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)     

Photosynthetic independence of initial light-caused increase in extractable nitrate reductase activity from maize seedlings

Comparisons of the effects of light on extractable nitrate reductase(EC 1.6.6.1 [EC] ) activity in Zea mays L. seedlings with and withoutnormally developing photosynthetic systems were made. Generally,plants lacking chlorophyll, due to either chemical treatment(fluridone) or genetic lesion (a chlorophyll and a carotenoidmutant), had as high or higher nitrate reductase (NR) activitiesas normally greening plants during the first 12 to 24 hr ofcontinuous white light (W) induction. This trend was especiallypronounced in mesocotyl tissues. In apical tissues (includingleaf, coleoptile and apical meristem) NR activity continuedto increase in normally greening plants between 24 and 48 hrwhile activities of achlorophyllous plants decreased markedly.These decreases could not be explained by a toxic accumulationof nitrite. Continuous far-red light (FR), which causes pronouncedphotomorphogenic development without significant greening, inducedabout one-half as much NR in the mesocotyls as did continuouswhite light. In apical tissues the effects of W and FR weresimilar throughout a 72 hr time course. Although trends weresimilar, the effects of light (W and FR) on nitrate concentrationwere kinetically different from effects on NR activity. Theseresults indicate that photosynthetic pigments are only secondarilyinvolved in NR induction. (Received June 12, 1979; )     

Ferredoxin Isolated from Plant Non-Photosynthetic Tissues. Purficaition and Characterization

A ferredoxin was isolated from non-photosynthetic tissues ofthe lower storage root of radish (Raphanus sativus L. var. acantiformiscultivar Miyashige) in a pure form by conventional means. Itshowed the characteristic features in its absorption spectrumof chloroplast-type ferredoxin. However, amino acid compositionand amino (N)- terminal sequence were different from those ofradish leaf ferredoxin. Root ferredoxin was able to transferelectrons from dithionite to nitrite reductase [EC 1.7.7.1 [EC] ]isolated from mung bean seedling roots and also to mediate NADP⁺photoreduction in spinach broken chloroplasts. It therefore is suggested that a set of distinctive molecularspecies of ferredoxin is present in non-photosynthetic tissuesand functions as a redox mediator in ferredox-independent enzymesystems. (Received October 18, 1985; Accepted January 16, 1986)     

The Development of Activities of Some Enzymes Concerned with Glycollate Metabolism in Greening Bean Leaves

The activities of phosphoglycollate phosphatase (EC 3.1.3.18 [EC] ),glycollate oxidase (EC 1.1.3.1 [EC] .). catalase (EC 1.11.1.6 [EC] ), theperoxisomal NADH-glyoxylate reductase (EC 1.1.1.26 [EC] ) which isconsidered to function as a hydroxypyruvate reductase in theperoxisomes, and the chloro-plastic NADPH-dependent glyoxylatereductaae, have been measured in extracts prepared from 14-d-olddark-grown bean leaves during the course of their greening inresponse to exposure to continuous illumination. All of theenzymes were found in the dark-grown leaves and on a per-leafbasis the activities increased from 6- to 12-fold with the exceptionof a 2–3-fold increase of NADPH-dependent glyoxylate reductaseduring 96-h greening, while the activities either remained constantor declined during similar periods in darkness. Initial lagperiods were evident before the illumination-induced increasesin enzyme activities. As D-threo-chloramphenicol did not affectthe increase in activity of any of these enzymes it would appearthat the increases were in no way dependent on protein synthesisby 70S ribosomes, or on the development of photosynthetic activity.     

Purification and Characterization of Molecular and Immunological Properties of Rice Ferredoxin-Nitrite Reductase

Ferredoxin-nitrite reductase [EC 1.7.7.1] has been purified to apparent homogeneity from rice (Oryza satira cv. Kinmaze) leaves by a procedure used for the spinach enzyme [S. Ida and B. Mikami, Biochim. Biophys. Acta, 681, 167 (1986)]. The rice enzyme consists of a single polypeptide of % molecular weight of 60,000 with 536 amino acid residues. The enzyme showed nearly identical absorption, circular dichroism, and magnetic circular dichroism spectra to those of the spinach enzyme, indicating the presence of the same prosthetic groups and protein conformation in both enzymes. The apparent Km values for nitrite and methyl viologen were 360 μm and 63 μm, respectively. The pH optimum was 7.6. These kinetic parameters are indistinguishable from those reported for spinach nitrite reductase. Monospecific antiserum against purified rice enzyme cross-reacted with nitrite reductases from a variety of higher plants and some phylogenetically divergent plants. Immunological comparisons indicated the rice enzyme is much more closely related to the other monocot enzymes in antigenic structure than to the dicot enzyme proteins. The results lend further support to our previous study [S. Ida, Plant Sci., 49, 111 (1987)] that spinach ferredoxin-nitrite reductase is serologically more related to the dicot enzymes than to the monocot nitrite reductases. Conspicuous differences between the rice and spinach enzymes were found in their molecular sizes and antigenicity. Relatedness of amino acid compositions of the enzyme proteins is discussed in relation to antigenic properties of ferredoxin-nitrite reductase.     

Effects of univalent cations on the formation of nitrate reductase and nitrite reductase in rice seedlings

An investigation was made to determine the effects of univalentcations as activators on the formation of nitrate reductaseand nitrite reductase in rice seedlings. K⁺ functioned moreeffectively as a univalent cation activator than did other univalentcations examined. Substitution of Rb⁺ for K⁺ resulted in stimulationof nitrate reductase formation at about half the rate obtainedwith K⁺. There was no effect on nitrite reductase formation.Na⁺ could be partially substituted for K⁺ in the formation ofboth enzymes. NH₄⁺ slightly inhibited formation of the enzymes.In the absence of univalent cations, enzyme formation proceededat a slower rate during the initial 15-hr period, but thereafterproceeded at a higher rate. This delayed formation was not observedin the presence of K⁺. Results from inhibitor experiments suggestthat K⁺ stimulates the formation of nitrate reductase and nitritereductase. In conclusion, when nitrate nitrogen is supplied to rice plantsutilization of the nitrogen may be accelerated by increasedformation of enzymes involved in nitrate assimilation in thepresence of K⁺. (Received February 21, 1969; )     

NO2 Suppression of Light-Induced Nitrate Reductase in Squash Cotyledons

NADH-nitrate reductase (NR) (EC 1.6.6.1 [EC] ) activity in the cotyledonsof squash (Cucurbita maxima Duch.) seedlings showed daily variationwhen the seedlings were subjected to an alternating light-darkcycle. When the seedlings were transferred into continuous darkness,NR activity rose at first and then decreased continuously. Irradiationafter continuous darkness induced a rapid increase in NR activity;this light induction of NR activity was inhibited completelyby fumigation with 4 ppm nitrogen dioxide (NO₂). This inhibitoryeffect of NO₂ was prominent even at 1 ppm and became more pronouncedas the concentration of NO₂ increased. NO₂ fumigation did notremarkably affect the content of reductant (NADH) in the cotyledons.The results of immunoblotting using anti-NR serum indicatedthat irradiation induced the increase in the NR-polypeptidecontent and NO₂ fumigation inhibited the increase, suggestingthat NO₂ put an inhibitory effect on the synthesis of NR inducedby irradiation. ⁴ Present address: College of Environmental Health, Azabu University,Fuchinobe, Sagamihara, Kanagawa 229, Japan ⁵ Present address: Faculty of Home Economics, Otuma Women'sUniversity, Sanban-cho, Chiyoda, Tokyo 102, Japan (Received October 21, 1987; Accepted January 13, 1988)     

Nitrate reductase inactivating factor from rice seedlings

A nitrate reductase inactivating factor was found in extractsof leaf blades, leaf sheaths, and roots of rice seedlings. Thefactor was nondialyzable, precipitable with (NH₄)₂SO₄, and heatlabile. The factor from rice roots inactivated NADH nitratereductase, FMNH2 nitrate reductase, and NADH cytochrome c reductasefrom rice shoots, but had no effect on the activities of NADHdiaphorase and nitrite reductase. The factors from rice shoots,rice roots, and maize roots inactivated NADH nitrate reductaseprepared from cultured rice cells. The factor from culturedrice cells also inactivated rice shoot NADH nitrate reductase. The activity of the inactivating factor showed a diurnal changein shoots of rice seedlings grown with NO₃– medium, althoughthe fluctuation was not large compared to that of NADH nitratereductase activity. When the seedlings were placed in darkness,the activity of the factor did not change during 20 hr withNO₃– medium. However, the activity of the factor fluctuatedwith NO₃– -free medium in light; its activity startedto increase at the 8th hour after transfer. NADH nitrate reductaseactivity from rice shoots declined rapidly during the first8 hr and gradually thereafter in both types of culture. (Received August 24, 1977; )     

Monodehydroascorbate Reductase in Spinach Chloroplasts and Its Participation in Regeneration of Ascorbate for Scavenging Hydrogen Peroxide

The primary reaction product of chloroplast ascorbate peroxidaseactivity was shown to be monodehydroascorbate radical (MDA).MDA reductase (EC 1.6.5.4 [EC] ) was localized in spinach chloroplaststroma. The MDA reductase activity of spinach chloroplasts,using NAD(P)H as electron donor, could account for the regenerationof ascorbate from MDA produced by ascorbate peroxidase activity.In the absence of MDA reductase, MDA disproportionated to ascorbate(AsA) and dehydroascorbate (DHA). The DHA was reduced to AsAby DHA reductase (EC 1.8.5.1 [EC] ) in chloroplasts. Both NADH andNADPH served as the electron donor of partially purified MDAreductase from spinach leaves. (Received September 24, 1983; Accepted January 23, 1984)     

Collapse of Peroxide-Scavenging Systems in Apple Flower-Buds Associated with Freezing Injury

Changes in the metabolic activities of peroxide-producing systemsand peroxide-scavenging systems after freezing and thawing inflower buds of the apple, Malus pumila Mill., were studied withspecial reference to freezing injury. In flower buds of the‘McIntosh’ apple that were frozen below lethal temperatures,the activity of NADH-Cyt c reductase (EC 1.6.99.3 [EC] ), one of theenzymes in the electron-transport chains that are related tothe peroxide-producing systems, decreased slightly, while thatof Cyt c oxidase (EC 1.9.3.1 [EC] ) hardly changed. By contrast, theactivities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49 [EC] ),dehydroascorbate reductase (EC 1.8.5.1 [EC] ) and ascorbate peroxidase(EC 1.11.1.11 [EC] ), which are involved in the peroxide-scavengingsystems, decreased to very low levels. The activity of glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12 [EC] ) also decreased markedly. However,little change was observed in the activities of hexokinase (EC2.7.1.1 [EC] ), glucosephosphate isomerase (EC 5.3.1.9 [EC] ), glutathionereductase (EC 1.6.4.2 [EC] ) and glutathione peroxidase (EC 1.11.1.9 [EC] ).Examination of substrates involved in the peroxide-scavengingsystems revealed that the levels of glucose-6-phosphate andfructoses-phosphate decreased to approximately 10^–4 to10^–5 M and 10^–5 M, respectively, and the levelsof GSH decreased to about 10^–5 M or became barely detectable.A decrease in the levels of GSSG also occurred while levelsof ascorbate rose slightly. Similar results were observed withflower buds from ‘Starking Delicious’ and ‘Jonathan’apple trees. These results suggest that the freezing injury to apple flower-budsis closely related to the collapse of the peroxide-scavengingsystems that are coupled with the pentose phosphate cycle. Theresults also suggest that the dysfunction of these peroxide-scavengingsystems is caused by H₂O₂, which may be produced during freezingand thawing. (Received March 14, 1992; Accepted June 5, 1992)     

Effect of shade treatment on biosynthesis of catechins in tea plants

When tea plants were shaded with black lawn cloth for severaldays in the field, the accumulations of (—)-epicatechin,(—)-epicatechin-3-gallate, (—)-epigallocatechinand (—)-epigallocatechin-3-gallate decreased in newlydeveloping tea shoots. Radioactive tracer studies showed thatthe conversions of glucose-U-¹⁴C, shikimic acid-G-¹⁴C and phenylalanine-U-¹⁴Cinto (—)-epicatechin and (—)-epigallocatechin moietieswere depressed by the shade treatment for tea plants but theincorporation of trans-cinnamic acid-3-¹⁴C was not affected.The treatment was found to have no significant effect on theactivities of phospho-2-keto-3-deoxy-heptonate. aldolase (EC.4.1.2.15 [EC] ), 3-dehydroquinate synthase (EC. 4.6.1.3 [EC] ), 3-dehydroquinatedehydratase (EC. 4.2.1.10 [EC] ), shikimate dehydrogenase (EC. 1.1.1.25 [EC] )and trans-cinnamate 4-monooxygenase (EC. 1.14.13.11 [EC] ) in theshoots, whereas the activity of phenylalanine ammonia-lyase(EC. 4.3.1.5 [EC] ) clearly decreased. (Received March 17, 1980; )     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings VI. Anabolic Pathway of 4-Thiouridine

In germinating radish seeds, [U-¹⁴C]-4-thiouridine was convertedto 4-thio-UMP, 4-thio-UDP, 4-thio-UTP, 4-thio-UDP glucose and4-thiouracil, of which 4-thiouracil accounted for 60–85%.4-Thio-UTP is incorporated into RNAs of radish seedlings [Shibataet al. (1980) FEBS Lett. 119: 85]. These same metabolites werelabeled following germination of radish seeds with [2-¹⁴C]-4-thiouracil.4-Thiouridine was hydrolyzed by the uridine nucleosidase (EC3.2.2.3 [EC] ) of radish seedlings as effectively as was uridine.The activity of uridine nucleosidase was increased by germinationwith 4-thiouridine. These results are a strong indication that4-thiouridine is converted to 4-thiouracil, then to 4-thio-UMPby uracil phosphoribosyltransferase (EC 2.4.2.9 [EC] ). The alternativeformation of 4-thio-UMP from 4-thiouridine by uridine kinase(EC 2.7.1.48 [EC] ) also was suggested. A possible mechanism whichmay cause inhibition of chloroplast biogenesis in 4-thiouridine-culturedseedlings is discussed. (Received October 12, 1981; Accepted January 14, 1982)     

Limitations to the Measurement of In Vitro Nitrate Assimilation by Exogenous Additives and Endogenous Interference Factors in the Leaves of Zea mays L. Seedlings

An evaluation of existing assay procedures for the measurementof nitrate assimilation in the leaves of Zea mays L. has highlightedlimitations in established in vitro assay techniques. Both exogenouslyadded compounds and endogenous leaf components affected theresults of an in vitro nitrate reductase (NADH: oxido-reductase,EC 1.6.6.1 [EC] .) assay. Reducing agents employed as enzyme protectantswere excluded from the assay in order to accurately measurethe concentration of nitrogen compounds by colorimetric andHPLC analysis. Endogenous nitrate levels in a leaf extract asmeasured by these two analytical techniques indicated significantinterference in the colorimetric method due to the presenceof various organic compounds. This interference was most apparentat low nitrate concentrations, however, changes in nitrate concentrationappeared to be more closely comparable between the two techniques.In addition, endogenous leaf components also interfered withthe precise determination of nitrite that had accumulated duringan in vitro nitrate reductase assay. These endogenous factorsacted directly upon the colorimetric assay of nitrite by a concentration-dependentreaction with the diazotizing reagent sulphanilamide. The interferingcomponents were of low molecular weight ( 5000 daltons) andeasily separable from nitrate reductase by molecular sieve chromatography.Their interference in the nitrite assay could only be partiallyprevented by heating or storage, while other treatments studied,including those frequently used to terminate an in vitro assaysuch as zinc acetate precipitation or chloroform extraction,had less effect in alleviating the interference. Similar endogenouscomponents which affected the colorimetric assay of nitritewere also found in leaf extracts from wheat, pea, soybean andsunflower seedlings. Zea mays L., nitrate reductase, reducing agents, plant interference factors     

Properties of S-adenosyl-L-methionine-magnesium-protoporphyrin IX methyltransferase from barley

S-Adenosyl-L-methionine-magnesium-protoporphyrin IX methyltransferase(EC 2.1.1.11 [EC] ) is present in greening barley seedlings associatedwith the particulate fraction. This enzyme was purified 20 foldusing protamine and ammonium sulfate precipitation. The enzymewas active over a wide pH range with highest activity at pH7.5. The Km values for Mg-protoporphyrin IX and S-adenosylmethioninewere 48 and 39 µM, respectively; S-adenosylethionine andS-adenosyihomocysteine were competitive inhibitors with respectto S-adenosylmethionine; hemin inhibition was non-competitivewith respect to Mg-protoporphyrin IX; thiol compounds exhibiteda stimulatory effect on enzyme activity. The properties of theenzyme are discussed and compared with the enzyme from otherorganisms. ¹ This research was supported in part by the Utah State AgriculturalExperiment Station. ² Present address: Department of Chemistry, Boston University,Boston, Massachusetts, U. S. A. ³ Present address: Department of Biochemistry and Microbiology,Faculty of Pharmacy, Comenius University, Bratislava, Czechoslovakia. (Received February 20, 1978; )     

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)     

The possibility of photo-induced induction of nitrate reductase in rice seedlings

Nitrate reductase activity in rice seedlings showed daily fluctuations.Seedlings placed in the dark slowly lost activity and quicklyregained it when exposed to sunlight. Etiolated seedlings alsoshowed rapid increases in activity when transferred to sunlight.This increase in activity by sunlight was inhibited by bothchloramphenicol and ethionine. Ethionine inhibition was reversedby methionine. Purification of nitrate reductase was carriedout by ammonium sulfate fractionation and DEAE-cellulose columnchromatography. Nitrate reductase was purified about 40-foldfrom plants placed in the dark and in sunlight. Incorporationof mediionine-S-¹⁴CH₃ into the nitrate reductase fraction wasstudied. In sunlight, the specific radioactivity of the nitratereductase fraction from the DEAE-cellulose column increased2-fold as compared with that of the crude extracts. Specificradioactivity did not increase in the dark. ¹Present address: Asahi Kasei Chemicals Industry Co., Ltd.,Sameshima 2-1, Fuji, Shizuoka, Japan (Received December 3, 1968; )     

Isourea and triazinone derivatives as related to their ability to stimulate rice seedling growth

TA [4-ethoxy-1-(p-tolyl)-s-triazine-2,6 (1H, 3H)-dione] wasprepared chemically by intramolecular cyclization of IU [2-ethyl-3-methoxycarbonyl-1-(p-tolylcarbamoyl)-isourea],which has been reported to be an effective GA-synergist. TAalone slightly stimulated the shoot growth of rice seedlings,and in combination with GA showed a distinctly synergistic effecton the growth of rice shoots. The results suggest that TA andIU are nearly equal in their physiological activity. From theresults of the rice seedling test of these two compounds andtheir analogs, structure-activity relationships of isoureaswere correlated with those of triazinones. The results of applicationof IU, at 100 mg/liter to rice seedlings followed by extractionand silica gel thin-layer chromatography, suggested that IUwas biologically converted into its closed-ring form, TA, whichis a stable and potent GA-synergist. Thus, isoureas like IUseem to be easily converted into their counterparts, triazinones,in rice tissues. ¹ This paper is Part III in the series "Plant growth-regulatingactivities of isourea derivatives and related compounds." ² Permanent address: Agricultural Chemicals Research Laboratories,Sankyo Co., Ltd., Hiro-machi, Shinagawa-ku, Tokyo 140, Japan. ³ Present address: Higashi-Osaka Junior College, Nishitsutsumigakuen,Higashi-Osaka 577, Japan. (Received February 21, 1977; )     

Appearance of Nitrate in Soybean Seedlings and Chlorella Caused by Nitrogen Starvation

Disinfected seeds of soybean and actively growing cultures ofChlorella vulgaris were grown on nitrogen-free media. The nitratecontent of both the soybean seedlings and algal cultures substantiallyincreased during nitrogen starvation. The nitrate level in soybeanseedlings was at least eight times greater than seeds aftertwo to three weeks. Nitrogen starvation also caused an increasein the nitrate content of the algal cultures. Nitrate reductaseactivity also increased, and its appearance was sensitive tocycloheximide. Tungstate, added during starvation, inhibitedthe induction of nitrate reductase with a concomitant increasein the level of nitrate. These data suggest that oxidation ofreduced nitrogen compounds can occur in higher plants and algae. ¹ A contribution of the Texas Agricultural Experiment Station (Received June 6, 1981; Accepted August 25, 1981)     

Regulation of Mo-cofactor, NADH- and NAD(P)H-specific nitrate reductase activities in the wild type and two nar-mutant lines of barley (Hordeum vulgare L.)

Seedlings of three genotypes of barley, Hordeum vulgare L.,cv. Winer, were grown in nutrient solutions for 12 d: (a) Wt,the wild type; (b) Chlo19 and (c) Chlo29, two nitrate reductase(NR) deficient nar-mutants. Nar-mutant plants grown in nitratedeveloped about 5–24% of NADH-NR (EC 1.6.6.1 [EC] .) activitylevel characteristic of the Wt. The NR in vitro assays in whichNADH or NADPH were used as electron donors showed that the twomutant lines contained a mixture of NADH-specific and NAD(P)H-bispecific(EC 1.6.6.2 [EC] .) NRs. Chlo19 had a very low level of MoCo activityas compared to Chlo29 and Wt. Chlo19 appeared to be mutatedin a MoCo gene rather than in the genes coding for the nitrateNR apoenzyme. NAD(P)H-NR was found in the shoots and roots of both mutantsbut only in the roots of Wt. Several aspects of the regulationof NADH and NAD(P)H specific NRs in plants of the barley cv.Winer genotypes are discussed. MoCo was a strong limiting factorfor NR biosynthesis in nitrate-fed plants of Chlo19, but lesslimited in N-starved and ammonium-fed plants. Biomass productionby the three genotypes was similar during first 12 d after germination,regardless of the level of NR detected in vitro. Mutant plantsmay be able to supply the nitrogen required for growth withonly 5–24% of the NR level of the WT. Key words: Hordeum vulgare, mutants, nitrate, nitrate reductase, molybdenum cofactor     

Accumulation of Demolybdo Nitrate Reductase during Induction and Its Separation from Active Nitrate Reductase in Chlorella

During induction of nitrate reductase in Chlorella vulgaris,synthesis of the precursor, demolybdo cytochrome c reductase,exceeds the synthesis of active enzyme. Evidence is also presentedwhich shows that the purification procedure of Funkhouser etal. [(1980) Plant Physiol. 65: 939] separates demolybdo cytochromec reductase from active nitrate reductase. ¹Supported in part by a grant to B. V. from the Deutsche Forschungsgemeinschaftand a contribution of the Texas Agricultural Experiment Station. (Received July 27, 1983; Accepted September 13, 1983)