Nitrate reductase from Dunaliella tertiolecta, purification and properties

The purification and properties of a nitrate reductase fromthe green alga Dunaliella tertiolecta are described. The enzymeis soluble, with a molecular weight greater than 500,000 andhas Km values of 0.26, 0.18, 0.10 and 0.06 m for NO₃^–,NADH, NADPH and FADH₂ respectively. Even at the highest specificactivity obtained, (0.86 µmoles NO₃^– reduced min^–1mg protein^–1) the enzyme retains the capacity to acceptelectrons from both NADH and NADPH. Unlike other nitrate reductasesit does not appear to be able to use reduced viologens as electrondonors. Its other properties are consistent with its being amolybdoflavoprotein of high molecular weight, which is alsoable to function as a cytochrome C reductase. ¹ Supported in part by the National Research Council of Canada. (Received June 18, 1972; )     

Purification and Characterization of Assimilatory Nitrate Reductase from the Cyanobacterium Plectonema boryanum

Assimilatory nitrate reductase (NR) was solubilized by acetonetreatment from Plectonema boryanum and was purified 7,700-foldby heat treatment, ammonium sulfate fractionation and chromatographyon DEAE-Sephacel and Sephadex G-150. Purified NR had a specificactivity of 85 µmol NO₂^– formed min^–1 mg^–1protein. The enzyme retained both ferredoxin (Fd)- and methylviologen (MV)-linked NR activities throughout the purificationprocedure. Molecular weight was 80,000. The pH optimum was 10.5in the MV-assay and 8.5 when assayed with enzymatically reducedFd as the electron donor. Apparent Km values for nitrate andMV were 700 µM and 2,500µM in the MVassay and 55µM and 75 µM for nitrate and Fd in the Fd-assay.The enzyme was inhibited by thiol reagents and metal-chelatingreagents. (Received October 1, 1982; Accepted March 8, 1983)     

Nitrate Reductase Activity and Nitrite in Native Pyrenoids Purified from the Green Alga Bryopsis maxima

The native, starchless pyrenoids purified from Bryopsis maximashowed NADH-nitrate reductase [NR, EC 1.6.6.1 [EC] ] activity andcontained nitrite. The specific activity of NR was 0.024 µmolNO₂ formed per min per mg of protein. The value was 80 timesgreater than that in the crude extract of chloroplasts. Theamount of nitrite in the pyrenoids was 2.37 µmol per mgof protein, showing that nitrite was concentrated by a factorof 66 times. These results suggest a physiological role forpyrenoids in the assimilation of nitrate. (Received November 15, 1989; Accepted February 27, 1990)     

Primary Structure and Properties of Glutathione Reductase from Arabidopsis thaliana

A cDNA encoding glutathione reductase (EC 1.6.4.2 [EC] ) from Arabidopsisthaliana was cloned by immunoscreening. The amino acid sequencededuced from the nucleotide sequence agrees with the N-terminalamino acid sequence of the major isozyme (GR II) purified fromleaves of A. thaliana. The predicted polypeptide comprises anN-terminal leader sequence of 74 amino acids, which has featuresof chloroplast-targeting peptides, and a mature polypeptideof 491 residues with a molecular mass of 52.7 kDa, which showshomology with glutathione reductases from other species. TheK_m for GSSG was 44 µM and that for NADPH was 5.0 µMfor GR II at 25C. The pH optimum for GR II was 7.5 to 8.0.The native molecular mass of GR II was 110 kDa, indicating thatGR II is a homodimer. GR II had an isoelectric point of 4.8.The cDNA hybridizes with a 2.1-kb poly(A)⁺RNA from leaves ofA. thaliana. Genomic Southern analysis indicates that the genecorresponding to the cDNA is likely a single-copy gene. (Received July 1, 1993; Accepted September 8, 1993)     

Activation Energies of Reactions Catalyzed by the Nitrate Reductase from Chlorella vulgaris

The thermal dependence of two of the reactions catalyzed bythe nitrate reductase from Chlorella vulgaris was determined.The activation energies for NADH:nitrate oxidoreductase (EC1.6.6.1 [EC] ) and NADH:Cytochrome c oxidoreductase (EC 1.6.99.3 [EC] )are 42.1 kJ?mol^–1 and 21.5 kJ?mol^–1, respectively.Since the thermal dependency of the two enzymes is different,ratios of the activities will vary with temperature. The importanceof both rigorous thermal control during nitrate reductase assaysas well as the need to specify the temperature at which theratio of activities for the enzyme are clearly established. ¹Present Address: Cropping Systems Research Laboratory, USDA-ARS,Route 3, Box 215, Lubbock, TX 79401, U.S.A. (Received November 25, 1987; Accepted March 2, 1988)     

Relationship between biomass and enzymatic activity of a bloom-forming dinoflagellate (Dinophyceae) in southern Chile (41{degrees} S): a field approach

In a coastal area of southern Chile (41° S), the major ammoniumassimilating enzyme glutamine synthetase (GS) was detected ina green dinoflagellate bloom during April 2003. High chlorophylla concentrations (1000 µg L^–1) attributable to Gymnodiniumcf. chlorophorum in surface waters were associated with highand very low nitrate reductase activities. Coincident with thebloom, dissolved inorganic nitrogen concentrations were nearthe detection limit (NO₃^– + NH₄⁺ <0.5 µM). SinceGS correlates with the use of ammonium as an external nitrogensource, we suggest that GS activity seems to be a good indicatorof ammonium utilization in a period dominated by a single dinoflagellatespecies.     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

Leaf Development in Lolium temulentum: Photosynthesis and Photosynthetic Proteins in Leaves Senescing under Different Irradiances

Changes in carbon fixation rate and the levels of photosyntheticproteins were measured in fourth leaves of Lolium temulentumgrown until full expansion at 360 µmol quanta m^–2s^–1 and subsequently at the same irradiance or shadedto 90 µmol m^–2 s^–1. Ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco), light-harvesting chlorophylla/b protein of photosystem II (LHCII), 65 kDa protein of photosystemI (PSI), cytochrome f (Cytf) and coupling factor 1 (CF₁) declinedsteadily in amount throughout senescence in unshaded leaves.In shaded leaves, however, the decrease in LHCII and the 65kDa protein was delayed until later in senescence whereas theamount of Cyt f protein decreased rapidly following transferto shade and was lower than that of unshaded leaves at the earlyand middle stages of senescence. Decreases in the Rubisco andCF₁ of shaded leaves occurred at slightly reduced rates comparedwith unshaded leaves. These results indicate that chloroplastproteins in fully-expanded leaves are controlled individually,in a direction appropriate to acclimate photosynthesis to agiven irradiance during senescence. (Received August 20, 1992; Accepted January 5, 1993)     

Purification and Properties of an Extracellular Endo-1,4-r{beta}-Glucanase from Suspension-Cultured Poplar Cells

An endo-1,4-rß-glucanase (EC 3.2.1.4 [EC] ) was purifiedto apparent homogeneity from the culture medium of poplar (Populusalba L.) cells by sequential anion-exchange, hydrophobic, andgel-filtration chromatography. The preparation of extracellularrß-glucanase was homogeneous on SDS-polyacrylamidegel electrophoresis (PAGE) and native PAGE. The molecular weight,as determined by SDS-PAGE was 50,000, whereas that determinedby gel filtration was 40,000. The isoelectric point (pI) was5.5. The purified enzyme catalyzed the endohydrolysis of carboxy-methylcellulosewith a pH optimum of 6.0 and a k_m of 1.0 mg ml^–1. Theenzyme specifically cleaved the 1,4-rß-glucosyl linkagesof carboxymethylcellulose, swollen cellulose, lichenan and xyloglucan,although the last was hydrolyzed more slowly than the othertested substrates. The activity of the endo-1,4-rß-glucanaseincreased up to the early stage of the mid-logarithmic phaseof growth and then decreased rapidly, suggesting that the rß-glucanaseis induced before cell development. (Received April 28, 1993; Accepted July 19, 1993)     

Purification and Characterization of Phosphoenolpyruvate Carboxylase of Photomixotrophically Cultured Green Tobacco Cells

Phosphoenolpyruvate (PEP) carboxylase (PEPCase, EC 4.1.1.31 [EC] )was purified to apparent electrophoretic homogeneity from photomixotrophicallycultured tobacco cells by ammonium sulfate fractionation, DEAE-Sephacel-,hydroxylapatite-, Phenyl-Sepharose CL-4B-, and Sepharose CL-6B-chromatography,and fast protein liquid chromatography on Mono Q. The purifiedenzyme had a specific activity of 32 units per mg protein, andits purity was determined by denaturing polyacrylamide gel electrophoresis.The native enzyme, with a molecular weight of about 440,000,was a tetramer of four identical subunits and showed maximumactivity at pH 8.5–9.0. Non-denaturing isoelectric focusingshowed a single band at pl 5.4. Substrate-saturation kineticsof the purified enzyme for PEP, bicarbonate, and Mg^2$ were typicalMichaelis-Menten type, with K_m-values of 60, 200, and 80µM,respectively. Most effectors which are known to influence theactivity of C₄- or bacterial PEPCase had only small effectson the activity of the purified enzyme at optimum pH, whilesome inhibitory effects by organic acids (malate, citrate andoxaloacetate) and.an activating effect by glucose-6-phosphatewere observed at a suboptimal pH of 7.5. (Received September 30, 1987; Accepted December 14, 1987)     

Nitrate Uptake and Reduction of Aseptically Cultivated Spruce Seedlings, Picea abies (L.) Karst

Spruce (Picea abies (L.) Karst.) seedlings were asepticallycultivated and the effects of different N-nutrition on net uptakeand reduction of nitrate were investigated. The characteristicsof nitrate uptake were calculated, K_s as 0?2 mol m^–3 andV_max as 18 µmol g^–1 d^–1. Low pH, and Al³⁺ in the medium caused adecrease in nitrate uptake rate. An in vivo assay was set upwhich allowed the measurement of NRA in both roots and needlesof spruce seedlings. The in vivo nitrate reductase activitywas repressed by ammonium and stimulated by nitrate. Nitratereduction was similar to nitrate uptake, negatively affectedby low pH and ammonium. Therefore, a limited N-supply to spruceseemed to occur when pH was low in the rhizosphere combinedwith the presence of Al³⁺ and . Key words: Spruce, nitrate uptake, nitrate reduction     

Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Effect of Plant Growth Regulators on Nitrate Utilization by Roots of Nitrogen-Depleted Dwarf Bean

We examined the effect of pretreatments (18 h at 5 µmoldm^–3) with abscisic acid, the ethylene-releasing substance‘Ethephon’, gibberellic acid, indoleacetic acid,kinetin and zeatin on nitrate uptake and in vivo nitrate reductaseactivity (NRA) in roots of nitrogen-depleted Phaseolus vulgarisL. Nitrate uptake showed an apparent induction pattern witha steady state after about 6 h, in all treatments. The nitrateuptake rate after 6 h was unaffected or at most 30% lower aftertreatments with the plant growth regulators. Gibberellic acid, kinetin and zeatin induced substantial NRAin roots in the absence of nitrate, whereas Ethephon enhancedNRA only during nitrate nutrition. Kinetin-induced NRA (Ki-NRA)was maximal after a pretreatment at 1 µmol dm^–3,and showed a lag phase of 6–8 h. Ki-NRA was additive tonitrate-induced NRA (NO^–₃-NRA) for at least 24 h, independentof the induction sequence. After full induction, Ki-NRA approximated20% of NO-₃-NRA. Abscisic acid counteracted the developmentof Ki-NRA, but not of NO^–₃-NRA. Cycloheximide and tungstatewere equally effective to suppress the development of nitratereductase activity after supply of kinetin or NO^–₃. Our data are consistent with the operation of two independentenzyme fractions (Ki-NRA and NO^–₃-NRA) with apparentlyidentical properties but with separate control mechanisms. Theabsence of major effects of plant growth regulators on the time-courseand rate of nitrate uptake suggests that exogenous regulators,and possibly endogenous phytohormones are of minor importancefor initial nitrate uptake. The differential effect of someregulators on nitrate uptake and root NRA furthermore indicatesthat the processes of uptake and reduction of NO^–₃ arenot obligatory or exclusively coupled to each other.     

Flavine nucleotide nitrate reductase from broad bean leaves

Hydrosulfite-reduced FMN served as an electron donor for nitratereductase purified from broad bean leaves. FMN was successfullyreplaced with BV. The flavine nucleotide nitrate reductase hadits pH optima at about 7.8 with phosphate buffer and at about7.4 with Tris-HCl buffer. The Km's for nitrate and FMN were3.7 ? 10^–4 M and 3.7 ? 10^–5 M, respectively. NADH₂: nitrate reductase activity was completely inhibited by0.1 mM p-CMB, whereas FMNH₂: nitrate reductase activity wasnot. Inhibited activity was restored by the addition of cysteine.A sulfhydryl enzyme is involved in the NADH₂: nitrate reductasesystem but not in the FMNH₂ : nitrate reductase system. NADH₂and FMNH₂ probably feed electrons into the electron transportchain at different sites. The nitrate reductase preparationhad an NADH₂-specific diaphorase activity which was almost completelyinhibited by 0.1 mM p-CMB. The NADH₂-specific diaphorase mayform the sulfhydryl enzyme which mediates electron transferbetween NADH₂ and nitrate. (Received May 6, 1969; )     

Characterisation of Acidic and Basic Apoplastic Peroxidases from Needles of Norway Spruce (Picea abies, L., Karsten) with Respect to Lignifying Substrates

Acidic and basic peroxidases, termed as POD-A and POD-B, wereisolated from the apoplastic space of spruce (Picea abies, L.)needles and purified by acetone precipitation and anion exchangechromatography to apparent homogeneity. The molecular massesof POD-A and POD-B were 39.6 and 29.0 kDa, respectively. ThepH optimum of both isozymes ranged from 4.5 to 6. The apparentK_m values of POD-A and POD-B were 460 and 210 µM for coniferylalcohol. Both isozymes acted also as NADH oxidases with apparentK_m-values of 103 µM (POD-A) and 70 µM (POD-B). NAD⁺but not NADH was found in the apoplastic space of lignifyingneedles. Based on the lignification rate, the contents and kineticproperties of PODs, NADH oxidation by POD is not the major sourceof H₂O₂ required for lignin polymerisation. (Received December 21, 1996; Accepted March 3, 1997)     

The Influence of the Concentration of Gaseous Ammonia on its Uptake by the Leaves of Italian Ryegrass, with and without an Adequate Supply of Nitrogen to the Roots

Whitehead, D. C. and Lockyer, D. R. 1986. The influence of theconcentration of gaseous ammonia on its uptake by the leavesof Italian ryegrass, with and without an adequate supply ofnitrogen to the roots.—J. exp. Bot. 38: 818–827. Plants of Italian ryegrass (Lolium multiflorum Lam.) were grownin pots of soil with two rates of ¹⁵N-labclled nitrate, oneproviding adequate, and the other less than adequate, N formaximum growth. After 25 d in a controlled environment cabinet,the plants were transferred to chambers and exposed for 33 dto NH₃in the air at one of nine concentrations ranging from14 to 709 µg NH₃ m^–3. Increasing the concentrationof NH₃ in the air increased the dry weight of the shoots ofplants grown at the lower but not the higher rate of nitrate.The content of total N in the plant shoots (% dry weight) waslinearly related to NH₃ concentration; at 709 µg NH₃ andin both sets of plants it was more than double the content at14 µg NH₃ m^–3. Calculations, based on ¹⁵N enrichment,indicated that the amount of N taken up from the NH₃ per unitleaf area increased linearly with increasing concentration ofNH₃ in the air uptake (µg dm^–2 h^–1) = 0.1009xat the lower rate of nitrate and 0-0829x at the higher rateof nitrate, where x is the concentration of NH₃ in the air expressedas µg NH₃m^–3. The proportion of the total plant N that was derived from theNH₃ ranged from 4?0% at a concentration of 14 µg NH₃ m^–3with the higher rate of nitrate addition to 77?5% at a concentrationof 709 µg m^–3 with the lower rate of nitrate addition.The proportions of the total N in the water-insoluble proteinof the leaf tissue that were derived from nitrate and gaseousNH₃ were similar to the proportions in the whole leaf material. Key words: Ammonia, nitrogen, leaf sorption, Lolium multiflorum     

Properties of a Large Complex with NADH Dehydrogenase Activity from Barley Thylakoids

When assays for NAD(P)H-ferricyanide oxidoreductases were performed,activities specific for NADH (0.23 unit (mg protein)^–1)and NADPH (0.68 unit (mg protein)^–1) were detected inchloroplasts isolated from leaves of barley (Hordeum vulgareL.). Activities of chloroplast NADH- and NADPH-ferricyanideoxidoreductase were 5-fold and 25-fold higher, respectively,than the maximum activity that could be attributed to mitochondrialcontamination. Moreover, most of the chloroplast NADH-ferricyanideoxidoreductase (60 to 80%) was solubilized by deoxycholate (DOC)from thylakoids as a single, high-molecular-mass complex thatwas distinguishable from the mitochondrial complex by its lowerelectrophoretic mobility in 3% polyacrylamide, as revealed byreduction of nitro blue tetrazolium (NBT) in the presence ofNADH or NADPH on gels after electrophoresis. The stroma yieldeda single band of a dehydrogenase (66 kDa) that used NADH asits electron donor. Several NADPH-dependent activities weredetected after electrophoresis of the stromal fraction. Moreover,chloroplast-specific activities could be distinguished frommitochondrial activities on the basis of the specificity ofthe donor and the acceptor of electrons, the dependence of theactivities on pH, and the sensitivity to various inhibitors.K_m values for NADH (26 µM) and NADPH (75 µM) werein the same range as those of mitochondrial activities. Mostof the NADPH-dependent activity probably corresponds to thechloroplast ferredoxin-NADP⁺ oxidoreductase. The possibilityis discussed that thylakoid NADH dehydrogenase(s) might be theproduct of chloroplast ndh genes and that this activity is involvedin chlororespiration. (Received April 25, 1994; Accepted December 5, 1994)     

Incorporation of radioactive molybdenum into protein during nitrate reductase formation and effect of molybdenum on nitrate reductase and diaphorase activities of spinach (Spinacea oleracea L.)

Spinach plants grown without molybdenum lack nitrate reductaseand when plants are deprived of nitrate existing activity islost. Transfer of molybdenum-deficient plants to a solutioncontaining (NH₄)₂⁹⁹MoO₄) or nitrate-starved plants to NaNO₃solution induced enzyme activity in 24 hr. After purificationby selective adsorption, precipitation and disc electrophoresis,the protein from molybdenum-deficient plants given ⁹⁹Mo showedradioactivity only where nitrate reductase was revealed on theacrylamide gel. Molybdenum was similarly selectively concentratedinto the enzyme as a result of induction by nitrate in plantsgrown with sub-optimal molybdenum supply in order to minimizeeffects of isotope dilution on measurement of ⁹⁹Mo incorporation. There was no exchange in vitro between ⁹⁹Mo and purified activeenzyme in the resting state over 18 hr at 4°C, or with functioningenzyme held at room temperature for 24 hr. There was evidenceeither for possible in vivo exchange of ⁹⁹Mo andenzyme boundMo or for slight synthesis of fresh enzyme under conditionsof net loss of enzyme in nitrate starved plants. Five NADH₂ and two NADPH₂ reactive diaphorases which could beseparated by electrophoresis were present in extracts. Onlyone of these having strong NADH₂ and weak NADPH₂ activity wasdirectly associated with nitrate reductase. The same complexalso showed the only benzyl viologen (BV.) reactive nitratereductase. Nitrate reductase in spinach is therefore considered to be amolybdenum-dependant and molybdenum-containing protein in whichNADH₂ (with weak NADPH₂) and BVelectron donor functions anddiaphorase/reductase activities remain closely associated duringpurification and electrophoresis. The techniques provide a simple means for the production andpurification of enzyme containing radioactively labelled Moapplicable to investigations on the structure of the enzyme. (Received January 16, 1971; )     

Acyl-CoA Synthetase in Maturing Safflower Seeds

Acyl-CoA synthetase in maturing seeds of safflower (Carthamustinctorius) was membranebound, and the highest specific activitywas associated with microsomes. Activity absolutely dependedon the concentrations of fatty acid, CoA, ATP and Mg²⁺. Theapparent K_m values were 4.2 µM for oleate, 24 µMfor CoA, and 250 µM for ATP. The optimum pH of the reactionwas 7.5. Triacsin C, a potent inhibitor of the animal and bacterialacyl-CoA synthetase, was ineffective for the safflower enzyme.The enzyme utilized C₁₆ and C₁₈ long-chain fatty acids preferentially,while medium-chain and very-long-chain fatty acids were poorsubstrates. The order of specificity for native fatty acidswas linoleate > oleate=palmitate > stearate. Althoughactivity per seed varied during seed maturation, it was enoughto account for the rate of triacylglycerol synthesis in vivo. (Received February 2, 1993; Accepted March 3, 1993)