NADH-Nitrate Reductase Inhibitor from Soybean Leaves

A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.

The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO₃. The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in V_max.

The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.

Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.