Molybdenum is absolutely required for the nitrate-reducing activity of the nicotinamide adenine dinucleotide nitrate reductase complex isolated from Chlorella fusca. The The assimilatory nitrate-reducing system of the green alga Chlorella has been thoroughly characterized in recent years (13-16, 21, 23, 24, 28, 29) and has been shown to be similar to that of higher plants (6,9 (6,8,9,17). However, although molybdenum was early identified by Nicholas and Nason (19) as the metal prosthetic group of nitrate reductase from soybean leaves, workers in the field (3, 6, 14) have until recently been unable to find evidence for either its presence or its function as an electron carrier in nitrate reductase preparations purified from a variety of green plants species. Hewitt and coworkers (1, 2, 9) have reported that, in molybdenum-deficient plants grown in the presence of nitrate, molybdenum is required for the synthesis of nitrate reductase and have suggested that it is involved in the induction process and acts not merely as the constituent metal. Heimer et al. (7) and Wray and Filner (27) have recently studied the effect of tungstate as a competitive inhibitor of molybdate on nitrate assimilation in higher plant tissues and have described the structural and functional relationships of enzyme activities induced by nitrate in barley. Their results support the idea that the nitrate reductase complex is formed in the presence of tungstate but is functional only with respect to NADH-diaphorase. By adding radioactive 9Mo (as molybdate) to a culture of Chlorella cells at the moment derepression of the enzymes of the nitrate-reducing system was initiated by removal of ammonia from the medium, we have recently been able to demonstrate that the metal becomes incorporated into nitrate reductase and remains associated with it during purification (3). Furthermore, we showed that after a mild heat treatment of the enzyme exogenous molybdate chemically reduced by hydrosulfite can be used as electron donor for the enzymatic reduction of nitrate (3).The present work is intended to clarify the role played by molybdenum in nitrate reduction by Chlorella. MATERIALS AND METHODSChlorella fusca Shihira et Kraus (pyrenoidosa, strain 211-15 from Pringsheim's culture collection at Gottingen) was grown autotrophically as described previously (29). When ammonia or ammonium nitrate replaced nitrate as the source of nitrogen, the molarity of each compound was also maintained at 8 mM. In the experiments where ammonium ions were involved, the culture media were buffered with 20 mm sodium phosphate, pH 7.5. In the experiments involving the effect of metals, molybdate was omitted from the standard nutrient solution and either sodium molybdate or sodium tungstate was added as indicated. The algae used for inoculation were grown on nitrate media lacking added molybdate in order to produce a low molybdenum inoculum. Growth was determined by meas-294 www.plantphysiol.org on March 22, 2019 -Published by Downloaded from
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Nitrate-grown Azotobacter chroococcum ATCC 4412 cells lack the ability to fix N2. Nitrogenase activity developed after the cells were suspended in a combined nitrogen-free medium and was paralleled by a concomitant decrease in nitrate assimilation capacity. In such treated cells exhibiting transitory nitrate assimilation and N2-fixation capacity, nitrate or nitrite caused a short-term inhibitory effect on nitrogenase activity which ceased once the anion was exhausted from the medium. The glutamate analog L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, prevented inhibition of nitrogenase activity by nitrate or nitrite without affecting the uptake of these antions, which were reduced and stoichiometrically released into the external medium as ammonium. Inhibition of nitrogenase by nitrate (nitrite) did not take place in A. chroococcum MCD1, which is unable to assimilate either. We conclude that the short-term inhibitory effect of nitrate (nitrite) on nitrogenase activity is due to some organic product(s) formed during the assimilation of the ammonium resulting from nitrate (nitrite) reduction. Repression of nitrogenase synthesis by different sources of nitrogen, such as ammonium, nitrate, or urea, has been known for many years (10,21,31,32). Furthermore, an immediate inhibition of nitrogenase activity in whole organisms, but not in extracts, in response to exogenous ammonium ions has been described to occur in photosynthetic bacteria (1, 2, 4, 33) and in some azotobacters (7,9,13,17). Inhibition and reactivation of nitrogenase takes place shortly after the addition or depletion of ammonium, respectively, and it is known as the switch-off/switch-on effect.To explain the short-term effect of ammonium on nitrogen fixation in Azotobacter vinelandii, Laane et al. (17) (7).Mid-logarithmic-growth cells (A560 of ca. 0.5) were used for each experiment. The cells were harvested at room temperatute by centrifugation at 1,000 x g for 10 min, washed with nitrogen-free medium, and resuspended to a cell density of about 70 pLg of cell protein per ml of the same medium.Analytical procedures. For nitrogenase activity and nitrate or nitrite uptake assays, three 20-ml samples of the abovedescribed cell suspension were preincubated at 30°C with shaking (100 strokes min-') in 50-ml sealed conical flasks. Two of the samples were used for nitrogenase assay and the third one was used for nitrate or nitrite uptake assay. At zero time the atmosphere in each flask was made 10% in acetylene. Nitrogenase activity was determined by analyzing the appearance of ethylene in the flask atmosphere. Nitrate and nitrite consumption and ammonium released were determined by estimating the concentration of the corresponding ion in the medium. This estimation was done with samples of the cell suspension after the cells had been rapidly removed by filtration in a Millipore system with glass microfiber Whatman paper.Nitrate reductase activity was determined in situ as follows. A 5-ml sample of cell suspension was centrifuged at 9,000 x g ...
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