Nitrate reductase inactivator from Spirodela polyrhiza (L.) Schleiden.

NADH:nitrate reductase (EC 1.6.6.1) activity in the crude extract from Spir odela polyrhiza was relatively labile in vitro. Inclusion of polyvinylpolyp yrrolidone into the extraction medium had only a slight effect on the stabi lity of the enzyme, whereas addition of 3 % casein, azocasein, or other pro teins to the extraction medium greatly increased the nitrate reductase (NR) activity. Various protease inhibitors were tested for their ability to pre vent the loss of NR activity in vitro. Iodoacetate and para-chloromercutric benzoate, the thiol-protease inhibitors, as well as pepstatin, the asparti c-protease inhibitor had no effect on stability of the nitrate reductase. E DTA had a slight stimulatory effect, whereas 5 mM o-phenantroline, another inhibitor of the metallo-proteases increased the activity of nitrate reduct ase. The highest enzyme activity was found in the presence of phenylmethyls ulphonyl fluoride and di-isopropyl phosphorofluoridate both being serine-pr otease inhibitors. The protease-like inactivator was separated from Spirodela polyrhiza by amm onium sulfate fractionation and acid treatment (pH 4.0). After centrifugati on the protein of inactivator in supernatant adjusted to pH 7.5 was removed . When this fraction was examined by electrophoresis in polyacrylamide whic h copolymerized with edestin, the protein of the nitrate reductase inactiva tor remained at the cathode. Fractions containing a protein of inactivator degraded casein to products soluble in trichloroacetic acid. Inhibition of the inactivator proteolytic activity by phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate but not by other reagents (thiol- and meta llo-protease inhibitors) suggested the involvement of a serine residue at i ts active site. The inactivator fraction from Spirodela polyrhiza resulted in a loss of the nitrate reductase activity in crude extracts from both cuc umber and corn seedlings. A biochemical nature a protein of the nitrate red uctase inactivator from S. polyrhiza is discussed.