Purification and Characterization of Nitrate Reductase from the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

Summary Factors affecting the activity of nitrate reductase (E.C.1.7.7.2) from the halotolerant cyanobacterium Aphanothece halophytica were investigated. Cells grown in nitrate-containing medium exhibited higher nitrate reductase activity than cells grown in medium in which nitrate was replaced by glutamine. When ammonium was present in the medium instead of nitrate, the activity of nitrate reductase was virtually non-detectable, albeit with normal cell growth. The enzyme was localized mainly in the cytoplasm. The enzyme was purified 406-fold with a specific activity of 40.6 μmol/min/mg protein. SDS-PAGE revealed a subunit molecular mass of 58 kDa. Gel filtration experiments revealed a native molecular mass of 61 kDa. The K _m value for nitrate was 0.46 mM. Both methyl viologen and ferredoxin could serve as electron donor with K _m values of 4.3 mM and 5.2 μM, respectively. The enzyme was strongly inhibited by sulfhydryl-reactive agents and cyanide. Nitrite, the product of the enzyme reaction, showed little inhibition. Chlorate, the substrate analog, could moderately inhibit the enzyme activity. NaCl up to 200 mM stimulated the activity of the enzyme whereas enzyme inhibition was observed at ≥300 mM NaCl.     

Nitrate reductase activity of two leafy vegetables as affected by nickel and different nitrogen forms

The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO₃ ⁻–N, NH₄ ⁺–N, NH₄NO₃). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in lettuce plants supplied with nitrate nitrogen (NO₃ ⁻–N) or mixed (NH₄NO₃) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants. At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with NO₃ ⁻–N or NH₄NO₃. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the enzyme activity in the roots of NO₃ ⁻-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution contained ammonium nitrogen (NH₄ ⁺–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs, especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated with NH₄ ⁺–N the enzyme activity in roots was even higher than in those supplied with NO₃ ⁻–N.     

Isolation and characterization of nitrate reductase from the halophilic sulfur-oxidizing bacterium <Emphasis Type="Italic">Thioalkalivibrio nitratireducens</Emphasis>

A novel nitrate reductase (NR) was isolated from cell extract of the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens strain ALEN 2 and characterized. This enzyme is a classical nitrate reductase containing molybdopterin cofactor in the active site and at least one iron-sulfur cluster per subunit. Mass spectrometric analysis showed high homology of NR with the catalytic subunit NarG of the membrane nitrate reductase from the moderately halophilic bacterium Halomonas halodenitrificans. In solution, NR exists as a monomer with a molecular weight of 130–140 kDa and as a homotetramer of about 600 kDa. The specific nitrate reductase activity of NR is 12 μmol/min per mg protein, the maximal values being observed within the neutral range of pH. Like other membrane nitrate reductases, NR reduces chlorate and is inhibited by azide and cyanide. It exhibits a higher thermal stability than most mesophilic enzymes.     

Effect of N oxyanions on anaerobic induction of nitrate reductase in subcellular fractions of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. (Lupinus)

Anaerobic induction of nitrate reductase in subcellular fractions of Bradyrhizobium sp. strain USDA 3045 showed fivefold increase of the enzyme activity in spheroplasts, considered as the source of intact-membrane-bound nitrate reductase, within a 3 h time frame after nitrate addition. Such a dynamics was confirmed at the protein level, with antibodies specific to membrane-bound nitrate reductase. Nitrate reductase activity in the periplasm was one order of magnitude lower and significant only at initial 3 h of induction, within a narrow range of nitrate added. Nitrite induced the membrane-bound nitrate reductase at least 70% as effectively as nitrate, as judged from its activity pattern and Western blot analysis. The limited ability of Bradyrhizobium sp. to dissimilate ≥5 mM nitrate is not due to direct inhibition of respiratory nitrate reductase by accumulated nitrite. Moreover, a synergistic induction of membrane-bound nitrate reductase by nitrate and nitrite was indicated due to a twofold higher protein synthesis after simultaneous addition of these N oxyanions than when they were given separately.     

Nitrate reduction associated with respiration in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> 2011 is performed by a membrane-bound molybdoenzyme

The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria–Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV–Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis–Menten mechanism (K _m = 97 ± 11 μM, V = 9.4 ± 0.5 μM min⁻¹, and k _cat = 12.1 ± 0.6 s⁻¹) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.     

The development of nitrate reductase in Chlorella and its repression by ammonium

Summary Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.     

Heterotrophic nitrogen removal by <Emphasis Type="Italic">Providencia rettgeri</Emphasis> strain YL

Providencia rettgeri strain YL was found to be efficient in heterotrophic nitrogen removal under aerobic conditions. Maximum removal of NH₄ ⁺–N occurred under the conditions of pH 7 and supplemented with glucose as the carbon source. Inorganic ions such as Mg²⁺, Mn²⁺, and Zn²⁺ largely influenced the growth and nitrogen removal efficiency. A quantitative detection of nitrogen gas by gas chromatography was conducted to evaluate the nitrogen removal by strain YL. From the nitrogen balance during heterotrophic growth with 180 mg/l of NH₄ ⁺–N, 44.5% of NH₄ ⁺–N was in the form of N₂ and 49.7% was found in biomass, with only a trace amount of either nitrite or nitrate. The utilization of nitrite and nitrate during the ammonium removal process demonstrated that the nitrogen removal pathway by strain YL was heterotrophic nitrification-aerobic denitrification. A further enzyme assay of nitrate reductase and nitrite reductase activity under the aerobic condition confirmed this nitrogen removal pathway.     

Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> S7

Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40°C. K _m values for NO₃ ⁻ (110 μM) and for ClO₃ ⁻ (138 μM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.     

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

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

Lithotrophic growth ofSulfurospirillum deleyianum with sulfide as electron donor coupled to respiratory reduction of nitrate to ammonia

Sulfurospirillum deleyianum grew in batch culture under anoxic conditions with sulfide (up to 5 mM) as electron donor, nitrate as electron acceptor, and acetate as carbon source. Nitrate was reduced to ammonia via nitrite, a quantitatively liberated intermediate. Four moles of sulfide were oxidized to elemental sulfur per mole nitrate converted to ammonia. The molar growth yield per mole sulfide consumed, Y_m, was 1.5 ± 0.2 g mol^–1 for the reduction of nitrate to ammonia. By this type of metabolism, S. deleyianum connected the biogeochemical cycles of sulfur and nitrogen. The sulfur reductase activity in S. deleyianum was inducible, as the activity depended on the presence of sulfide or elemental sulfur during cultivation with nitrate or fumarate as electron acceptor. Hydrogenase activity was always high, indicating that the enzyme is constitutively expressed. The ammonia-forming nitrite reductase was an inducible enzyme, expressed when cells were cultivated with nitrate, nitrite, or elemental sulfur, but repressed after cultivation with fumarate. Received: 13 March 1995 / Accepted: 29 May 1995     

Aluminium effect on nitrate assimilation in cucumber (Cucumis sativus L.) roots

In vivo effect of aluminium on nitrate uptake and reduction by cucumber seedlings was investigated. The high-performance liquid chromatography was used to analyse the rate of nitrate uptake. Low (0.5 mM) concentration of AlCl₃ in the nutrient solution stimulated nitrate uptake during the first 3 h. On the other hand, 6 h exposure of the cucumber seedlings to 1 or 5 mM of AlCl₃ resulted in inhibition of nitrate uptake and at 5 mM concentration of AlCl₃ the efflux of nitrate was observed. Furthermore, the amount of nitrate accumulated in cucumber roots after aluminium treatment was decreased. The noteworthy fact was observed, that at all concentrations of aluminium tested on increase of the nitrate reductase activity. This stimulation was concentration depended, but independent of the source of the enzyme. The activity of both the cytosolic and the plasma membrane bound nitrate reductase activity was enhanced in vivo. On the other hand, AlCl₃ applied in vitro only slighty decreased nitrate reductase activity.     

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.     

Effect of phytohormones and tungsten on light-induced and nitrate-induced nitrate reductase activity of mustard cotyledons

Mustard (Brassica juncea Coss cv. T-59 ‘Varuna’) seedlings pretreated with gibberellic acid (GA) and kinetin (KiN) were grown in light. In vivo nitrate reductase (NR) activity was estimated and effect of tungsten on light-induced and NO ₃ ^su− -induced NR activity was investigated. Different concentrations of GA did not show any effect on induction of light-induced NR; addition of nitrate promoted in vivo NR activity but no concentration effect of GA was evident. Light-induced NR was promoted by KiN and like in GA treatment, addition of nitrate increased NR activity. Addition of Na-tungstate inhibited NO ₃ ⁻ induced NR while light-induced NR was not much affected in both GA and KiN treated seedlings. The two forms of NRs were further characterized by studying the decay kinetics using Na-tungstate. In light-induced NR, tungstate did not affect NR activity up to 11 h, while at later periods, a slight decay was observed. On the other hand, NO ₃ ⁻ -induced NR activity increased up to 4 h and subsequently a rapid fall was observed. It was therefore apparent that light-induced NR had a very low turnover rate as compared to NO ₃ ⁻ -induced NR. These results further support the earlier conclusion that in mustard seedlings two distinct types of NR enzyme exist and that nitrate requirement for NR induction is not absolute.     

Increase in Nitrate Reductase Activity in Bean Leaves by Light Involves a Regulator Protein

Nitrate reductase activity, assayed either in vivo or in vitro was considerably higher in bean (Phaseolus vulgaris L.) leaves from 7-day-old light grown seedlings than those from dark grown, both in the absence as well as presence of nitrate. Cytochrome c reductase activity was however similar in both regimes, while peroxidase was lower in light than in dark. The light stimulated increase in nitrate reductase activity in leaf segments from dark grown seedlings was inhibited by cycloheximide, DNP, chloramphenicol, and sodium tungstate and was unaffected by lincomycin and DCMU. Under similar conditions, the increase in total chlorophyll was inhibited completely by cycloheximide and DNP, partially by chloramphenicol and lincomycin, and was unaffected by tungstate and DCMU. A supply of 1~5 mm reduced glutathione increased enzyme activity in the dark and also to some extent in light. The substrate induction of enzyme activity started after a lag of one hr in light or dark and continued for either 5 hr in the dark or 8 hr in light. Two proteinaceous inhibitors (Factors I and II) of nitrate reductase were isolated by ammonium sulfate precipitation and Sephadex gel filtration. The amount of Factor I was higher in the dark than in light. The amount and activity of Factor II was however, almost equal in light and dark. The inhibition of enzyme activity by these inhibitors increased with their concentration. It is proposed that light increases nitrate reductase activity by decreasing the amount of a nitrate reductase inhibitor.     

Effect of nitrate concentration and UVR on photosynthesis,respiration, nitrate reductase activity,and phenolic compounds in <Emphasis Type="Italic">Ulva rigida</Emphasis> (Chlorophyta)

Seaweeds growing in the intertidal zone are exposed to fluctuating nitrate and ultraviolet radiation (UVR) levels. While it has been shown that elevated UVR levels and the decrease of nitrate concentration can reduce photosynthetic levels in seaweeds, less is known about the combined effect of nitrate levels and UVR on metabolism and photoprotection mechanisms of intertidal species. Consequently, the objective of this study was to evaluate the effect of nitrate concentration and UVR treatments on photosynthesis, respiration, nitrate reductase activity and phenolic compound levels of Ulva rigida (Chlorophyta). There was a two- to threefold increase in maximal gross photosynthesis (GP_max) and respiration rates, as nitrate increased from 0 to 50 μM NO₃⁻. Similarly, nitrate reductase activity increased linearly from low values in algae incubated at 0 μM NO₃ to high values in tissue incubated at 50 μM NO₃⁻. Phenolic compounds in the tissue of U. rigida increased approximately 60% under 50 μM NO₃⁻ relative to those incubated at 0 μM NO₃⁻. Algae exposed to UVR (8 h) showed a significant decrease in the effective quantum yield and respiration, however, no effect was observed in the phenolic compounds levels. Full recovery of effective quantum yield was observed after U. rigida was transferred for 48 h to low PAR. Nitrate reductase also decreased after an 8-h UVR exposure, but no differences were observed among the nitrate treatments. This study shows that high nitrate levels reduced the negative effect of UVR on the effective quantum yield and increased the recovery of key metabolic enzymes. It is possible that the increase of phenolic compounds in the thallus of U. rigida under high nitrate levels provide a photoprotective mechanism when exposed to high UV levels during low tides.     

Extracellular production of <Emphasis Type="Italic">Streptomyces exfoliatus</Emphasis> poly(3-hydroxybutyrate) depolymerase in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. T104: determination of optimal biocatalyst conditions

The phaZ _Sex gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T _m) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine–histidine–carboxylic acid in the active site. High-performance liquid chromatography (HPLC)–mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.     

Nitrate content and nitrate reductase activity in Rumex obtusifolius L.

Summary The aim of this work was to investigate the effect of nitrogen starvation and subsequent fentilization with nitrate or ammonium on nitrate content and nitrate reductase activity of Rumex obtusifolius L. under natural conditions.When plants were transplanted to nitrate-poor media, endogenous nitrate was reduced within a few days. In parallel, nitrage reductase activities dropped to about 25% of the initial values. As a consequence of nitrate fertilization (1; 10 or 100 mmol KNO₃/l substrate), endogenous nitrate content of the plant abruptly increased within one day. In extreme cases, nitrate concentrations of up to 10% of plant dry weight could be observed without being lethal. High external nitrate concentrations caused an inhibition of nitrate reductase within the leaves, while low external concentrations provoked an increase in the enzyme activity of about 450% within one day. Ammonium fertilization (5 mmol (NH₄)₂SO₄/l substrate) also caused an increase in nitrate reductase activity and nitrate content within leaf blades. This observation indicates a rapid nitrification of ammonium in the substrate. When plants were fertilized with ammonium plus nitrate (2.5 mmol (NH₄)₂SO₄+ 5 mmol KNO₃/l substrate), an extremely high and long term increase in nitrate reduction could be observed. Due to an intensive enzymatic nitrate turnover, the nitrate content of leaf blades then remained relatively low. Our observations do not point to an inhibition of nitrate reductase activity in leaves of Rumex obtusifolius by ammonium. Despite temporarily high endogenous nitrate concentrations, Rumex obtusifolius may not be termed as a nitrate storage plant, since the accumulation of nitrate is a short term process only.     

Characteristics of protection by MgADP and MgATP of α3β3γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis

MgADP and MgATP binding to catalytic sites of βY341W-α₃β₃Γ subcomplex of F₁-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α₃β₃Γ sub-complex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 μM and 12 mM, and 46 μM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K _d of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α₃β₃Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F₁-ATPases.     

Purification and characterization of the sunflower seed (Helianthus annuus L.) major aminopeptidase

The sunflower seed (Helianthus annuus L.) major peptidase was purified to molecular homogeneity. It is an 80 kDa enzyme with pI of 4.6 and optimal activity at pH 7.5–8.0 and 45–50°C. It is a thiol-dependent aminopeptidase hydrolyzing peptides in a step-by-step manner as cleaving after the N-terminal amino acid residue of the substrate. It requires substrate acyl parts with a free amino group in either α- or β-position and l-configuration of the adjacent carbon atom. The enzyme prefers amino acid residues with bulky hydrophobic side chains at P₁-position and its catalytic efficacy is affected by the structure of both P₁ and P₁′ parts of the substrate.     

Characterization of the Reversible Inactivation of Ankistrodesmus braunii Nitrate Reductase by Hydroxylamine

The photoreversible nature of the regulation of nitrate reductase is one of the most interesting features of this enzyme. As well as other chemicals, NH₂OH reversibly inactivates the reduced form of nitrate reductase from Ankistrodesmus braunii. From the partial activities of the enzyme, only terminal nitrate reductase is affected by NH₂OH. To demonstrate that the terminal activity was readily inactivted by NH₂OH, the necessary reductants of the terminal part of the enzyme had to be cleared of dithionite since this compound reacts chemically with NH₂OH. Photoreduced flavins and electrochemically reduced methyl viologen sustain very effective inactivation of terminal nitrate reductase activity, even if the enzyme was previously deprived of its NADH-dehydrogenase activity. The early inhibition of nitrate reductase by NH₂OH appears to be competitive versus NO₃⁻. Since NO₃⁻, as well as cyanate, carbamyl phosphate and azide (competitive inhibitors of nitrate reductase versus NO₃⁻), protect the enzyme from NH₂OH inactivation, it is suggested that NH₂OH binds to the nitrate active site. The NH₂OH-inactivated enzyme was photoreactivated in the presence of flavins, although slower than when the enzyme was previously inactivated with CN⁻. NH₂OH and NADH concentrations required for full inactivation of nitrate reductase appear to be low enough to potentially consider this inactivation process of physiological significance.