Effects of Univalent Cations on the Inductive Formation of Nitrate Reductase

An investigation has been made to determine the effectiveness of univalent cations as cofactors for the inductive synthesis of nitrate reductase. In these experiments K(+) functions more effectively as the univalent cation activator than other univalent cations. Substitution of Rb(+) for K(+) resulted in enzyme formation at a rate of about one-half of that obtained with K(+). Sodium, Li(+), or NH(4) (+) either failed to stimulate or completely inhibited the inductive formation of the enzyme. When no univalent cations were present in the induction medium, enzyme formation was delayed for an initial 3-hour period in contrast to the normal one-hour delay in enzyme formation where adequate K(+) was present in the induction medium.During the period of inductive formation of nitrate reductase the activity of pyruvic kinase, a constitutive enzyme, was assayed under conditions where adequate K(+) was present. Results indicate that the presence of the different univalent cations in the induction medium had no striking effect on the activity of this enzyme during the induction period.     

Comparative stability of ammonium- and nitrate-induced nitrate reductase activity in maize leaves

Ammonium sulfate (5 mM) had no effect on nitrate reductase activity during a 3 hr dark incubation, but the enzyme was increased 2.5-fold during a subsequent 24 hr incubation of the maize leaves in light. The enzyme activity induced by ammonium ion declined at a slower rate under non-inducing conditions than that induced by nitrate. The decline in ammonium stimulated enzyme activity in the dark was also slower than that with nitrate. Further. cycloheximide accelerated the dark inactivation of the ammonium-enzyme while it had no effect on the nitrate-enzyme. The experiments demonstrate that increase in nitrate reductase activity by ammonium ion is different from the action of nitrate action.     

Nitrate reductase activity in heme-deficient mutants of Staphylococcus aureus.

Mutants H-14 and H-18 of Staphylococcus aureus require hemin for growth on glycerol and other nonfermentable substrates. H-14 also responds to delta-aminolevulinate. Heme-deficient cells grown in the presence of nitrate do not have lactate-nitrate reductase activity but gain this activity when incubated with hemin in buffer and glucose. Lactate-nitrate reductase activity is also restored to the membrane fraction from such cells by incubation with hemin and dithiothreitol; addition of adenosine 5'-triphosphate has no effect upon the restoration. Cells grown with nitrate in the absence of hemin have two to five times more reduced benzyl viologen-nitrate reductase activity than do those grown with hemin. The activity increases throughout the growth period in the absence of hemin, but with hemin present enzyme formation ceases before the end of growth. There was no evidence of enzyme destruction. The distribution of nitrate reductase activity between membrane and cytoplasm was similar in cells grown with and without hemin; 70 to 90% was in the cytoplasm. It is concluded that heme-deficient staphylococci form apo-cytochrome b, which readily combines in vitro with its prosthetic group to restore normal function. The avaliability of the heme prosthetic group influences the formation of nitrate reductase.     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.     

Monovalent cation-induced conformational change in glucose oxidase leading to stabilization of the enzyme

Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).     

Inhibition of nitrate reductase induction by canavanine in maize roots

The induction of nitrate reductase activity in maize root tips was inhibited by canavanine and the inhibition increased with increasing concentration of canavanine between 0·1 and 1 mM. Addition of canavanine to the induced enzyme had little effect on the disappearance of the enzyme when nitrate was removed, and it is likely that the canavanine reduces the activity of the nitrate reductase by inhibiting its synthesis rather than by accelerating its breakdown.     

Nitrate Reductase Activity of Wheat Seedlings during Exposure to and Recovery from Water Stress and Salinity

Nitrate reductase activity was inhibited as a result of reduced soil moisture potentials or application of NaCI to nutrient solutions. The decrease in enzyme activity of wheat seedlings exposed to salinity, was found 24 hours after exposure to stress. The effect of stress on nitrate reductase was found in cell-free extracts as well as in riro in assays of intact leaf sections. A recovery in enzyme activity was found after irrigation or after removal of seedlings from salinity. While relative water content of the leaves was restored within 3 hours after removal of stress, full recovery of enzyme activity occurred only after 24 hours. Cycloheximide and chloramphenicol suppressed the activity of nitrate reductase in non-stressed seedlings, but had no effect on the activity of plants exposed to salinity. However, during removal of stress, cycloheximide prevented completely the recovery of nitrate reductase, while chloramphenicol did not interfere with the recovery of the inhibited enzyme activity. It is concluded that a fraction of nitrate reductase may be located in the cytoplasm and lost activity during stress, probably due to inhibited protein synthesis. Another fraction which may be associated with chloroplasts, was inhibited by stress due to conformational changes or partial denaturation.     

Differential effect of tungsten on the development of endogenous and nitrate-induced nitrate reductase activities in soybean leaves

The effect of tungsten on the development of endogenous and nitrate-induced NADH- and FMNH₂-linked nitrate reductase activities in primary leaves of 10-day-old soybean (Glycine max [L.] Merr.) seedlings was studied. The seedlings were grown with or without exogenous nitrate. High levels of endogenous nitrate reductase activities developed in leaves of seedlings grown without nitrate. However, no endogenous nitrite reductase activity was detected in such seedlings. The FMNH₂-linked nitrate reductase activity was about 40% of NADH-linked activity. Tungsten had little or no effect on the development of endogenous NADH- and FMNH₂-linked nitrate reductase activities, respectively. By contrast, in nitrate-grown seedlings, tungsten only inhibited the nitrate-induced portion of NADH-linked nitrate reductase activity, whereas the FMNH₂-linked activity was inhibited completely. Tungsten had no effect on the development of nitrate-induced nitrite reductase activity. The complete inhibition of FMNH₂-linked nitrate reductase activity by tungsten in nitrate-grown plants was apparently an artifact caused by the reduction of nitrite by nitrite reductase in the assay system. The results suggest that in soybean leaves either the endogenous nitrate reductase does not require molybdenum or the molybdenum present in the seed is preferentially utilized by the enzyme complex as compared to nitrate-induced nitrate reductase.     

Phytochrome regulation of nitrate reductase in wheat

In excised wheat leaves, the activity of nitrate reductase was enhanced by a brief pulse of red light and this increase was reversed by far-red light irradiation. Even under continuous far-red light, nitrate reductase activity increased by 258% after 18 h. When leaves were kept in distilled water during exposure to red light and then transferred to potassium nitrate, there was no difference in endogenous nitrate concentration. The nitrate reductase activity was the same whether leaves were floated in potassium nitrate or in distilled water during irradiation. Partial to complete inhibition of enzyme activity was observed when leaves were incubated in actinomycin-D and cycloheximide respectively, following 4 h of red light irradiation.In vitro irradiation of extract had no significant effect on nitrate reductase activity     

NaCl Effects on Root Plasma Membrane ATPase of Salt Tolerant Wheat

Wheat seedlings of a salt tolerant cultivar were grown hydroponically in presence and absence of 100 mM NaCl. Roots were harvested, and the plasma membrane (PM) fraction was purified. PM ATPase required a divalent cations for activity (Mg > Mn > Ca > Co > Zn > Ni > Cu), and it was further stimulated by monovalent cations (K > Rb > NH₄ > Li > choline > Cs). The pH optima were 6.0 and 5.6 in absence and presence of 25 mM KCl, respectively. The enzyme was sensitive to vanadate and DCCD but insensitive to azide, oligomycine and nitrate. The enzyme displayed a high preference for ATP but was also able to hydrolyze other nucleotide tri- and diphosphates. The enzyme activity showed a simple Michaelis-Menten kinetics for the substrate Mg²⁺-ATP in both control and salt exposed roots. The polypeptide patterns of control and salt stressed PM fractions, detected by SDS-PAGE, were very similar. NaCl substantially reduced the PM ATPase specific activity, whereas it had little effect on the apparent K_m for Mg²⁺-ATP. Since the root PM ATPase of salt sensitive and resistant genotypes responded similarly to salinity stress, it seems unlikely that the mechanism of salt tolerance in wheat is primarily based on differences in PM ATPase characteristics.     

Variation in the nitrate reductase of rice seedlings

Summary Nitrate reductase was induced in rice seedlings by nitrate and by chloramphenicol. During the induction period the different enzyme activities associated with nitrate reductase increased to different degrees. Nitrate induced high NADH-nitrate reductase activity and a great increase in the NADH-cytochrome c reductase activity which was associated with the nitrate reductase in a sucrose gradient. Chloramphenicol induced a nitrate reductase which had higher activity with NADPH than NADH. Chloramphenicol also induced a marked increase in NADPH-cytochrome c reductase activity as well as in NADH-cytochrome c reductase activity. Both activities were associated with the nitrate reductase in a sucrose gradient.After partial purification by sucrose gradient sedimentation or by starch gel electrophoresis, the nitrate reductase of rice induced by nitrate and chloramphenicol showed the same preference in pyridine nucleotide cofactors as was shown by the crude enzyme extracts.     

In Vitro Studies of Nitrate Reductase Activity in Cotton Cotyledons: Effects of Dowex 1-Cl and BSA

Germinating cotton (Gossypium hirsutum L. cv. Deltapine 16) cotyledons developed two peaks of in vitro nitrate reductase activity; the first was stable in vitro and appeared 24 hours after imbibition; and the second, which was extremely labile in vitro, began to develop after the seedlings had emerged and developed chlorophyll. Nitrite reductase activity peaked only after the seedlings had emerged. Dowex 1-Cl (10%, w/v) and bovine serum albumin (3%, w/v) significantly improved the activity of extracted enzyme; greater improvement occurred as expansion of the cotyledons progressed. The major effect of bovine serum albumin on nitrate reductase activity in cotyledon extracts appeared to be that of making the extracted enzyme more active rather than increasing the amount of nitrate reductase extracted or improving the stability of the extracted enzyme.     

Effects of asparagine,Irradiation and NADH on the nitrate reductase inWolffia microscopica

Nitrate concentration required for maximal extractable level of nitrate reductase (NR) inWolffia varies with the conditions prior to the nitrate treatment. Maximal enzyme activity is obtained at 2 mM nitrate concentration with asparagine grown plants and at 15 mM with nitrate grown. Both the level of enzyme activity and nitrate uptake by the tissue are increased by irradiation. The radiant energy induced increase in enzyme activity is not due to photosynthetic activity alone. An effect of radiant energy at the membrane level is sug gested. The extracted enzyme, which is labile, is protected and activated by NADH at 0 °C.     

Alterations in the Cytoplasmic Membrane Proteins of Various Chlorate-Resistant Mutants of Escherichia coli

Chlorate-resistant mutants corresponding to each known genetic locus (chlA, chlB, chlC, chlD, chlE) were isolated from Escherichia coli K-12. All these mutants showed decreased amounts of membrane-bound nitrate reductase, cytochrome b, and formic dehydrogenase, but all had normal succinic dehydrogenase activity. Proteins from the cytoplasmic membranes of these mutants were compared to those of the wild type-on polyacrylamide gels. The addition of nitrate to wild-type anaerobic cultures caused increased formation of three membrane proteins. These same proteins, along with one other, were missing in varying patterns in mutants altered at the different genetic loci. One of the missing proteins was found to be the enzyme nitrate reductase, although this protein was present in some mutants lacking nitrate reductase activity. None of the others has been identified.     

Effects of Cysteine on Cellular Activity of Dissimilatory Nitrate Reductase in Pseudomonas denitrificans

Dissimilatory nitrate reductase activity in Pseudomonas denitrificans grown under denitrifying conditions was markedly decreased upon incubation of the cells with Cys or GSH. The activity of the enzyme was also low in the membrane fraction isolated from the Cys-treated cells, indicating that the decrease in enzyme activity was due to the inactivation of the enzyme. Cys appeared to exert its effect, at least in part, after conversion to GSH as judged by the stimulation of the Cys effect by concomitant addition of Glu, although even a trace of GSH could not be detected in the cells presumably owing to the high activity of GSH consumption. The inactivating effect of Cys on the enzyme was found to compete with the activating effect of Na⁺ that had been reported previously, suggesting the presence of a natural mechanism controlling nitrite accumulation in the environment.     

Isolation of Capsicum annuum leaf nitrate reductase and characterization of the effect of adenine nucleotides and NADH on its activity

Abstract. In the preliminary purification of Capsicum leaf nitrate reductase (EC 1.6.6.1), treatment of the crude extract on Sephadex G-25 was necessary to prevent a gelling of the extract and sedimentation of the enzyme. Its K_m values for NADH and nitrate were estimated to be 9.3 and 105mmol m⁻³ ADP and ATP gave hyperbolic competitive inhibition, with respect to NADH, while the inhibition by AMP was linear competitive. K_i values calculated were: ADP and ATP approximately lmol m⁻³ and AMP 2.3 mol m⁻³. Inhibition by ADP was not altered by reduced glutathione.
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity.     

Purification and further characterization of the second nitrate reductase of Escherichia coli K12

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.     

Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora.

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 muM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH2-nitrate reductase of Neurospora suggests some differences in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 mu M) markedly enhanced the action of NAD(P)H on the maize enzyme; Cyanide at a higher level (6 mu M) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no effect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.     

Effect of salicylic acid on nitrite reductase and glutamate dehydrogenase activities in maize roots

The effects of 0.01 to 5 m M salicyclic acid on the increase in nitrite reductase or glutamate dehydrogenase activities in maize roots by nitrate or ammonium respectively, were examined. Nitrite reductase activity was inhibited by the highest concentration of the acid. The activity of NADH-glutamate dehydrogenase was stimulated slightly (but consistently) by the lowest concentration and was inhibited by higher concentrations. Total protein content was also inhibited at high concentrations. When the crude enzyme extract was stored at 25°C in light, the glutamate dehydrogenase activity in the control decreased after 4 h of incubation. Low concentrations of the acid had no effect on this decrease but higher concentration accelerated the process. The divalent cations Caz²⁺, Mn²⁺, Mg²⁺ and Zn²⁺ protected against loss of enzyme activity during storage, both in the absence and presence of the acid. The inhibitory effect of 5 m M salicylic acid on glutamate dehydrogenase activity is apparent due to interference with the activity of the enzyme rather than with its synthesis.