Nitrate reductase from rice seedlings: Partial purification, characterization and the effects of in situ and in vitro NaCl salinity

Abstract

Summary The effect of NaCl salinity in situ on NADH-nitrate reductase (EC 1.6.6.1) activity was studied in rice seedlings grown in sand cultures. NaCl salinity levels of 7 dS m−1 and 14 dS m−1 caused significant inhibition in NR activity in 10- and 20-day-grown seedlings. The enzyme was partially purified from leaves of 15-day-grown non-salinized seedlings using ammonium sulphate (25–45 percent) precipitation and affinity chromatography on a Blue-Sepharose CL-6B column. The purification achieved was 303-fold with 53 percent yield and the purified enzyme showed a specific activity of 3.6 μmoles NO2− min−1 mg−1 protein. NR activity was inhibited in vitro with an increase in NaCl in the assay medium. With 1000 mmol/L NaCl in the assay medium almost 85 percent NR activity was lost. The purified enzyme showed optimum activity at pH 7.5 and temperature optima of 30°C for 30 min. Preincubation of NR with 0.4 mmol/L NADH caused a significant increase in its activity. Apparent Km values of the enzyme for nitrate and NADH were 0.286 mmol/L and 4 mmol/L, respectively. The enzyme NR thus appears to be fairly sensitive to in situ as well as in vitro effects of NaCl.