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Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties.
J Biol Chem. 1975 Jun 10; 250(11):4120-7.JB

Abstract

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.

Authors

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Pub Type(s)

Journal Article

Language

eng

PubMed ID

165192

Citation

Solomonson, L P., et al. "Reduced Nicotinamide Adenine Dinucleotide-nitrate Reductase of Chlorella Vulgaris. Purification, Prosthetic Groups, and Molecular Properties." The Journal of Biological Chemistry, vol. 250, no. 11, 1975, pp. 4120-7.
Solomonson LP, Lorimer GH, Hall RL, et al. Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties. J Biol Chem. 1975;250(11):4120-7.
Solomonson, L. P., Lorimer, G. H., Hall, R. L., Borchers, R., & Bailey, J. L. (1975). Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties. The Journal of Biological Chemistry, 250(11), 4120-7.
Solomonson LP, et al. Reduced Nicotinamide Adenine Dinucleotide-nitrate Reductase of Chlorella Vulgaris. Purification, Prosthetic Groups, and Molecular Properties. J Biol Chem. 1975 Jun 10;250(11):4120-7. PubMed PMID: 165192.
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TY - JOUR T1 - Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties. AU - Solomonson,L P, AU - Lorimer,G H, AU - Hall,R L, AU - Borchers,R, AU - Bailey,J L, PY - 1975/6/10/pubmed PY - 1975/6/10/medline PY - 1975/6/10/entrez SP - 4120 EP - 7 JF - The Journal of biological chemistry JO - J Biol Chem VL - 250 IS - 11 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/165192/Reduced_nicotinamide_adenine_dinucleotide_nitrate_reductase_of_Chlorella_vulgaris__Purification_prosthetic_groups_and_molecular_properties_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(19)41394-X DB - PRIME DP - Unbound Medicine ER -