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. 1985 May;78(1):80–84. doi: 10.1104/pp.78.1.80

Nitrate Reductases from Wild-Type and nr1-Mutant Soybean (Glycine max [L.] Merr.) Leaves 1

I. Purification, Kinetics, and Physical Properties

Luc Streit 1,2,2, Richard S Nelson 1,2, James E Harper 1,2
PMCID: PMC1064680  PMID: 16664214

Abstract

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr1-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c1NR, and the form eluted with NADH was designated c2NR. Nitrate-grown nr1 mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c1NR and c2NR had similar pH optima of 6.5, sedimentation behavior (s20,w of 5.5-6.0), and electrophoretic mobility. However, c1NR was more active with NADPH than with NADH, while c2NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c1NR) and 0.19 millimolar (c2NR). The iNR from the mutant had a pH optimum of 7.5, s20,w of 7.6, and was less mobile on polyacrylamide gels than c1NR and c2NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

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Selected References

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