Comparison of the Kinetic Behavior toward Pyridine Nucleotides of NAD-Linked Dehydrogenases from Plant Mitochondria

In this article we compare the kinetic behavior toward pyridine nucleotides (NAD⁺, NADH) of NAD⁺-malic enzyme, pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine decarboxylase extracted from pea (Pisum sativum) leaf and potato (Solanum tuberosum) tuber mitochondria. NADH competitively inhibited all the studied dehydrogenases when NAD⁺ was the varied substrate. However, the NAD⁺-linked malic enzyme exhibited the weakest affinity for NAD⁺ and the lowest sensitivity for NADH. It is suggested that NAD⁺-linked malic enzyme, when fully activated, is able to raise the matricial NADH level up to the required concentration to fully engage the rotenone-resistant internal NADH-dehydrogenase, whose affinity for NADH is weaker than complex I.     

Pyridine nucleotide transhydrogenases of parasitic helminths.

Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD⁺ and NADH:NADP⁺ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD⁺ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD⁺ system was approximately five times more active than the NADH:NADP⁺ system. The NADH:NADP⁺ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD⁺ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD⁺ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD⁺ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD⁺ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD⁺ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD⁺ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.     

Effects of NADH and thiol compounds on wheat leaf nitrate reductase

The initial activity of wheat leaf nitrate reductase was depressed on inclusion of the following thiol compounds; dithiothreitol, dithioerythreitol or mercaptoethanol, but not cysteine and glutathione. This thiol effect simply resulted from an interference with the chemical determination of nitrite. Preincubation of the enzyme with NAD⁺ and these thiols enhanced the inhibition of nitrate reductase activity. This effect was mediated by NADH production by the thiol reduction of NAD⁺. The inactivation by NAD⁺ in the presence of thiol compounds which was enhanced by cyanide ions could be reversed by ferricyanide, as has been observed previously for NADH-mediated inactivation of nitrate reductase.     

Properties of Bromphenol Blue as an Electron Donor for Higher Plant NADH: Nitrate Reductase

Bromphenol blue, which was reduced with dithionite, was found to support nitrate reduction catalyzed by squash NADH:nitrate reductase at a rate about 5 times greater than NADH with freshly prepared enzyme and 10 times or more with enzyme having been frozen and thawed. Kinetic analysis of bromphenol blue as a substrate for squash nitrate reductase yielded apparent K_m values of 60 micromolar for bromphenol blue at 10 millimolar nitrate and 500 micromolar for nitrate at 0.2 millimolar bromphenol blue. With the same preparation of enzyme the apparent K_m values were 9 micromolar for NADH at 10 millimolar nitrate and 50 micromolar nitrate at 0.1 millimolar NADH. Bromphenol blue was found to be a noncompetitive inhibitor versus NADH with a K_i of 0.3 millimolar. When squash NADH:nitrate reductase activity was inactivated with p-hydroxymercuribenzoate or denatured by heating at 40°C, the bromphenol blue nitrate reductase activity was not lost. These results were taken to indicate that bromphenol blue and NADH donated electrons to nitrate reductase at different sites. When monoclonal antibodies prepared against corn and squash nitrate reductases were used to inhibit the nitrate reductase activities supported by NADH, bromphenol blue, and methyl viologen, differential inhibition was found which tended to indicate that the three electron donors were interacting with the enzyme at different sites. One monoclonal antibody prepared against squash nitrate reductase inhibited all three activities of both corn and squash nitrate reductase. It appears this antibody may bind to a highly conserved antigenic site in the nitrate binding region of the enzyme.     

Complexes of NADH with betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus L.

The kinetic mechanism of betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus is ordered with NAD⁺ adding first. NADH is a noncompetitive inhibitor against NAD⁺, which was interpreted before as evidence of an iso mechanism, in which NAD⁺ and NADH binds to different forms of free enzyme. With the aim of testing the proposed kinetic mechanism, we have now investigated the ability of NADH to form different complexes with the enzyme. By initial velocity and equilibrium binding studies, we found that the steady-state levels of E.glycine betaine are negligible, ruling out binding of NADH to this complex. However, NADH readily bind to E.betaine aldehyde, whose levels most likely are kinetically significant given its low dissociation constant. Also, NADH combined with E.NADH and E.NAD⁺. Finally, NADH was not able to revert the hydride transfer step, what suggest that there is no acyl-enzyme intermediate, i.e. the release of the reduced dinucleotide takes place after the deacylation step. Although formation of the complex E.NAD⁺.NADH would produce an uncompetitive effect in the inhibition of NADH against NAD⁺, the iso mechanism cannot be conclusively discarded.     

Nitrate reductase from Spinacea oleracea. Reversible inactivation by NAD(P)H and by thiols

The enzymatic complex nitrate reductase from Spinacea oleracea is inactivated by NADH or NADPH and by simple thiols. The inactivation affects FNH₂-nitrate reductase but not NADH-diaphorase. Reactivation can be achieved by addition of ferricyanide. The extent of inactivation by dithioerythritol is increased by NAD⁺, but not by NADP⁺. Nitrate protects against inactivation by NADH or NADPH, and abolishes the effect of NAD⁺ on the inactivation by dithioerythritol. The NAD(P)H-inactivation of nitrate reductase requires that the diaphorase moiety of the complex be functional. However, there is no proportionality between NADH-diaphorase or NADH-nitrate reductase activities and the susceptibility of the enzymatic preparation to NADH or NADPH. It seems likely that the nitrate reductase complex contains a specific regulatory site, different from the catalytic site, the reduction of which is accompanied by the production of an inactive form of the complex.     

cDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P):Glyceraldehyde-3-Phosphate Dehydrogenase : Characterization of the Clones and Analysis of the Expression of the Genes in Leaves as Influenced by Nitrate in the Light and Dark

cDNA clones were selected from a corn (Zea mays L.) leaf lambda gt11 expression library using polyclonal antibodies for corn leaf NADH:nitrate reductase. One clone, Zmnrl, had a 2.1 kilobase insert, which hybridized to a 3.2 kilobase mRNA. The deduced amino acid sequence of Zmnrl was nearly identical to peptide sequences of corn leaf NADH:nitrate reductase. Another clone, Zm6, had an insert of 1.4 kilobase, which hybridized to a 1.4 kilobase mRNA, and its sequence coded for chloroplastic NAD(P)⁺:glyceraldehyde-3-phosphate dehydrogenase based on comparisons to sequences of this enzyme from tobacco and corn. When nitrate was supplied to N-starved, etiolated corn plants, nitrate reductase, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels in leaves increased in parallel. When green leaves were treated with nitrate, only nitrate reductase mRNA levels were increased. Nitrate is a specific inducer of nitrate reductase in green leaves, but appears to have a more general effect in etiolated leaves. In the dark, nitrate induced nitrate reductase expression in both etiolated and green leaves, indicating light and functional chloroplast were not required for enzyme expression.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Purification and characterization of malate dehydrogenase from the phototrophic bacterium,Rhodopseudomonas capsulata

Malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37) has been purified about 480-fold from crude extract of the facultative phototrophic bacterium, Rhodopseudomonas capsulata by only two purification steps, involving Red-Sepharose affinity chromatography. The enzyme has a molecular mass of about 80 kDa and consists of two subunits with identical molecular mass (35 kDa). The enzyme is susceptible to heat inactivation and loses its activity completely upon incubation at 40°C for 10 min. Addition of NAD⁺, NADH and oxaloacetate, but not l-malate, to the enzyme solution stabilized the enzyme. The enzyme catalyzes exclusively the oxidation of l-malate, and the reduction of oxaloacetate and ketomalonate in the presence of NAD⁺ and NADH, respectively, as the coenzyme. The pH optima are around 9.5 for the l-malate oxidation, and 7.75–8.5 and 4.3–7.0 for the reduction of oxaloacetate and ketomalonate, respectively. The K_m values were determined to be 2.1 mM for l-malate, 48 μM for NAD⁺, 85 μM for oxaloacetate, 25 μM for NADH and 2.2 mM for ketomalonate. Initial velocity and product inhibition patterns of the enzyme reactions indicate a random binding of the substrates, NAD⁺ and l-malate, to the enzyme and a sequential release of the products: NADH is the last product released from the enzyme in the l-malate oxidation.     

l-proline dehydrogenase of Triticum vulgare germ: Purification,properties and cofactor interactions

The enzyme catalysing the l-proline-dependent reduction of NAD⁺has been purified over 600-fold from wheat germ acetone powder extracts. l-Proline, 3,4 dehydro-dl-proline, thiazolidine-4-carboxylate were the only substrates utilized readily. The K_m for l-proline was 1·0 mM and for NAD⁺ 0·8 mM. The enzyme was highly specific for NAD⁺ with NADP⁺ and NADPH acting as effective competitive inhibitors with a K_i of 1·8 and 0·4 μM, respectively. All ribonucleoside triphosphates tested were good non-competitive inhibitors, in particular UTP. The purified enzyme could reduce pyrroline-5-carboxylate, either chemically synthesized or generated in a linked assay system from ornithine by a highly-purified ornithine transaminase. In the latter case both NADH and NADPH were utilized equally well as the reductant. With chemically synthesized dl-pyrroline-5-carboxy-late as the substrate. NADPH was used at only 25% the rate of NADH, and NADPH strongly inhibited the oxidation of NADH.     

Stability of nitrate reductase and source of its reductant in Sorghum seedlings

Pre-incubation of nitrate reductase from Sorghum seedlings with NADH increased enzyme activity by 25%. Ferricyanide had no effect. NADH protected the enzyme from inactivation during storage. Malonate inhibited in vivo nitrate reduction in Sorghum leaves by 95%. The inhibitory effect of malonate was reversed by fumarate. Sodium fluoride in the presence of phosphate also inhibited in vivo nitrate reduction by 60%. It is suggested that NADH generated via the citric acid cycle is utilized for nitrate reduction in Sorghum seedlings.     

Purification and characterization of pyruvate dehydrogenase complex from borccoli floral buds.

The pyruvate dehydrogenase complex (PDC) was purified from Brassica oleracea var. italica floral buds to a specific activity of approximately 6 μmol of NADH formed/min/ mg of protein. The PDC had cofactor requirements for NAD⁺, thiamine pyrophosphate, coenzyme A, and a divalent cation (Mg²⁺, Ca²⁺, or Mn²⁺). The enzyme catalyzed the oxidative decarboxylation of pyruvate at a rate threefold faster than 2-oxobutyrate but was inactive toward 2-oxoglutarate. The PDC was competively inhibited by acetyl-CoA against CoA and NADH against NAD⁺. The enzyme was shown to be more sensitive to regulation by NADH than acetyl-CoA.     

Glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes. Kinetic mechanism and competitive substrate inhibition by glyceraldehyde 3-phosphate

Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was isolated from human erythrocyte ghosts by a simple procedure utilizing ammonium sulfate precipitation and affinity chromatography on NAD⁺-Sepharose 4B. The purified enzyme had a specific activity of 98 units/mg protein. The kinetic mechanism of GAPD was studied by product and deadend inhibition using NADH, α-glycerophosphate, nitrate, and 2,3-diphosphoglycerate. The results indicated that the human erythrocyte GAPD-catalyzed reaction follows an ordered ter bi mechanism characterized by the sequential addition of NAD⁺, glyceraldehyde 3-phosphate (GAP), and phosphate to the enzyme and the sequential release of 1,3-diphosphoglycerate and NADH from the enzyme. This contrasts with the mechanism (rapid equilibrium random ter bi) proposed by Oguchi (1970, J. Biochem. (Tokyo)68, 427–439) who based his conclusion on the initial rate data alone. Since the Michaelis-Menten kinetics were not applicable to this enzyme because of the competitive substrate inhibition by GAP, we devised a new kinetic approach for determining the parameters of the GAPD-catalyzed reaction. Results of this study indicate that the GAPD-catalyzed reaction is regulated by both ATP and GAP. We propose that GAP acts as an “amplifier” for the feedback inhibition effect of ATP. We discuss the effect this may have played in causing controversy over the regulatory role of this enzyme in glycolysis.     

Trichomonas vaginalis: NADH oxidase activity.

NADH oxidase activity was detected in the 105,000g supernatant (“soluble”) fraction of Trichomonas vaginalis and the enzyme was purified 50-fold by centrifugation, ammonium sulfate precipitation, Sephadex G-200, and DEAE-Sephadex A-25 chromatography. The ratio of oxygen uptake to NADH oxidation was approximately one-half. Addition of catalase did not affect the rate of oxygen uptake elicited by NADH. Since the purified fraction was free from interfering enzymes, the postulated reaction is as follows: $\text{NADH}\text{+}\text{H}{}^{\mathrm{+}}\text{+}\text{1}\text{2}\text{= NAD}{}^{\mathrm{+}}\text{+ H}{}_2\text{O}$. Among numerous substances tested, only NADH was a functional substrate, whereas NADPH was not oxidized. The purified enzyme had a V_max of 16.5 μmole of oxygen consumed/min/mg protein, and the apparent K_m for NADH was 7.4 μM. Substrate inhibition was observed at 3.7 mM NADH. The purified NADH oxidase was competitively inhibited by NAD⁺ as well as by NADP⁺ with 50% inhibition at 1 and 5 mM, respectively. The enzyme was also markedly inhibited by p-chloromercuribenzoate, hydrogen peroxide, and transient metal-chelators such as bathophenanthroline or o-phenanthroline. A flavoprotein antagonist, atebrin was slightly less inhibitory. Various quinones, flavin nucleotides and artificial dyes, except for p-benzoquinone, ferricyanide and cytochrome c, did not function in accepting electrons from NADH oxidase. These three compounds, however, were still poor electron acceptors in the enzymatic reaction suggesting that the trichomonad NADH oxidase has little diaphorase activity. All of these findings indicate that T. vaginalis has an unique NADH oxidizing enzyme in that H₂O seems to be the prdouct of oxygen reduction. This NADH oxidase appears important in the aerobic metabolism of this parasite.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

A pH-Dependent Kinetic Model of Dihydrolipoamide Dehydrogenase from Multiple Organisms

Dihydrolipoamide dehydrogenase is a flavoenzyme that reversibly catalyzes the oxidation of reduced lipoyl substrates with the reduction of NAD⁺ to NADH. In vivo, the dihydrolipoamide dehydrogenase component (E3) is associated with the pyruvate, α-ketoglutarate, and glycine dehydrogenase complexes. The pyruvate dehydrogenase (PDH) complex connects the glycolytic flux to the tricarboxylic acid cycle and is central to the regulation of primary metabolism. Regulation of PDH via regulation of the E3 component by the NAD⁺/NADH ratio represents one of the important physiological control mechanisms of PDH activity. Furthermore, previous experiments with the isolated E3 component have demonstrated the importance of pH in dictating NAD⁺/NADH ratio effects on enzymatic activity. Here, we show that a three-state mechanism that represents the major redox states of the enzyme and includes a detailed representation of the active-site chemistry constrained by both equilibrium and thermodynamic loop constraints can be used to model regulatory NAD⁺/NADH ratio and pH effects demonstrated in progress-curve and initial-velocity data sets from rat, human, Escherichia coli, and spinach enzymes. Global fitting of the model provides stable predictions to the steady-state distributions of enzyme redox states as a function of lipoamide/dihydrolipoamide, NAD⁺/NADH, and pH. These distributions were calculated using physiological NAD⁺/NADH ratios representative of the diverse organismal sources of E3 analyzed in this study. This mechanistically detailed, thermodynamically constrained, pH-dependent model of E3 provides a stable platform on which to accurately model multicomponent enzyme complexes that implement E3 from a variety of organisms.     

Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K_m values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K_m values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.     

Chloride inhibition of spinach nitrate reductase

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V_max and K_m for NO₃⁻ yielding V_max values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K_m values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K_m of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K_i of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO₃⁻ complex. Binding of Cl⁻ to the enzyme-NO₃⁻ complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.     

Purification of Squash NADH:Nitrate Reductase by Zinc Chelate Affinity Chromatography

NADH:nitrate reductase (EC 1.6.6.1) was isolated and purified from the green cotyledons of 5-day-old squash seedlings (Cucurbita maxima L.). The 10-hour purification procedure consisted of two steps: direct application of crude enzyme to blue Sepharose and specific elution with NADH followed by direct application of this effluent to a Zn²⁺ column with elution by decreasing the pH of the phosphate buffer from 7.0 to 6.2. The high specific activity (100 micromoles per minute per milligram protein) and high recovery (15-25%) of electrophoretically homogeneous nitrate reductase show that the enzyme was not damaged by exposure to the bound zinc. With this procedure, homogeneous nitrate reductase can be obtained in yields of 0.5 milligram per kilogram cotyledons.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.