NADH-nitrate reductase in barley leaves: Identification and amino acid composition of subunit protein

Abstract

NADH-nitrate reductase (NADH:nitrate oxidoreductase, EC 1.6.6.1) isolated from barley ( Hordeum vulgate L. cv. Steptoe) leaves was identified on a native slab gel after electrophoresis by staining for reduced methyl viologen nitrate reductase activity, NO 2 − production and diaphorase activity. The nitrate reductase protein, when subjected to a second-dimensional SDS-polyacrylamide gel electrophoresis, migrated as a single protein band of M r 110000. Thus, barley nitrate reductase (native molecular weight 221000) consists of two identical subunits. Preparative SDS-polyacrylamide gel electrophoresis was used to isolated highly homogeneous nitrate reductase subunit protein. Fluorograms of peptide maps indicated no homology among the nitrate reductase subunit and other polypeptides (60 kDa diaphorase, 41 kDa, 35 kDa and 25 kDa species) which are common contaminants of the affinity-purified nitrate reductase. Thus, the 60 kDa diaphorase and 41 kDa polypeptide observed under our experimental conditions are not breakdown products of the 110 kDa nitrate reductase subunit. The amino acid composition of the barley nitrate reductase subunit was similar to that reported for the nitrate reductase of Chlorella vulgaris and Ankistrodesmus braunii.