| NADH nitrate reductase was studied in suspension cultures of the XD cell line of Nicotiana tabacum L. cv. Xanthi. When cultured on suboptimal concentrations of either nitrate or sulfate, the overt nitrate reductase, i.e. that which is assayable without an activation treatment, decreases with decreasing initial concentrations of the suboptimal nutrient.;In vitro, nitrate reductase can be partially inactivated by incubation with NADH or cyanide, but essentially complete inactivation can be achieved by incubation with cyanide plus NADH. Incubation with ferricyanide can reverse this inactivation. In many respects, these characteristics resemble those of the nitrate reductase system in green algae (L. Solomonson, Biochim. Biophys. Acta, 334, 297-308 (1974)) in which cyanide binds reversibly to reduced nitrate reductase thereby inactivating it. However, a kinetic test indicates that inactivation by cyanide cannot fully account for the latent form of the enzyme extractable from XD cells.;Addition of ammonium to nitrate grown XD cells results in a 60% decrease in the overt activity after 11 hours without concomitant formation of latent activity. This is also at variance with the algal system.;Future studies of the "induction" or "repression" of nitrate reductase in plants will have to take this phenomenon into account.;As XD cells growing on nitrate nitrogen progress from exponential phase into stationary phase due to depletion of nitrate, the overt nitrate reductase activity decreases and a latent, i.e. activable, form becomes detectable. The latent enzyme spontaneously activates when stored at -20(DEGREES)C in 0.1 M potassium phosphate, 1 mM ethyleneglycol-bis ((beta)-amino ethyl ether) N,N'-tetraacetic acid, 1 mM L-cysteine, 1 (mu)M flavin adenine dinucleotide, pH 7.5. Incubation with ferricyanide also activates the latent form of the enzyme. Nine-fold activations were observed in extracts from stationary phase cultures. Replacement of overt by latent activity closely parallels depletion of nitrate from the medium. Whereas overt activity per culture decreases, the sum of overt plus latent activities per culture increases.;A latent but ferricyanide-activable form of nitrate reductase was also observed during sulfur starvation of XD cells.;A method for maintaining constant but suboptimal concentrations of nitrate was devised employing the equilibrium between the insoluble salt of nitrate, nitron nitrate, and free nitrate. |
