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. 1985 Jan;77(1):124–128. doi: 10.1104/pp.77.1.124

Spinach Nitrate Reductase

Purification, Molecular Weight, and Subunit Composition

Hiroki Nakagawa 1,2, Yasuhiro Yonemura 1,2, Hiroyasu Yamamoto 1,2, Takahide Sato 1,2, Nagao Ogura 1,2, Ryo Sato 1,2
PMCID: PMC1064469  PMID: 16663994

Abstract

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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