Immunochemical Characterization of Nitrate Reductase Forms from Wild-Type (cv Williams) and nr1 Mutant Soybean

Abstract

Soybean (Glycine max L. Merr.) leaves contain two forms of nitrate reductase (NR)—NAD(P)H:NR and NADH:NR. Wild-type (cv Williams), nr1 mutant and an unrelated cultivar (Prize) were grown with either no N source or with nitrate. Crude extracts were assayed for NR activities and the enzyme forms were purified on blue Sepharose. Analyses were done by polyacrylamide gel electrophoresis and `Western blotting' using antibodies specific for NR. NAD(P)H:NR was identified as the constitutive NR present in wild-type and Prize, but was absent from the mutant. All three soybean lines contained nitrate-inducible NADH:NR with highest activity at pH 7.5. The results showed that NAD(P)H:NR and constitutive NR were one in the same and confirmed the presence of NADH:NR with pH 7.5 optimum.