Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties.
NADH:nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris has been purified 640-fold with an over-all yield of 26% by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, gel chromatography, density gradient centrifugation, and DEAE-chromatography. The purified enzyme is stable for more than 2 months when stored at minus 20 degrees in phosphate buffer (pH 6.9) containing 40% (v/v) glycerol. After the initial steps of the purification, a constant ratio of NADH:nitrate reductase activity to NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities was observed. One band of protein was detected after polyacrylamide gel electrophoresis of the purified enzyme. This band also gave a positive stain for heme, NADH dehydrogenase, and reduced methyl viologen:nitrate reductase. One band, corresponding to a molecular weight of 100, 000, was detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains FAD, heme, and molybdenum in a 1:1:0.8 ratio. One "cyanide binding site" per molybdenum was found. No non-heme-iron or labile sulfide was detected. From a dry weight determination of the purified enzyme, a minimal molecular weight of 152, 000 per molecule of heme or FAD was calculated. An s20, w of 9.7 S for nitrate reductase was found by the use of sucrose density gradient centrifugation and a Stokes radius of 89 A was estimated by gel filtration techniques. From these values, and the assumption that the partial specific volume is 0.725 cc/g, a molecular weight of 356, 000 was estimated for the native enzyme. These data suggest that the native enzyme contains a minimum of 2 molecules each of FAD, heme, and molybdenum and is composed of at least three subunits.
D001665:Binding Sites; D002501:Centrifugation, Zonal; D002708:Chlorella / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003486:Cyanides / Q000494:pharmacology; D005415:Flavins / Q000032:analysis; D006418:Heme / Q000032:analysis; D007501:Iron / Q000032:analysis; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis; D009243:NAD; D009565:Nitrate Reductases / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013440:Sulfides / Q000032:analysis
D003486:Cyanides; D005415:Flavins; D013440:Sulfides; D009243:NAD; D006418:Heme; D008982:Molybdenum; D007501:Iron; D009565:Nitrate Reductases