NITRATE REDUCTASE IN PHOTOSYNTHETIC BACTERIUM, RHODOSPIRILLUM RUBRUM. ADAPTIVE FORMATION OF NITRATE REDUCTASE

1. A method was discovered for adapting the cells of Rhodospirillumrubrum to grow on a nitrate medium, a capacity initially lackingin the organism. The adapted cells were able to grow with nitrateas the sole source of nitrogen. The growth responses of theadapted cells towards various nitrogenous sources were investigatedunder various conditions of incubation (aero- and anaerobiosis,light and dark).
2. The adapted cells were found to have simultaneouslyacquiredthe capacity for reducing nitrite and hydroxylamineas wellas nitrate. The path of nitrogen in the adapted cellswas assumedto be as follows: NO₃^– NO₂^– NH₂OH CellularNitrogen.
3. Nitrate metabolism of the adapted cells was investigatedundervarious conditions. In the light, nitrate was reducedand furtherassimilated, leaving insignificant amounts of nitritein themedium. In this case, consumption of nitrate was markedlyinhibitedby other forms of nitrogen (e.g., nitrite, hydroxylamine,aminoacids and ammonium salts). In the dark, nitrate was reducedas the terminal hydrogen acceptor in the oxidative breakdownof organic substances (e.g., malate) in the medium (i.e., nitraterespiration). More nitrite was accumulated in this case thanin the light. Molecular oxygen inhibited the reduction of, aswell as the growth on, nitrate in any of the above cases.
4. Theeffects on the rate of nitrate reduction (and respiratoryoxygenuptake) caused by various experimental factors (pH, nitrateconcentration, electron donors, and addition of hydroxylamine)were investigated, using the resting cells of the adapted organism.

¹ This paper was submitted to the University of Tokyo to fulfillthe requirement for the author's doctorate. ² Present Address: Botanical Institute, Kyoto University, Sakyo-ku,Kyoto. (Received February 14, 1963; )     

COMPARATIVE EFFECTS OF SOME ANTIMETABOLITES ON RIBONUCLEIC ACID SYNTHESIS AND INDUCTION OF NITRATE REDUCTASE IN CAULIFLOWER LEAF TISSUES

The effect of three antibiotics, actidione, patulin and polymyxinB, one synthetic antimetabolite, l-2-dichloro-4(p-nitrobenzenesulphonylamido)-5-nitrobenzene (DCDNS) and ribonuclease on the induction of nitratereductase, gross ribonucleic acid content and the incorporationof phosphorus into the ribonucleic acid of cauliflower leaveshas been studied. The effects of inhibitor concentration and duration of incubationon the inhibition of enzyme production were tested. The induction of the enzyme by molybdenum was inhibited by allcompounds tested. Induction by nitrate was inhibited by actidione,patulin and ribonuclease. Gross ribonucleic acid was decreasedby ribonuclease, patulin and DCDNS in nitrate-starved tissue. Phosphorus incorporation into ribonucleic acid was inhibitedby actidione, patulin, polymyxin B and DCDNS when infiltratedwith nitrate into nitrate starved tissue and by patulin, polymyxinB and DCDNS with molybdenum as the inducer in molybdenum deficienttissue. Ribonuclease in nitrate starved tissue increased theincorporation of phosphorus. Some possible explanations of theseresults are advanced. ¹Present address: Aligarh Muslim University, U. P., India.     

NITRATE REDUCTASE IN SUGARCANE TISSUES

Nitrate reductase purified from extracts of sugarcane leaf tissuesshowed an absolute requirement for NADH and a partial dependenceon the presence of FAD and Mo⁺⁺⁺. The purified enzyme had poorstability. Activity of nitrate reductase increased toward theyounger nodal regions of the stalk but the enzyme appeared tobe inhibited in the tissues of the apical meristem. Roots showedlow nitrate reductase activity compared to leaf tissue. ¹ Published with the approval of the Director as Paper No. 198in the Journal Series of the Experiment Station, Hawaiian SugarPlanters' Association, Honolulu, Hawaii, U. S. A. This investigationwas supported in part with funds provided by U. S. Departmentof Agriculture (ARS) Contract No. 12-14-100-7788 (34) to theExperiment Station of the Hawaiian Sugar Planters' Association.     

Solubilization of nitrate reductase from the blue-green alga Anabaena cylindrica

Nitrate reductase was solubilized and purified from Anabaenacylindrica by Triton X-100 treatment of particulate preparationsfollowed by adsorption on calcium phosphate gel. Reduced methylviologen, FAD or FMN, but not ferredoxin, served as an effectiveelectron donor for the nitrate reduction by solubilized nitratereductase. ¹This work was supported by a grant (4061) from the Ministryof Education (Received June 25, 1970; )     

REDUCTION OF NITRATE AND NITRITE BY SUBCELLULAR PREPARATIONS OF ANABAENA CYLINDRICA II. REDUCTION OF NITRATE TO NITRITE

Reduction of nitrate to nitrite by particulate preparationsof Anabaena cylindrica was investigated. Preparations whichshowed high activity of nitrate reductase were obtained by sonication(preparation A) or acetone treatment (preparation B). The preparationA also showed a high activity of DPIP-ascorbate photooxidation.The nitrate reductase system accepted electrons from eitherreduced ferredoxin (preparation A & B) or NADH (preparationB), but not directly from NADPH. Ferredoxin was active whenreduced either by action of photochemical system I or by NADPHand NADP-reductase, but dithionite-reduced ferredoxin was completelyinactive. Ferredoxin could be replaced with methyl viologen,benzyl viologen and diquat. Reduced FMN and FAD could serveas electron donors, but the affinity of the reductase towardthese flavin compounds was very low. ¹ This work was supported by grants from the Ministry of Education(4093 and 95612) and from the National Institutes of Health,U.S. (GM-11300).     

Properties of the Respiratory NAD(P)H Dehydrogenase Isolated from the Cyanobacterium Synechocystis PCC6803

Activity staining with NADPH-nitroblue tetrazolium after native-PAGEof membrane proteins of Synechocystis PCC6803, solubilized with3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),revealed four NAD(P)H dehydrogenase (NDH) activities; an NDHcomplex of the respiratory chain, a ferredoxin NADP⁺ reductase(FNR), a drgA product which oxidized both NADH and NADPH, andan uncharacterized NADH-specific enzyme. The NDH complex waspurified with anion exchange and gel filtration chromatographies.The purified complex had a molecular mass of 376 kDa and wascomposed of 9 subunits. Western analysis showed that the complexcontained the NDH-H subunit, but not NDH-A or B. The enzymereduced ferricyanide much faster than plastoquinone and usedNADPH as its prefered electron donor rather than NADH. The enzymaticactivity was inhibited by diphenyleneiodonium chloride and salicylhydroxamicacid, but not by rotenone, p-chloromercuribenzoate, N-ethylmaleimide,flavon, dicumarol, or antimycin A. These results suggest thatthe purified complex is a hydrophilic subcomplex which containsan NADPH binding site and flavin, and is dissociated from ahydrophobic subcomplex, which contains quinone binding site. ¹Present address: Division of Applied Life Sciences, GraduateSchool of Agriculture, Kyoto University, Sakyo, Kyoto, 606-8502Japan ³Present address: Department of Biotechnology, Faculty of Engineering,Fukuyama University, 1 Gakuencho, Fukuyama, Hiroshima, 729-0292Japan     

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)     

PHOTOSYNTHETIC NITRITE REDUCTASE FROM SPINACH

Photosynthetic nitrite reductase (PNiR) was purified extensivelyfrom spinach leaves by the method including acetone precipitation,ammonium sulfate fractionations and DEAE-cellulose column chromatography.The purified PNiR had the highest specific activity thus farobtained. Absorption spectrum of the purified PNiR indicatedthat it had no flavin component. There was no difference inthe rate of photosynthetic nitrite reduction between aerobicand anaerobic conditions. In chloroplasts two reductase systems related with ferredoxinhad been recognized, which were ferredoxin-nitrite and ferredoxin-NADPreductase system. The relationship between the two systems wasinvestigated by using the reconstituted enzyme system. Whenthe reduction of NADP was taking place, the reduction of nitritewas strongly inhibited. Some physiological significance of thephenomenon was discussed. ¹This work was mainly perfomed in the Department of Cell Physiology,University of California, during the author's stay, and waspresented in the 6th annual meeting of the Japanese Societyof Plant Physiologists on April 4-7, 1964 in Tokyo.     

COMPARATIVE STUDIES OF PHOTOCHEMICAL OXIDATION-REDUCTION REACTIONS IN LAMELLAR FRAGMENTS OF VARIOUS ALGAE AND SPINACH

The activity of various electron carriers, including DPIP, spinachplastocyanin, mammalian cytochrome c, and Anabaena cytochrome553, as donor in the reaction induced by the photochemical systemI was examined with lamellar fragments of various algae andspinach. Reduced DPIP was an effective electron donor irrespective ofthe organisms, when it was supplied at a high concentration(10^–3 M). Spinach plastocyanin was effective in the reactionswith the lamellae of green algae, Euglena, diatom Phaeodactyrumand red algae Porphyra yezoensis and Porphyra sp. Yamamoto II,whereas it was inactive in the lamellae of blue-green algae.Horse-heart cytochrome c and Anabaena cytochrome 553 were activein the reaction with the lamellae of bluegreen algae. The formercytochrome was also active in the reactions in Porphyridiumand Cyanidium. The cytochromes were less active in the reactionsin which spinach plastocyanin acted as effective electron donor. The data were interpreted as that the photochemical system Iin bluegreen algae differs from that of other photosyntheticorganisms with respect to the properties of the site of theelectron-input. ¹ Present address: Nomura Research Institute for Technologyand Economics, Kamakura, Kanagawa. ² Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo.     

FLAVOPROTEIN IN CHLORELLA ELLIPSOIDEA CATALYZING TPNH-LINKED REDUCTION OF PLASTOCYANIN

1. An enzyme possessing a capacity of catalyzing reduction of thecopper protein, plastocyanin, with reduced pyridine nucleotides(TPNH-plastocyanin reductase) was isolated from Chlorella ellipsoidea.The procedures of purification and the properties of the purifiedenzyme are described.
2. From the results of chromatographicand enzymic tests, the prostheticgroup of the enzyme was identifiedas FAD. No evidence was obtainedto indicate the participationof metal ions in the reaction.
3. The enzyme utilizes both TPNHand DPNH as electron donors, butthe reaction is about 12 timesfaster with TPNH than with DPNH.
4. The enzyme, with TPNH aselectron donor, catalyzes the reductionof Chlorella plastocyanin,cytochrome c, 2,6-dichlorophenolindophenol and oxygen in adecreasing order of reaction rate.The reaction with oxygenas electron acceptor was found to bemuch more strongly acceleratedby the addition of higher concentrationsof flavins as comparedwith the reaction with other acceptors.FAD and FMN added tothe reaction mixture are not appreciablyreduced.
5. The propertiesof the enzyme are compared with those of alliedchloroplastenzymes reported by various investigators and thepossible roleof the new enzyme in photosynthesis is discussed.

(Received January 18, 1961; )     

185W-labelled nitrate reductase from Chlorella

By adding ¹⁸⁵W-tungstate to a Chlorella culture, it has beenpossible to incorporate this metal into the nitrate reductasecomplex. The W-labelled enzyme was completely inactive as nitratereductase, but maintained unaffected its diaphorase activity.In vivo incorporation of tungsten into the enzyme was competitivelyhindered by molybdenum. ¹ This work was supported by a grant from the Instituto de EstudiosNucleares, J.E.N., Spain. (Received July 6, 1971; )     

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)     

Nitrogen-13 Studies of Nitrate Fluxes in Barley Roots: II. EFFECT OF PLANT N-STATUS ON THE KINETIC PARAMETERS OF NITRATE INFLUX

Influx of nitrate into the roots of intact barley plants wasfollowed over periods of 1–15 min using nitrogen-13 asa tracer. Based on measurements taken over 15 min from a rangeof external nitrate concentrations (0·2–250 mmolm^–3), the kinetic parameters of influx, I_max and K_m, werecalculated. Compared with plants grown in the presence of nitrate throughout,plants that had been starved of N for 3 d showed a significantlygreater value ofI_max for ¹³N-nitrate influx (by a factor of1·4–1·8), but a similar value of K_m (12–14mmol m^–3). Pre-treating N-starved plants with nitratefor about 5 h further increased the subsequent rate of ¹³N-nitrateinflux, but had little effect in the unstarved controls. Allowingfor this induction of additional nitrate transport, the differencein rates of nitrate influx in control and N-starved plants wassufficient to account for the previously-observed differencein net uptake by the two groups of plants. In barley plants grown without any exposure to nitrate, butwith ammonium as N-source, both I_max and K_m for subsequent ¹³N-nitrateinflux were significantly decreased (by about one-half) comparedwith the corresponding nitrate-grown controls. The importance of changes in the rate of influx in the regulationof net uptake of nitrate is discussed. Key words: Ion transport, nitrate, influx, kinetic parameters, N-deficiency     

Periplasmic location of dissimilatory nitrate and nitrite reductases in a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans

The localization of dissimilatory nitrate and nitrite reductasesof a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroidesforma sp. denitrificans, was investigated. Nitrate and nitritereductases were located in the periplasmic space of the bacteriumgrown anaerobically in the presence of nitrate either in lightor in darkness. Chromatophores showed nitrate and nitrite reductaseactivities when dithionite-reduced benzyl viologen was an electrondonor; this suggests that the enzymes were trapped inside thevesicles. ¹Present address: Japanese Red Cross Central Blood Center, Hiroo4-1-31, Shibuyaku, Tokyo 150, Japan. ²Present address: Plant Growth Laboratory, University of California,Davis, California 95616, U.S.A. (Received November 7, 1979; )     

STUDIES ON HOMOSERINE DEHYDROGENASE IN PEA SEEDLINGS

Partially purified homoserine dehydrogenase was prepared frompea seedlings. The optimum pH for this enzyme is approximately 5.4. The K_mvaluesfor ASA and TPNH are 4.6xl0^–4Af and 7.7xl0^–5M, respectively.This enzyme can also utilize DPNH but less effectively thanTPNH. In contrast with yeast homoserine dehydrogenase whichis insensitive to — SH reagents, the pea enzyme is inhibitedalmost completely by 10^–4MPCMB and 10^–5MHgCl₂, theinhibition being removed by 10^–2M thioglycolate. Homoserinedehydrogenase was found not only in decotylized seedlings, butalso in cotyledons. The significance of this enzyme in homoserine biosynthesis ingerminating pea seeds has been discussed. (Received February 20, 1961; )     

ELECTRON TRANSPORT SYSTEMS OF RHIZOBIUM GROWN IN NODULES AND IN LABORATORY MEDIUM

The electon transport systems of Rhizobium japonicum were studied,comparing cells harvested from effective nodules with thosefrom artificial culture. Participation of the cytochrome systemwas confirmed in both forms of cells. Absorption peaks of thecytochromes of cultured cells were a, b, c type, resemblingthose of Bacillus subtilis, yeast and mammalian tissue. Cytochromea could not be detected in the absorption spectrum of symbioticcells, although the CO binding difference spectrum showed apeak at about 438 mµ, which can be attributed to a componenta₃ or a₁. CO difference spectrum also showed a shoulder at about416 mµ. Cells cultivated under the insufficient supply of oxygen showedthe cytochrome absorption spectrum closely resembled that ofsymbiotic cells. Diaphorase activity was lower in symbioticcells. These results are considered to be due to the insufficientsupply of oxygen within nodule tissue. Succinate oxidation bythe symbiotic cell paniculate was shown to be carbon monooxideresistant. NADH₂ oxidation by the supernatant fraction of symbioticcells was accelerated by flavin mononucleotide, 2, 6-dichiorophenolindophenol, methylene blue and vitamin K₃. ¹Present address: Faculty of Agriculture, Tôhoku University,Sendai. ²Present address: Central Agricultural Experiment Station, Kitamoto.     

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; )     

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)     

Sodium Requirement of Monocotyledonous C4 Plants for Growth and Nitrate Reductase Activity

The effects of Na application on growth and nitrate reductaseactivity of seven C₄ plant species, Zea mays, Echinochloa crus-galli,Panicum miliaceum, Panicum coloratum, Panicum dichotomiflorum,Panicum maximum and Chloris gayana were studied. Except forZ. mays and P. miliaceum, Na application enhanced growth significantly,and concurrent increases in nitrate reductase activities weredetected in Panicum coloratum, Panicum dichotomiflorum, Panicummaximum and Chloris gayana. ¹Present address: International Research Institute, Ciba GeigyJapan Ltd., Takarazuka, Hyogo 665, Japan. ²Present address: Photobiology Lab., Research Institute forFood Science, Kyoto Univ., Uji, Kyoto 611, Japan. (Received May 2, 1988; Accepted August 22, 1988)     

Nitrogen Economy of the Main Shoot of Field-Grown Barley (Hordeum vulgare L.): II. IN VIVO NITRATE REDUCTASE ACTIVITY DURING GROWTH

The method for assay of in vivo nitrate reductase (NR) activitywas standardized for barley (Hordeum vulgare L.). NR activitywas determined in the various organs of the main shoot of field-grownJyoti barley at 40 kg N ha_–1. Total nitrate reductaseactivity (TNRA) of each organ for the period it was metabolicallyactive was calculated. The NR activity was highest in the laminae,followed by the sheaths, reproductive organs; and internodes.The NR activity was high in the first-formed laminae and itshowed a decline in the ones formed subsequently. The valuesvaried from 43.2 ± 4.33 to 7.2 ± 1.49 µmolNO^–₃ reduced g^–1 dry wt. h^–1. Maximum TNRAin the laminae, sheath, and internodes was at 49, 84, and 84–93d after sowing, respectively. The TNRA of the main shoot asa whole showed three peaks, one around 49 d, a second around63 d, and a third around 84 d after sowing. Correlation coefficient(r) between NR and NO3 concentration was highly significantin the laminae and sheath viz. 0.76^*** and 0.62^***, respectively.The results are discussed in relation to alteration in managementpractices to maximize nitrate assimilatory activity and theamount of reduced N harvested.