Purification and properties of a nitrate reductase inactivating factor from rice cells in suspension culture

The nitrate reductase inactivating factor in cultured rice cellswas purified 320-fold. The purification procedure involved precipitationwith (NH₄)₂SO₄, fractionation at pH 4.0, adsorption on CM-cellulose,and gel filtration on Sephadex G-200. The molecular weight wasestimated to be 200,000 from the Sephadex G-200 gel filtration. The inactivating factor shows maximal activity at pH 8.0 andappears to be located in the cytoplasm of the cultured ricecells. The inactivating factor was more stable to heat treatmentthan NADH nitrate reductase. The factor inactivated nitratereductase complex except for reduced methylviologen nitratereductase. It had no influence on the activity of nitrite reductase,glutamate dehydrogenase, and NADH diaphorase, but inactivatedxanthine oxidase. The inactivating factor had no protease activitywhen casein, bovine serum albumin, or nitrate reductase fractionwas used as the substrate. The type of inactivation of nitratereductase by the inactivating factor was noncompetitive. Inhibitionof the inactivating factor by o-phenanthroline, EDTA, and p-chloromercuribenzoicacid suggested the involvement of a metal and sulfhydryl groupat its active site. (Received January 28, 1977; )