Partial purification and properties of the assimilatory nitrate reductase of the food yeast Candida utilis.
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.
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
D008670:Metals; D013438:Sulfhydryl Compounds; D012402:Rotenone; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D009565:Nitrate Reductases; D050901:Nitrate Reductase