Effects of hydroxylamine and molybdate on the formation of nitrate reductase in the yeast Rhodotorula

NADPH-nitrate reductase (NR) and NADPH-cytochrome c reductase(CR) activities of Rhodotorula glulinis var. salinaria cellsgrown in nitrate medium supplied with hydroxylamine (0.2 mM)were respectively 1.6- and 3.1-fold higher than those of cellsgrown without hydroxylamine. NR formed in nitrate plus hydroxylaminemedium is almost totally in an inactive form which is reactivatedin vitro by K₃Fe(CN)₆. When molybdate (10^–7 M) was suppliedto this medium, total (active plus inactive) NR activity increasedfurther about twofold but CR activity somewhat decreased. Inordinary nitrate medium, such molybdate effects were small.Most of the CR derepressed (induced) excessively in the nitrateplus hydroxylamine medium had a molecular size similar to NRon the basis of Bio-Gel A 1.5 m chromatography. Some other propertiesof CR formed in this medium were the same as those of the CRmoiety of NR. Adding molybdate to the nitrate plus hydroxylamine medium aftergrowing the cells for 20 hr induced the development of NR activitywithout any increase in CR activity. This induction was completelyblocked by cycloheximide, puromycin and L-canavanine but notcompletely by 6-methylpurine. Ammonium repressed this inductionwith markedly decreasing CR activity. The roles of hydroxylamine and molybdate in the formation ofNR are discussed. (Received May 26, 1980; )     

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)     

Purification and Characterization of Sucrose Synthase from Peach (Prunus persica) Fruit

Sucrose synthase (EC 2.4.1.13 [EC] ) was purified from peach fruit(Prunus persica) to a single band of protein on SDS-PAGE byammonium sulfate fractionation, DEAE-cellulose (DE-52) chromatography,Sepharose CL-6B gel filtration, PBA-60 affinity chromatographyand Sephadex G-200 gel filtration. The molecular weight wasestimated to be 360,000 by gel filtration. The enzyme was foundto be a tetramer of identical 87-kDa subunits. The maximum activityfor the synthesis and cleavage of sucrose was observed at pH8.5 and pH 7.0, respectively. The enzymatic reaction followedtypical Michaelis-Menten kinetics in both directions, with thefollowing parameters: K_m(fructose), 4.8 mmM; K_m(UDPglucose),0.033 mM; K_m(sucrose), 62.5 mM; K_m(UDP), 0.080 mM. Other properties,such as substrate specificity and the effects of divalent cations,were also investigated. The relationship between the enzymeand the accumulation of sucrose in peach fruit is discussed. Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 2, 1988; Accepted September 14, 1988)     

Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)     

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)     

Studies on the change of the respiratory system in roots in relation to the growth of plants II. Activity of iron-containing oxidases in roots of cereal crops with the aging of plants

Distribution of iron-containing oxidases in aging nodal rootsof rice and wheat was studied. Activities of cytochrome c oxidase(1.9.3.1 [EC] , cytochrome c : O₂ oxidoreductase), catalase (1.11.1.6 [EC] ,H₂O₂: H₂O₂ oxidoreductase) and peroxidase (1.11.1.7 [EC] , donor:H₂O₂ oxidoreductase) in wheat roots were comparatively higherthan were those in rice roots at corresponding stages. Cytochromec oxidase in roots remained active throughout the lives of therice and wheat crops. In rice roots, catalase seemed to playa distinct role around the panicle formation stage. Decay ofcatalase activity took place earlier than did that of peroxidaseand cytochrome c oxidase activities. In wheat roots similarenzyme activity changes were not observed. Data may suggestthat the high activity of iron containing oxidases at the panicleformation stage (I) may be chiefly due to catalase activityin rice roots. ¹Paper presented at the 14th Annual Meeting of the Society ofthe Science of Soil and Manure, Japan (1968). (Received November 21, 1968; )     

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)     

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; )     

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)     

Characterization of Porphobilinogen Synthase from an Aerobic Photosynthetic Bacterium, Erythrobacter sp. Strain OCh 114

Porphobilinogen synthase (formerly 5-aminolevulinic acid dehydratase,EC 4.2.1.24 [EC] ) was purified 7,405-fold from an aerobic photosyntheticbacterium, Erythrobacter sp. strain OCh 114. The molecular weightof the enzyme was determined to be 260,000 by Sephadex G-200gel filtration. The enzyme had a single pH optimum at 8.0 andshowed no requirement for metal ion and thiol compound for itsmaximum activity. The K_m value for 5-aminolevulinic acid was0.29 mM. 4,5-Dioxovaleric acid and levulinic acid were foundto be competitive inhibitors of the enzyme, with K_i values of0.65 and 0.80 mM, respectively. The enzyme was extremely labilein acidic pH and almost completely lost its activity within1 h at pH 6.0 and 30?C. This Erythrobacter enzyme seems to besimilar to the enzyme from the anaerobic photosynthetic bacteriumRhodobacter capsulatus in its molecular and catalytic properties. (Received February 17, 1988; Accepted May 9, 1988)     

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; )     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

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     

Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves

A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31 [EC] ) and NADP-dependent malic enzyme (EC1.1.1.40 [EC] ) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)_–1 min_–1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.K_m values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)_–1 min_–1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP⁺ and requireda divalent metal ion for activity. K_m values of 0.33 and 0.008mM for L-malate and NADP⁺, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989)     

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     

Inhibition of Porphobilinogen Synthase Activity by 4,5-Dioxovalerate

The effect of 4,5-dioxovaleric acid on the activity of porphobilinogen(PBG) synthase (formerly 5-aminolevulinic acid dehydratase,EC 4.2.1.24 [EC] ) of the porphyrin synthetic pathway was studiedwith the enzyme purified from Chlorella regularis. 4,5-Dioxovalericacid, a metabolite of 5-aminolevulinic acid, competitively inhibitedPBG synthase with a K_i value of 1.4 mM. The concentration forthe half inhibition of 4,5-dioxovaleric acid (7 mM) was slightlylower than that for the known competitive inhibitor, levulinicacid (12 mM). (Received October 8, 1984; Accepted December 13, 1984)     

Inhibition of nitrate reductase from tomato leaves by adenosine-5'-diphosphate

ADP was found to inhibit the activity of nitrate reductase fromtomato leaves in vitro. No effects of ATP, AMP, adenosine andadenine could be detected. Orthophosphate promoted activityonly in the presence of high concentrations of NADH₂. It is suggested that nitrate reductase possesses characteristicsof a regulatory enzyme and that ADP brings about a transformationin the conformation of the enzyme, with a resulting decreasein its activity. (Received February 29, 1968; )     

A heme-C-containing enzyme complex that exhibits nitrate and nitrite reductase activity from the dissimilatory iron-reducing bacterium Geobacter metallireducens

Nitrate reduction in the dissimilatory iron-reducing bacterium Geobacter metallireducens was investigated. Nitrate reductase and nitrite reductase activities in nitrate-grown cells were detected only in the membrane fraction. The apparent K _m values for nitrate and nitrite were determined to be 32 and 10 μM, respectively. Growth on nitrate was not inhibited by either tungstate or molybdate at concentrations of 1 mM or less, but was inhibited by both at 10 and 20 mM. Nitrate and nitrite reductase activity in the membrane fraction was not, however, affected by dialysis with 20 mM tungstate. An enzyme complex that exhibited both nitrate and nitrite reductase activity was solubilized from membrane fractions with CHAPS and was partially purified by preparative gel electrophoresis. It was found to be composed of four different polypeptides with molecular masses of 62, 52, 36, and 16 kDa. The 62-kDa polypeptide [a low-midpoint potential (–207 mV), multiheme cytochrome c] exhibited nitrite reductase activity under denaturing conditions. No molybdenum was detected in the complex by plasma-emission mass spectrometry. Received: 26 March 1999 / Accepted: 16 August 1999     

Participation of Hydrogen Peroxide in the Inactivation of Calvin-Cycle SH Enzymes in SO2-Fumigated Spinach Leaves

In SO₂-fumigated spinach leaves under light, chloroplast SHenzymes, glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD)(EC 1.2.1.13 [EC] ), ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19 [EC] )and fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11 [EC] ) weremore remarkably inactivated than other chloroplast enzymes.Their activities recovered after removal of SO₂. The inactivationparalleled light-dependent CO₂-fixation in spinach leaves. Inilluminated chloroplasts isolated from SO₂-fumigated spinachleaves, NADP-GAPD and Ru5PK were more specifically in activatedthan other chloroplast enzymes. These two enzymes could be protectedfrom the inactivation by adding catalase. The NADP-GAPD inactivationwas suppressed by DCMU, cytochrome c or anaerobic conditions.By adding thiol compounds, the NADP-GAPD inactivation was dischargedand the activity increased. In chloroplasts or crude extractsfrom non-fumigated spinach leaves, NADP-GAPD and Ru5PK weremore strongly inhibited by externally added H₂O₂ than otherchloroplast enzymes. All results supported the idea that thesuppression of photosynthesis at the beginning of SO₂ fumigationwas caused by the reversible inhibition of chloroplast SH enzymewith H₂O₂. (Received October 7, 1981; Accepted June 16, 1982)