Ammonium-grown cultures of Sporobolomyces roseus developed the capacity to assimilate nitrate when they were incubated in nitrate or nitrogen-free medium. Cycloheximide, 6-methyl purine and antimycin A inhibited this process. Nitrate assimilation required aerobic energy metabolism and was inhibited completely by ammonium. The rapid inhibition of nitrate assimilation by ammonium was not the result of an inhibition of nitrate reductase (NR) activity. Nitrite also inhibited nitrate assimilation. NR in cell-free extracts of S . roseus was NADPHspecific and its activity was repressed in cultures containing ammonium and derepressed during nitrogen starvation. Nitrate stimulated the appearance of NR in these cultures. Nitrate assimilation was not limited by apparent (potential) NR activity.
NAD(P)H nitrate reductase (EC 1 . 6 . 6 . 2 ) from the yeast Candida nitratophila has been purified to electrophoretic homogeneity. The concentrated enzyme has a molecular mass of 365 kDa and consists of four subunits each of 95 kDa, but appears to dissociate into dimers after dilution. The absorption spectrum of the homogeneous protein is typical of a b-type cytochrome and its isoelectric point is pH 5.4. The enzyme utilizes both NADH and NADPH but is more active with NADH. Preincubation of pure enzyme with NAD(P)H and cyanide, but not NAD(P)H and ADP, leads to a reversible redox inactivation of the enzyme. Preincubation with NAD(P)H alone activates the enzyme.
S L: M M A R yNitrite uptake in the nitrate-assimilating yeast, Candida nitratophila, was inhibited completely by addition of nitrate or ammonium to cultures at pH SO. Nitrite assimilation m these cultures was also completely dependent on the availability of a suitable carbon source (glucose). In cultures at pH 5'0, neither nitrate nor ammonium inhibited nitrite uptake completely. In these cultures, some nitrite was assimilated in the absence of a carbon source. Carbon-deficient cultures at pH 5'0 were able to reduce nitrite to ammonium but excreted this ammonium without further assimilation. E\idence for the uptake of nitrite by passive diffusion and by active transport via the nitrate uptake system is discussed.
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