Characterization of the reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase of Aspergillus nidulans.
The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans was purified over 200-fold by use of salt fractionation, gel filtration, and ion-exchange chromatography. The purified enzyme was specific for NADPH and catalyzed reduction of nitrate, cytochrome c from isolated mitochondria of Aspergillus, and mammalian cytochrome c. An S(0.725) (20, w) of 7.8 was derived with sucrose density gradient centrifugation, and a Stokes radius of 6.4 nm was derived by gel filtration on Sephadex G-200. From these values, a molecular weight of 197,000 was computed, assuming v = 0.725 cm(3)/g. The spectral properties of the purified enzyme suggested a flavine component was present but revealed no pattern indicative of a hemoprotein. A cytochrome c, similar to the cytochrome c from isolated mitochondria, was found unassociated with the nitrate reductase after ion-exchange chromatography. No NADPH-nitrate reductase activity was detected in isolated mitochondria. Spectrally discernable reduction of the flavine component of the enzyme at 450 nm was noted after reaction with NADPH. This reduction was inhibited by p-chloromercuribenzoate but not by KCN. The addition of nitrate to NADPH reduced enzyme caused a reoxidation of the flavine component via a reaction which was inhibited by KCN but not by p-chloromercuribenzoate. The half-life of the purified enzyme at 37 C was 20 min for NADPH-nitrate reductase and 35 min for NADPH-cytochrome c reductase.
D000179:Acrylates; D001230:Aspergillus / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003124:Colorimetry; D003486:Cyanides / Q000494:pharmacology; D003580:Cytochromes / Q000378:metabolism; D004586:Electrophoresis; D005415:Flavins / Q000378:metabolism; D005782:Gels; D008928:Mitochondria; D008970:Molecular Weight; D009249:NADP; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D013053:Spectrophotometry; D013395:Sucrose
D000179:Acrylates; D002729:Chloromercuribenzoates; D003486:Cyanides; D003580:Cytochromes; D005415:Flavins; D005782:Gels; D009566:Nitrates; D009249:NADP; D013395:Sucrose; D010088:Oxidoreductases; D011188:Potassium