Nitrate Reductases from Wild-Type and nr₁-Mutant Soybean (Glycine max [L.] Merr.) Leaves

Abstract

Soybean (Glycine max IL.I Merr.) leaves have been shown to contain three forms of nitrate reductase (NR). Two of the forms, which are present in leaves of wild-type (cv. Williams) plants grown in the absence of NO3-, are termed constitutive and designated c,NR and c2NR. The third form, which is present in N03-grown mutant (nrj) plants lacking the constitutive forms, is termed inducible and designated iNR. Samples of cjNR, c2NR, and iNR obtained from appropriately treated plants were analyzed for the presence of partial activities, response to inhibitors, and ability to complement a barley NR which lacks the molybdenum cofactor (MoCo) but is otherwise active.The three forms were similar to most assimilatory NR enzymes in that they (a) exhibited NADH-cytochrome c reductase, reduced flavin mononucleotide-NR, and reduced methyl viologen-NR partial activities; (b) were inhibited by p-hydroxymercuribenzoate at the site of initial electron transport through each enzyme; (c) were more inhibited by CN-in their reduced enzyme state as compared with their oxidized state; and (d) complemented a MoCo-defective NR ( e.g. contained cofactors with characteristics similar to the MoCo found in barley NR and commercial xanthine oxidase). However, among themselves, they showed dissimilarities in their response to treatment with HC03-and CN-, and in their absolute ability to complement the barley NR. The site of effect for these treatments was the terminal cofactor-containing portion of each enzyme. This indicated that, although a terminal cofactor (presumably a MoCo) was present in each form, structural or conformational differences existed in the terminal cofactor-protein complex of each form.Assimilatory NR3 from higher plants has been characterized as a flavin-and heme-containing molybdoenzyme which catalyzes several partial reactions (NADH-Cyt c reductase, reduced flavin-NR, MV°-NR) in addition to the NAD(P)H-NR reaction (7,9). The NADH-Cyt c reductase (diaphorase) activity, associ-

The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H-Nitrate Reductase Enzyme from Soybean

“…The Blue Sepharose-purified enzyme had the ability to use FMNH2 and reduced MV as electron donors, in addition to NAD(P)H, for the NO(x) evolution activity (Tables III and IV in the absence of inhibitor). As determined before by Nelson et al (15), these same electron donors will support cjNR activity (Tables III and IV). However, as seen in Tables III and IV in the absence of inhibitor, the efficiency of each electron donor was different for each activity.…”

A Method for the Separation and Partial Purification of the Three Forms of Nitrate Reductase Present in Wild-Type Soybean Leaves

“…2), migration on native PAGE and sucrose density gradients was markedly different (14,20). Inhibitor studies also point to differences at the nitrate reducing site of the native enzyme (10). We conclude that differences affecting charge and hydrophobicity of the apoprotein, rather than subunit size, are responsible for the different kinetic and physical properties among the three soybean NRs.…”

“…Moreover, a separation of NADH pH 6.5 and pH 7.5 NR forms was partially achieved. Previously published procedures failed to separate all three isozymes from soybean (4,7,14,20), and only through use of the nr1 mutant and two subsequent mutants was tentative identification of the three NR forms possible (10,19,20 (Fig. 3).…”

Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase

“…A mutation in LNR-5 and LNR-6 affecting the molybdenum cofactor per se can be ruled out, since the presence of both c1NR and iNR necessitates the presence of an active molybdenum cofactor (8). The mutation in both the LNR-5 and LNR-6 mutants appears to affect the whole c2NR enzyme, since the Cyt c reductase and FMNH2:NR activities are affected as well as NADH:NR activity.…”

“…Nitrate reductase activity was extracted as described by Scholl et al (13) and assayed for reduced pyridine nucleotide activity as described by Streit et al (14). FMNH2:NR activity was determined as described by Nelson et al (8) and Cyt c reductase activity was measured according to Wray and Filner (15). For Cyt c reductase assays, 0.5 ml extract was dialyzed against two changes (0.5 L each) of extraction medium minus cysteine.…”