Purification and partial characterization of nitrate reductase from barley leaves

Abstract

NADH-nitrate reductase from barley leaves ( Hordeum vulgare L. cv. Steptoe) was purified 570-fold with over 50% yield by a procedure using ammonium sulfate fractionation and blue dextran-agarose affinity chromatography. The purified enzyme had NADH-, FMNH 2-, and reduced methyl viologen-nitrate reductase and NADH-cytochrome c reductase activities, but not NADPH nitrate reductase activity. The NADH-nitrate reductase has a pH optimum at 7.5 and a Michaelis constant ( K m) for nitrate of 2.4 × 10 −4 M. The specific activity of the purified enzyme was 8 μ mol NO 2 −/min/mg protein. A single protein band corresponding to reduced methyl viologen-nitrate reductase activity was observed by disc gel electrophoresis. A S 20,w of 7.9 S was found by a sucrose density centrifugation and a Stokes radius of 68 Å was determined by gel filtration. From these values, a molecular weight of 221 000 was estimated for the native enzyme. SDS-gel electrophoresis of the native enzyme indicated a subunit molecular weight of approximately 100 000. Thus, the barley nitrate reductase contains two subunits of the same size.