Purification and general properties of spinach leaf nitrite reductase

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Nitrite reductase was isolated from spinach leaves. The enzyme was purified 168-fold by a procedure involving extraction with phosphate buffer, gel filtration on Sephadex G-200, ion-exchange chromtography on DEAE-Sephadex A-50, and adsorption on hydroxyapatite. The preparation was homogeneous in the ultracentrifuge with sedimentation coefficient at infinite dilution (s°_<20,w>) of 4.57 S. Disc electrophoresis revealed some small bands together with a major protein band. The molecular weight of the spinach nitrite reductase was estimated to be 60,000 by gel filtration on Sephadex G-100 while a molecular weight of 72,000 was obtained from the sedimentation-diffusion coefficients of the protein. Results of sodium dodecyl sulfate gel electrophoresis suggested that the enzyme molecule consists of two subunits of molecular size of 37,000. After close examination of assay systems based on sodium dithionite-viologen dye procedures, we developed a more elaborate, improved chemical assay method. Some enzymatic properties of the purified nitrite reductase were examined.
日本植物生理学会の論文