Further Purification and Properties of Neurospora Nitrate Reductase

作者：Reginald H. Garrett, Alvin Nason · 发表于：Journal of Biological Chemistry · 年份：1969 · DOI：10.1016/s0021-9258(18)91707-2 · 被引用次数：265 · 研究领域：Enzyme Catalysis and Immobilization、Wastewater Treatment and Nitrogen Removal、Plant nutrient uptake and metabolism

Abstract Neurospora crassa (5297a) NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2), a soluble, sulfhydryl-containing protein with FAD, a cytochrome b designated cytochrome b557 (N. crassa), and molybdenum as prosthetic groups, has been purified 500-fold by procedures including pH adjustment, ammonium sulfate fractionation of the resultant supernatant solution, phase separation to remove nucleic acids, diethylaminoethyl cellulose chromatography, hydroxylapatite chromatography, and, finally, Sephadex G-200 gel filtration. Other enzymatic activities associated with NADPH-nitrate reductase throughout the purification and maintaining a proportional relationship with it are NADPH-cytochrome c reductase, FADH2-nitrate reductase, and reduced methyl viologen-nitrate reductase. Polyacrylamide gel electrophoresis of the most highly purified enzyme preparations yielded a major buffalo black-staining zone containing the above enzymatic activities, a somewhat smaller zone, and several faint zones. In unstained gels, a red zone, which was apparently due to the presence of cytochrome b557, was visible at the position corresponding to the major buffalo black-staining zone. By the use of sucrose density gradient centrifugation, a relative s20, w0.725 value of 8.0 for the nitrate reductase was found, and with Sephadex G-200 gel filtration techniques, a Stokes radius of 70 A was determined. From the relationship, mol wt = 6πeNas/(1 - vp), a molecular weight of 228,000 was calc...