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[24] Nitrate reductase from higher plants
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power, is nearly axial, with g values of 2.04 and 1.93 typical o f a tetranuclear (Fe4S4*) ironsulfur center-containing protein. Chemical analysis for total iron and acid-labile S ~- confirms the presence of 3 moles of iron and 2 moles of $2-/61,000 gm of protein. However, a reinterpretation of these data leads them to suggest that the enzyme contains 6 moles of iron and 4 moles of acid-labile S2-/mol of siroheme. The EPR results show that there is one iron-sulfur center/siroheme. Rapid kinetic studies show that the high spin ferriheme and the iron-sulfur center of spinach nitrite reductase are reduced by dithionite on a comparable time scale (k = 3 to 4 sec-'). The iron-sulfur center of the enzyme prereduced with dithionite is rapidly reoxidized upon addition of nitrite (k = I00 sec-a), what strongly supports a role for the iron-sulfur center in the mechanism of nitrite reduction. Finally, they proposed an extinction coefficient of E3a6 = 7.6 × 104 cm -1 (M active center)-'.
[24] N i t r a t e R e d u c t a s e f r o m H i g h e r P l a n t s 1
By R. H. HAGEMANand A. J. REED NO3- + NAD(P)H + H ÷ ~ NO2- + NAD + H20
This article is an update of the same topic published in 1971.2 Consequently limited details of standard extraction and assay procedures will be given and major emphasis placed on new developments. Preparation
Material. Chlorophyllous lamina tissue from illuminated plants well s u p p l i e d w i t h n i t r a t e is n o r m a l l y u s e d a s s o u r c e m a t e r i a l b e c a u s e o f i t s high activity. However, nonchlorophyllous organs, such as corn scutella 3 or roots, 4 have been used. Soybean leaves, 5 corn scutella, 3 and cultured rice seedlings 6 are the best known sources of NAD(P)H nitrate reductase. E x t r a c t i o n . A s t a n d a r d p r o c e d u r e f o r t h e e x t r a c t i o n o f t h e e n z y m e is a s f o l l o w s : T h e m a t e r i a l is h o m o g e n i z e d f o r 30 t o 9 0 s e c i n a m e d i u m o f 1 m M E D T A , 1 t o 25 m M c y s t e i n e , a n d 25 m M p o t a s s i u m p h o s p h a t e ,
t EC 1.6.6.1 nitrate oxidoreductase is the most prevalent enzyme in plants; however, EC 1.6.6.2 NAD(P)H nitrate oxidoreductase is present in some tissues. z R. H. Hageman and D. P. Hucklesby, Vol. 23, p. 491. 3 j. E. Eisner, D. P. Hucklesby, and R. H. Hageman, Agron. Abstr. p. 20 (1971). 4 W. Wallace, Plant Physiol. 55, 774 (1975). 5 S. A. Jolly, W. H. Campbell, and N. E. Tolbert, Arch. Biochem. Biophys. 174, 431 (1976). e T. C. Shen, Plant Physiol. 49, 546 (1972).
METHODS IN ENZYMOLOGY,VOL. 69
Copyright© 1980by AcademicPress, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181969-8
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adjusted to a final pH of 8.8 with KOH. Six milliliters of grinding medium are added for each gram of fresh weight of tissue. The homogenate is pressed through four layers of cheesecloth or a single layer of Miracloth (Chicopee Mills, New York, New York) and the filtrate centrifuged for 15 min at 30,000 g. The supernatant fluid is then decanted through glass wool and used for assays. The homogenates and extracts are kept cold (2° to 3°) throughout. The optimum concentration of cysteine must be verified for each type of tissue and homogenization technique. Glutathione and dithiothreitol are usually slightly more effective (10%) than cysteine. A marked improvement in the extraction of nitrate reductase from soybean leaf tissue was obtained with the following procedure. 7 One gram fresh tissue is frozen with liquid N2 in a precooled mortar. After evaporation of the N2, the frozen tissue is rapidly (15 sec) ground to a powder. The extraction medium (20 volumes of 25 mM potassium phosphate, pH 7.8, 1 mM cysteine, 5 mM KNOa, 5 mM EDTA and 25/xM FAD) is added and allowed to freeze in the mortar. The tissue is suspended by grinding as the mixture thaws. The homogenate is then treated as previously described. Special Protectants. With tobacco leaves, the addition of 0.1% (w/v) bovine serum albumin to sucrose (plus other additives) extraction medium prevented the indiscriminate and variable binding of nitrate reductase to various organelles during subsequent sucrose density separation 8 and enhanced (twofold) the total recovery of activity. The enhanced activity observed with the use of exogenous protein was reported to be due to protection from a small-molecular-weight, heat-stable inhibitor. The addition of casein or bovine serum albumin (1 to 3%) to the standard extraction medium results in increased (up to 15-fold) recovery and stability of nitrate reductase from leaves of corn, oat, and tobacco plants a and corn roots. 4 It was suggested that the added protein protected the enzyme from proteolytic enzymes. With seedling tissue from corn or soybeans, extraction with added protein often slightly decreases recoverable activity (unpublished data). With cotton cotyledons, the addition of bovine serum albumin (3%, w/v) or Dowex 1-CI, anion exchange resin (10%, w/v) to the extraction medium significantly improved the activity of the extracted enzyme. 1° Neither protein nor resin enhanced stability r R. L. Scholl, J. E. Harper, and R. H. Hageman, Plant Physiol. 53, 825 (1974). 8 M. J. Dalling, N. E. Tolbert, and R. H. Hageman, Biochim. Biophys. Acta 283, 505 (1972). 9 L. E. Schrader, D. E. Cataldo, and D. M. Peterson, Plant Physiol. 53, 688 (1974). 10 A. C. Purvis, L. R. Tischler, and R. C. Fites, Plant Physiol. 58, 95 (1976).
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COMPONENTS
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of the enzyme. The protective hction was attributed to phenolic binding rather than decreased degradation by proteases. The inclusion of NADH (1 mg/ml) in the crude extract of beans 11 or rice TM stabilized nitrate reductase activity. The variation in interpretation given for the mode of action of the various protectants is attributed to the variation in inhibitors, proteases, and inherent stability (dissociation into inactive components) of the enzyme among the different plant species and tissues utilized. Polyvinylpyrrolidone may be the best protectant against the inhibitory effects of phenolics. 13 No reports were found where two or more protectants were added concurrently.
Assay Method (in Vitro) NOz- + AH2 ---*NO2- (+ A + H20
Principle. Nitrate reductase is capable of utilizing the reduced form of pyridine nucleotides, flavins, or benzyl viologen as electron donors for reduction of nitrate to nitrite. Because NADH-dependent nitrate reductase is most prevalent in plants, NADH is the most frequently used donor. Activity is usually measured by colorimetric determination 14 of nitrite produced; however, it can also be measured by following the oxidation of pyridine nucleotides at 340 nm. The stoichiometric relationship between nitrite produced and pyridine nucleotide or flavin oxidized has been established. 15"~6
Reagents for Reaction Potassium phosphate buffer, 0.1 M (pH 7.5) Potassium nitrate, 0.1 M NADH, 2 mM
Reagents for Nitrite Assay Sulfanilamide, 1% (w/v) in 1.5 to 3.0 N HC1 N-(1-Naphthyl)ethylenediamine dihydrochloride, 0.02% (w/v) Procedure (NADH). The assay mixture contains in micromoles, potassium phosphate, 50; KNO3, 20; NADH, 0.8; and enzyme, 0.1 to 0.2 11 C. M. Sluiters-Scholten, Planta 123, 175 (1975). 12 A. P. Gandhi, S. K. Sawhney, and M. S. Naik, Biochem. Biophys. Res. Commun. 55, 291 (1973). 13 W. D. Loomis and J. Battaile, Phytochemistry 5, 423 (1966). 14 F. D. Snell and C. T. Snell, "Colorimetric Methods of Analysis." Van Nostrand Reinhold, Princeton, New Jersey (1949). 1~ H. J. Evans and A. Nason, Plant Physiol. 28, 233 (1953). 16 L. E. Schrader, G. L. Ritenour, G. L. Eilrich, and R. H. Hageman, Plant Physiol. 43, 930 (1%8).
[24]
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ml of crude extract (equivalent to 0.2 to 1.0 mg of trichloroacetic acid precipitable protein) in a final volume of 2.0 ml. The optimum pH is usually 7.5; however, with crude extracts from soybean leaves the optimum pH is 6.5. The reaction can be initiated either by addition of NADH or enzyme. A zero time and minus NADH reaction mixture are used for controls. After incubation at 30° for 15 min the reaction is terminated by rapid addition of 1.0 ml of sulfanilamide reagent followed by 1.0 ml of the N-(1-naphthyl)ethylenediamine dihydrochloride reagent. The color is allowed to develop for 30 min prior to reading at 540 nm. The enzyme reaction rate is linear over a 30-min period. The same procedure is used when NADH oxidation is followed spectrophotometrically at 340 nm. The linear initial reaction rate is used to compute the activity. Activity is expressed as /zmoles of NOR- produced per minute per gram fresh or dry tissue weight or per milligram of protein. The protein is precipitated by 5% trichloroacetic acid from the extract which is determined by any of several standard protein methods. Details for preparation of other reductants and other details are as previously published. 2 Coupled Assays. In addition to the coupled enzyme reactions previously reported, 2 nitrate reduction can be coupled via NADH generated by the addition of malate (20 mM) and NAD (2 mM) as a substitute for NADH (0.4 mM) in the standard assay. 17 With crude extracts of corn leaves containing high levels of malate dehydrogenase, the activity of the coupled reaction was 80% of the standard (NADH) assay. Precautions. Residual NADH at the end of the assay, and unknown factors present in the crude extract, interfere with NO2- color development. While NADH can be oxidized enzymatically,TM this does not eliminate the extract factors. Postassay treatments have been found that minimize these problems, r The reaction is stopped by addition of zinc acetate (50/zmoles/ml reaction mixture) or by placing the reaction tubes in boiling water. After clarification by centrifugation (1000 g for 10 min), aliquots of the cooled (25°) supernatant liquid are treated with phenazine methosulfate (15 nmoles/ml reaction mixture) to oxidize the residual NADH. After 20 min, color is developed in the aliquot as previously described. The enzyme from most plant species is very unstable even at 0°. Therefore, minimum time should elapse between extraction and assay even when stabilizers of enzyme activity have been added. ,7 C. A. N e y r a and R. H. H a g e m a n , Plant Physiol. 58, 726 (1976). 18 D. Spencer, Aust. J. Biol. Sci. 12, 181 (1959).
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Assay Method (in Vivo) Principle. Sections of plant tissue, containing adequate amounts of endogenous nitrate and substrates for NADH generation, produce and accumulate NO2- wJaen placed under dark anaerobic conditions. The assay as originally developed TM has been adapted to provide a more accurate estimate of nitrate reduction in situ 2° or to study nitrate assimilation in tissue from species having high inhibitor content that prevents extraction of active enzymeY ,21 Rates of activity obtained with the in vivo assay are lower (50 to 80%) than in vitro rates, however, they are correlated. 20,22 Procedure. z°'23 Sections or discs of fresh tissue (0.2 to 0.5 gm) are placed in a test tube or small flask containing the incubation medium. The medium is composed of 0.1 M potassium phosphate, pH 7.5, 1% (v/ v) 1-propanol or 0.04% (v/v) Neutronyx 600 (Onyx Chemical Co., Jersey City, New Jersey) and 0.03 to 0.05 M KNO3. Stainless steel screens may be placed in each tube to hold the tissue below the solution surface. The samples are then evacuated (5 mm Hg) in a vacuum desiccator. Nitrogen gas is bled into the desiccator and the process repeated. The tubes may be stoppered before transferring to a shaking water bath and incubated (0.25 to 4 hr) at 30° in the dark. Aliquots can be removed from the same reaction tube at timed intervals to establish linearity of NO2- production. Usually there is a 5- to 10-min lag period prior to establishment of a linear rate. The reaction is usually linear for 60 to 90 min. Normally the reaction is stopped by transferring the tubes to a boiling water bath for 2 min to release NO2- remaining in the tissue. After cooling, aliquots are removed for colorimetric determination of NO2-. The difference between the amount of NO2- produced at 30 min minus the production at 5 or 10 min, with a comparable set of materials, is used to calculate the rate of NO2- production (equated with NO3- reduction). Comments. The amount of nitrite produced slightly underestimates nitrate reduction, as some nitrite disappears under dark anaerobic conditions. 21 However, very little nitrite appears to be assimilated to aminoN under these conditionsY 4 Anaerobiosis is essential, 24 and other systems (continuous N2 bubbling) can be devised to establish the anaerobic condition. 19 E. G. Mulder, R. Borma, and W. L. VanVeen, Plant Soil Sci. 10, 335 (1959). 20 N. Brunetti and R. H. Hageman, Plant Physiol. 58, 583 (1976). 21 j. W. Radin, Plant Physiol. 51,332 (1973). 22 j. G. Streeter and M. E. Bosler, Plant Physiol. 49, 448 (1972). 23 j. C. Nicholas, J. E, Harper, and R. H. Hageman, Plant Physiol. 58, 731 (1976). 24 D. T. Canvin and C, A. Atkins, Planta !16, 207 (1974).
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With tissue deficient in sugars or organic acids (prolonged exposure of plants to dark prior to sampling), marked stimulation of nitrate reduction can be obtained by addition of sugar phosphates or 3-phosphoglyceraldehyde (6 mM) to the incubation medium. 25 With dark pretreated soybean leaf tissue, the addition of 10 and 100 mM glucose to the incubation medium increased activity 4.5- to 5.2-fold, respectively, over the control, z6 Addition of 10 mM pyruvate, citrate, succinate, or malate was without effect, while activity was increased (two- to threefold) when added at 100 mM. Purification Nitrate reductase extracted from higher plants is extremely unstable and difficult to purify to homogenity with reasonable recoveries. These difficulties are illustrated by a comparison of specific activities of 0.02, 0.2, 0.7, 0.7 and 2.5/~moles NO3- reduced/min/mg protein for enzymes from soybeans, ~ maize, 27 marrow, In maize, 16 and spinach, ~8 respectively with the specific activity of 83.1 for Chlorella,29 when conventional purification techniques were used. By combining conventional purification procedures with the recently developed blue-dextran agarose affinity chromatography, 3° a homogenous nitrate reductase (specific activity 24.1 and 7% recovery) was prepared from spinach leaves, al This procedure is detailed as follows. Extraction. Freshly harvested and chilled spinach leaves (1 kg) were homogenized in 5 mM Tris-HC1, pH 7.5, and 1 mM EDTA (2 ml buffer/ gm leaf). The macerate was filtered through two layers of cheesecloth and centrifuged at 60,000 g for 20 min. The supernatant was used as the crude extract. Purification. Nucleic acids were removed by adding streptomycin (5 mg/gm leaf) to the crude extract. After 1 hr, the precipitate was removed (60,000 g for 20 min) and discarded. Solid (NH,)2SO4 (ARISTAR grade, British Drug House, Poole, England) was added to the supernatant to attain 50% saturation. After 20 min the precipitated protein was collected (60,000 g for 20 min) and saved. The protein was redissolved in 0.1 M 25 L. Klepper, D. Flesher, and R. H. Hageman, Plant Physiol. 48, 580 (1971). 26 j. C. Nicholas, J. E. Harper, and R. H. Hageman, Plant Physiol. 58, 736 (1976). zr j. Roustan, M. Neuberger, and A. Fourey, Physiol. Veg. 12, 527 (1974). 28 B. A. Notton and E. J. Hewitt, Plant Cell Physiol. 12, 465 (1971). 29 L. P. Solomonson, G. H. Lorimer, R. L. Hall, R. Borchers, and J. L. Bailey, J. Biol. Chem. 250, 4120 (1975). zo L. P. Solomonson, Plant Physiol. 56, 853 (1975). 3~ B. A. Notton, R. J. Fido, and E. J. Hewitt, Plant Sci. Lett. 8, 165 (1977).
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phosphate, pH 7.5, and desalted on a Sephadex G-25 (Pharmacia, Uppsala, Sweden) column equilibrated with the same buffer. Fractions containing the enzyme were transferred to a column of hydroxylapatite (BioRad Laboratories, Richmond, California) equilibrated with 0.05 M phosphate pH 7.5. The column was then washed with buffer until absorbance (280 nm) of the eluate was less than 0.01. The enzyme was eluted from the column using the washing buffer fortified with 5% (NH4)2SO4. The appropriate fractions were combined and the enzyme precipitated by adding solid (NH4)2SO4 to attain 50% saturation. After 20 min the precipitate was collected (60,000 g for 20 min) and redissolved in 0.1 M phosphate, (pH 7.5) containing 0.1 M KCI. This solution was transferred to a BioGel A (0.5 m, 100 to 200 mesh, Bio-Rad Laboratories) column. Column equilibration and elution were with the dissolving solution. Fractions containing the enzyme were bulked and reprecipitated with (NH4)2SO4 at 50% saturation. The precipitate was collected by centrifugation and dissolved in 0.08 M phosphate, pH 7.5, and the solution transferred to a blue-dextran Sepharose column equilibrated with the same buffer. (The blue-dextran Sepharose was made as previously described.32). The column was washed with the same buffer until the eluate gave negligible absorbance at 280 nm. Nitrate reductase was eluted by applying a 0 to 3 M KCI gradient (in 0.08 M phosphate, pH 7.5). Fractions containing the enzyme were bulked and concentrated with (NH4)zSO4 (50% saturation) and dissolved in a small amount of 0.08 M phosphate, pH 7.5. The BioGel A and blue-dextran Sepharose steps increased specific activity 4- and 22-fold, but lost 42 and 30% of the total activity, respectively. Unlike the enzyme from Chlorella, 3° nitrate reductase from higher plants cannot be eluted, effectively, from the blue-dextran Sepharose with NADH. However, this problem was circumvented by the use of an alternative affinity medium, blue-Sepharose (Cibacron blue F3GA, Polysciences, Inc., Warrington, Pennsylvania, coupled to Sepharose CL4B, Pharmica, Uppsala, Sweden, as described33). The blue-Sepharose was added to the clarified homogenate and stirred for 1 hr at 4 ° to bind the nitrate reductase. 34 (The homogenate was prepared by grinding 100 gm squash cotyledons in 100 ml of 100 mM phosphate, pH 7.5, 1 mM EDTA, 1 mM cysteine, and 20 gm polyvinylpyrrolidone.) The Sepharose was then collected by filtration and washed with the grinding medium prior 3z L. D. Ryan and C. S. Vestling, Arch. Biochem. Biophys. 160, 179 (1974). 33 H. J. Brhme, G. Kopperschlager, J. Schulz, and E. Hofmann, J. Chromatogr. 69, 209 (1972). 34 W. H. Campbell and J. Smarrelli, Jr., Plant Physiol. 57, 637 (1976).
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to transfer to a column (2.5 cm diameter). The enzyme was eluted with the extraction buffer containing 0.1 mM NADH, and the appropriate fractions combined. The enzyme was concentrated by precipitation with 45% saturated (NH4)2SO4 and desalted on a Sephadex G-25 column with 100 mM phosphate, pH 7.5, 5 mM EDTA and 1 mM cysteine. Activity was 2 and 12/xmoles NO3- reduced/min/mg protein for the bulked sample and maximum fraction, respectively. Recovery in the pooled sample was 30% of the initial activity. This procedure was not as effective with the enzyme extracted from corn leaves and the bulk of the activity was not eluted with NADH, but was washed off with 300 mM KNO3. Characteristics The properties of nitrate reductases from higher plants have been recently reviewed. 3~,36 Molecular Weight. Values reported are 160,000 (maize2), 197,000 (spinach36), 230,000 (barleyar), 220,000 (NADPH-enzyme, soybeanS), 330,000 (NADH-enzyme, soybean 5) and 500,000 (spinach3S). For the spinach enzyme 36 the following properties were found: Stokes radius, 60 A; S value, 8.1; diffusion constant, d20.w of 3.4 × 10r; frictional ratio, f / f o of 1.55; asymmetry, rl/r2 of 10; isoelectric point, pH 5.0; and an E0.1~ of 1.62. 280 Substrate Affinity. A Michaelis constant (Kin) for nitrate of 0.2 mM has been reported for several plant species. 2 Km's for nitrate of 0.11 and 4.5 mM were reported for the NADH and NAD(P)H enzymes from soybeans .5.39 Pyridine Nucleotide Specificity. The enzyme extracted from most higher plants preferentially utilizes NADH as the electron donor; however, the specificity is not absolute. The Km for NADH is approximately 2.5 /zM. 4° A phosphatase, that converts NADPH to NADH and is not completely removed by conventional purification procedures, may account for some apparent NADPH activity. In maize, leaves, but not in maize scutella, the NADPH activity in crude or partially purified extracts
35 E. J. Hewitt, D. P. Hucklesby, and B. A. Notton, in "Plant Biochemistry" (J. Bonner and J. E. Varner, eds.), 3rd ed., p. 633. Academic Press, New York, 1976. 38 B. A. Notton and E. J. Hewitt, in Nitrogen Assimilation of Plants (E. J. Hewitt and C. V. Cutting, eds.) p. 708. Academic Press, New York, 1979. 37 j. L. Wray and P. Filner, Biochem. 3. 119, 715 (1970). 3s A. M. Relimpio, P. J. Aparicio, A., Panaque, and M. Losada, FEBS Lett. 7,226 (1971). 39 W. H. Campbell, Plant Sci. Lett. 7, 239 (1976). 4o L. Beevers, D. Flesher, and R. H. Hageman, Biochim. Biophys. Acta 89, 453 (1964).
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can be attributed to phosphatase activity. 3,41 Two enzymes, NAD(P)H (EC 1.6.6.2) and NADH (EC 1.6.6.1) have been separated from soybean leaves. The Km's for NADPH and NADH for the NAD(P)H enzyme were 1.5 and 39 IzM and for the NADH enzyme 200 and 8 izM, respectively. 5 In the crude extracts the total NADPH activity accounted for approximately 10% of the total. 3a Flavin Requirement. FAD is a constituent of the enzyme. 15,36Addition of FAD often but not always stimulates the in vitro reduction of nitrate. It has been proposed that this may reflect the strength of attachment of the indigenous FAD and be species dependent. Reduced FAD or FMN can also serve as electron donors for nitrate reduction, however, there is disagreement over the apparent Km value (0.02 to 1.0 mM) and whether the flavins are physiological or incidental electron donors. 16'42 Supplemental FAD protects nitrate reductase from heat inactivation, 43 and stabilizes the enzyme during sucrose density centrifugation or dilution. 36 Involvement of Metals. Molybdenum has been shown 44 to be a constituent of nitrate reductase of spinach by the use of aaMo. A b-type cytochrome with absorption bands at 560, 528, and 425 nm has been shown to be a functional constituent of the spinach enzyme. 31 Tungsten can be incorporated into the enzyme as a replacement for molybdenum; however, the analog will not reduce nitrate. 45 Sulfhydryl. The protective effect of sulfhydryl compounds on nitrate reductase during extraction and purification is well established, z The sulfhydryl group on the enzyme from higher plants is considered to be involved in binding of the pyridine nucleotide. ~6 Phosphate Requirement. Nitrate reductase extracted from higher plant tissue with a media devoid of phosphate is stimulated by the addition of phosphate to the assay medium. 2 The reason for the stimulation is not known; however, it has been proposed that it may complex with the molybdenum of the enzyme thereby facilitating its reduction. Sequence of Electron Transfer. The sequence of electron transfer of higher plant nitrate reductase is thought to be similar if not identical with that of Neurospora. 2,35,36
41 G. N. Wells and R. H. Hageman, Plant Physiol. 54, 136 (1974). 42 A. Panaque, F. F. Del Campo, and M. L. Losada, Biochim. Biophys. Acta 109, 79 (1965). 43 W. G. Zumft, P. J. Aparicio, A. Panaque, and M. L. Losada, FEBS Lett. 9, 157 (1970). 44 B. A. Notton and E. J. Hewitt, Biochern. Biophys. Res. Commun. 44, 702 (1971). ~ A. R. J. Eaglesham and E. J. Hewitt, Biochern. J. 122, 18 (1971).
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Alternate electron donors (reduced FMN, benzyl or methyl viologen)
l
NAD(P)H---~FAD--~Cyt b--*Mo protein--~NO~-
Alternate electron acceptors (Cyt c, DCPIP)
The enzyme exhibits two functions: as a dehydrogenase in the reduction of cytochrome c and as a reductase for nitrate. 36 It is not known whether the cytochrome b is associated with the dehydrogenase reaction or not. Subunits and Proposed Structure. The holoenzyme (8.1 S fraction from sucrose density centrifugation) is composed of a pyridine nucleotide-cytochrome c reductase (3.7 S) bonded noncovalently to a molybdenum-containing subunit (minimum effective molecular weight of 30,000). se TheSe functional units can be physically separated and recombined. It has been proposed 3n that the holoenzyme has four subunits having cytochrome c reductase activity and one molybdenum-containing complex. Kinetics. The reaction is essentially irreversible to the right. With purified spinach enzyme, an ordered sequential ping-pong mechanism was reported. 45 Work with nitrate reductase purified from squash and corn suggested standard ping-pong kinetics (initial velocity plots) or a two-sided ping-pong mechanism (product inhibition plots). 34 Inhibitors. Nitrate reductase is sensitive to inhibition by PCMB (1 ~ M to 1 mM) especially when pyridine nucleotides rather than FMNH2 is the electron donor. Sulfhydryl compounds protect the enzyme from PCMB inhibition. 2,1~ The enzyme is sensitive to reagents that react with metals (8-hydroxyquinoline and o-phenanthroaline) and cyanide and azide are especially effective. Atabrin inhibits marrow nitrate reductase at 5 mM. z Neither carbon monoxide nor fluoride (1 mM) inhibit soybean nitrate reductase. 15 Nitrate reductase from Chlorella inactivated by NADH and HCN can be reactivated by oxidation with ferricyanide.46 This system has been proposed as a mechanism for regulation of nitrate reductase in situ. 4r Purified nitrate reductase from corn leaves can be inactivated by addition of NADH and HCN in vitro; however, the inactivation can be measured only when the assays are done under suboptimal 46 L. P. Solomonson, Biochim. Biophys. Acta 334, 287 (1974). 47 L. P. Solomonson and A. M. Spehar, Nature (London) 265, 373 (1977).
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(0.2 mM) levels of nitrate. The presence of the normal level (10 mM) nitrate in the assay system apparently causes a rapid activation of the enzyme. 48,49 Cellular Location. Although there is disagreement over the cellular location of nitrate reductase, the bulk of the evidence strongly suggests that it is located in the cytoplasm. 35 In plants with C4-type photosynthesis, nitrate reductase is confined primarily to the mesophyll cells. 35 Induction. Nitrate reductase is inducible by its substrate, nitrate, z and the increased activity is due to de novo synthesis. 5° Nitrite, the product, will induce nitrate reductase in rice seedlings 6 and beans51; however, it is not an effective inducer with many plant species. 52"53Chloramphenicol and certain other organic nitro compounds will preferentially induce NAD(P)H nitrate reductase in rice seedlings. 6 In contrast, nitrate or nitrite will preferentially induce the NADH enzyme. 6 The two types of enzymes were separated by means of blue dextran Sepharose chromatography. 54 Stability. Nitrate reductase is unstable both in vivo and in vitro. In excised whole maize seedlings at 30°, the half-life of the enzyme was estimated to be 4 hr. With intact maize plants placed in the dark at 25 ° the half-life was 12 to 14 hr. z This decrease in activity follows first-order kinetics, in Using triple labeling techniques, it was shown that synthesis and degradation of the enzyme were concurrent. 5° The stability in vitro and in vivo varies greatly with plant age, species, and tissue. 4a D. L o u s s a e r t and R. H. H a g e m a n , Plant Physiol. 57S, 539 (1976). 49 R. H. H a g e m a n , in Nitrogen Assimilation of Plants, (E. J. Hewitt and C. V. Cutting, eds.) p. 708. A c a d e m i c Press, N e w York, 1979. 5o H. R. Zielke and P. Filner, J. Biol. Chem. 246, 1772 (1971), 51 S. H. Lips, D. Kaplan, and N. Roth-Bejerano, Eur. J. Biochem. 37, 589 (1973). 52 M. M. R. K. Afridi, and E. J. Hewitt, J. Exp. Bot. 16, 628 (1965). L. Beevers, L. E. Schrader, D. Flesher, and R. H. H a g e m a n , Plant Physiol. 40, 691 (1%5). 54 T. C. Shen, E. A. F u n k h o u s e r , and M. G. Guerrero, Plant Physiol. 58, 292 (1976).
[25] P 7 0 0 D e t e c t i o n By T. V. MARSHO and B. KOK
The pigment designated P7001 is the photoreactive center of photosystem I and as such is an obligate component in the overall photochemical transport of electrons from an appropriate donor (H20 or artificial 1 B. Kok, Biochim. Biophys. Acta 22, 399 (1956). METHODS IN ENZYMOLOGY, VOL. 69
Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181969-8
COMPONENTS
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power, is nearly axial, with g values of 2.04 and 1.93 typical o f a tetranuclear (Fe4S4*) ironsulfur center-containing protein. Chemical analysis for total iron and acid-labile S ~- confirms the presence of 3 moles of iron and 2 moles of $2-/61,000 gm of protein. However, a reinterpretation of these data leads them to suggest that the enzyme contains 6 moles of iron and 4 moles of acid-labile S2-/mol of siroheme. The EPR results show that there is one iron-sulfur center/siroheme. Rapid kinetic studies show that the high spin ferriheme and the iron-sulfur center of spinach nitrite reductase are reduced by dithionite on a comparable time scale (k = 3 to 4 sec-'). The iron-sulfur center of the enzyme prereduced with dithionite is rapidly reoxidized upon addition of nitrite (k = I00 sec-a), what strongly supports a role for the iron-sulfur center in the mechanism of nitrite reduction. Finally, they proposed an extinction coefficient of E3a6 = 7.6 × 104 cm -1 (M active center)-'.
[24] N i t r a t e R e d u c t a s e f r o m H i g h e r P l a n t s 1
By R. H. HAGEMANand A. J. REED NO3- + NAD(P)H + H ÷ ~ NO2- + NAD + H20
This article is an update of the same topic published in 1971.2 Consequently limited details of standard extraction and assay procedures will be given and major emphasis placed on new developments. Preparation
Material. Chlorophyllous lamina tissue from illuminated plants well s u p p l i e d w i t h n i t r a t e is n o r m a l l y u s e d a s s o u r c e m a t e r i a l b e c a u s e o f i t s high activity. However, nonchlorophyllous organs, such as corn scutella 3 or roots, 4 have been used. Soybean leaves, 5 corn scutella, 3 and cultured rice seedlings 6 are the best known sources of NAD(P)H nitrate reductase. E x t r a c t i o n . A s t a n d a r d p r o c e d u r e f o r t h e e x t r a c t i o n o f t h e e n z y m e is a s f o l l o w s : T h e m a t e r i a l is h o m o g e n i z e d f o r 30 t o 9 0 s e c i n a m e d i u m o f 1 m M E D T A , 1 t o 25 m M c y s t e i n e , a n d 25 m M p o t a s s i u m p h o s p h a t e ,
t EC 1.6.6.1 nitrate oxidoreductase is the most prevalent enzyme in plants; however, EC 1.6.6.2 NAD(P)H nitrate oxidoreductase is present in some tissues. z R. H. Hageman and D. P. Hucklesby, Vol. 23, p. 491. 3 j. E. Eisner, D. P. Hucklesby, and R. H. Hageman, Agron. Abstr. p. 20 (1971). 4 W. Wallace, Plant Physiol. 55, 774 (1975). 5 S. A. Jolly, W. H. Campbell, and N. E. Tolbert, Arch. Biochem. Biophys. 174, 431 (1976). e T. C. Shen, Plant Physiol. 49, 546 (1972).
METHODS IN ENZYMOLOGY,VOL. 69
Copyright© 1980by AcademicPress, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181969-8
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adjusted to a final pH of 8.8 with KOH. Six milliliters of grinding medium are added for each gram of fresh weight of tissue. The homogenate is pressed through four layers of cheesecloth or a single layer of Miracloth (Chicopee Mills, New York, New York) and the filtrate centrifuged for 15 min at 30,000 g. The supernatant fluid is then decanted through glass wool and used for assays. The homogenates and extracts are kept cold (2° to 3°) throughout. The optimum concentration of cysteine must be verified for each type of tissue and homogenization technique. Glutathione and dithiothreitol are usually slightly more effective (10%) than cysteine. A marked improvement in the extraction of nitrate reductase from soybean leaf tissue was obtained with the following procedure. 7 One gram fresh tissue is frozen with liquid N2 in a precooled mortar. After evaporation of the N2, the frozen tissue is rapidly (15 sec) ground to a powder. The extraction medium (20 volumes of 25 mM potassium phosphate, pH 7.8, 1 mM cysteine, 5 mM KNOa, 5 mM EDTA and 25/xM FAD) is added and allowed to freeze in the mortar. The tissue is suspended by grinding as the mixture thaws. The homogenate is then treated as previously described. Special Protectants. With tobacco leaves, the addition of 0.1% (w/v) bovine serum albumin to sucrose (plus other additives) extraction medium prevented the indiscriminate and variable binding of nitrate reductase to various organelles during subsequent sucrose density separation 8 and enhanced (twofold) the total recovery of activity. The enhanced activity observed with the use of exogenous protein was reported to be due to protection from a small-molecular-weight, heat-stable inhibitor. The addition of casein or bovine serum albumin (1 to 3%) to the standard extraction medium results in increased (up to 15-fold) recovery and stability of nitrate reductase from leaves of corn, oat, and tobacco plants a and corn roots. 4 It was suggested that the added protein protected the enzyme from proteolytic enzymes. With seedling tissue from corn or soybeans, extraction with added protein often slightly decreases recoverable activity (unpublished data). With cotton cotyledons, the addition of bovine serum albumin (3%, w/v) or Dowex 1-CI, anion exchange resin (10%, w/v) to the extraction medium significantly improved the activity of the extracted enzyme. 1° Neither protein nor resin enhanced stability r R. L. Scholl, J. E. Harper, and R. H. Hageman, Plant Physiol. 53, 825 (1974). 8 M. J. Dalling, N. E. Tolbert, and R. H. Hageman, Biochim. Biophys. Acta 283, 505 (1972). 9 L. E. Schrader, D. E. Cataldo, and D. M. Peterson, Plant Physiol. 53, 688 (1974). 10 A. C. Purvis, L. R. Tischler, and R. C. Fites, Plant Physiol. 58, 95 (1976).
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of the enzyme. The protective hction was attributed to phenolic binding rather than decreased degradation by proteases. The inclusion of NADH (1 mg/ml) in the crude extract of beans 11 or rice TM stabilized nitrate reductase activity. The variation in interpretation given for the mode of action of the various protectants is attributed to the variation in inhibitors, proteases, and inherent stability (dissociation into inactive components) of the enzyme among the different plant species and tissues utilized. Polyvinylpyrrolidone may be the best protectant against the inhibitory effects of phenolics. 13 No reports were found where two or more protectants were added concurrently.
Assay Method (in Vitro) NOz- + AH2 ---*NO2- (+ A + H20
Principle. Nitrate reductase is capable of utilizing the reduced form of pyridine nucleotides, flavins, or benzyl viologen as electron donors for reduction of nitrate to nitrite. Because NADH-dependent nitrate reductase is most prevalent in plants, NADH is the most frequently used donor. Activity is usually measured by colorimetric determination 14 of nitrite produced; however, it can also be measured by following the oxidation of pyridine nucleotides at 340 nm. The stoichiometric relationship between nitrite produced and pyridine nucleotide or flavin oxidized has been established. 15"~6
Reagents for Reaction Potassium phosphate buffer, 0.1 M (pH 7.5) Potassium nitrate, 0.1 M NADH, 2 mM
Reagents for Nitrite Assay Sulfanilamide, 1% (w/v) in 1.5 to 3.0 N HC1 N-(1-Naphthyl)ethylenediamine dihydrochloride, 0.02% (w/v) Procedure (NADH). The assay mixture contains in micromoles, potassium phosphate, 50; KNO3, 20; NADH, 0.8; and enzyme, 0.1 to 0.2 11 C. M. Sluiters-Scholten, Planta 123, 175 (1975). 12 A. P. Gandhi, S. K. Sawhney, and M. S. Naik, Biochem. Biophys. Res. Commun. 55, 291 (1973). 13 W. D. Loomis and J. Battaile, Phytochemistry 5, 423 (1966). 14 F. D. Snell and C. T. Snell, "Colorimetric Methods of Analysis." Van Nostrand Reinhold, Princeton, New Jersey (1949). 1~ H. J. Evans and A. Nason, Plant Physiol. 28, 233 (1953). 16 L. E. Schrader, G. L. Ritenour, G. L. Eilrich, and R. H. Hageman, Plant Physiol. 43, 930 (1%8).
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ml of crude extract (equivalent to 0.2 to 1.0 mg of trichloroacetic acid precipitable protein) in a final volume of 2.0 ml. The optimum pH is usually 7.5; however, with crude extracts from soybean leaves the optimum pH is 6.5. The reaction can be initiated either by addition of NADH or enzyme. A zero time and minus NADH reaction mixture are used for controls. After incubation at 30° for 15 min the reaction is terminated by rapid addition of 1.0 ml of sulfanilamide reagent followed by 1.0 ml of the N-(1-naphthyl)ethylenediamine dihydrochloride reagent. The color is allowed to develop for 30 min prior to reading at 540 nm. The enzyme reaction rate is linear over a 30-min period. The same procedure is used when NADH oxidation is followed spectrophotometrically at 340 nm. The linear initial reaction rate is used to compute the activity. Activity is expressed as /zmoles of NOR- produced per minute per gram fresh or dry tissue weight or per milligram of protein. The protein is precipitated by 5% trichloroacetic acid from the extract which is determined by any of several standard protein methods. Details for preparation of other reductants and other details are as previously published. 2 Coupled Assays. In addition to the coupled enzyme reactions previously reported, 2 nitrate reduction can be coupled via NADH generated by the addition of malate (20 mM) and NAD (2 mM) as a substitute for NADH (0.4 mM) in the standard assay. 17 With crude extracts of corn leaves containing high levels of malate dehydrogenase, the activity of the coupled reaction was 80% of the standard (NADH) assay. Precautions. Residual NADH at the end of the assay, and unknown factors present in the crude extract, interfere with NO2- color development. While NADH can be oxidized enzymatically,TM this does not eliminate the extract factors. Postassay treatments have been found that minimize these problems, r The reaction is stopped by addition of zinc acetate (50/zmoles/ml reaction mixture) or by placing the reaction tubes in boiling water. After clarification by centrifugation (1000 g for 10 min), aliquots of the cooled (25°) supernatant liquid are treated with phenazine methosulfate (15 nmoles/ml reaction mixture) to oxidize the residual NADH. After 20 min, color is developed in the aliquot as previously described. The enzyme from most plant species is very unstable even at 0°. Therefore, minimum time should elapse between extraction and assay even when stabilizers of enzyme activity have been added. ,7 C. A. N e y r a and R. H. H a g e m a n , Plant Physiol. 58, 726 (1976). 18 D. Spencer, Aust. J. Biol. Sci. 12, 181 (1959).
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Assay Method (in Vivo) Principle. Sections of plant tissue, containing adequate amounts of endogenous nitrate and substrates for NADH generation, produce and accumulate NO2- wJaen placed under dark anaerobic conditions. The assay as originally developed TM has been adapted to provide a more accurate estimate of nitrate reduction in situ 2° or to study nitrate assimilation in tissue from species having high inhibitor content that prevents extraction of active enzymeY ,21 Rates of activity obtained with the in vivo assay are lower (50 to 80%) than in vitro rates, however, they are correlated. 20,22 Procedure. z°'23 Sections or discs of fresh tissue (0.2 to 0.5 gm) are placed in a test tube or small flask containing the incubation medium. The medium is composed of 0.1 M potassium phosphate, pH 7.5, 1% (v/ v) 1-propanol or 0.04% (v/v) Neutronyx 600 (Onyx Chemical Co., Jersey City, New Jersey) and 0.03 to 0.05 M KNO3. Stainless steel screens may be placed in each tube to hold the tissue below the solution surface. The samples are then evacuated (5 mm Hg) in a vacuum desiccator. Nitrogen gas is bled into the desiccator and the process repeated. The tubes may be stoppered before transferring to a shaking water bath and incubated (0.25 to 4 hr) at 30° in the dark. Aliquots can be removed from the same reaction tube at timed intervals to establish linearity of NO2- production. Usually there is a 5- to 10-min lag period prior to establishment of a linear rate. The reaction is usually linear for 60 to 90 min. Normally the reaction is stopped by transferring the tubes to a boiling water bath for 2 min to release NO2- remaining in the tissue. After cooling, aliquots are removed for colorimetric determination of NO2-. The difference between the amount of NO2- produced at 30 min minus the production at 5 or 10 min, with a comparable set of materials, is used to calculate the rate of NO2- production (equated with NO3- reduction). Comments. The amount of nitrite produced slightly underestimates nitrate reduction, as some nitrite disappears under dark anaerobic conditions. 21 However, very little nitrite appears to be assimilated to aminoN under these conditionsY 4 Anaerobiosis is essential, 24 and other systems (continuous N2 bubbling) can be devised to establish the anaerobic condition. 19 E. G. Mulder, R. Borma, and W. L. VanVeen, Plant Soil Sci. 10, 335 (1959). 20 N. Brunetti and R. H. Hageman, Plant Physiol. 58, 583 (1976). 21 j. W. Radin, Plant Physiol. 51,332 (1973). 22 j. G. Streeter and M. E. Bosler, Plant Physiol. 49, 448 (1972). 23 j. C. Nicholas, J. E, Harper, and R. H. Hageman, Plant Physiol. 58, 731 (1976). 24 D. T. Canvin and C, A. Atkins, Planta !16, 207 (1974).
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With tissue deficient in sugars or organic acids (prolonged exposure of plants to dark prior to sampling), marked stimulation of nitrate reduction can be obtained by addition of sugar phosphates or 3-phosphoglyceraldehyde (6 mM) to the incubation medium. 25 With dark pretreated soybean leaf tissue, the addition of 10 and 100 mM glucose to the incubation medium increased activity 4.5- to 5.2-fold, respectively, over the control, z6 Addition of 10 mM pyruvate, citrate, succinate, or malate was without effect, while activity was increased (two- to threefold) when added at 100 mM. Purification Nitrate reductase extracted from higher plants is extremely unstable and difficult to purify to homogenity with reasonable recoveries. These difficulties are illustrated by a comparison of specific activities of 0.02, 0.2, 0.7, 0.7 and 2.5/~moles NO3- reduced/min/mg protein for enzymes from soybeans, ~ maize, 27 marrow, In maize, 16 and spinach, ~8 respectively with the specific activity of 83.1 for Chlorella,29 when conventional purification techniques were used. By combining conventional purification procedures with the recently developed blue-dextran agarose affinity chromatography, 3° a homogenous nitrate reductase (specific activity 24.1 and 7% recovery) was prepared from spinach leaves, al This procedure is detailed as follows. Extraction. Freshly harvested and chilled spinach leaves (1 kg) were homogenized in 5 mM Tris-HC1, pH 7.5, and 1 mM EDTA (2 ml buffer/ gm leaf). The macerate was filtered through two layers of cheesecloth and centrifuged at 60,000 g for 20 min. The supernatant was used as the crude extract. Purification. Nucleic acids were removed by adding streptomycin (5 mg/gm leaf) to the crude extract. After 1 hr, the precipitate was removed (60,000 g for 20 min) and discarded. Solid (NH,)2SO4 (ARISTAR grade, British Drug House, Poole, England) was added to the supernatant to attain 50% saturation. After 20 min the precipitated protein was collected (60,000 g for 20 min) and saved. The protein was redissolved in 0.1 M 25 L. Klepper, D. Flesher, and R. H. Hageman, Plant Physiol. 48, 580 (1971). 26 j. C. Nicholas, J. E. Harper, and R. H. Hageman, Plant Physiol. 58, 736 (1976). zr j. Roustan, M. Neuberger, and A. Fourey, Physiol. Veg. 12, 527 (1974). 28 B. A. Notton and E. J. Hewitt, Plant Cell Physiol. 12, 465 (1971). 29 L. P. Solomonson, G. H. Lorimer, R. L. Hall, R. Borchers, and J. L. Bailey, J. Biol. Chem. 250, 4120 (1975). zo L. P. Solomonson, Plant Physiol. 56, 853 (1975). 3~ B. A. Notton, R. J. Fido, and E. J. Hewitt, Plant Sci. Lett. 8, 165 (1977).
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phosphate, pH 7.5, and desalted on a Sephadex G-25 (Pharmacia, Uppsala, Sweden) column equilibrated with the same buffer. Fractions containing the enzyme were transferred to a column of hydroxylapatite (BioRad Laboratories, Richmond, California) equilibrated with 0.05 M phosphate pH 7.5. The column was then washed with buffer until absorbance (280 nm) of the eluate was less than 0.01. The enzyme was eluted from the column using the washing buffer fortified with 5% (NH4)2SO4. The appropriate fractions were combined and the enzyme precipitated by adding solid (NH4)2SO4 to attain 50% saturation. After 20 min the precipitate was collected (60,000 g for 20 min) and redissolved in 0.1 M phosphate, (pH 7.5) containing 0.1 M KCI. This solution was transferred to a BioGel A (0.5 m, 100 to 200 mesh, Bio-Rad Laboratories) column. Column equilibration and elution were with the dissolving solution. Fractions containing the enzyme were bulked and reprecipitated with (NH4)2SO4 at 50% saturation. The precipitate was collected by centrifugation and dissolved in 0.08 M phosphate, pH 7.5, and the solution transferred to a blue-dextran Sepharose column equilibrated with the same buffer. (The blue-dextran Sepharose was made as previously described.32). The column was washed with the same buffer until the eluate gave negligible absorbance at 280 nm. Nitrate reductase was eluted by applying a 0 to 3 M KCI gradient (in 0.08 M phosphate, pH 7.5). Fractions containing the enzyme were bulked and concentrated with (NH4)zSO4 (50% saturation) and dissolved in a small amount of 0.08 M phosphate, pH 7.5. The BioGel A and blue-dextran Sepharose steps increased specific activity 4- and 22-fold, but lost 42 and 30% of the total activity, respectively. Unlike the enzyme from Chlorella, 3° nitrate reductase from higher plants cannot be eluted, effectively, from the blue-dextran Sepharose with NADH. However, this problem was circumvented by the use of an alternative affinity medium, blue-Sepharose (Cibacron blue F3GA, Polysciences, Inc., Warrington, Pennsylvania, coupled to Sepharose CL4B, Pharmica, Uppsala, Sweden, as described33). The blue-Sepharose was added to the clarified homogenate and stirred for 1 hr at 4 ° to bind the nitrate reductase. 34 (The homogenate was prepared by grinding 100 gm squash cotyledons in 100 ml of 100 mM phosphate, pH 7.5, 1 mM EDTA, 1 mM cysteine, and 20 gm polyvinylpyrrolidone.) The Sepharose was then collected by filtration and washed with the grinding medium prior 3z L. D. Ryan and C. S. Vestling, Arch. Biochem. Biophys. 160, 179 (1974). 33 H. J. Brhme, G. Kopperschlager, J. Schulz, and E. Hofmann, J. Chromatogr. 69, 209 (1972). 34 W. H. Campbell and J. Smarrelli, Jr., Plant Physiol. 57, 637 (1976).
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to transfer to a column (2.5 cm diameter). The enzyme was eluted with the extraction buffer containing 0.1 mM NADH, and the appropriate fractions combined. The enzyme was concentrated by precipitation with 45% saturated (NH4)2SO4 and desalted on a Sephadex G-25 column with 100 mM phosphate, pH 7.5, 5 mM EDTA and 1 mM cysteine. Activity was 2 and 12/xmoles NO3- reduced/min/mg protein for the bulked sample and maximum fraction, respectively. Recovery in the pooled sample was 30% of the initial activity. This procedure was not as effective with the enzyme extracted from corn leaves and the bulk of the activity was not eluted with NADH, but was washed off with 300 mM KNO3. Characteristics The properties of nitrate reductases from higher plants have been recently reviewed. 3~,36 Molecular Weight. Values reported are 160,000 (maize2), 197,000 (spinach36), 230,000 (barleyar), 220,000 (NADPH-enzyme, soybeanS), 330,000 (NADH-enzyme, soybean 5) and 500,000 (spinach3S). For the spinach enzyme 36 the following properties were found: Stokes radius, 60 A; S value, 8.1; diffusion constant, d20.w of 3.4 × 10r; frictional ratio, f / f o of 1.55; asymmetry, rl/r2 of 10; isoelectric point, pH 5.0; and an E0.1~ of 1.62. 280 Substrate Affinity. A Michaelis constant (Kin) for nitrate of 0.2 mM has been reported for several plant species. 2 Km's for nitrate of 0.11 and 4.5 mM were reported for the NADH and NAD(P)H enzymes from soybeans .5.39 Pyridine Nucleotide Specificity. The enzyme extracted from most higher plants preferentially utilizes NADH as the electron donor; however, the specificity is not absolute. The Km for NADH is approximately 2.5 /zM. 4° A phosphatase, that converts NADPH to NADH and is not completely removed by conventional purification procedures, may account for some apparent NADPH activity. In maize, leaves, but not in maize scutella, the NADPH activity in crude or partially purified extracts
35 E. J. Hewitt, D. P. Hucklesby, and B. A. Notton, in "Plant Biochemistry" (J. Bonner and J. E. Varner, eds.), 3rd ed., p. 633. Academic Press, New York, 1976. 38 B. A. Notton and E. J. Hewitt, in Nitrogen Assimilation of Plants (E. J. Hewitt and C. V. Cutting, eds.) p. 708. Academic Press, New York, 1979. 37 j. L. Wray and P. Filner, Biochem. 3. 119, 715 (1970). 3s A. M. Relimpio, P. J. Aparicio, A., Panaque, and M. Losada, FEBS Lett. 7,226 (1971). 39 W. H. Campbell, Plant Sci. Lett. 7, 239 (1976). 4o L. Beevers, D. Flesher, and R. H. Hageman, Biochim. Biophys. Acta 89, 453 (1964).
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can be attributed to phosphatase activity. 3,41 Two enzymes, NAD(P)H (EC 1.6.6.2) and NADH (EC 1.6.6.1) have been separated from soybean leaves. The Km's for NADPH and NADH for the NAD(P)H enzyme were 1.5 and 39 IzM and for the NADH enzyme 200 and 8 izM, respectively. 5 In the crude extracts the total NADPH activity accounted for approximately 10% of the total. 3a Flavin Requirement. FAD is a constituent of the enzyme. 15,36Addition of FAD often but not always stimulates the in vitro reduction of nitrate. It has been proposed that this may reflect the strength of attachment of the indigenous FAD and be species dependent. Reduced FAD or FMN can also serve as electron donors for nitrate reduction, however, there is disagreement over the apparent Km value (0.02 to 1.0 mM) and whether the flavins are physiological or incidental electron donors. 16'42 Supplemental FAD protects nitrate reductase from heat inactivation, 43 and stabilizes the enzyme during sucrose density centrifugation or dilution. 36 Involvement of Metals. Molybdenum has been shown 44 to be a constituent of nitrate reductase of spinach by the use of aaMo. A b-type cytochrome with absorption bands at 560, 528, and 425 nm has been shown to be a functional constituent of the spinach enzyme. 31 Tungsten can be incorporated into the enzyme as a replacement for molybdenum; however, the analog will not reduce nitrate. 45 Sulfhydryl. The protective effect of sulfhydryl compounds on nitrate reductase during extraction and purification is well established, z The sulfhydryl group on the enzyme from higher plants is considered to be involved in binding of the pyridine nucleotide. ~6 Phosphate Requirement. Nitrate reductase extracted from higher plant tissue with a media devoid of phosphate is stimulated by the addition of phosphate to the assay medium. 2 The reason for the stimulation is not known; however, it has been proposed that it may complex with the molybdenum of the enzyme thereby facilitating its reduction. Sequence of Electron Transfer. The sequence of electron transfer of higher plant nitrate reductase is thought to be similar if not identical with that of Neurospora. 2,35,36
41 G. N. Wells and R. H. Hageman, Plant Physiol. 54, 136 (1974). 42 A. Panaque, F. F. Del Campo, and M. L. Losada, Biochim. Biophys. Acta 109, 79 (1965). 43 W. G. Zumft, P. J. Aparicio, A. Panaque, and M. L. Losada, FEBS Lett. 9, 157 (1970). 44 B. A. Notton and E. J. Hewitt, Biochern. Biophys. Res. Commun. 44, 702 (1971). ~ A. R. J. Eaglesham and E. J. Hewitt, Biochern. J. 122, 18 (1971).
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Alternate electron donors (reduced FMN, benzyl or methyl viologen)
l
NAD(P)H---~FAD--~Cyt b--*Mo protein--~NO~-
Alternate electron acceptors (Cyt c, DCPIP)
The enzyme exhibits two functions: as a dehydrogenase in the reduction of cytochrome c and as a reductase for nitrate. 36 It is not known whether the cytochrome b is associated with the dehydrogenase reaction or not. Subunits and Proposed Structure. The holoenzyme (8.1 S fraction from sucrose density centrifugation) is composed of a pyridine nucleotide-cytochrome c reductase (3.7 S) bonded noncovalently to a molybdenum-containing subunit (minimum effective molecular weight of 30,000). se TheSe functional units can be physically separated and recombined. It has been proposed 3n that the holoenzyme has four subunits having cytochrome c reductase activity and one molybdenum-containing complex. Kinetics. The reaction is essentially irreversible to the right. With purified spinach enzyme, an ordered sequential ping-pong mechanism was reported. 45 Work with nitrate reductase purified from squash and corn suggested standard ping-pong kinetics (initial velocity plots) or a two-sided ping-pong mechanism (product inhibition plots). 34 Inhibitors. Nitrate reductase is sensitive to inhibition by PCMB (1 ~ M to 1 mM) especially when pyridine nucleotides rather than FMNH2 is the electron donor. Sulfhydryl compounds protect the enzyme from PCMB inhibition. 2,1~ The enzyme is sensitive to reagents that react with metals (8-hydroxyquinoline and o-phenanthroaline) and cyanide and azide are especially effective. Atabrin inhibits marrow nitrate reductase at 5 mM. z Neither carbon monoxide nor fluoride (1 mM) inhibit soybean nitrate reductase. 15 Nitrate reductase from Chlorella inactivated by NADH and HCN can be reactivated by oxidation with ferricyanide.46 This system has been proposed as a mechanism for regulation of nitrate reductase in situ. 4r Purified nitrate reductase from corn leaves can be inactivated by addition of NADH and HCN in vitro; however, the inactivation can be measured only when the assays are done under suboptimal 46 L. P. Solomonson, Biochim. Biophys. Acta 334, 287 (1974). 47 L. P. Solomonson and A. M. Spehar, Nature (London) 265, 373 (1977).
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(0.2 mM) levels of nitrate. The presence of the normal level (10 mM) nitrate in the assay system apparently causes a rapid activation of the enzyme. 48,49 Cellular Location. Although there is disagreement over the cellular location of nitrate reductase, the bulk of the evidence strongly suggests that it is located in the cytoplasm. 35 In plants with C4-type photosynthesis, nitrate reductase is confined primarily to the mesophyll cells. 35 Induction. Nitrate reductase is inducible by its substrate, nitrate, z and the increased activity is due to de novo synthesis. 5° Nitrite, the product, will induce nitrate reductase in rice seedlings 6 and beans51; however, it is not an effective inducer with many plant species. 52"53Chloramphenicol and certain other organic nitro compounds will preferentially induce NAD(P)H nitrate reductase in rice seedlings. 6 In contrast, nitrate or nitrite will preferentially induce the NADH enzyme. 6 The two types of enzymes were separated by means of blue dextran Sepharose chromatography. 54 Stability. Nitrate reductase is unstable both in vivo and in vitro. In excised whole maize seedlings at 30°, the half-life of the enzyme was estimated to be 4 hr. With intact maize plants placed in the dark at 25 ° the half-life was 12 to 14 hr. z This decrease in activity follows first-order kinetics, in Using triple labeling techniques, it was shown that synthesis and degradation of the enzyme were concurrent. 5° The stability in vitro and in vivo varies greatly with plant age, species, and tissue. 4a D. L o u s s a e r t and R. H. H a g e m a n , Plant Physiol. 57S, 539 (1976). 49 R. H. H a g e m a n , in Nitrogen Assimilation of Plants, (E. J. Hewitt and C. V. Cutting, eds.) p. 708. A c a d e m i c Press, N e w York, 1979. 5o H. R. Zielke and P. Filner, J. Biol. Chem. 246, 1772 (1971), 51 S. H. Lips, D. Kaplan, and N. Roth-Bejerano, Eur. J. Biochem. 37, 589 (1973). 52 M. M. R. K. Afridi, and E. J. Hewitt, J. Exp. Bot. 16, 628 (1965). L. Beevers, L. E. Schrader, D. Flesher, and R. H. H a g e m a n , Plant Physiol. 40, 691 (1%5). 54 T. C. Shen, E. A. F u n k h o u s e r , and M. G. Guerrero, Plant Physiol. 58, 292 (1976).
[25] P 7 0 0 D e t e c t i o n By T. V. MARSHO and B. KOK
The pigment designated P7001 is the photoreactive center of photosystem I and as such is an obligate component in the overall photochemical transport of electrons from an appropriate donor (H20 or artificial 1 B. Kok, Biochim. Biophys. Acta 22, 399 (1956). METHODS IN ENZYMOLOGY, VOL. 69
Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181969-8