Studies on the enzymatic reduction of C-nitroso compounds. V. Molecular properties of porcine heart C-nitrosoreductase and identity of this enzyme with NAD(P)H dehydrogenase.
NAD(P)H-dependent C-nitrosoreductase of porcine heart cytosol was purified 12,000-fold in the presence of NADH with an overall yield of 2.2%. The purification procedure included ammonium sulfate fractionation, gel filtration with Sephadex G-100, ion-exchange chromatography on DEAE-Sephadex A-50, hydrophobic chromatography on Octyl-Sepharose CL-4B, and gel filtration with Sephadex G-200. The purity of the preparation was approximately 90% and the molecular weight of the enzyme estimated by gel filtration was about 60,000. The purified enzyme was composed of two molecular forms, nitrosoreductases 1 and 2, having isoelectric points of 8.45 and 8.6, respectively. A significant amount of zinc was found in the preparation by X-ray fluorescence analysis. The enzyme as it was prepared was colorless, but, after oxidation with p-nitrosophenol followed by gel filtration in the absence of NADH, it showed the absorption spectrum of a flavoprotein. Spectral data indicated the presence of 1 mol of flavin per mol of the enzyme. The molecular turnover number was calculated to be 10,000 nmol p-nitrosophenol reduced to p-aminophenol per min per nmol enzyme at pH 5.8 and 22 degrees C. The activity was inhibited by p-chloromercuribenzoate by 50% at a concentration of 3 x 10(-5) M. Besides the nitrosoreductase activity, the purified preparation showed NAD(P)H-dependent menadione reductase activity. The activities were both strongly inhibited by dicumarol and markedly activated by serum albumin and by Tween 20. These results indicate the probable identity of this enzyme with soluble NAD(P)H dehydrogenase (quinone) [EC 1.6.99.2].
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003579:Cytochrome Reductases / Q000378:metabolism*; D005420:Flavoproteins / Q000032:analysis; D007525:Isoelectric Focusing / Q000379:methods; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000378:metabolism*; D011808:Quinone Reductases / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine
D005420:Flavoproteins; D000429:Alcohol Oxidoreductases; C021880:C-nitrosoreductase (NADH); D003579:Cytochrome Reductases; D011808:Quinone Reductases; D009245:NADH Dehydrogenase