.8, which was similar to the ratio in barley using the data of Wray and Filner [7] . The pH opt ima for cy tochrome c reductase was 7.5 with either NADPH or NADH as electron donor (Fig. 2). The Km's for NADPH and NADH as electron donor for cy tochrome c reductase with 80 pM cyto- chrome c were 1.7 and 4 pM, respectively. However, the Vmax'S were equal. The Vmax for NADPH nitrate reduction is 1.5 times the Vmax for NADH, which is a similar result to that previously found for soybean NAD(P)H: nitrate reductase [4] .

DISCUSSION

A review of the literature reveals that to achieve a high level of purification of nitrate reductase, one must utilize a multi-step procedure which generally results in a poor yield of purified enzyme [9] . Two recent papers [10,11] have applied affinity media to the purification of nitrate reductase. Solomon- son [10] has purified Chlorella nitrate reductase by affinity binding to blue dextran-Sepharose, providing a homogeneous enzyme with 60% yield. Solomonson [10] has suggested that blue dextran-Sepharose can be used as a general tool for purification of assimilatory nitrate reductases. Heimer et al. [11] have used NADH-Sepharose to purify an alga and a higher plant nitrate reductase. They [ 11 ] obtained barley nitrate reductase in a yield and purity similar to that which had been obtained for spinach nitrate reductase by a multi-step procedure [12] . The procedure I used to separate and purify soybean nitrate reductases utilizes blue dextran-Sepharose in a manner similar to Solomonson [10] and results in specific activities for the soybean nitrate reductases which are equal or nearly equal to those achieved for spinach nitrate reductase [12] .

Since blue dextran-Sepharose appears to bind proteins at the site of pyridine nucleotide binding [5] , the separation of the two soybean nitrate reductases is apparently achieved as a result of the differences in their affinities for NADPH. The large differences of Km's for NADPH of the

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NAD(P)H: and NADH: nitrate reductases appear to reflect this difference in affinities [4].

The fold purification and yields of soybean nitrate reductases separated by blue dextran-Sepharose affinity chromatography are greater than those obtained by DEAE-cellulose column chromatography [4]. However, the properties of the soybean nitrate reductases purified by blue dextran- Sepharose are nearly identical to those reported for these same proteins purified by DEAE-cellulose [4]. I have described the properties of the cytochrome c reductase activity of the soybean NAD(P)H: nitrate reductase which was not done in the earlier study [4].

Little data has been reported on the cytochrome c reductase activity associated with the nitrate reductase of higher plants. Wray and Filner [7] studied the cytochrome c reductase activity of barley leaves at pH 7.5. They [7] showed that this activity had the same induction kinetics as the nitrate reductase activity and that the sedimentation characteristics of the nitrate induced cytochrome c reductase and nitrate reductase were identical. Garrett and coworkers [13,14] have studied the properties of cytochrome c reductase activity of the nitrate reductase of Neurospora crassa and Thalassiosira pseudonana. Their research showed the pH optimum of the cytochrome c reductase activity to be the same as the nitrate reductase activity, namely pH 7.5 [13,14]. Furthermore, they showed that the cytochrome c reduction had the same cofactor requirements as the nitrate reduction [13,14]. The ratio of cytochrome c reductase activity to nitrate reductase activity for the nitrate reductases of four organisms range from 2.4 to 5.9 [13--15] ; none of which are as high as that reported here for soybean NAD(P)H: nitrate reductase. Since this NAD(P)H: nitrate reductase was prepared without the benefit of cysteine [4] and previous studies on spinach nitrate reductase have indicated the cytochrome c reductase activity to be more stable than the complete nitrate reductase [16], it can be suggested that the high ratio of cytochrome c reductase to nitrate reductase is a result of the partial denaturation of the NAD(P)H: nitrate reductase.

In other nitrate reductase enzyme complexes where the nitrate reductase activity was found to be stimulated by FAD, the cytochrome c reductase activity was also stimulated [13,14]. This does not appear to be the case with soybean NAD(P)H: nitrate reductase. The FAD stimulation of NAD(P)H: nitrate reductase is different for each preparation and will require further study.

If denaturation of the NAD(P)H: nitrate reductase has occurred during isolation, it appears unlikely to have disrupted the structure of the cytochrome c reductase component of nitrate reductase. This is based on the similarity of Km's for the pyridine nucleotides for cytochrome c reduction and nitrate reduction and suggestions from the literature that the cytochrome c reductase component is more stable than the FADH2 nitrate reductase component of the nitrate reductase [9,16]. Thus, the demonstration of a pH optimum for the cytochrome c reductase of 7.5, while the complete nitrate reductase

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reaction and the partial reactions of FADH2 and reduced methyl viologen have pH optima of 6.5 (see Fig. 2) is significant. This would suggest that the rate limiting aspect of the nitrate reductase's catalysis of nitrate reduct ion is located in the FADH2 nitrate reductase component and not in the cytochrome c reductase component . This also might be inferred from the fact that cy tochrome c reductase activities are generally greater than either the NADH: or FADH~ : nitrate reductase activities [13--15] . Furthermore, this difference in pH optima for the partial reactions, indicates that the structural alteration responsible for the pH 6.5 opt ima of the soybean nitrate reductases is in the FADH2 : nitrate reductase component . Soybean nitrate reductases are the only nitrate reductases with pH opt ima below pH 7.0 [9] .

Shen [17] has shown that an NADPH utilizing nitrate reductase is present in the extracts of rice seedlings. Wallace [18] has shown the presence of NADPH utilizing nitrate reductase in corn scutella extracts, which had been investigated earlier by Elsner [19] . I have recently shown the separation of NADPH and NADH nitrate reductase activities of corn scutella [20] . NADPH utilizing nitrate reductases are not unique to the extracts of soybean leaves and it appears that these activities probably do not result from an artifact of the preparation.

Tingey et al. [21] have provided evidence for the in vivo presence of both NADH and NADPH utilizing nitrate reduction systems in the leaves of soybean. Assuming that the nitrate reductases isolated from the leaves of soybean have the same properties in vivo and in vitro, one can suggest that the physiological roles of these two nitrate reductases may be related to their kinetic properties. NAD(P)H: nitrate reductase functions optimally at concen- trations of nitrate of 5 mM or higher and can utilize both NADPH and NADH. NADH: nitrate reductase functions optimally at concentrations of nitrate in the range of 1 to 10 mM and can utilize only NADH. Jolly et al. [4] found in the earlier s tudy of these enzymes that NADH: nitrate reductase was inhibited at high concentrat ions of KNO3. Therefore, NAD(P)H: nitrate reductase would only be operational at high concentrations of nitrate bu t would be able to scavenge any available excesses of both NADPH and NADH. Thus, at the high concentrat ions of nitrate that are probably found in the leaves of soybean seedlings [ 22] , NAD(P)H: nitrate reductase could be making a significant contr ibut ion to the total nitrate reducing ability of the soybean leaf.

REFERENCES

1 H.J. Evans and A. Nason, Plant Physiol., 28 (1953) 233. 2 L. Beevers, D. Flesher and R.H. Hageman, Bioehim. Biophys. Acta, 89 (1964) 453. 3 G.N. Wells and R.H. Hageman, Plant Physiol., 54 (1974) 136. 4 S.O. Jolly, W.H. Campbell and N.E. Tolbert, Arch. Biochem. Biophys., (1976) in press. 5 S.T. Thompson, K.H. Kass, and E. Stellwagen, Proc. Nat. Acad. Sci. USA, 72 (1975)

669. 6 L.D. Ryan and C.S. Vestling, Arch. Biochem. Biophys., 160 (1974) 279.

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7 J.L. Wray and P. Filner, Biochem. J., 119 (1970) 715. 80.H. Lowry, N.J. Rosebrough, A.L. Farr, and R.J. Randall, J. Biol. Chem.,

193 (1951) 265. 9 E.J. Hewitt, Annu. Rev. Plant Physiol., 26 (1975) 73.

10 L.P. Solomonson, Plant Physiol., 56 (1975) 853. 11 Y.M. Helmet, S. Krashin and E. Riklis, FEBS Lett., 62 (1976) 30. 12 A. Paneque, F. DeICampo, J. Ramirez and M. Losada, Bioehim. Biophys. Acta,

109 (1965) 79. 13 R. Garrett and A. Nason, J. Biol. Chem., 244 (1969) 2870. 14 N. Amy and R. Garrett, Plant Physiol., 54 (1974) 629. 15 L. Solomonson, G. Lorimer, R. Hall, R. Botchers and J. Bailey, J. Biol. Chem.,

250 (1975) 4120. 16 E. Palacian, F. DeLaRosa, F. Castiilo and C. Gomez-Moreno, Arch. Biochern. Biophys.,

161 (1974) 441. 17 T.C. Shen, Planta, 108 (1972) 21. 18 W. Wallace, Plant Physiol., 52 (1973) 191. 19 J.E. Eisner, Ph.D. Thesis, Univ. of Illinois, Urbana, Ill., 1969. 20 W.H. Campbell, Plant Physiol. Supplement, 57 (1976) 70. 21 D. Tingey, R. Fires and J. Baharsjah, New Phytol., 73 (1974) 21. 22 P.S. Thibodeau and E.G. Jaworski, Planta, 127 (1975) 133.