NADH‐dependent nitrate reductase from two‐row barley roots: Purification, characteristics and comparison with leaf enzyme

Abstract

NADH‐nitrate reductase (EC 1.6.6.1) was purified 800‐fold from roots of two‐row barley (Hordeum vulgare L. cv. Daisen‐gold) by a combination of Blue Sepharose and zinc‐chelate affinity chromatographies followed by gel filtration on TSK‐gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)−1 min−1 at 30°C.Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH‐cytochrome c reductase, NADH‐ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH2‐nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent Km for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent Km for nitrate as well as Vmax whereas it did not alter the Km for NADH.