Spinach Nitrate Reductase

Abstract

Nitrate reductase was purified .apprx. 3000-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 on 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 80-130 units/m protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S [Svedberg] and a Stokes radius of 6.3 nm, respectively. From these values, a MW 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 MW of 114,000 together with a very faint band possessing a somewhat smaller MW. Spinach nitrate reductase apparently is composed of 2 identical subunits possessing a MW of 110,000-120,000.