Purification and Characterization of NADH-Nitrate Reductase from Leaves of 2-Row Barley, and Its Activity as Affected by Some Metabolites

Abstract

Summary NADH-nitrate reductase (EC 1.6.6.1) was purified 4260-fold from leaves of 2-row barley ( Hordeum vulgare L.). The purification procedure involved extraction with Tris-HCl buffer, ammonium sulfate fractionation, adsorption on Phenyl Sepharose CL-4B and hydroxyapatite, affinity chromatography on Blue Sepharose CL-6B and followed by gel filtration on TSK-Gel (G-4000SW) by using high-performance liquid chromatography. The purified preparation was nearly homogenous as revealed by disc gel electrophoresis. It had a specific activity of 37 μmol nitrite formed min −1 · mg protein −1 at 30°C; the highest specific activity among nitrate reductases so far isolated from a variety of barley cultivars. Its native molecular weight was estimated as 225,000. The 130,000 daltons subunit found on SDS gels seemed to be the actual nitrate reductase subunit, the smaller proteins seeming to be degradation products. The respective apparent Km values for NADH and nitrate at pH 7.5 (optimum for the activity) were 3.8 and 270 μM. FAD was required for the maximal activity of the enzyme; the apparent Km for FAD was 8 nM. Carbamyl phosphate behaved as a competitive inhibitor with respect to nitrate. Among adenine derivatives, only ADP inhibited the activity, the inhibition being competitive with NADH. Inhibition by pyridoxal-5′-phosphate was uncompetitive with nitrate. No inhibition was observed with pyridoxal itself, or with pyridoxine or pyridoxamine-5′-phosphate. Other metabolites tested were without effect on the activity. It seems that barley leaf nitrate reductase has a lysine residue at its active site. Heavy metals (Cu 2+ , Zn 2+ , Co 2+ , {ie1} and {ie2}) caused a strong inhibition at 1 mM. {ie3} (10 mM) activated the activity.