Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase

Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M₂ seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH₂-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c₁NR), constitutive NADH:NR (c₂NR), and nitrate-inducible NR (iNR).     

Soybean mutants lacking constitutive nitrate reductase activity : I. Selection and initial plant characterization

The objectives of this study were to select and initially characterize mutants of soybean (Glycine max L. Merr. cv Williams) with decreased ability to reduce nitrate. Selection involved a chlorate screen of approximately 12,000 seedlings (progeny of mutagenized seed) and subsequent analyses for low nitrate reductase (LNR) activity. Three lines, designated LNR-2, LNR-3, and LNR-4, were selected by this procedure.

In growth chamber studies, the fully expanded first trifoliolate leaf from NO₃⁻-grown LNR-2, LNR-3, and LNR-4 plants had approximately 50% of the wild-type NR activity. Leaves from urea-grown LNR-2, LNR-3, and LNR-4 plants had no NR activity while leaves from comparable wild-type plants had considerable activity; the latter activity does not require the presence of NO₃⁻ in the nutrient solution for induction and on this basis is tentatively considered as a constitutive enzyme. Summation of constitutive (urea-grown wild-type plants) and inducible (NO₃⁻-grown LNR-2, LNR-3, or LNR-4 plants) leaf NR activities approximated activity in leaves of NO₃⁻-grown wild-type plants. Root NR activities were comparable in wild-type and mutant plants grown on NO₃⁻, and roots of both plant types lacked constitutive NR activity when grown on urea. In both growth chamber- and field-grown plants, oxides of nitrogen [NO_(x)] were evolved from young leaves of wild-type plants, but not from leaves of LNR-2 plants, during in vivo NR assays. Analysis of leaves from different canopy locations showed that constitutive NR activity was confined to the youngest three fully expanded leaves of the wild-type plant and, therefore, on a total plant canopy basis, the NR activity of LNR-2 plants was approximately 75% that of wild-type plants. It is concluded that: (a) the NR activity in leaves of NO₃⁻-grown wild-type plants includes both constitutive and inducible activity; (b) the missing NR activity in LNR-2, LNR-3, and LNR-4 leaves is the constitutive component; and (c) the constitutive NR activity is associated with NO_(x) evolution and occurs only in physiologically young leaves.

     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

     

Immunochemical Characterization of Nitrate Reductase Forms from Wild-Type (cv Williams) and nr(1) Mutant Soybean

Soybean (Glycine max L. Merr.) leaves contain two forms of nitrate reductase (NR)—NAD(P)H:NR and NADH:NR. Wild-type (cv Williams), nr₁ mutant and an unrelated cultivar (Prize) were grown with either no N source or with nitrate. Crude extracts were assayed for NR activities and the enzyme forms were purified on blue Sepharose. Analyses were done by polyacrylamide gel electrophoresis and `Western blotting' using antibodies specific for NR. NAD(P)H:NR was identified as the constitutive NR present in wild-type and Prize, but was absent from the mutant. All three soybean lines contained nitrate-inducible NADH:NR with highest activity at pH 7.5. The results showed that NAD(P)H:NR and constitutive NR were one in the same and confirmed the presence of NADH:NR with pH 7.5 optimum.     

A method for the separation and partial purification of the three forms of nitrate reductase present in wild-type soybean leaves

A rapid and simple purification method was used to separate and purify nitrate reductases (NR) from Williams soybean leaves. Blue Sepharose columns were sequentially eluted with 50 millimolar NADPH and 50 millimolar NADH, thus separating NAD(P)H:NR from NADH:NRs. Subsequent purification of the collected peaks on a fast protein liquid chromatography-Mono Q column enabled separation of two NADH:NRs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the subunit relative molecular mass for all three NR forms (constitutive NAD(P)H:NR [pH 6.5], EC 1.6.6.2; constitutive NADH:NR [pH 6.5], EC not assigned; and inducible NADH:NR [pH 7.5], EC 1.6.6.1) was approximately 107 to 109 kilodaltons. All three NRs showed similar spectra with absorption maxima at 413 and 273 nanometers in the oxidized state, and with the characteristics of a cytochrome b type heme upon reduction with NADH (absorption maxima at 556, 527, and 424 nanometers). The technique developed provides an improved separation of the three NR forms from soybean leaves. The similarity of the NRs with regard to their cytochrome b₅₅₆ type heme content and in relative molecular mass indicated that other differences must exist to account for the different kinetic and physical properties previously reported.     

Soybean Mutants Lacking Constitutive Nitrate Reductase Activity : II. Nitrogen Assimilation, Chlorate Resistance, and Inheritance

Nitrogen assimilation in three nitrate reductase (NR) mutants of soybean (Glycine max L. Merr. cv Williams) was studied in the growth chamber and in the field. These mutants, LNR-2, LNR-3, and LNR-4, lack the non-NO₃⁻-inducible or constitutive fraction of leaf NR activity found in wild-type plants, but this had no effect on the concentration of nitrogen accumulated when grown on NO₃⁻ in the growth chamber. Dry weight accumulation of two of the mutants (LNR-3 and LNR-4) was decreased relative to LNR-2 and wild type. In the field, LNR-2 had dry weights and nitrogen concentrations similar to the wild type at 34 and 61 days after planting, and at maturity. Acetylene reduction activities were also similar at 61 days.     

Purification and properties of an F420-dependent NADP reductase from methanobacterium thermoautotrophicum

The F₄₂₀-dependent NADP reductase of Methanobacterium thermoautotrophicum has been purified employing a combination of DEAE-cellulose ion-exchange chromatography, affinity chromatography with Blue Sepharose, Sephadex G-200 column chromatography and Red Sepharose affinity chromatography. The enzyme, which requires reduced F₄₂₀ as an electron donor, has been purified over 3000-fold with a recovery of 65%. A molecular weight of 112000 was determined by Sephadex G-200 chromatography. A subunit molecular weight of 28 500 was determined by Sephadex G-200 chromatography. A subunit native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 60°C with a pH optimum of 8.0. The NADP reductase had an apparent K_m of 128 μMJ for reduced F420 and 40 μM for NADP. The enzyme was stable for at least 4 h at 65°C and pH 7.5. No loss of enzyme activity was detected when purified enzyme was stored aerobically in buffer containing 2-mercaptoethanol for 10 days at 4°C. Neither FMNH₂ nor FADH₂ could serve as electron donors; NAD was not utilized as electron acceptor.     

Evolution of Nitrogen Oxide(s) during In Vivo Nitrate Reductase Assay of Soybean Leaves

Studies were conducted to quantitate the evolution of nitrogen oxides (NO_(x)) from soybean [Glycine max (L.) Merr.] leaves during in vivo nitrate reductase (NR) assays with aerobic and anaerobic gas purging. Anaerobic gas purging (N₂ and argon) consistently resulted in greater NO_(x) evolution than did aerobic gas purging (air and O₂). The evolution of NO_(x) was dependent on gas flow rate and on NO₂⁻ formation in the assay medium; although a threshold level of NO₂⁻ appeared to exist beyond which the rate of NO_(x) evolution did not increase further.     

Comparison between NO(x) Evolution Mechanisms of Wild-Type and nr(1) Mutant Soybean Leaves

The nr₁ soybean (Glycine max [L.] Merr.) mutant does not contain the two constitutive nitrate reductases, one of which is responsible for enzymic conversion of nitrite to NO_x (NO + NO₂). It was tested for possible nonenzymic NO_x formation and evolution because of known chemical reactions between NO₂⁻ and plant metabolites and the instability of nitrous acid. It did not evolve NO_x during the in vivo NR assay, but intact leaves did evolve small amounts of NO_x under dark, anaerobic conditions. Experiments were conducted to compare NO₃⁻ reduction, NO₂⁻ accumulation, and the NO_x evolution processes of the wild type (cv Williams) and the nr₁ mutant. In vivo NR assays showed that wild-type leaves had three times more NO₃⁻ reducing capacity than the nr₁ mutant. NO_x evolution from intact, anerobic nr₁ leaves was approximately 10 to 20% that from wild-type leaves. Nitrite content of the nr₁ mutant leaves was usually higher than wild type due to low NO_x evolution. Lag times and threshold NO₂⁻ concentrations for NO_x evolution were similar for the two genotypes. While only 1 to 2% of NO_x from wild type is NO₂, the nr₁ mutant evolved 15 to 30% NO₂. The kinetic patterns of NO_x evolution with time weré completely different for the mutant and wild type. Comparisons of light and heat treatments also gave very different results. It is generally accepted that the NO_x evolution by wild type is primarily an enzymic conversion of NO₂⁻ to NO. However, this report concludes that NO_x evolution by the nr₁ mutant was due to nonenzymic, chemical reactions between plant metabolites and accumulated NO₂⁻ and/or decomposition of nitrous acid. Nonenzymic NO_x evolution probably also occurs in wild type to a degree but could be easily masked by high rates of the enzymic process.     

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.     

Determination of the distribution of three nitrate reductase isoforms in soybean seedlings by chromatography and a simple method based on assay conditions

Blue Sepharose affinity chromatography was used to study the distribution of the constitutive NAD(P)H-nitrate reductase (EC 1.6.6.2: Cl-NR) and of the constitutive and inducible NADH-nitrate reductases (EC 1.6.6.1; C2-NR and i-NR, respectively), in the unifoliolate leaf (F0), the first and the second trifoliolate leaves (F1 and F2) and the roots of urea- and nitrate-grown soybean ( Glycine max [L.] Merr.) plants. The C1-NR eluted by NADPH is present in the F0 and F1 leaves and nearly absent in the F2 leaf. The activity pattern of this isoform is not modified by nitrate nutrition. The C2-NR eluted by NADH is high in the F0 leaf, low in the F1 leaf and nearly absent in the F2 leaf of urea-grown plants. The NADH elution from leaves of nitrate-grown plants is a mixture of C2-NR and i-NR, requiring careful interpretation of results. However, i-NR appears the principal isoform in the leaves especially in the F2 leaf. This i-NR is the only NR present in the roots.
The pH effect on the assay of the 3 partially purified isoforms was studied using LNR2 and LNR5 soybean mutants to remove the cross contamination. It appears that C1-NR and C2-NR activities are negligible at pH 8.5, which allows the assay of only the i-NR in a crude extract at this pH, even when C1-NR and C2-NR are present. It appears also that the assay of C1-NR activity at pH 6.5 with NADPH is free of interference by the i-NR. To estimate the C2-NR activity with NADH at pH 6.5 in a crude extract in the presence of C1-NR and i-NR, we propose a simple calculation using the coefficient from the pH responses. These calculations are used to compare the development of C1-NR, C2-NR and i-NR activities in the F0 and F1 leaves of plants previously grown on urea and transferred to nitrate. Only the activity of the inducible isoform is modified by the nitrogen treatment. Activity of the constitutive isofroms appear stable during the 48 h treatment, with only a slight decrease in C1-NR activity being observed with time.     

Differential effect of tungsten on the development of endogenous and nitrate-induced nitrate reductase activities in soybean leaves

The effect of tungsten on the development of endogenous and nitrate-induced NADH- and FMNH₂-linked nitrate reductase activities in primary leaves of 10-day-old soybean (Glycine max [L.] Merr.) seedlings was studied. The seedlings were grown with or without exogenous nitrate. High levels of endogenous nitrate reductase activities developed in leaves of seedlings grown without nitrate. However, no endogenous nitrite reductase activity was detected in such seedlings. The FMNH₂-linked nitrate reductase activity was about 40% of NADH-linked activity. Tungsten had little or no effect on the development of endogenous NADH- and FMNH₂-linked nitrate reductase activities, respectively. By contrast, in nitrate-grown seedlings, tungsten only inhibited the nitrate-induced portion of NADH-linked nitrate reductase activity, whereas the FMNH₂-linked activity was inhibited completely. Tungsten had no effect on the development of nitrate-induced nitrite reductase activity. The complete inhibition of FMNH₂-linked nitrate reductase activity by tungsten in nitrate-grown plants was apparently an artifact caused by the reduction of nitrite by nitrite reductase in the assay system. The results suggest that in soybean leaves either the endogenous nitrate reductase does not require molybdenum or the molybdenum present in the seed is preferentially utilized by the enzyme complex as compared to nitrate-induced nitrate reductase.     

Simultaneous isolation of protein C activator,fibrin clot promoting enzyme (fiprozyme) and phospholipase A2 from the venom of the southern copperhead snake

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A₂.     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

Association of NADP- and NAD-linked malic enzyme acitivities in Zea mays: relation to C4 pathway photosynthesis.

Two of the three metabolic subtypes of species utilizing C₄-pathway photosynthesis are defined by high activities of either NADP malic enzyme (NADP malic enzyme type) or a coenzyme A (CoA)- and acetyl-CoA-activated NAD malic enzyme (NAD malic enzyme type). These enzymes function to decarboxylate malate as an integral part of the photosynthetic process. Leaves of NADP malic enzyme-type species also contain significant NAD-dependent malic enzyme activity. The purpose of the present study was to examine the nature and photosynthetic role of this activity. With Zea mays, this NAD-dependent activity was found to vary widely in fresh leaf extracts. Incubating extracts at 25 °C resulted in a disproportionate increase in NAD activity so that the final ratio of NADP to NAD activity was always about 5. Strong evidence was provided that the NADP and NAD malic enzyme activities in Z. mays extracts were catalyzed by the same enzyme. These activities remained associated during purification and were coincident after polyacrylamide gel electrophoresis. The pH optimum for NAD-dependent activity was about 7.1, compared with 8.3 for NADP malic enzyme activity. Other properties of the NAD-dependent activity are described, a particularly notable feature being the inhibition of this activity by less than 1 μm NADP and NADPH. Evidence is provided that the NADP malic enzyme of several other NADP malic enzyme-type C₄ species also has associated activity toward NAD. We concluded that the NAD-dependent malic enzyme activity would have no significant function in photosynthesis.     

Some characteristics of nitrate reductase from higher plants

With respect to cofactor requirements, NADH, and FMNH₂ were equally effective as electron donors for nitrate reductase obtained from leaves of maize, marrow, and spinach, when the cofactors were supplied in optimal concentrations. The concentration of FMNH₂ required to obtain half-maximal activity was from 40- to 100-fold higher than for NADH. For maximal activity with the corn enzyme, 0.8 millimolar FMNH₂ was required. In contrast, NADPH was functional only when supplied with NADP:reductase and exogenous FMN (enzymatic generation of FMNH₂).

All attempts to separate the NADH₂- and FMNH₂-dependent nitrate reductase activities were unsuccessful and regardless of cofactor used equal activities were obtained, if cofactor concentration was optimal. Unity of NADH to FMNH₂ activities were obtained during: A) purification procedures (4 step, 30-fold); B) induction of nitrate reductase in corn seedlings with nitrate; and C) inactivation of nitrate reductase in intact or excised corn seedlings. The NADH- and FMNH₂-dependent activities were not additive.

A half-life for nitrate reductase of approximately 4 hours was estimated from the inactivation studies with excised corn seedlings. Similar half-life values were obtained when seedlings were incubated at 35° in a medium containing nitrate and cycloheximide (to inhibit protein synthesis), or when both nitrate and cycloheximide were omitted.

In those instances where NADH activity but not FMNH₂ activity was lost due to treatment (temperature, removal of sulfhydryl agents, addition of p-chloromercuribenzoate), the loss could be explained by inactivation of the sulfhydryl group (s) required for NADH activity. This was verified by reactivation with exogenous cysteine.

Based on these current findings, and previous work, it is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH₂ as cofactor. However the high concentration of FMNH₂ required for optimal activity suggests that in vivo NADH is the electron donor and that nitrate reductase in higher plants should be designated NADH:nitrate reductase (E.C. 1.6.6.1).

     

Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris,a purple non-sulfur bacterium producing hopanoids and tetrahymanol

The squalene-hopene cyclase of the hopanoid- and tetrahymanol-producing Rhodopseudomonas palustris was released from the isolated membranes by CHAPS and purified to homogeneity by succesive chromatography on DEAE Sephacel, Octyl Sepharose, and Blue Sepharose. The enzyme has a molecular weight of 70 kDa as determined by SDS-PAGE and an isoelectric point at about pH 5.O. The enzyme activity has a maximum at 30°C and at pH 6.5. No production of tetrahymanol could be demonstrated by using either crude or purified cyclase preparations.     

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.     

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.     

Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.