Purification and characterization of nitrate reductase from nodule cytosol of soybean plants

Nodulated soybean (Glycine max [L.] Merr.) plants were grown in a nitrogen-free liquid culture medium prepared with distilled water. The cytosol fraction from root nodules showed a significant level of NADH-dependent nitrate reductase activity, even when the root did not show activity. This nitrate reductase was purified by column chromatography and native polyacrylamide gel electrophoresis (PAGE). The purified protein showed a main band at 100 kDa on sodium dodecyl sulfate (SDS)-PAGE. The K(m) value for nitrate was 0.16 mM, and the highest activity was obtained at around pH 7.5. These characteristics are very similar to the inducible type of nitrate reductase, previously purified from soybean leaves. The developmental change in activity of this enzyme corresponded to that in nitrogenase activity.