NADH-Nitrate Reductase Inhibitor from Soybean Leaves

Abstract

A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Meff. 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 sul-fate precipitation foUowed 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 elec-trophoresis 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 dinucle-otide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The hibitor did not appear to be a protease. Some form of association of the active hibitor 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 noncom-petitive with nitrate but caused a decrease in V,,. The Isolated Inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity retrned. 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. Photoinac-tivation of yellow impure inhibitor required no additional components, but inactivation of the puri 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. NR: nitrate reductase; DCPIP: dichlorophenolindo-phenol. sively reviewed (2), and our manuscript is concerned with the discovery, isolation, and partial characterization of an inhibitory protein for NR which may account for part of these phenomena. In addition, preliminary evidence is also presented for the presence of a stimulatory protein for NR. Contrary to previous hypotheses involving a rapid loss of NR in the dark, the enzyme and the inhibitor content from young soybean leaves does not change rapidly between conditions of light and dark, but rather regulation seems to be achieved by a reversible change in the inhibitor from an active form in the dark to an inactive form in the light. NR inhibitors isolated from corn roots (25, 32-34) and from rice roots (13) have been identified as proteases which attack NR from either roots or leaves. The inhibitor which we have isolated from leaves does not seem to be a protease, but brings about a reversible inhibition of the enzyme. MATERIALS AND METHODS Plant Material. Soybeans (Glycine max L. Merr. var. Amsoy) were germinated in Vermiculite in a dark room for 3 days and grown for 5 additional days at 28 C in a chamber with about 360 ,uE m-2 sec' of continuous light from fluorescent lamps supplemented with tungsten lamps. Then part of the plants were exposed to darkness for an additional 54 hr, and others were kept continuously in the light. NR was induced by watering with 50 mM KNO3 in Hoagland nutrient solution every day after the 4th day of light. Young first leaves were used for enzyme and inhibitor preparations. NR and Related Assays. NR (EC 1.6.6.2) with NADH, NADPH, FADH2, or reduced methylviologen was measured by a colorimetric determination of nitrite formation (8), as used previously by us (12). A unit of activity was I ,umol of nitrite production/min. The unique feature of this assay was the use of varying amounts of KNO3 to distinguish between the NADH-and NADPH-NR. In the present work FAD was not in the reaction mixture for the routine NR assays and I mM cysteine was used throughout the isolation procedure for the inhibitor. NADH-DCPIP reductase was measured by a decrease in A at 600 nm (26). NADH-Cyt c reductase activity was monitored at 550 nm in a recording spectrophotometer (7). These partial reactions of NR have been described in more detail (12). The in vivo NR activity was measured as the rate of nitrite excretion during a 30-min period by 10 leaf discs of 10-mm diameter (8). Assay for NR Inhibitor. The rate of nitrite formation by a stock preparation of NR was measured with and without the inhibitor. An aliquot of the inhibitor was added to a reaction mixture which contained in I ml 25 pimol of K-phosphate at pH 6.5, 10 ,umol of KNO3, and a specified amount of NADH-NR from soybean leaves. Without preincubation, the reaction was started by adding 0.2 jmol of NADH and was run for 30 min at 30 C. The amount of inhibitor was preselected by trial and error to give 30 to 55% inhibition, a range in which the inhibition was approximately linear with the amount of inhibitor. Data are expressed as a per cent inhibition or as the nmol of NR that were inhibited. The ,g of protein in the inhibitor fraction was measured by the Lowry 197