Quaternary structure and composition of squash NADH:nitrate reductase

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.     

Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties.

NADH:nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris has been purified 640-fold with an over-all yield of 26% by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, gel chromatography, density gradient centrifugation, and DEAE-chromatography. The purified enzyme is stable for more than 2 months when stored at minus 20 degrees in phosphate buffer (pH 6.9) containing 40% (v/v) glycerol. After the initial steps of the purification, a constant ratio of NADH:nitrate reductase activity to NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities was observed. One band of protein was detected after polyacrylamide gel electrophoresis of the purified enzyme. This band also gave a positive stain for heme, NADH dehydrogenase, and reduced methyl viologen:nitrate reductase. One band, corresponding to a molecular weight of 100, 000, was detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains FAD, heme, and molybdenum in a 1:1:0.8 ratio. One "cyanide binding site" per molybdenum was found. No non-heme-iron or labile sulfide was detected. From a dry weight determination of the purified enzyme, a minimal molecular weight of 152, 000 per molecule of heme or FAD was calculated. An s20, w of 9.7 S for nitrate reductase was found by the use of sucrose density gradient centrifugation and a Stokes radius of 89 A was estimated by gel filtration techniques. From these values, and the assumption that the partial specific volume is 0.725 cc/g, a molecular weight of 356, 000 was estimated for the native enzyme. These data suggest that the native enzyme contains a minimum of 2 molecules each of FAD, heme, and molybdenum and is composed of at least three subunits.     

Synthesis of wheat leaf nitrite reductase de novo following induction with nitrate and light

Nitrite reductase has been purified almost 3000-fold, in 35% yield, to a specific activity of 77 units (mg protein)-1 from wheat leaves using a multi-step procedure with affinity chromatography on ferredoxin-Sepharose as the final step. The purified enzyme, although not homogeneous, exhibited absorption maxima at 278, 390, 568 and 687 nm. Minor contaminants were removed by gel filtration in the presence of sodium dodecyl sulphate to yield a single polypeptide of Mr 60 500 as judged by polyacrylamide gel electrophoresis. Antibodies raised against this polypeptide were shown to cross-react with native nitrite reductase and were used to study the synthesis of nitrite reductase in vivo and in vitro. The increase in nitrite reductase activity following exposure of dark-grown plants to nitrate and light was shown by immunodecoration of Western blots to be due to synthesis de novo. Poly(A)-rich RNA isolated from plants actively synthesising nitrite reductase was shown to direct the synthesis in a rabbit reticulocyte lysate of a polypeptide of Mr 64000 which was immunoprecipitated by antibodies to nitrite reductase.     

Purification and properties of nitrate reductase from Mitsuokella multiacidus

Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).     

Reversible inactivation of maize leaf nitrate reductase

Preincubation of maize leaves crude extracts with NADH resulted in a progressive accumulation of nitrite which mimicked a rapid and lineal activation of nitrate reductase. Nevertheless, in partially purified preparations it was found that preincubation at pH 8.8 with NADH promoted a gradual inactivation of nitrate reductase. At pH 7.5, the enzyme was not inactivated by the presence of NADH alone, but, with the simultaneous presence of a low concentration of cyanide, a fast inactivation took place. The NADH-cyanide-inactivated nitrate reductase remained inactive after removing the excess of NADH and cyanide by filtration through Sephadex G-25. However, it could be readily reactivated by incubation with ferricyanide or by a short exposure to light in the presence of FAD. Prolonged irradiation caused a progressive inactivation of the photoreactivated enzyme.     

Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate-Nitrate Reductase of Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans was purified over 200-fold by use of salt fractionation, gel filtration, and ion-exchange chromatography. The purified enzyme was specific for NADPH and catalyzed reduction of nitrate, cytochrome c from isolated mitochondria of Aspergillus, and mammalian cytochrome c. An S(0.725) (20, w) of 7.8 was derived with sucrose density gradient centrifugation, and a Stokes radius of 6.4 nm was derived by gel filtration on Sephadex G-200. From these values, a molecular weight of 197,000 was computed, assuming v = 0.725 cm(3)/g. The spectral properties of the purified enzyme suggested a flavine component was present but revealed no pattern indicative of a hemoprotein. A cytochrome c, similar to the cytochrome c from isolated mitochondria, was found unassociated with the nitrate reductase after ion-exchange chromatography. No NADPH-nitrate reductase activity was detected in isolated mitochondria. Spectrally discernable reduction of the flavine component of the enzyme at 450 nm was noted after reaction with NADPH. This reduction was inhibited by p-chloromercuribenzoate but not by KCN. The addition of nitrate to NADPH reduced enzyme caused a reoxidation of the flavine component via a reaction which was inhibited by KCN but not by p-chloromercuribenzoate. The half-life of the purified enzyme at 37 C was 20 min for NADPH-nitrate reductase and 35 min for NADPH-cytochrome c reductase.     

Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes.

1. The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants (s20,w) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 106. 3. In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 Mr subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. The 52 000 Mr subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. p-Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p-chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine.     

Purification and properties of trimethylamine N-oxide reductase from aerobic photosynthetic bacterium Roseobacter denitrificans.

Trimethylamine N-oxide (TMAO) reductase was purified from an aerobic photosynthetic bacterium Roseobacter denitrificans. The enzyme was purified from cell-free extract by ammonium sulfate fractionation, DEAE ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme was composed of two identical subunits with molecular weight of 90,000, as identified by SDS-polyacrylamide gel electrophoresis, containing heme c and a molybdenum cofactor. The molecular weight of the native enzyme determined by gel filtration was 172,000. The midpoint redox potential of heme c was +200 mV at pH 7.5. Absorption maxima appeared at 418,524, and 554 nm in the reduced state and 410 nm in the oxidized state. The enzyme reduced TMAO, nicotine acid N-oxide, picoline N-oxide, hydroxylamine, and bromate, but not dimethyl sulfoxide, methionine sulfoxide, chlorate, nitrate, or thiosulfate. Cytochrome c2 served as a direct electron donor. It probably catalyzes the electron transfer from cytochrome b-c1 complex to TMAO reductase. Cytochrome c552, another soluble low-molecular-weight cytochrome of this bacterium, also donated electrons directly to TMAO reductase.     

Purification and Characterization of Nitrate Reductase from the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

Summary Factors affecting the activity of nitrate reductase (E.C.1.7.7.2) from the halotolerant cyanobacterium Aphanothece halophytica were investigated. Cells grown in nitrate-containing medium exhibited higher nitrate reductase activity than cells grown in medium in which nitrate was replaced by glutamine. When ammonium was present in the medium instead of nitrate, the activity of nitrate reductase was virtually non-detectable, albeit with normal cell growth. The enzyme was localized mainly in the cytoplasm. The enzyme was purified 406-fold with a specific activity of 40.6 μmol/min/mg protein. SDS-PAGE revealed a subunit molecular mass of 58 kDa. Gel filtration experiments revealed a native molecular mass of 61 kDa. The K _m value for nitrate was 0.46 mM. Both methyl viologen and ferredoxin could serve as electron donor with K _m values of 4.3 mM and 5.2 μM, respectively. The enzyme was strongly inhibited by sulfhydryl-reactive agents and cyanide. Nitrite, the product of the enzyme reaction, showed little inhibition. Chlorate, the substrate analog, could moderately inhibit the enzyme activity. NaCl up to 200 mM stimulated the activity of the enzyme whereas enzyme inhibition was observed at ≥300 mM NaCl.     

Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa

Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium Pseudomonas aeruginosa. The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography. The molecular weight of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography. Subunit molecular weights were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The active enzyme monomer, with a molecular weight of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with molecular weights of approximately 64,000 and 118,000. The monomer aggregated to form an inactive tetramer of about 800,000 molecular weight. Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5. Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 microM for dithionite-reduced benzyl viologen. Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 microM. Azide inhibition was competitive with nitrate (Ki = 2.0 microM) but uncompetitive with reduced benzyl viologen (Ki = 25 microM). Based upon spectral evidence, the purified molybdo-enzyme had no associated cytochromes but did contain nonhaem iron that responded to dithionite reduction and nitrate oxidation. The enzyme that was purified after being heat solubilized from membranes had properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization.     

Constitutive and nitrate-induced,membrane-bound nitrate reductase fromBradyrhizobium japonicum

Nitrate reductase (NR) activity was detected in membranes from cells ofBradyrhizobium japonicum cultured in defined medium either with glutamate or nitrate as the only nitrogen source. With gel filtration, the relative molecular mass (Mr) of the NR in cells growth with glutamate was estimated to be about 78 kDa. The enzyme from cells grown aerobically with nitrate had an Mr of 236 kDa, the same as that of the NR from microaerobically nitrate-grown cells. When cells that had been grown with glutamate were incubated microaerobically in both the absence and the presence of nitrate, the enzyme from each source resembled that of nitrate-grown cells in having an Mr of 236 kDa. In glutamate-grown cells that were further incubated, both microaerobiosis and nitrate were required for fully expression of the activity of the enzyme.     

Purification and properties of the NAD(P)H:nitrate reductase of the yeast Rhodotorula glutinis.

The assimilatory nitrate reductase from the yeast Rhodotorula glutinus has been purified 740-fold, its different catalytic activities have been characterized and some inhibitors studied. The purified enzyme (150 units per mg protein) contains a cytochrome of the b-557 type. An S20,w of 7.9 S was found by the use of sucrose density gradient centrifugation, and a Stokes radius of 7.05 nm was determined by gel filtration. From these values, a molecular weight of 230 000 was estimated for the native enzyme. The purified preparation consisted of two electrophoretically distinguishable proteins, both of which exhibited nitrate reductase activity. The species with the higher electrophoretic mobility which represented the great majority of the total nitrate reductase gave a positive stain for heme and was shown to be composed of subunits with a molecular weight of about 118 000. Thus the molecule contains two subunits of the same size.     

Purification and properties of low-Km aldehyde reductase from ox brain

A low-Km aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), which may be identical with aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21), has been purified from ox brain to homogeneity. It was shown to be a monomer with Mr values of 31 000 and 35 100 being obtained by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, respectively. The enzyme catalyses the NADPH-dependent reduction of a number of aromatic and sugar aldehydes. The activity of the enzyme with 133 microM NADH was about one-third of that with 120 microM NADPH. Activity with both these coenzymes was optimum at pH 6.2 and was inhibited by increasing the ionic strength with KCl, NaCl or NaNO3. In contrast, the activity was stimulated by sodium phosphate. The activity with NADH as the coenzyme was more sensitive to stimulation by phosphate and to inhibition by increasing ionic strength than that determined with NADPH.     

NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

A soluble beta-cyanoalanine synthase from the gut of the variegated grasshopper Zonocerus variegatus (L.)

Beta-cyanoalanine synthase (beta-cyano-l-alanine synthase; l-cysteine: hydrogen sulphide lyase (adding hydrogen cyanide (HCN)); EC 4. 4.1.9) was purified from the cytosolic fraction of the gut of grasshopper Zonocerus variegatus (L.) by ion-exchange chromatography on DEAE-Cellulose and gel filtration on Sephadex G-100 columns. The crude enzyme had a specific activity of 2.16nmol H2S/min/mg. A purified enzyme with a specific activity, which was seventeen times higher than that of the crude extract, was obtained. A molecular weight of about 55.23+/-1.00Kd was estimated from its elution volume on Sephadex G-100. The fraction when subjected to sodium dodecyl sulphate-polyacrylamide elel electrophoresis revealed the presence of a protein band with Mr of 23.25+/-0.25Kd. The enzyme exhibited Michaelis-Menten kinetics having Km of 0.38mM for l-cysteine and Km of 6.25mM for cyanide. The optimum temperature and pH for activity were determined to be at 30 degrees C and pH 9.0, respectively. This enzyme might be responsible for the ability to detoxify cyanide in this insect pest and hence its tolerance of the cyanogenic cassava plant. Biophysical, biochemical and kinetic properties of this enzyme, which will reveal how this ability can possibly be compromised by enzyme inhibition, may lead, in the long term, to the potential use of this enzyme as drug target for pest control.     

Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora.

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 muM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH2-nitrate reductase of Neurospora suggests some differences in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 mu M) markedly enhanced the action of NAD(P)H on the maize enzyme; Cyanide at a higher level (6 mu M) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no effect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.     

Rapid Modulation of Spinach Leaf Nitrate Reductase Activity by Photosynthesis : I. Modulation in Vivo by CO(2) Availability

It has been shown recently that in spinach leaves (Spinacia oleracea) net photosynthesis and nitrate reduction are closely linked: when net photosynthesis was low because of stomatal closure, rates of nitrate reduction decreased (WM Kaiser, J Förster [1989] Plant Physiol 91: 970-974). Here we present evidence that photosynthesis regulates nitrate reduction by modulating nitrate reductase activity (NRA, EC 1.6.6.1). When spinach leaves were exposed to low CO₂ in the light, extractable NRA declined rapidly with a half-time of 15 minutes. The inhibition was rapidly reversed when leaves were brought back to air. NRA was also inhibited when leaves were wilted in air; this inhibition was due to decreased CO₂ supply as a consequence of stomatal closure. The modulation of NRA was stable in vitro. It was not reversed by gel filtration. In contrast, the in vitro inhibition of nitrate reductase (NR) by classical inhibitors such as cyanide, hydroxylamin, or NADH disappeared after removal of free inhibitors by gel filtration. The negative modulation of NRA in —CO₂-treated leaves became manifest as a decrease in total enzyme activity only in the presence of free Mg²⁺ or Ca²⁺. Mg²⁺ concentrations required for observing half-maximal inhibition were about 1 millimolar. In the presence of EDTA, the enzyme activity was always high and rather independent of the activation status of the enzyme. NRA was also independent of the pH in the range from pH 7 to pH 8, at saturating substrate and Mg²⁺ concentrations. The apparent substrate affinities of NR were hardly affected by the in vivo modulation of NR. Only V_max changed.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Isolation and Some Properties of a 115-Kilodalton Nitrate Reductase-Inactivator Protein from Spinacia oleracea

A nitrate reductase inactivator protein in spinach leaves waspurified (90-fold). The purification involved precipitationwith ammonium sulfate, treatment at pH 4, CM-cellulose chromatog-raphyand gel filtration on a Toyopearl HW-55F column. From the ToyopearlHW-55F gel filtration step the molecular weight of the inactivatorwas estimated to be 115 kDa. The inactivator was particularly sensitive to EDTA, o-phenanthrolineand pronase. The inactivator was more stable to heat treatmentthan NADH-nitrate reductase. Incubation of purified spinachnitrate reductase with the inactivator results in a loss ofNADH-nitrate reductase and the associated partial activities,NADH-ferricyanide reductase, NADH-cytochrome c reductase, butnot in no loss in nitrate reducing activity with reduced methylviologen as the electron donor. The molecular weight of thenitrate reductase-inactivator protein complex was estimatedby gel filtration on Toyopearl HW-55F to be 460 kDa, comparedto an apparent molecular weight of 240 kDa for the untreatedcontrol estimated under the same conditions. These results indicatethat spinach nitrate reductase inactivator protein acts by bindingto nitrate reductase. The stoichiometry of binding is 2 moleculesof the inactivator protein to one dimeric molecule of nitratereductase. The action of the inactivator protein was partiallyprevented by NADH. (Received September 21, 1987; Accepted January 8, 1988)