NADPH- and NADH-nitrate reductases from soybean leaves

Abstract

Two nitrate reductase activities from young first leaves of soybeans have been isolated and separated by DEAE-cellulose column chromatography. They have different mobilities when electrophoresed on polyacrylamide gels. These two enzymes have been designated NADH-nitrate reductase and NADPH-nitrate reductase because of their differing affinities for reduced pyridine nucleotides. Five to ten millimolar cysteine was required in the extraction medium to achieve maximum activity of the two nitrate reductases. NADPH-nitrate reductase was about 50% as active as NADH-nitrate reductase. NADPH-nitrate reductase was detected in extracts prepared without cysteine, whereas the NADH enzyme was not found in the absence of cysteine. Both nitrate reductases utilized NADH, NADPH, FADH 2 (reduced flavin-adenine dinucleotide), reduced methyl viologen, and reduced benzyl viologen as electron donors. When NADPH was used as the electron donor for NADPH-nitrate reductase, FAD (flavin-adenine dinucleotide) was required for maximum activity, but FAD did not enhance the activity of NADH-nitrate reductase. NADH-nitrate reductase had a molecular weight of 330,000 by gel filtration, a pH optimum of 6.5 with NADH, K m (KNO 3) of 0.11 m m, and K m (NADH) of 8.1 μ m. NADPH-nitrate reductase had a 220,000 molecular weight as determined by gel filtration, a pH optimum of 6.5 in phosphate and of 6.2 in morpholinoethane sulfonic acid buffer, a K m (KNO 3) of 4.5 m m, and a K m (NADPH) of 1.5 μ m. Potassium cyanide and sodium azide inhibited both nitrate reductases, while potassium fluoride did not inhibit either.