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A novel enzyme that catalyses the oxygen-dependent oxidation of 3-nitropropionic acid (3NPA) to malonate semialdehyde, nitrate, nitrite and H2O2 has been purified from leaf extracts of the horseshoe vetch, Hippocrepis comosa, and named 3NPA oxidase. The enzyme is a flavoprotein with a subunit molecular mass of 36 kDa containing 1 molecule of FMN and exhibits little specificity for all nitroalkanes tested other than 3NPA (apparent Km 620 microM). The maximum enzyme activity in vitro was expressed at pH4.8 and was inhibited strongly by the products nitrate and nitrite. 3NPA oxidase activity was detected in green shoots, which also contain high concentrations of 3NPA, from plants grown with nitrate, ammonium or N2 as sources of nitrogen. Enzyme activity was absent from roots and cell cultures, neither of which accumulate high levels of 3NPA.

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Synthesis of nitrate reductase protein and increases in nitrate reductase activity occurred in cultures of the yeast Candida nitratophila when they were incubated in medium containing ammonium nitrate. Similar treatment with glutamine plus nitrate resulted in little increase in nitrate reductase activity, in cultures grown previously with reduced nitrogen compounds, and decreases in enzyme activity, in cultures adapted to nitrate. Labelling studies conducted in vivo revealed a rapid cessation of de novo nitrate reductase synthesis when glutamine was supplied to nitrate-adapted cultures in the presence of nitrate. Intracellular glutamine concentrations increased rapidly under these conditions and these cultures exhibited high glutamine: glutamate ratios. As nitrate was taken up in the presence of glutamine in these experiments, it is concluded that the glutamine-stimulated inhibition of nitrate reductase synthesis is a consequence of repression and rapid turnover of nitrate reductase mRNA and not inducer (nitrate) exclusion.

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Nitrate reductase from the yeast Candida nitratophila was found to contain one molecule of cytochrome b557 and one atom of molybdenum per subunit. FAD/haem-dependent diaphorase activity (haem domain) was associated with a 40 kDa tryptic fragment of the subunit. The 50 amino-terminal residues of this fragment were determined, and the sequence did not show significant similarity to deduced sequences of other nitrate reductases previously published. Increasing ionic strength in vitro had a stimulatory effect on enzymic activity via stimulation of the molybdenum-dependent terminal nitrate-reducing activity. Stimulation of activity by exogenous protein (bovine serum albumin or casein) also appeared to be an ionic effect. Stimulation of catalytic activity by phosphate was a separate effect.

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Pro-phenol oxidase was purified from the haemocytes of the cockroach Blaberus discoidalis by Blue Sepharose chromatography, hydrophobic-interaction chromatography on a Phenyl-Superose column and, finally, gel filtration on a Superose 6 column. Results suggest that the molecule exists as a polymer of identical 76 kDa monomeric units. The enzyme is a glycoprotein with pI of 5.2 and can be converted by trypsin into phenol oxidase.

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Visible spectra of oxidized and reduced Candida nitratophila assimilatory NAD(P)H:nitrate reductase yielded absorbance maxima of 413 nm and 423 nm, and 525 nm and 555 nm respectively, characteristic of a b5-type cytochrome. E.p.r. spectra of the partially reduced enzyme revealed a single Mo(V) species (g1 = 1.9957, g2 = 1.9664 and g3 = 1.9658) exhibiting superhyperfine coupling to a single proton [A(1H)av. = 1.4 mT]. Oxidation-reduction midpoint potentials (E'0) (25 degrees C, pH 7) for the haem and Mo-pterin prosthetic groups were determined by visible and e.p.r. potentiometric titrations and yielded values of E'0 = -174 mV (n = 1) for the haem and E'0 = -3 mV and E'0 = -27 mV for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples respectively. Comparison of initial rates of the NADH-oxidizing and nitrate-reducing partial activities at various ionic strengths indicated electron transfer from reduced haem to Mo was rate-limiting during turnover. These results suggest a close similarity between Candida nitratophila and Chlorella vulgaris nitrate reductases.

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In the yeast, Candida nitratophila Shifrine et Phaff, the intracellular concentrations of α-amino acids decreased rapidly during N-deprivation, with the ratio of glutamine: glutamate (Gln: Glu) falling from 07 in NH4 + -grown cells, or 0.5 in NO3 - -grown cells, to 0.1 after 1 h. Addition of NH4 + to N-deprived cultures resulted in rapid increases in glutamate, glutamine and alanine with Gln: Glu exceeding 1.5 within 30 min. Recovery of other amino acids, such as arginine, was much slower. Addition of NO3 , resulted in a less rapid increase in the concentration of some intracellular amino acids, including glutamate and glutamine, while levels of arginine continued to fall for 30 min after addition of this N-source. Gln: Glu was slow to rise in NO3 - pulsed cells. Addition of NH4 + to cells growing on NO3- produced little change over the following 2 h other than decreases in arginine and histidine. Carbon deprivation resulted in a rapid decrease in levels of glutamate, glutamine and alanine, but not of aspartate (which this yeast is unable to use as a sole C-source for growth) or arginine. Gln: Glu increased during C-deprivation but fell within 10 min to normal levels on addition of glucose. It is concluded that, in C. nitratophila, Gln:Glu values correlate well with C-N status.

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Ammonium-nitrogen was assimilated rapidly by nitrogen-replete cultures of the nitrate-utilizing yeast, Candida nitratophila as long as a suitable source of carbon was available. These cultures contained high activities of an NADPH-dependent glutamate dehydrogenase with a relatively high affinity for ammonium (Km = 0.27 mM) and high glutamine synthetase activity. Both enzyme activities were apparently derepressed when glutamine-grown cultures were starved of nitrogen or transferred to nitrate medium. Nitrogen-deficient cultures also contained NADH-dependent glutamate synthase activity that was inhibited by azaserine in vitro. Ammonium assimilation in vivo, was inhibited by methionine sulphoximine whilst addition of azaserine resulted in an accumulation of intracellular glutamine and an inhibition of glutamate production. Our results suggest that, in C. nitratophila, there is a potential for ammonium assimilation via both the glutamate dehydrogenase pathway and the glutamine synthetase/glutamate synthase pathway with the latter pathway predominating in nitrogen-deficient cells.

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In vivo labelling and in vitro translation studies were used to study the regulation of the synthesis of nitrate reductase in the yeast Candida nitratophila. These studies showed that synthesis of the enzyme subunit took place when ammonium-grown cells were nitrogen-starved and this was stimulated by subsequent addition of nitrate. Ammonium-grown cultures did not contain mRNA that could be translated into the nitrate reductase subunit in an in vitro system. Nitrate reductase mRNA could be extracted from nitrogen-starved and nitrate cultures. Synthesis of the enzyme is apparently controlled at the level of transcription in this yeast.

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Ammonium (NH 4 (+) ) assimilation by Chlamydomonas reinhardii was inhibited when cultures were incubated with methionine sulphoximine (MSO). Methionine sulphoximine inhibited glutamine synthetase acitvity in vitro in extracts from wild-type (2192) and mutant (CC419) cultures. Mutant cultures were insensitive to MSO inhibition in vivo. Nitrogen-starved, wild-type cultures excreted ammonium when they were incubated with MSO in light or in darkness. Ammonium generation was stimulated by glutamine, inhibited by CO2 and stoichiometrically related to loss of protein. Notrogen replete cultures treated with MSO excreted ammonium in light but little was excreted in darkness. Ammonium excretion in darkness, in the presence of MSO, was enhanced by either a period of nitrogen deprivation or by the addition of acetate. Nitrogen deprivation also diminished the lag before ammonium excretion commenced.

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

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

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Enzymic activities have been measured in cell-free extracts from nitrogen-starved cultures ofAnkistrodesmus braunii. During ten hours of nitrogenstarvation the activities of the enzymes nitrite reductase (E.C.1.6.6.4), glutamic dehydrogenase (E.C.1.4.1.4), glutamine synthetase (E.C.6.3.1.2) and urea amidolyase (E.C.3.5.1.5) were derepressed while the activities of the enzymes malate dehydrogenase (E.C.1.1.1.37) and hexokinase (E.C.2.7.1.1) remained more or less unchanged. In contrast, the photosynthetic capacity of the nitrogen-starved cultures declined rapidly and accompanying this decline were losses in the activities of ribulose diphosphate carboxylase (E.C.4.1.1.39) and triose phosphate-NADP-dehydrogenase (E.C.1.2.1.13).

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Nitrogen-limited chemostat cultures of Chlorella fusca var. vacuolata, when given nitrogen in the inorganic forms of nitrate, nitrite and ammonium divert photo-generated electrons, from CO2 fixation to nitrogen assimilation. Addition of nitrate or nitrite, but not ammonium, stimulates rate of oxygen evolution. All but the most severely nitrogen-deficient culture have increased dark respiration rates after addition of inorganic nitrogen. The nitrite reduction step of nitrogen assimilation is the most light-dependent reaction.

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Nitrate reductase activity was detectable in ammonium-grown cells of Ankistrodesmus braunii after 50 minutes of nitrogen starvation. The rate of formation of nitrate reductase was stimulated by addition of nitrate and inhibited completely by cycloheximide (20 µg/ml). Nitrogen-starved cells assimilated added nitrate or nitrite rapidly and no nitrite or nitrate was detectable in either cells or culture medium from cultures subjected to nitrogen starvation. It is concluded that nitrate is not obligatory for the formation of nitrate reductase.