NADH: nitrate reductase and NAD(P)H: nitrate reductase activities in mustard seedlings

Mustard (Brassica juncea L. Czern and Coss cv. Pusa Jai Kisan) seedlings were grown for 10 days in nutrient solutions containing different concentrations of nitrate (10, 20, 30, 40 and 50 mM). Nitrate reductase activity was determined in the presence of either NADH or NADPH as electron donor. NADH- and NADPH-supported nitrate reductase activity varied significantly in the seedlings grown at different nitrate concentrations. The NR activity was higher with NADH than NADPH in the seedlings grown at 10 and 20 mM nitrate levels. With the increase in nitrate concentration in nutrient solution, NADPH-supported NR activity gradually increased, while NADH-supported NR activity remained nearly constant. Seedlings grown at 30 mM nitrate had almost equal NR activity when either NADH or NADPH was used as electron donor. When NR activity was assayed in the presence of an equimolar concentration of both NADH and NADPH, it was greater than the one observed either with NADH or NADPH alone. By the use of affinity chromatography using blue-Sepharose, two nitrate reductases, viz. NADH:NR (EC 1.6.6.1) and NAD(P)H:NR (EC 1.6.6.2) were partially purified. The trend of activities of purified NADH:NR and NAD(P)H:NR were almost similar to that observed in the crude extract. The NAD(P)H:NR has a K-m of 2.5 mM for KNO3, K-m of 0.05 mM for NADPH and K-m of 0.20 mM for NADH. The NADH:NR has a K-m of 0.10 mM for KNO3, K-m of 0.01 mM For NADH. The pH optima for both the NRs was 6.8. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.