Monoclonal antibodies to a higher-plant nitrate reductase: Differential inhibition of enzyme activities

A set of monoclonal antibodies has been raised against NADH-nitrate reductase (NR; EC 1.6.6.1) from spinach (Spinacea oleracea L.) leaves. Antibodies were screened by enzyme-linked immunosorbent assay and by their ability to inhibit various activities of the enzyme. The six monoclonals selected (AFRC MAC 74 to 79) are all gamma globulins; four (MAC 74 to 77) inhibit all terminal donating activities (NADH-NR; flavin mononucleotide, reduced form (FMNH₂)-NR; and methyl viologen, reduced form (MV)-NR) and two (MAC 78 and 79) inhibit the acceptor activities (NADH-NR, and NADH-cytochrome c reductase). MAC 74 to 77 inhibit the NADH-NR activity of crude extracts of a variety of species (mono- and dicotyledoneae) while MAC 78 and 79 are effective against spinach and marrow, but not oil-seed rape, cucumber, oats, wheat and barley.Abbreviations Cyt c Rase cytochrome c reductase - ELISA enzyme-linked immunosorbent assay - FAD(H₂) flavin adenine dinucleotide (reduced form) - FMN(H₂) flavin mononucleotide (reduced form) - McAb monoclonal antibody - MV methyl viologen reduced form - NR nitrate reductase     

Pyridine nucleotide specificity and other properties of purified nitrate reductase from Chlorella variegata

Nitrate reductase (NR) (EC 1.6.6.2) from Chlorella variegata 211/10d has been purified by blue sepharose affinity chromatography. The enzyme can utilise NADH or NADPH for nitrate reduction with apparent K _m values of 11.5 M and 14.5 M, respectively. Apparent K _m values for nitrate are 0.13 mM (NADH-NR) and 0.14 mM (NADPH-NR). The diaphorase activity of the enzyme is inhibited strongly by parachloromercuribenzoic acid; NADH or NADPH protects the enzyme against this inhibition. NR proper activity of the enzyme is partially inactive after extraction and may be activated after the addition of ferricyanide. The addition of NAD(P)H and cyanide causes a reversible inactivation of the NR proper activity although preincubation with either NADH or NADH and ADP has no significant effect.Abbreviations NR Nitrate reductase - FAD Flavin-adenine dinucleotide - FMN Riboflavin 5-phosphate - p-CMB para-Chloromercuribenzoic - BV Benzyl viologen     

Nitrate reductase is not accumulated in chloroplast-ribosome-deficient mutants of higher plants

The activities of nitrite reductase (EC 1.7.7.1) are 60–70% of wild-type activity in pigment-deficient leaves of the chloroplast-ribosomedeficient mutants albostrians (Hordeum vulgare) and iojap (Zea mays). The activity and apoprotein of nitrate reductase (EC 1.6.6.1.) are lacking in the barley mutant. Only very low activities of nitrate reductase can be extracted from leaves of the maize mutant. The molybdenum cofactor of nitrate reductase and xanthine dehydrogenase (EC 1.2.3.2) is present in maize and barley mutant plants. However, it is not inducible by nitrate in pigment-deficient leaves of albostrians. From these results we conclude: (i) Nitrite reductase (a chloroplast enzyme) is synthesized in the cytoplasm and does not need the presence of nitrate reductase for the induction and maintenance if its activity. (ii) The loss or low activity of nitrate reductase is a consequence of the inability of the mutants to accumulate the apoprotein of this enzyme. (iii) The chloroplasts influence the accumulation (i.e. most probably the synthesis) of the nonchloroplast enzyme, nitrate reductase. The accumulation of nitrate reductase needs a chloroplast factor which is not provided by mutant plastids blocked at an early stage of their development.Abbreviations CRM cross-reacting material - Mo-co molybdenum cofactor - NiR nitrite reductase - NR nitrate reductase     

Studies on the regulation of assimilatory nitrate reductase in Ankistrodesmus braunii

In the green alga Ankistrodesmus braunii, all the activities associated with the nitrate reductase complex (i.e., NAD(P)H-nitrate reductase, NAD(P)H-cytochrome c reductase and FMNH₂-or MVH-nitrate reductase) are nutritionally repressed by ammonia or methylamine. Besides, ammonia or methylamine promote in vivo the reversible inactivation of nitrate reductase, but not of NAD(P)H-cytochrome c reductase. Subsequent removal of the inactivating agent from the medium causes reactivation of the inactive enzyme. Menadione has a striking stimulation on the in vivo reactivation of the inactive enzyme. The nitrate reductase activities, but not the diaphorase activity, can be inactivated in vitro by preincubating a partially purified enzyme preparation with NADH or NADPH. ADP, in the presence of Mg²⁺, presents a cooperative effect with NADH in the in vitro inactivation of nitrate reductase. This effect appears to be maximum at a concentration of ADP equimolecular with that of NADH.Abbreviations ADP Adenosine-5-diphosphate - AMP Adenosine-5-monophosphate - ATP Adenosine-5-triphosphate - FAD Flavin adenine dinucleotide - FMNH₂ Flavin adenine mononucleotide, reduced form - GDP Guanosine-5-diphosphate - MVH Methyl viologen, reduced form - NADH Nicotinamide adenine dinucleotide, reduced form - NADPH Nicotinamide adenine dinucleotide phosphate, reduced form     

Biochemical characterization of nitrate and nitrite reduction in the wild-type and a nitrate reductase mutant of soybean

Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild-type (cv. Williams) and mutant ( nr₁ ) soybean [ Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr₁ soybean mutant (formerly LNR-2), had decreased NADH-NR, FMNH₂-NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH₂-NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH₂ as a reductant source. Nitrite reductase activity was normal in the nr₁ plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate reductases from other plants, presence of xanthine dehydrogenase activity and loss of cytochrome c reductase activity indicated that the apoprotein and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea-grown wild-type plants had 1) greater ability to use FMNH₂ as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO₃⁻ and HCO₃⁻, compared with inducible NR from NO₃⁻-grown nr₁ plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity.     

Characterization of nitrate reductase-deficient barley mutants

Summary Ten nitrate reductase-deficient Hordeum vulgare mutants were characterized for NADH and FMNH₂ nitrate reductase (NR), cytochrome C reductase (CR) and nitrite reductase (NiR) activities. The mutants sort into four major groups. Group I represented by mutants Az 12, Az 23, Az 29 and Az 30 have low Nr and Cr activities. Group II represented by mutants Az 13, Az 31, Az 33 and Az 34 have low NR activities but intermediate CR activities. Group III represented by mutant Az 28 has low NR activity, but above normal CR activity. Group IV represented by Az 32 has low NADH-NR, low CR, but above normal FMNH₂-NR activity. All ten mutants have elevated NiR activities. None of the ten mutants were constitutive for nitrite reductase activity. Only Az 34 showed a definite high temperature sensitivity when the NADH nitrate reductase activity was compared in the 12 to 26° C range. The mutants Az 12, Az 13, Az 23, Az 28, Az 29, Az 30, Az 31, Az 32 and Az 33 are allelic and were assigned the locus designation nar1. Mutant Az 34 represents a different genetic locus designated nar2. The nar1 gene is codominant and the nar2 gene is recessive.Scientific Paper No. 5463. College of Agriculture Research Center, Washington State University, Pullman, Project Nos. 0233 and 0430. Supported in part by National Science Foundation Grants PCM 78-07649 and PCM 78-16025     

Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase

Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M₂ seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH₂-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c₁NR), constitutive NADH:NR (c₂NR), and nitrate-inducible NR (iNR).     

Action of Corn and Rice-inactivating Proteins on a Purified Nitrate Reductase from Chlorella vulgaris

When nitrate reductase (NR) purified from Chlorella was incubated with NR-inactivating proteins purified from corn roots and rice cell suspension cultures or with trypsin there was a loss in NADH-NR and NADH cytochrome c reductase (NADH-CR) activities with time whereas the reduced methylviologen NR (MV-NR) remained active. When NADH-NR and NADH-CR activities were inactivated completely by the incubation with corn protein, the major protein band obtained by polyacrylamide gel electrophoresis shifted from an R_F value of 0.12 to an R_F of 0.25 and reduced MV-NR activity moved to the new position on the gel. When NADH-NR and NADH-CR activities were partially inactivated by the corn protein, NADH-NR activity was detected in an intermediate position (R_F value of 0.18). Incubation with trypsin also caused a change in the NR protein migration pattern (R_F value of 0.20). This protein band also had reduced MV-NR activity. Thus, the corn inactivator degrades NR in a fashion similar to but not identical with trypsin. The incubation of NR with rice inactivating protein resulted in a loss of NADH-NR but had no effect on the migration of NR protein or on the reduced MV-NR activity or mobility suggesting that the rice protein binds to Chlorella NR.     

Occurrence of xanthine dehydrogenase in Chlamydomonas reinhardii: A common cofactor shared by xanthine dehydrogenase and nitrate reductase

Wild-type Chlamydomonas reinhardii cells have xanthine dehydrogenase activity when grown with nitrate, nitrite, urea, or amino acid media. Mutant strains 102, 104, and 307 of Chlamydomonas, lacking both xanthine dehydrogenase and nitrate reductase activities, were incapable of restoring the NADPH-nitrate reductase activity of the mutant nit-1 of Neurospora crassa, whereas wild type cells and mutants 203 and 305 had xanthine dehydrogenase and were able to reconstitute the nitrate reductase activity of nit-1 of Neurospora. Therefore, it is concluded that in Chlamydomonas a common cofactor is shared by xanthine dehydrogenase and nitrate reductase. Xanthine dehydrogenase is repressed by ammonia and seems to be inessential for growth of Chlamydomonas.     

Biochemical and genetical characterization of nitrate reductase deficient mutants of Petunia

Four NR^– lines were selected by their resistance to 100 mM chlorate from X-ray irradiated protoplasts of haploid Petunia hybrida var. Mitchell. The four cell lines were characterized by the presence of xanthine dehydrogenase activity and by complementation tests via protoplast fusion. One mutant (line 1) was classified as defective in the NR apoprotein (tentatively, nia-type) and the other three (lines 2, 3, 4) in the molybdenum cofactor (tentatively, cnx-type). Some NR activity (15 %) could be restored by adding unphysiologically high concentrations of molybdate to the culture medium in two of the cnx-lines (lines 3 and 4). The third cnx-line (line 2) had no NR activity. A complementation analysis via protoplast fusion confirmed that the mutants comprised 3 non-allelic groups. From these results it can be concluded that these NR^– mutants are recessive and that two of the cnx-mutants (lines 3, 4) are allelic.Abbreviations MS Murashige and Skoog medium (Murashige and Skoog 1962) - MG Müller and Grafe medium (Müller and Grafe 1978), containing amino acids - V47 protoplast medium (Binding 1974) - MS-413-medium (McCormack and Hanson 1980) - IAA indoleacetic acid - BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - AA amino acids - XDH xanthine dehydrogenase - PEG polyethylene glycol - NR nitrate reductase     

Metabolic activators of spinach leaf nitrate reductase: Effects on enzymatic activity and dephosphorylation by endogenous phosphatases

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L.) leaves was inactivated in the dark and reactivated by light in vivo. When extracted from dark leaves, NR activity was lower and more strongly inhibited by Mg²⁺ relative to the enzyme extracted from leaves harvested in the light. When dark extracts were desalted at pH 6.5 and preincubated at 25° C prior to assay, enzyme activity (assayed either in the presence or absence of Mg²⁺) remained essentially constant, i.e. there was no spontaneous reactivation in vitro. However, addition of certain metabolites resulted in a time- and concentration-dependent activation of NR in vitro. Effective activators included inorganic phosphate (Pi), 5-AMP, and certain of its derivatives such as FAD and pyridine nucleotides (both oxidized and reduced forms). All of the activators increased NR activity as assayed in the absence of Mg²⁺, whereas some activators (e.g. Pi, 5-AMP and FAD) also reduced Mg²⁺ inhibition. The reduction of Mg²⁺ inhibition was also time-dependent and was almost completely prevented by a combination of okadaic acid plus KF, suggesting the involvement of dephosphorylation catalyzed by endogenous phosphatase(s). In contrast, the activation of NR (assayed minus Mg²⁺) was relatively insensitive to phosphatase inhibitors, indicating a different mechanism was involved. Compounds that were not effective activators of NR included sulfate, ribose-5-phosphate, adenosine 5-monosulfate, coenzyme A, ADP and ATP. We postulate that NR can exist in at least two states that differ in enzymatic activity. The activators appear to interact with the NR molecule at a site distinct from the NADH active site, and induce a slow conformational change (hysteresis) that increases NR activity (assayed in the absence of Mg²⁺). Possibly as a result of the conformational change caused by certain activators, the regulatory phospho-seryl groups are more readily dephosphorylated by endogenous phosphatases, thereby reducing sensitivity to Mg²⁺ inhibition. Preliminary results suggest that light/dark transitions in vivo may alter the distribution of NR molecules between the low- and high-activity forms.Abbreviations AP₅A P¹, P⁵-di(adenosine-5)pentaphosphate - DTT dithiothreitol - Mops 3-(N-morpholino)propanesulfonic acid - NR NADH:nitrate reductase - NRA nitrate reductase activity Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. This work was also supported in part by grants from the U.S. Department of Energy (Grant DE-AIO5-91 ER 20031) and USDA-NRI (Grant 93-373-5-9231). The authors thank Dr. W.M. Kaiser (Lehrstuhl Botanik I der Universität, Würzburg, Germany) for discussions and Dr. C. Lillo (Rogaland University Center, Stavanger, Norway) for sharing results prior to publication.     

Fumarate reductase of Clostridium formicoaceticum

When Clostridium formicoaceticum was grown on fumarate or l-malate crude cell extracts contained a high fumarate reductase activity. Using reduced methyl viologen as electron donor the specific activity amounted to 2–3.5 U per mg of protein. Reduced benzyl viologen, FMNH₂ and NADH could also serve as electron donors but the specific activities were much lower. The NADH-dependent activity was strictly membrane-bound and rather labile. Its specific activity did not exceed 0.08 U per mg of particle protein. Fumarate reductase activity was also found in cells of C. formicoaceticum grown on fructose, gluconate, glutamate and some other substrates.The methyl viologen-dependent fumarate reductase activity could almost completely be measured with intact cells whereas only about 25% of the cytoplasmic acetate kinase activity was detected with cell suspensions. The preparation of spheroplasts from cells of C. formicoaceticum in 20 mM HEPES-KOH buffer containing 0.6 M sucrose and 1 mM dithioerythritol resulted in the specific release of 88% of the fumarate reductase activity into the spheroplast medium. Only small amounts of the cytoplasmic proteins malic enzyme and acetate kinase were released during this procedure. These results indicate a peripheral location of the fumarate reductase of C. formicoaceticum on the membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - O.D optical density - DTE dithioerythritol     

The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H-Nitrate Reductase Enzyme from Soybean

A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO_(x)) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO_(x) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent K_m for nitrite of 0.49 millimolar, and an apparent K_m for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO_(x) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH₂) and reduced methyl viologen (MV) can serve as electron donors for NO_(x) evolution activity. The NADPH-, FMNH₂-, and reduced MV-NO_(x) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymer-curibenzoate, whereas, the FMNH₂ and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO_(x). NADPH eluted both NR and NO_(x) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean.     

Selenium requirement for active xanthine dehydrogenase from Clostridium acidiurici and Clostridium cylindrosporum

The xanthine dehydrogenase of Clostridium acidiurici and C. cylindrosporum was assayed with methyl viologen as acceptor. In C. acidiurici the basal activity level was about 0.3 mol/min x mg of protein. Cells grown on uric acid in the presence of 10^-7 M selenite showed a 14-fold increase in xanthine dehydrogenase activity, which decreased with higher selenite concentrations (10^-5 M). The supplementation with 10^-7 M molybdate or tungstate was without effect. High concentrations of tungstate decreased the xanthine dehydrogenase if selenite was also present. In comparison, high concentrations of molybdate affected only a small decrease in activity level at the optimal concentration for selenite and relieved to some degree the inhibitory effect of 10^-5 M selenite. With hypoxanthine and xanthine as substrates for growth again only the addition of selenite was necessary to show a similar increase in xanthine dehydrogenase activity. C. acidiurici could be grown in a mineral medium. Both xanthine dehydrogenase and formate dehydrogenase exhibited the highest level of activity if selenite and tungstate were present in that medium.In C. cylindrosporum the basal activity level of xanthine dehydrogenase was about 0.95 mol/min x mg of protein. The addition of 10^-7 M selenite to the growth medium increased the activity level about 3-fold, but the highest level (3.7 U/mg) was reached if 10^-7 M molybdate was also added. The presence of tungstate resulted in a decreased enzyme activity.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Summary The wild-type line and 14 nitrate reductase-deficient mutant cell lines of Nicotiana tabacum were tested for the presence of nitrate reductase partial activities, and for nitrite reductase and xanthine dehydrogenase activity. Data characterizing the electron donor specificity of nitrate reductase (EC 1.6.6.1., NADH:nitrate oxidoreductase) and nitrite reductase (EC 1.7.7.1., ferredoxin:nitrite oxidoreductase) of the wild-type line are presented. Three lines (designated cnx) simultaneously lack NADH-, FADH₂-, red. benzyl viologen-nitrate reductase, and xanthine dehydrogenase activities, but retain the nitrate reductase-associated NADH-cytochrome c reductase activity. These mutants are, therefore, interpreted to be impaired in gene functions essential for the synthesis of an active molybdenum-containing cofactor. For cnx-68 and cnx-101, the sedimentation coefficient of the defective nitrate reductase molecules does not differ from that of the wild-type enzyme (7.6S). In 11 lines (designated nia) xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities, including NADH-cytochrome c reductase. However, one line (nia-95) was found to possess a partially active nitrate reductase molecule, retaining its FADH₂- and red. benzyl viologen nitrate reductase activity. It is likely that nia-95 is a mutation in the structural gene for the apoprotein. Both, the nia and cnx mutant lines exhibit nitrite reductase activity, being either nitrate-inducible or constitutive. Evidence is presented that, in Nicotiana tabacum, nitrate, without being reduced to nitrite, is an inducer of the nitrate assimilation pathway.     

Interaction between electron transport at the plasma membrane and nitrate uptake by maize (Zea mays L.) roots

Summary In the present study nitrate uptake by maize (Zea mays L.) roots was investigated in the presence or absence of ferricyanide (hexacyanoferrate III) or dicumarol. Nitrate uptake caused an alkalization of the medium. Nitrate uptake of intact maize seedlings was inhibited by ferricyanide while the effect of dicumarol was not very pronounced. Nitrite was not detected in the incubation medium, neither with dicumarol-treated nor with control plants after application of 100 M nitrate to the incubation solution. In a second set of experiments interactions between nitrate and ferricyanide were investigated in vivo and in vitro. Nitrate (1 or 3 mM) did neither influence ferricyanide reductase activity of intact maize roots nor NADH-ferricyanide oxidoreductase activity of isolated plasma membranes. Nitrate reductase activity of plasma-membrane-enriched fractions was slightly stimulated by 25 M dicumarol but was not altered by 100 M dicumarol, while NADH-ferricyanide oxidoreductase activity was inhibited in the presence of dicumarol. These data suggest that plasma-membrane-bound standard-ferricyanide reductase and nitrate reductase activities of maize roots may be different. A possible regulation of nitrate uptake by plasmalemma redox activity, as proposed by other groups, is discussed.Abbreviations ADH alcohol dehydrogenase - HCF III hexacyanoferrate III (ferricyanide) - ME NADP-dependent malic enzyme - NR nitrate reductase - PM plasma membrane - PM NR nitrate reductase copurifying with plasma membranes     

Nitrate reductase-deficient mutants in barley : immunoelectrophoretic characterization

Nitrate reductase-deficient barley (Hordeum vulgare L.) mutants were assayed for the presence of a functional molybdenum cofactor determined from the activity of the molybdoenzyme, xanthine dehydrogenase, and for nitrate reductase-associated activities. Rocket immunoelectrophoresis was used to detect nitrate reductase cross-reacting material in the mutants. The cross-reacting material levels of the mutants ranged from 8 to 136% of the wild type and were correlated with their nitrate reductase-associated activities, except for nar 1c, which lacked all associated nitrate reductase activities but had 38% of the wild-type cross-reacting material. The cross-reacting material of two nar 1 mutants, as well as nar 2a, Xno 18, Xno 19, and Xno 29, exhibited rocket immunoprecipitates that were similar to the wild-type enzyme indicating structural homology between the mutant and wild-type nitrate reductase proteins. The cross-reacting materials of the seven remaining nar 1 alleles formed rockets only in the presence of purified wild-type nitrate reductase, suggesting structural modifications of the mutant cross-reacting materials. All nar 1 alleles and Xno 29 had xanthine dehydrogenase activity indicating the presence of functional molybdenum cofactors. These results suggest that nar 1 is the structural gene for nitrate reductase. Mutants nar 2a, Xno 18, and Xno 19 lacked xanthine dehydrogenase activity and are considered to be molybdenum cofactor deficient mutants. Cross-reacting material was not detected in uninduced wild-type or mutant extracts, suggesting that nitrate reductase is synthesized de novo in response to nitrate.     

Regulation of nitrate reductase and phosphoenolpyruvate carboxylase activities in barley leaf protoplasts

Barley leaf protoplasts were incubated in light or darkness in the presence of various inhibitors, metabolites or weak acids/bases. Nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPCase) were rapidly extracted from the protoplasts and assayed under sub-optimal conditions, i.e. in the presence of Mg²⁺ and malate, respectively. Under these conditions changes in activities are thought to reflect changes in the phosphorylation states of the enzymes. The NR was activated by illumination to 90% of its maximal activity within 10 min. Photosynthetic electron transport appeared necessary for light activation of NR since activation was inhibited by the photosynthetic electron-transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and, additionally, an electron acceptor (HCO ₃ ^- ) was required. The PEPCase was also activated by light. However, this activation was not prevented by DCMU or lack of HCO ₃ ^- . Loading of protoplasts in the dark with a weak acid resulted in activation of both NR and PEPCase. For NR, full activation was completed within 5 min, whereas for PEPCase a slower, modest activation continued for at least 40 min. Incubation of protoplasts with a weak base also gave activation of PEPCase, but not of NR. On the contrary, base loading counteracted light activation of NR. Since several treatments tested resulted in the modulation of either NR or PEPCase activity, but not both, signal transduction cascades leading to changes in activities appear to be very different for the two enzymes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - DMO 5,5-dimethyl-2,4 oxazolidinedione - NR nitrate reductase - PEPCase Phosphoenolpyruvate carboxylase This work was supported by the Norwegian Research Council by a Grant to C.L: L.H.S. was supported by the Biotechnology and Biological Sciences Research Council.     

Isolation of biochemical mutants using haploid mesophyll protoplasts of Hyoscyamus muticus

A population of 3070 clones derived from N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-treated mesophyll protoplasts of haploid Hyoscyamus muticus was tested for amino-acid auxotrophy without enrichment. One clone (MA-2) was stably and specifically dependent on casein hydrolysate and could be fed also by a number of single amino acids or by other reduced nitrogen sources. MA-2 was found to be chlorate resistant and devoid of in vivo nitrate reductase activity under inductive conditions. Permissive and restrictive growth conditions for MA-2 were investigated more closely and media were found promoting morphogenesis. Selection and testing of clones were complicated by an unspecific growth stimulation of some wild type cultures by amino acids, thiamine and m-inositol.Abbreviations NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - MNNG N-methyl-N-nitro-N-nitrosoguanidine - NR nitrate reductase - CH casein hydrolysate