NAD(P)H Bispecific nitrate reductase in barley leaves: Partial purification and characterization

Abstract

A nitrate reductase was isolated from a barley (Hordeum vulgare L.) mutant (nar la) deficient in NADH nitrate reductase (EC 1.6.6.1). The nitrate reductase from nar la exhibited activity with both NADH and NADPH as electron donors. The enzyme was purified 200 fold with a recovery of 49 %. Based upon kinetic, catalytic, and immunological characteristics the enzyme was determined to be a NAD(P)H bispecific nitrate reductase (EC 1.6.6.2) with an NADPH to NADH activity ratio of 1.8. The bispecific enzyme cross-reacted with antiserum raised against the NADH nitrate reductase from the wild type but with a much lower specificity than the NADH enzyme. The Km for nitrate of the NAD(P)H enzyme was 0.61 mM which was about five times greater than that of the NADH enzyme. The Km for NADPH and NADH of the bispecific enzyme were 10 μM and 68 μM, respectively, while the Km for NADH of the NADH enzyme was 10 μM. Unlike the NADH enzyme, the NAD(P)H bispecific enzyme was inhibited by dithionite and NADPH. These data support the conclusion that the nitrate reductase in the nar la mutant of barley is a single NAD(P)H bispecific enzyme.