Determination of the distribution of three nitrate reductase isoforms in soybean seedlings by chromatography and a simple method based on assay conditions

Blue Sepharose affinity chromatography was used to study the distribution of the constitutive NAD(P)H-nitrate reductase (EC 1.6.6.2; C1-NR) and of the constitutive and inducible NADH-nitrate reductases (EC 1.6.6.1; C2-NR and i-NR, respectively), in the unifoliolate leaf (F0), the first and the second trifoliolate leaves (F1 and F2) and the roots of urea- and nitrate-grown soybean (Glycine max [L.] Merr.) plants. The C1-NR eluted by NADPH is present in the F0 and F1 leaves and nearly absent in the F2 leaf. The activity pattern of this isoform is not modified by nitrate nutrition. The C2-NR eluted by NADH is high in the F0 leaf, low in the F1 leaf and nearly absent in the F2 leaf of urea-grown plants. The NADH elution from leaves of nitrate-grown plants is a mixture of C2-NR and i-NR, requiring careful interpretation of results. However, i-NR appears the principal isoform in the leaves especially in the F2 leaf. This i-NR is the only NR present in the roots. The pH effect on the assay of the 3 partially purified isoforms was studied using LNR2 and LNR5 soybean mutants to remove the cross contamination. It appears that C1-NR and C2-NR activities are negligible at pH 8.5, which allows the assay of only the i-NR in a crude extract at this pH, even when C1-NR and C2-NR are present. It appears also that the assay of C1-NR activity at pH 6.5 with NADPH is free of interference by the i-NR. To estimate the C2-NR activity with NADH at pH 6.5 in a crude extract in the presence of C1-NR and i-NR, we propose a simple calculation using the coefficient from the pH responses. These calculations are used to compare the development of C1-NR, C2-NR and i-NR activities in the F0 and F1 leaves of plants previously grown on urea and transferred to nitrate. Only the activity of the inducible isoform is modified by the nitrogen treatment. Activity of the constitutive isoforms appear stable during the 48 h treatment, with only a slight decrease in C1-NR activity being observed with time.