Light/dark regulation of higher plant nitrate reductase related to hysteresis and calcium/magnesium inhibition

Examination of nitrate reductase (NR, EC 1.6.6.1) activity in crude extracts made from squash leaves before and after a light/dark transition, indicates the existence of two different forms of nitrate reductase; a 'light form' with a pH optimum of 7.8 that is not inhibited by calcium or magnesium, and a 'dark form' with a pH optimum of 7.6 that is strongly inhibited by calcium or magnesium. The same properties also characterise purified NR. The 'light and dark forms' of NR correspond to the two kinetically different forms of purified NR showing (1) linear product formation and (2) delayed product formation, i.e. hysteretic behaviour.     

Identification of a Protein That Inhibits the Phosphorylated Form of Nitrate Reductase from Spinach (Spinacia oleracea) Leaves

The low-activity, phosphorylated form of nitrate reductase (NR) became activated during purification from spinach (Spinacia oleracea) leaves harvested in the dark. This activation resulted from its separation from an approximately 110-kd nitrate reductase inhibitor protein (NIP). Readdition of NIP inactivated the purified phosphorylated NR, but not the active dephosphorylated form of NR, indicating that the inactivation of NR requires its interaction with NIP as well as phosphorylation. Consistent with this hypothesis, NR that had been inactivated in vitro in the presence of NR kinase, ATP-Mg, and NIP could be reactivated either by dephosphorylation with protein phosphatase 2A or by dissociation of NIP from NR.     

Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K_m values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K_m values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

     

Low- and high-activity forms of glutamine synthetase from Rhodospirillum rubrum: sensitivity to feed-back effectors and activation of the low-activity form.

Glutamine synthetase from Rhodospirillum rubrum can be isolated in two forms, with low and high activity, respectively, depending on the concentration of combined nitrogen in the medium before harvest. The two forms have been studied with respect to their dependence on Mn2+ and Mg2+ in both the transferase and the biosynthetic assay. There is no difference in pH optimum between the forms in the biosynthetic assay. In addition the pH-optima for the two cations studied are very close, 7.4 (Mg2+) and 7.2 (Mn2+). It also shows that the activity of the low-activity form is higher than that of the high-activity form in the Mn(2+)-dependent biosynthetic assay. The two forms of Rsp. rubrum glutamine synthetase have also been studied with respect to their sensitivity towards feed-back effectors. In the transferase assay both forms are inhibited to essentially the same degree by alanine, glycine, histidine, AMP, CTP and UTP, CTP being the most effective of the nucleotides and of the amino acids alanine causes the highest inhibition. In the biosynthetic assay these effectors show different degrees of inhibition on the two different forms; the high-activity form being the most sensitive. The results are discussed in relation to properties of glutamine synthetase from Escherichia coli and other phototropic bacteria in which regulation of glutamine synthetase is known to be due to adenylylation. It is also shown that the low-activity form of Rsp. rubrum glutamine synthetase can be activated in crude extracts in a reaction that is inhibited by glutamine.     

Evidence for the role of sulfhydryl groups in a pH-dependent transition of ferredoxin:NADP oxidoreductase.

The pH profile of diaphorase activity of ferredoxin:NADP oxidoreductase suggests a pH-dependent transition between two forms of the enzyme. The apparent pH for this transition is about 8.0. The high-activity form, measured at pH 9.0, is strongly inhibited by increased salt concentration, whereas the low-activity form, measured at pH 7.0, is only weakly inhibited. Likewise, treatment of the enzyme with N-ethylmaleimide (NEM) inhibits the high-activity form more severely than the low-activity form. The data presented suggest that either high salt or NEM treatment converts the enzyme into a low-activity, pH-independent form. The data indicate that an ionized sulfhydryl group may be necessary for the high-activity form. With the exception of the ferredoxin-cytochrome c acceptor system, all other electron acceptors showed a pH dependence similar to that of the diaphorase activity.     

Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes.

1. The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants (s20,w) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 106. 3. In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 Mr subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. The 52 000 Mr subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. p-Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p-chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine.     

In vitro stability of nitrate reductase from wheat leaves: I. Stability of highly purified enzyme and its component activities

NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH₂)-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes—methylviologen-nitrate reductase and NADH-ferricyanide reductase—also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH₂-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH₂-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t_½ [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH₂ do not donate electrons to the same site of the nitrate reductase protein.     

Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.     

Factors affecting in vivo nitrate reductase activity in triticale

Factors influencing in vivo nitrate reductase activity in triticale (×Triticosecale Wittmack) primary leaves were investigated. Nitrate reductase activity was found to be a function of reaction time or tissue weight. In the range of 1–10 mm, the optimum slice width for nitrate reductase activity in triticale was found to be 1–2 mm. The optimum exogenous nitrate concentration is 300 mM. Substantial nitrite production was obtained even when exogenous nitrate was omitted from the assay. Of the five low molecular weight organic solvents tested, n-propanol is the most effective in enhancing enzyme activity. The optimum n-propanol concentration is 1% (v/v). The concentration of phosphate buffer (pH 6) does not affect nitrate reductase activity. Enzyme activity drops significantly below or above pH 6. In our system, nitrite production is enhanced by incubating under nitrogen, instead of air. The highest level of in vivo activity of nitrate reductase was found to be 10–15 cm from tip, which is close to the basal meristem of triticale primary leaves. Younger but physiologically mature leaves have higher nitrate reductase activity than old leaves.     

In Vitro Formation of Nitrate Reductase Using Extracts of the Nitrate Reductase Mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum

In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH-nitrate reductase, FADH(2)-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10(-4) M Na(2)MoO(4) and 10(-2) M NaNO(3) to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase.     

Functional groups involved in the nitrate reductase activity of milk xanthine oxidase

Milk xanthine oxidase possesses the nitrate reductase activity at pH 5.2; the pH optimum of the xanthine oxidase activity for the enzyme lies at 9.6. After removal of FAD and binding of Mo and Fe with a simultaneous measurement at the pH optima of the above activities it was found that only the Mo-containing site is necessary for the nitrate reductase activity. The switch-over of the enzyme from the xanthine oxidase to the nitrate reductase activity is associated with considerable conformational changes of the enzyme molecule.     

Spinach Nitrate Reductase : Effects of Ionic Strength and pH on the Full and Partial Enzyme Activities

Initial velocity studies of immunopurified spinach nitrate reductase have been performed under conditions of controlled ionic strength and pH and in the absence of chloride ions. Increased ionic strength stimulated NADH:ferricyanide reductase and reduced flavin:nitrate reductase activities and inhibited NADH:nitrate reductase, NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities. NADH:dichlorophenolindophenol reductase activity was unaffected by changes in ionic strength. All of the partial activities, expressed in terms of micromole 2 electron transferred per minute per nanomole heme, were faster than the overall full, NADH:nitrate reductase activity indicating that none of the partial activities included the rate limiting step in electron transfer from NADH to nitrate. The pH optimum for NADH:nitrate reductase activity was determined to be 7 while values for the various partial activities ranged from 6.5 to 7.5. Chlorate, bromate, and iodate were determined to be alternate electron acceptors for the reduced enzyme. These results indicate that unlike the enzyme from Chlorella vulgaris, intramolecular electron transfer between reduced heme and Mo is not rate limiting for spinach nitrate reductase.     

Studies on nitrate reductase from Cyanidium caldarium

Summary A nitrate reductase from the thermophilic acidophilic alga, Cyanidium caldarium, was studied. The enzyme utilises the reduced forms of benzyl viologen and flavins as well as both NADPH₂ and NADH₂ as electron donors to reduce nitrate.Heat treatment has an activating effect on the benzyl viologen (FMNH₂, FADH₂) nitrate reductase. At 50°C the activation of the enzyme is complete in about 20 min of exposure, whereas at higher temperatures (until 75°C) it is virtually an instantaneous phenomenon. The observed increase in activity is very low in extracts from potassium nitrate grown cells, whereas it is 5 or more fold in extracts from ammonium sulphate supplied cells. The benzyl viologen nitrate reductase is stable at 60°C and is destroyed at 75°C after 3 min; the NADPH₂ nitrate reductase is destroyed at 60°C. The pH optimum for both activities was found in the range 7.8–8.2.Ammonium nitrate grown cells possess a very low level of nitrate reductase: when they are transferred to a nitrate medium a rapid synthesis of enzyme occurs. By contrast, when cells with fully induced activity are supplied with ammonia, a rapid loss of NADPH₂ and benzyl viologen nitrate reductase occurs; however, activity measured with heated extracts shows that the true level of benzyl viologen nitrate reductase is as high as before ammonium addition. It is suggested that the presence of ammonia causes a rapid inactivation but no degradation of the enzyme.Cycloheximide inhibits the formation of the enzyme; the drug is without effect on the loss of nitrate reductase activity induced by ammonium. The nitrate reductase is reactivated in vivo by the removal of the ammonium, in the absence as well as in the presence of cycloheximide.     

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.     

Reduction of ferric citrate catalyzed by NADH:nitrate reductase

We show that NADH:nitrate reductase from squash cotyledons can catalyze the reduction of ferric citrate. When nitrate reductase was purified to homogeneity using a two-step affinity chromatography procedure, an NADH:Fe(III)-citrate reductase activity copurified with it and had identical electrophoretic mobility to it. The iron reductase activity was optimum near pH 6.3, had an apparent Km for Fe(III)-citrate of 0.02 mM, and was inhibited by monospecific anti-nitrate reductase rabbit sera. Differential inhibition of the enzyme's activities indicated iron and nitrate were reduced at different sites. In addition to its role in nitrogen assimilation, nitrate reductase catalyzes ferric citrate reduction and could have a role in iron assimilation.     

Cloning, expression, and purification of the functional 2,4-dienoyl-CoA reductase from rat liver mitochondria.

The mitochondrial 2,4-dienoyl-CoA reductase (EC 1.3.1.34) is an auxiliary enzyme for the beta-oxidation of unsaturated fatty acids. Import of this enzyme into the mitochondria requires a mitochondrial signal sequence at the amino terminus of the polypeptide chain which is processed/removed once inside the mitochondria. The cDNA of the full-length 2,4-dienoyl-CoA reductase was previously cloned as pRDR181. PCR methodologies were used to subclone the gene encoding the functional 2,4-dienoyl-CoA reductase from pRDR181. The PCR product was inserted into a pET15b expression vector and overexpressed in Escherichia coli. The soluble expressed protein can be separated into high- and low-activity fractions. The low-activity fraction can be converted to the high specific activity form by thermal annealing, suggesting it is a metastable misfolded form of the enzyme. Using ion-exchange and affinity chromatography, the enzyme has been purified to homogeneity and exhibits a single band on Coomassie blue-stained SDS-PAGE. The molecular mass of 32,413 Da determined by electrospray ionization-mass spectrometry indicates that the amino-terminal methionine had been removed. The Michaelis constants for trans-2, trans-4-hexadienoyl-CoA and NADPH were determined to be 0.46 and 2.5 microM, respectively; a turnover number of 2.1 s(-1) was calculated.     

Reduced nicotinamide–adenine dinucleotide–nitrite reductase from Azotobacter chroococcum

1. The assimilatory nitrite reductase of the N(2)-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH(3) with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH(3) is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.