Model experiments on nitrite and nitrate in simulated primeval conditions

In experiments on the prebiotic formation of nitric oxides, anoxic mixtures of N₂ and water vapour were sparked in contact with phosphate buffer solutions at various pH values. Nitrite was found in the aqueous phase, and nitrate grew from it, presumably by reaction with H₂O₂. In acid solutions, these anions were reduced and destroyed by Fe²⁺, and the same was true of nitrite in solutions kept at a pH value similar to that of the contemporary ocean (8.2) with HEPES buffer. Nitrate was not destroyed in short-term experiments, but as in sparking nitrate is formed only via nitrite, neither anion could accumulate. In further sparking experiments with alkaline sulphide, both nitrite and nitrate were reduced entirely. It is concluded that it is unlikely that the primeval ocean contained appreciable concentrations of nitrite or nitrate either at the reducing or at the redox-neutral stage.     

The synthesis of some lipid-like structures in simulated primeval earth conditions

Starting from a simple gas mixture rich in methane, suggested as being a model of the primeval atmosphere for a certain time period, we succeeded in synthesizing lipid-like compounds. The hydrophobic nature of the products was demonstrated by IR, NMR, MS, elementary analysis, gel filtration measurements and solubility tests. The hydrophobic products self-assemble in stable, uniformly sized microspheres possessing membrane-like properties. They can selectively retain biologically active compounds. The wall-membrane of the microspheres shows a supra-molecular order.     

Effects of simulated microgravity on arterial nitric oxide synthase and nitrate and nitrite content.

The aim of the present work was to investigate the alterations in nitric oxide synthase (NOS) expression and nitrate and nitrite (NOx) content of different arteries from simulated microgravity rats. Male Wistar rats were randomly assigned to either a control group or simulated microgravity group. For simulating microgravity, animals were subjected to hindlimb unweighting (HU) for 20 days. Different arterial tissues were removed for determination of NOS expression and NOx. Western blotting was used to measure endothelial NOS (eNOS) and inducible NOS (iNOS) protein content. Total concentrations of NOx, stable metabolites of nitric oxide, were determined by the chemiluminescence method. Compared with controls, isolated vessels from simulated microgravity rats showed a significant increase in both eNOS and iNOS expression in carotid arteries and thoracic aorta and a significant decrease in eNOS and iNOS expression of mesenteric arteries. The eNOS and iNOS content of cerebral arteries, as well as that of femoral arteries, showed no differences between the two groups. Concerning NOx, vessels from HU rats showed an increase in cerebral arteries, a decrease in mesenteric arteries, and no change in carotid artery, femoral artery and thoracic aorta. These data indicated that there were differential alterations in NOS expression and NOx of different arteries after hindlimb unweighting. We suggest that these changes might represent both localized adaptations to differential body fluid redistribution and other factors independent of hemodynamic shifts during simulated microgravity.     

The effect of oxygen on nitrate and nitrite assimilation in leaves of Zea mays L. under dark conditions

The assimilation of nitrate under dark-N₂ and dark-O₂ conditions in Zea mays leaf tissue was investigated using colourimetric and ¹⁵N techniques for the determination of organic and inorganic nitrogen. Studies using ¹⁵N indicated that nitrate was assimilated under dark conditions. However, the rate of nitrate assimilation in the dark was only 28% of the rate under non-saturating light conditions. No nitrite accumulated under dark aerobiosis, even though nitrate reduction occurred under these conditions. The pattern of nitrite accumulation in leaf tissue in response to dark-N₂ conditions consisted of three phases: an initial lag phase, followed by a period of rapid nitrite accumulation and finally a phase during which the rate of nitrite accumulation declined. After a 1-h period of dark-anaerobiosis, both nitrate reduction and nitrite accumulation declined considerably. However, when O₂ was supplied, nitrate reduction was stimulated and the accumulated nitrite was rapidly reduced. Anaerobic conditions stimulated nitrate reduction in leaf tissue after a period of dark-aerobic pretreatment.     

Effects of electron donors on nitrate removal by nitrate and nitrite reductases

Effects of artificial electron donors to deliver reducing power on enzymic denitrification were investigated using nitrate reductase and nitrite reductase obtained fromOchrobactrum antropi. The activity of nitrite reductase in the soluble portion was almost the same as that in the precipitated portion of the cell extract. Nitrate removal efficiency was higher with benzyl viologen than with methyl viologen or NADH as an artificial electron donor. The turn-over numbers of nitrate and nitrite reductase were 14.1 and 1.9 μmol of nitrogen reduced/min·mg cell extracts, respectively when benzyl viologen was used as an electron donor.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Blood plasma response and urinary excretion of nitrite and nitrate in milk-fed calves after oral nitrite and nitrate administration

There is marked endogenous production of nitrate in young calves. Here we have studied the contribution of exogenous nitrate and nitrite to plasma concentrations and urinary excretion of nitrite and nitrate in milk-fed calves. In experiment 1, calves were fed 0 or 200 &mgr;mol nitrate or nitrite/kg(0.75) or 100 &mgr;mol nitrite plus 100 &mgr;mol nitrate/kg(0.75) with milk for 3 d. In experiment 2, calves were fed 400 &mgr;mol nitrate or nitrite/kg(0.75) with milk for 1 d. Plasma nitrate rapidly and comparably increased after feeding nitrite, nitrate or nitrite plus nitrate. The rise of plasma nitrate was greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. Plasma nitrate decreased slowly after the 3-d administration of 200 &mgr;mol nitrate or nitrite/kg(0.75) and reached pre-experimental concentrations 4 d later. Urinary nitrate excretions nearly identically increased if nitrate, nitrite or nitrite plus nitrate were administered and excreted amounts were greater if 400 than 200 &mgr;mol nitrate or nitrite/kg(0.75) were fed. After nitrite ingestion plasma nitrite only transiently increased after 2 and 4 h and urinary excretion rates remained unchanged. Plasma nitrate concentration remained unchanged if milk was not supplemented with nitrite or nitrate. Nitrate concentrations were stable for 24 h after addition of nitrite to full blood in vitro, whereas nitrite concentrations decreased within 2 h. In conclusion, plasma nitrate concentrations and urinary nitrate excretions are enhanced dose-dependently by feeding low amounts of nitrate and nitrite, whereas after ingested nitrite only a transient and small rise of plasma nitrite is observed because of rapid conversion to nitrate.     

Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus.

Staphylococcus carnosus reduces nitrate to ammonia in two steps. (i) Nitrate was taken up and reduced to nitrite, and nitrite was subsequently excreted. (ii) After depletion of nitrate, the accumulated nitrite was imported and reduced to ammonia, which again accumulated in the medium. The localization, energy gain, and induction of the nitrate and nitrite reductases in S. carnosus were characterized. Nitrate reductase seems to be a membrane-bound enzyme involved in respiratory energy conservation, whereas nitrite reductase seems to be a cytosolic enzyme involved in NADH reoxidation. Syntheses of both enzymes are inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite, respectively. In whole cells, nitrite reduction is inhibited by nitrate and also by high concentrations of nitrite (> or = 10 mM). Nitrite did not influence nitrate reduction. Two possible mechanisms for the inhibition of nitrite reduction by nitrate that are not mutually exclusive are discussed. (i) Competition for NADH nitrate reductase is expected to oxidize the bulk of the NADH because of its higher specific activity. (ii) The high rate of nitrate reduction could lead to an internal accumulation of nitrite, possibly the result of a less efficient nitrite reduction or export. So far, we have no evidence for the presence of other dissimilatory or assimilatory nitrate or nitrite reductases in S. carnosus.     

The Paracoccus denitrificans NarK‐like nitrate and nitrite transporters—probing nitrate uptake and nitrate/nitrite exchange mechanisms

Nitrate and nitrite transport across biological membranes is often facilitated by protein transporters that are members of the major facilitator superfamily. Paracoccus denitrificans contains an unusual arrangement whereby two of these transporters, NarK1 and NarK2, are fused into a single protein, NarK, which delivers nitrate to the respiratory nitrate reductase and transfers the product, nitrite, to the periplasm. Our complementation studies, using a mutant lacking the nitrate/proton symporter NasA from the assimilatory nitrate reductase pathway, support that NarK1 functions as a nitrate/proton symporter while NarK2 is a nitrate/nitrite antiporter. Through the same experimental system, we find that Escherichia coli NarK and NarU can complement deletions in both narK and nasA in P. denitrificans, suggesting that, while these proteins are most likely nitrate/nitrite antiporters, they can also act in the net uptake of nitrate. Finally, we argue that primary sequence analysis and structural modelling do not readily explain why NasA, NarK1 and NarK2, as well as other transporters from this protein family, have such different functions, ranging from net nitrate uptake to nitrate/nitrite exchange.     

The effect of nitrate and nitrite on oxygen evolution and carbon-dioxide assimilation and the reduction of nitrate and nitrite by intact chloroplasts

Summary Intact chloroplasts isolated from spinach reduced NO₃ ^- and NO₂ ^- in the light without the addition of either co-factors or added enzymes. The maximum rate observed, however, for the reduction of NO₃ ^- was approximately 3Moles hr^-1 mg^-1 (chlorophyll) and for NO₂ ^- 6 Moles hr^-1 mg^-1 (chlorophyll). These rates were consistent with the enzyme content of whole chloroplasts, but much lower than those found in whole leaf extracts.The addition of both NO₃ ^- and NO₂ ^- in low concentrations resulted in transient increases in both O₂ evolution and CO₂ fixation. The increases in oxygen evolution were not consistent in amount and bore no relation to the amount of substrate reduced. Similar transients were observed in a number of experiments when NaCl or NH₄Cl were added.The addition of NO₂ ^- at concentrations of 10^-4 M and above resulted in marked inhibition of both O₂ evolution and CO₂ fixation. NO₂ ^- appears to inhibit by blocking the reduction of NADP. NO₃ ^- at similar concentrations had no such effect.An increase in the soluble amino nitrogen content of the chloroplasts was observed when NO₃ ^- or NO₂ ^- was reduced. There was, however, no increase in the incorporation of ¹⁴C from ¹⁴CO₂ into amino acids under these conditions. Even with the addition of ammonia the amount of ¹⁴C incorporated into the amino acids was not changed from less than 5% of the total ¹⁴C fixed. We conclude that while intact chloroplasts do have the ability to reduce both NO₃ ^- and NO₂ ^- at low rates, they do not synthesize appreciable amounts of amino acid directly, and this fact must be considered when formulating any pathways for nitrogen metabolism during photosynthesis.Supported in part by the National Research Council of Canada.     

A spectrophotometric assay for nitrate in an excess of nitrite.

Miranda et al. have developed a method for simultaneous evaluation of nitrate and nitrite concentrations using reduction of nitrate by vanadium(III) combined with detection by the acidic Griess reaction [K.M. Miranda, M.G. Espey, D.A. Wink, A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite, Nitric Oxide 5 (2001) 62-71]. The sensitivity of the nitrate assay decline if the mixture analyzed contains a large excess of nitrite relative to nitrate, for instance, in the case of oxidation products of nitric oxide (NO) in aerated solutions, or in sweat. By this reason nitrite should be removed before the nitrate assay, if [NO2-]>[NO3-]. Here we lay out an improved method allowing the above limitation to be erased, using sulfamic acid for nitrite removal. We also describe some modifications that enhance the reproducibility of the assay.     

Effect of nitrite and nitrate on chlorophyll fluorescence in green algae

Summary The influence of nitrite and nitrate on chlorophyll fluorescence, a very sensitive indicator for the redox state of the primary acceptor of photosystem II of photosynthesis, was studied in green algae (several species of Chlorella, and Ankistrodesmus braunii). In phosphate solution under an atmosphere of nitrogen (i.e., in the absence of O₂ and CO₂, and without nitrite or nitrate), fluorescence shows a pronounced induction and then rises to a high steady-state level. In the presence of nitrite, however, fluorescence decreases after a rather short induction peak to a much lower steady-state. Nitrate, on the other hand, does not have any influence on either induction or steady-state of fluorescence. These results clearly demonstrate that nitrite reduction in the light is very closely coupled to the photosynthetic electron transport system, whereas nitrate is not reduced photosynthetically in vivo.     

Effects of nitrite, chlorate, and chlorite on nitrate uptake and nitrate reductase activity

Effects of NO₂⁻, ClO₃⁻, and ClO₂⁻ on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO₃⁻ influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m⁻³ NO₂⁻ fully induced NO₃⁻ transport but failed to induce NRA. Similar pretreatments with ClO₃⁻ and ClO₂⁻ induced neither NO₃⁻ transport nor NRA. Net ClO₃⁻ uptake was induced by NO₃⁻ but not by ClO₃⁻ itself, indicating that NO₃⁻ and ClO₃⁻ transport occur via the NO₃⁻ carrier. At the uptake step, NO₂⁻ and ClO₂⁻ strongly inhibited NO₃⁻ influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO₃⁻ proved to be a weak inhibitor of NO₃⁻ influx (K_i = 16 mol m⁻³) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO₃⁻ analogs as screening agents for the isolation of mutants defective in NO₃⁻ transport.     

Induction of nitrate and nitrite reductases in Anabaena cylindrica

Induction of nitrate and nitrite reductases in Anabaena cylindrica(FOGG strain) was investigated. At various stages of algal growthin the presence of nitrate or nitrite, the levels of these enzymeswere determined using cell-free preparations. Nitrate and nitritereductases were induced by the respective substrates. Nitratedid not act either as an inducer or as a repressor of nitritereductase. ¹This work was supported by grant No. 8814 from the Ministryof Education ²Department of Biology, Faculty of Science, Tokyo MetropolitanUniversitySetagaya-ku, Tokyo, Japan (Received June 18, 1970; )