Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.     

Purification and Characterization of Thiol Proteinase as a Nitrate Reductase-Inactivating Factor from Leaves of Hordeum distichum L.

A thiol proteinase was purified 6,400-fold from leaves of Hordeumdistichum L. by a sequence of ammonium sulfate fractionation,gel filtration, ion exchange chromatography, hydrophobic chromatographyand chromatofocusing. This enzyme also had nitrate reductase(NR)-inactivating activity, which was associated with proteolyticactivity in almost constant proportions during the purificationprocedures. Its molecular weight was estimated as 74,000 bygel filtration, and it had an isoelectric point of 4.05 andan apparent K_m of 0.83 mg ml^–1 for azocasein. The respectiveoptimum pH for proteolytic and NR-inactivating activities were6.0 and 7.0. On electrophoresis, the proteinase gave a majorband that coincided with both activities; it also produced afaint band associated with no activity. Our purified thiol proteinase inactivated FMNH₂-NR and MVH-NRas well as NADH-NR, but it had only a slight effect on NADHcytochrome c reductase activity. This enzyme also inactivatedglutamine synthetase. (Received September 16, 1983; Accepted January 26, 1984)     

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; )     

Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)     

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)     

Purification and Properties of NADH:Nitrate Reductase from the Red Alga Porphyra yezoensis

Assimilatory nitrate reductase (NADH) (EC 1.6.6.1 [EC] ) from thered alga Porphyra yezoensis was purified 5,700-fold by a combinationof polyethylene glycol (PEG) treatment, ammonium sulfate fractionation,chromatography on columns of butyl-Toyopearl 650-M, Blue SepharoseCL-6B, DEAE-cellulose (DE 52), and hydroxyapatite, gel filtrationon Sephacryl S-400. The purest preparation of the enzyme hada specific activity of 12.5 units mg^–1 protein. A singleband of protein was detected after polyacrylamide gel electrophoresisunder nondenaturing conditions. This band corresponded to aband that stained positive for reduced methyl viologen-nitratereductase activity. The molecular weight of the native enzymewas estimated to be 220,000. A single band of a protein witha molecular weight of 100,000 was detected after sodium dodecylsulfate-polyacrylamide gel electrophoresis. These results indicatethat the native nitrate reductase is composed of two identicalsubunits. The homogeneous preparation of nitrate reductase hada UV/visible spectrum typical of a b-type cytochrome. The K_mvalues for NADH and KNO₃ were 23 µM and 64 µM, respectively.The pH optimum for the reaction catalyzed by the nitrate reductasewas 8.3, while pH values that supported maximum partial activitiesranged from 7.0 to 8.5. Sulfhydryl reagents, such as p-HMB andNEM, inhibited full and NADH-utilizing partial activities, whilecyanide and azide were effective inhibitors of full and nitrate-reducingpartial activities. (Received March 3, 1993; Accepted September 6, 1993)     

Isolation and some properties of copper-binding proteins found in a copper-resistant strain of yeast

Copper-binding proteins were extracted from a copper-resistantstrain of Saccharomyces cerevisiae which was obtained by repeatedsubculturing in a copper-containing medium. They were separatedinto three types through purification steps such as salt fractionation,gel filtration and preparative polyacrylamide gel electrophoresis.They resembled each other in amino acid composition. Acidicamino acids, lysine, serine, glycine and half-cystine constituteda large part of the protein, with a small amount of hydrophobicamino acids. Aromatic amino acids and methionine were almostabsent. The molecular weight of the components was estimatedto be about 10,000 by Sephadex gel filtration and electrophoresison polyacrylamide gel (slope method). Absorption spectra ofthe components exhibited a broad band at 275 nm, but none inthe visible region, thus resembling that of copper-thionein.Moreover, the absorption band at 275 nm changed markedly onaddition of Ag⁺, Hg2⁺, CN^– or H₂O₂, which are well knownas thiol reagents. These components were abo produced in theparent cells, if they could grow in a copper-containing medium.Based the results of experiments using various culture conditionsand some other yeast species, a possible role of the componentsis discussed. (Received July 13, 1976; )     

Ferredoxin-sulfite Reductase from Spinach

Ferredoxin-sulfite reductase (Fd-SiR) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from spinach leaves has been purified to homogeneity by a new procedure. Subunit analysis by sodium dodecyl sulfate gel electrophoresis yielded a single protein band with a molecular weight of 71,000. Gel electrophoresis in non-denaturing media at different acrylamide concentrations gave a molecular weight of 270,000, suggesting that the native enzyme was composed of four identical subunits. In the presence of 0.2 m sodium chloride, however, gel filtration produced a value of 136,000, indicating the presence of dimer in this ionic environment. A plot of substrate (sulfite) concentration versus enzymatic (Fd-SiR) activity yielded a sigmoidal curve, giving a Hill coefficient (n?) of 2.1. Purified Fd-SiR, in the oxidized form, had absorption maxima at 279, 385, 588 and 714nm. Thus the enzyme has the property of a siroheme-containing protein.     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

A Nitrate Reductase Inactivator Protein from Spinach. Purification, Molecular Weight and Subunit Composition

A nitrate reductase-inactivator protein has been purified 16,000-foldfrom spinach leaves by pH 5 treatment, chromatography on SE53,Con A-Sepharose, and chromatofocusing. The yield was 12%, thespecific activity was 115 units mg^–1. Polyacrylamide gelelectrophoresis of the final purified inactivator fraction yielded2 major protein bands and both bands exhibited nitrate reductase-inactivatoractivity. Analysis of this inactivator protein by gel filtrationand SDS-gel electrophoresis revealed protein stainable materialonly in a molecular weight range of 110,000–115,000. SDSgel electrophoresis under reducing conditions yielded 2 proteinbands corresponding to molecular weights of 51,000 and 53,000.The proteolytic mapping for the two separated subunits appearedsimilar and possibly identical. (Received October 28, 1991; Accepted February 24, 1992)     

Purification and Properties of Soluble Chlorophyllase from Tea Leaf Sprouts

Soluble chlorophyllase (chlorophyll-chlorophyllido-hydrolase,EC 3.1.1.14 [EC] ) was purified 650-fold from tea leaf sprouts byammonium sulfate fractionation and gel filtration through SephadexG-200 and Sepharose CL-6B. The purified enzyme showed two bandson polyacrylamide gel electrophoresis and the specific activitywas 2.6 µmol chlorophyll a hydrolyzed min^–1 mg^–1of protein. The molecular weights determined by Sepharose CL-6Bwere 910,000 and 350,000, indicating high molecular aggregates.The subunit molecular weight estimated by sodium lauryl sulfate-polyacrylamidegel electrophoresis was 38,000. The isoelectric point was 3.9.The optimum pH was 5.5 in acetate buffer and the Km value forchlorophyll a was 10 µM. This enzyme did not require athiol compound nor metal ion such as Mg²⁺. (Received January 26, 1981; Accepted April 3, 1981)     

Purification and properties of a nitrate reductase inactivating factor from rice cells in suspension culture

The nitrate reductase inactivating factor in cultured rice cellswas purified 320-fold. The purification procedure involved precipitationwith (NH₄)₂SO₄, fractionation at pH 4.0, adsorption on CM-cellulose,and gel filtration on Sephadex G-200. The molecular weight wasestimated to be 200,000 from the Sephadex G-200 gel filtration. The inactivating factor shows maximal activity at pH 8.0 andappears to be located in the cytoplasm of the cultured ricecells. The inactivating factor was more stable to heat treatmentthan NADH nitrate reductase. The factor inactivated nitratereductase complex except for reduced methylviologen nitratereductase. It had no influence on the activity of nitrite reductase,glutamate dehydrogenase, and NADH diaphorase, but inactivatedxanthine oxidase. The inactivating factor had no protease activitywhen casein, bovine serum albumin, or nitrate reductase fractionwas used as the substrate. The type of inactivation of nitratereductase by the inactivating factor was noncompetitive. Inhibitionof the inactivating factor by o-phenanthroline, EDTA, and p-chloromercuribenzoicacid suggested the involvement of a metal and sulfhydryl groupat its active site. (Received January 28, 1977; )     

Ferredoxin-dependent Nitrite Reductase from Spinach Leaves

A ferredoxin-dependent nitrite reductase from Spinacea oleracea was purified approximately 180-fold, with a specific activity of 285 units/mg protein. This purified enzyme also had methyl viologen-dependent nitrite reductase activity, with a specific activity of 164 units/mg protein. After disc electrophoresis with polyacrylamide gel, the purified enzyme showed one major and one minor protein band.

The molecular weight of the enzyme was estimated to be 86,000 from Ultrogel filtration. This purified enzyme in oxidized form had absorption peaks at 278, 390, 573 and 690 nm. The absorbance ratios, A₃₉₀: A₂₇₈ and A₆₇₃: A₃₉₀ were 0.61 and 0.37, respectively.

By applying the purified enzyme to DEAE-Sephadex A–50 column chromatography, the ferredoxin-dependent nitrite reductase activity was selectively decreased. However, the methyl viologen-dependent nitrite reductase activity was increased, with a specific activity of 391 units/mg protein. This modified enzyme was homogeneous by disc electrophoresis with polyacrylamide gel.     

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 of Sulphite-Cytochrome c Reductase of Thiobacillus novellus and Reconstitution of Its Sulphite Oxidase System with the Purified Constituents

Sulphite-cytochrome c reductase (sulphite: ferricytochrome coxidoreductase, EC 1.8.2.1 [EC] ) derived from Thiobacillus novelluswas purified by chromatography on a DEAE-cellulose column andby gel filtration with a Sephadex G-100 column. Although thereductase thus purified moved as a single band both in gel filtrationand in isoelectric focusing it was always split into two bandsby polyacrylamide gel electrophoresis; the one had the enzymaticactivity and showed absorption spectrum of cytochrome, whilethe other had no activity and was colourless, in contrast withthe results reported by Charles and Suzuki [(1966) Biochim.Biophys. Acta 128: 522]. The enzymatic properties of the purifiedreductase were almost the same as those of the enzyme obtainedby Charles and Suzuki. Cytochrome c-551 free of the reductase activity was obtained.Its molecular weight was determined to be 23,000 by polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate.The cytochrome seemed to exist in the organism as a complexwith the reductase or a subunit of the enzyme. In the stateof the complex with the enzyme, the cytochrome was reduced veryquickly on addition of sulphite, while the cytochrome free ofthe reductase activity was hardly reduced by the enzyme withsulphite. A sulphite oxidase system was reconstituted with the reductase,cytochrome c-550 and cytochrome oxidase highly purified fromthe bacterium. ¹ Present address: Water Research Institute, Nagoya University,Nagoya 464, Japan ² Present address: Institute for Biological Science, SumitomoChemical Co., Ltd., Takarazuka, Hyogo 665, Japan (Received January 23, 1981; Accepted March 9, 1981)     

Purification and properties of a dissimilatory nitrite reductase of a denitrifying phototrophic bacterium

Nitrite reductase [nitric-oxide : (acceptor) oxidoreductase,EC 1.7.2.1 [EC] ] from a denitrifying phototrophic bacterium, Rhodopseudomonassphaeroides forma sp. denitrificans, was purified. The molecularweight of the enzyme, estimated by gel-filtration, was 80,000.Sodium dodecyl sulfate polyacrylamide gel electrophoresis ofthe purified enzyme showed a single 39,000 molecular weightband, indicating that the enzyme was composed of two subunitsof identical molecular weight. The oxidized form of the enzymeexhibited maximum absorption at 280 nm, 450 nm and 590 nm, andthe reduced form only at 280 nm. The ESR spectrum of a frozensolution of the oxidized enzyme showed a typical spectrum patternof a copper protein, suggesting that two types of Cu²⁺ existedwithin the enzyme. Estimates with an atomic absorption spectrophotometer,revealed two copper atoms per molecule. The optimum pH of theenzyme was 7.0. Km for nitrite was estimated to be 51 µM,and the optimum temperature, 30?C. The enzyme was inhibitedby CO, potassium cyanide and diethyldithiocarbamate and activatedby monoiodoacetate. Phenazine methosulfate, 2,6-dichlorophenolindophenol,horse heart cytochrome c, and cytochrome c₂ from this bacteriumwere suitable electron donors. The enzyme also showed cytochromec oxidase activity. (Received May 4, 1978; )     

R plasmid dihydrofolate reductase with subunit structure.

Dihydrofolate reductase, specified by the type II plasmid of a trimethoprim-resistant Escherichia coli, was purified 40-fold to homogeneity using a combination of gel filtration, DEAE-Sephacel chromatography, and hydrophobic chromatography. The final product shows a single protein band on polyacrylamide gel electrophoresis and has a specific activity of 1.0 unit/mg. The molecular weight of the purified enzyme is 36,000 as determined both by gel filtration and Ferguson analysis of polyacrylamide gel electrophoresis. In contrast, a single polypeptide with a molecular weight of 8,500 was observed on sodium dodecyl sulfate-gel electrophoresis. These experiments suggest that, unlike any bacteria or vertebrate dihydrofolate reductase previously examined, the type II R plasmid reductase is a tetramer composed of four identical subunits. A partial amino acid sequence determination shows no heterogeneity of the subunits and also no clear homology with any reductase sequence previously reported.     

Purification and some properties of polyphenol oxidase from root-forming carrot callus tissues

1. Polyphenol oxidase (o-diphenol : O₂ oxidoreductase; E.C.1.10.3.1 [EC] ) was isolated from the other phenolases which werepresent in root-forming carrot callus, and its properties wereexamined. 2. The enzyme was purified about 45-fold over crudeextracts (precipitates between 40–70% saturation widiammonium sulfate) by a combination of Bio-gel filtration, protein-bagfiltration, and carboxymethyl cellulose chromatography. Thepurified oxidase was homogeneous according to polyacrylamidegel electrophoresis and Sephadex gel filtration. It was confirmedby CM-cellulose chromatography that the enzyme was absent incallus tissues without accompanying redifferentiation. 3. Themolecular weight of this oxidase was estimated to be 110,000-120,000 from molecular weight-mobility profiles on polyacrylamidegels containing sodium dodecyl sulfate and molecular size-elutionvolume correlations on Sephadex G-150 columns. 4. The enzymeoxidized o-diphenols but showed no detectable activity againstmonophenols. Pyrocatechol, dopamine, caffeic acid, and chlorogenicacid were effectual substrates of the enzyme with K_m valuesranging from 10^–3 M to 10^–5M. The enzyme effectivelycatalyzed the oxidation of o-diphenols over the range of pH6.0 to 7.0 and was readily inactivated by heating. The enzymeactivity was slightly influenced by increasing ionic strength.The initial rate of the enzymic reaction was enhanced by additionof Cu²⁺, Co²⁺ and Mn²⁺ ions, and was reduced in the presenceof DTT, PCMPS, glycylglycine, and DIECA. (Received June 17, 1978; )     

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)     

Effects of Cyclic AMP on the Activity of Soluble Protein Kinases in Lemna paucicostata

Three protein kinases which phosphorylate histone were isolatedfrom cellular extract of Lemna plants. They were separated byelution from DEAE-Sephacel column and referred to as PI, PITand PHI. The PI protein kinase activity was partially inhibitedby 10µM cyclic AMP, cyclic GMP or cyclic IMP, while thePII enzyme was activated in the presence of these cyclic nucleotides.The PIII enzyme was cAMPindependent, but slightly inhibitedby cyclic CMP and cyclic UMP. The molecular weights of thesethree protein kinases were 165,000, 85,000 and 145,000, respectively,as estimated from Sephacryl S-300 gel filtration. A single cyclicAMP-binding protein was detected in the PII enzyme fractionby using the photoaffinity cAMP-analogue, 8-N₃-cAMP. The proteinwhich specifically bound [³H]-8-N₃-cAMP had an apparent molecularweight of 48,000 as determined by SDS-polyacrylamide gel electrophoresis.The phosphorylation of cellular proteins in Lemna was examinedby SDS-polyacrylamide gel electrophoresis. Four phosphorylatedpolypeptides were detected, the phosphorylations of which werestimulated by cAMP. The molecular weights of these four polypeptideswere 59,000, 19,000, 16,000 and 14,000, respectively. (Received January 26, 1983; Accepted April 13, 1983)