Purification and properties of a nitrate reductase-inactivating enzyme

Abstract

The NADH nitrate reductase-inactivating enzyme in the maize root has been purified 460-fold. It was precipitated by 40–60% saturation with (NH 4) 2SO 4 and was soluble at pH 4.0. On a CM-32 cellulose column, equilibrated with 10 mM acetate (ph 5.0), it was eluted after a lag as a single peak with 10 mM acetate containing 50 mM NaCl (pH 5.0). It was then characterized on a Sephadex G-200 column and its molecular weight estimated to be 44 000. The inactivating enzyme, like nitrate reductase, has its optimum activity in the neutral pH range and appears to be located in the cytoplasm of the mature-root cell. It was more stable to heat treatment than nitrate reductase. Inhibition of the inactivating enzyme by phenylmethylsulphonyl fluoride suggested the involvement of a serine residue at its active site. When phenylmethylsulphonyl fluoride was used in the extraction medium for the mature root, the in vitro inactivation of nitrate reductase was prevented. The fraction containing the inactivating enzyme degraded casein by a phenylmethylsulphonyl fluoride-sensitive reaction but it had no peptidase activity.