title: Spinach nitrate reductase: purification, molecular weight, and subunit composition.
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.
authors: Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, Matsudo, Chiba 271, Japan.
authors: H
authors: H
authors: Nakagawa
authors: Y
authors: Y
authors: Yonemura
authors: H
authors: H
authors: Yamamoto
authors: T
authors: T
authors: Sato
authors: N
authors: N
authors: Ogura
authors: R
authors: R
authors: Sato