title: Partial purification and properties of the assimilatory nitrate reductase of the food yeast Candida utilis.
abstract: The addition of nitrate, EDTA and dithiothreitol to the enzyme extraction buffer resulted in improved stability of the assimilatory nitrate reductase activity from the food yeast Candida utilis at both 4 degrees C and -10 degrees C. By incorporating this critical step in the following sequence the yeast NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) was purified approximately 68-fold by protamine sulphate precipitation, calcium gel adsorption, ion exchange chromatography and gel filtration. Both NADPH-nitrate reductase and NADH-nitrate reductase activities remained in constant association and ratio (2:3) during the entire course of purification. The enzyme showed an absolute requirement of NADPH or NADH for its activity. Maximal enzyme activity was obtained with 10-120 micrograms protein in a 10 min assay at 30 degrees C at pH 6.5, with an apparent Michaelis constant of 0.69 mM for nitrate as substrate. The enzyme is a molybdoflavo-protein involving sulphydryl groups, and is highly sensitive to free reducing agents, heavy metal ions and electron-transfer inhibitors. The results also suggested possible involvement of a second metal ion, perhaps iron, which was hypothesized to participate in the electron transfer scheme catalysed by this enzyme.
mesh_terms: D002175:Candida / Q000201:enzymology*; D004579:Electron Transport / Q000187:drug effects; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000302:isolation & purification / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013696:Temperature
chemical_list: D008670:Metals; D013438:Sulfhydryl Compounds; D012402:Rotenone; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D009565:Nitrate Reductases; D050901:Nitrate Reductase