Purification and properties of assimilatory nitrate reductase [NAD(P)H] from Ankistrodesmus braunii.
Assimilatory nitrate reductase [NAD(P)H] (EC 1.6.6.2) from Ankistrodesmus braunii has been purified to homogeneity by a simple procedure that utilizes as the main step affinity chromatography on Blue-Sepharose. The best enzyme preparation has a specific activity of 61.25 units/mg protein. The enzyme has a sedimentation coefficient of 10.9 S by sucrose-density-gradient centrifugation, and a Stokes radius of 9.8 nm was estimated by gel filtration techniques. Its molecular weight is 460000, but only one single band of 58000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of eight subunits. The nitrate reductase absorption spectrum shows wavelengths maxima at 280 and 416 nm and a broad shoulder at 450 nm. Reduced enzyme shows maxima at 424 (Soret), 527 (beta) and 557 (alpha) nm, and a bleaching at 450 nm. The reduced extracted heme chromophore, in pyridine and KOH, shows absorption bands at 414, 522 and 552 nm. These properties indicate the presence of a b-type cytochrome and flavin as prosthetic groups of A. braunii nitrate reductase. A minimum of four molecules of heme has been calculated per molecule of the enzyme complex. Redox titration of the enzyme shows a midpoint potential for the heme of -73 mV at pH 7.0. In the presence of p-hydroxymercuribenzoate, which inhibits the NAD(P)H-dependent activities of the complex, the enzyme-bound heme can be reduced with dithionite, but not with NAD(P)H.
D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D000460:Chlorophyta / Q000201:enzymology*; D006418:Heme / Q000032:analysis; D008970:Molecular Weight; D050896:Nitrate Reductase (NAD(P)H); D009565:Nitrate Reductases / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis
D013438:Sulfhydryl Compounds; D006418:Heme; D009565:Nitrate Reductases; D050896:Nitrate Reductase (NAD(P)H)