The Role of Nitrogen Sources in the Regulation of Nitrate Reductase and Nitrite Reductase Levels in the Yeast Hansenula wingei

SUMMARY: The ascomycete yeast Hansenula wingei was able to grow with nitrate, ammonium, glutamate, glutamine or hypoxanthine as sole nitrogen source. Conditions for assay of nitrate reductase (EC 1.6.6.2; NR)and nitritereductase (EC 1.6.6.4; NiR) wereoptimized incell-freeextracts from nitrate-grown cells. NR utilized NADH and NADPH as electron donor and required FAD for maximum activity; NiR was NADPH-specific. Glutamate-grown cells possessed low levels of both enzyme activities and, of the nitrogen sources tested only nitrate was able to induce both enzyme activities above this low basal level of constitutive expression. Ammonium, glutamate and glutamine each prevented nitrate induction of the activities in glutamate-grown cells. Addition of ammonium, glutamate or glutamine to nitrate-grown cells which possessed appreciable levels of NR and NiR caused loss of activity, even if added with nitrate. Loss of both activities under these conditions occurred faster than in cells in which activity loss occurred solely due to nitrate depletion. Both increases and decreases in NR and NiR activity were dependent on protein synthesis.